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variants showing a more benign motor disease course compared to risk variants. These observed clinical differences could reflect a lower pathogenicity for p.G2019S mutation, however additional genetic and environmental factors beyond mutational status might contribute to these different manifestations. LRRK gene variants and Parkinson disease: Pathogenic mechanisms As a major player in the ALP, pathogenic LRRK2 mutations have been shown to alter lysosomal activity, including late stage endocytosis, lysosome trafficking and synaptic vesicle endocytosis (161). In primary mouse astrocytes, G2019S, R1441C, and Y1699C reduce lysosomal capacity and increase lysosome size, and G2019S also reduces lysosomal pH, which is associated with dysfunctional lysosomal activity (162). Some reports also suggest a gain-of-function mechanism for G2019S involving ER stress and UPR, although the precise mechanisms and how they may underlie PD are poorly understood (163-165). G2019S heterozygous and homozygous mice are reported to exhibit impaired extracellular release of dopamine and profound abnormalities of mitochondria in the striatum (166). More recent studies show that G2019S knock-in mice exhibit increased dopamine transporter levels, dopamine uptake and phosphorylation of α-synuclein from 9 months of age, while LRRK2-KO mice show slight elevation of total αsynuclein immunoreactivity at 23 months of age (167, 168). In addition, G2019S also alter glutaminergic synaptic transmission in midbrain dopaminergic neurons of 10-12 month old (middle-aged) mice which reflects aging before the onset of motor symptoms in PD (169). In astrocyte-dopaminergic neuron co-cultures from G2019S LRRK2-carrying PD patients, astrocytes accumulate α-synuclein and the neurons display shortened neurites and neurodegeneration which are not seen in co-cultures with control-patient-derived astrocytes (170).

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Collectively, this suggests that gain-of-function LRRK2 variants may increase the susceptibility of dopaminergic neurons to degeneration and implicates LRRK2 in α-synuclein clearance and homeostasis in PD pathology. Although most studies focus on gain-of-function LRRK2 variants, large-scale genetic sequencing suggests that loss-offunction variants can also reduce LRRK2 protein levels in around 82% of heterozygous carriers. However, loss-of-function variants may not be strongly associated with a specific PD phenotype (171). This not only further emphasizes the link between increased kinase activity and familial PD, but also highlights the importance of additional research to elucidate both the physiological functions of LRRK2 as well as the precise mechanisms in which LRRK2 variants influence PD risk, onset and progression. Rab proteins linked to LRRK in Parkinson disease LRRK2 kinase has been shown to phosphorylate a subset of GTPases, called Rab GTPases (172). Rab proteins play important roles in vesicle trafficking, regulating the formation, transport, tethering and fusion of vesicles specific to each specific Rab, and dysfunction in Rab-mediated vesicle trafficking has been implicated in PD pathology (173). Although G2019S has been shown to increase phosphorylation of Rab proteins, in vivo assays show that other mutations such as R1441G also enhance Rab phosphorylation by up to 20-fold (172, 174). However, dysfunctional mutant T1348N LRRK2 demonstrates reduced kinase activity, suggesting the importance of GTP-binding in downstream signaling events (175). RAB29, also known as RAB7L1, is contained within the PD-linked PARK16 locus (176,177). RAB29 is thought to be the master regulator of LRRK2, recruiting LRRK2 to the trans-Golgi network and stimulating kinase activity. The

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R1441G/C and Y1699C pathogenic variants have been shown to enhance this recruitment (175), and GTP-binding is thought to be crucial for RAB29-mediated activation of LRRK2. This then triggers downstream phosphorylation of various Rab proteins, such as RAB8A/B and RAB10 (124,178). RAB8A/B and RAB10 have been shown to be involved in primary ciliogenesis, although direct links between LRRK2 and ciliogenesis in PD have yet to be established (124). RAB29, RAB8A, and RAB10 are all implicated in maintaining lysosome homeostasis, and Liu et al. reported that phosphorylated RAB10 may also play a role in phagocytic immune response (179), further supporting any links between LRRK2 and lysosomal dysfunction in PD (180). LRRK gene variants and Parkinson disease: Current and future therapeutic strategies There have been many recent developments in LRRK2targeted strategies in PD, with a strong focus on small molecule LRRK2 kinase inhibitors which has been shown to trigger neuroprotective effects (181, 182). The majority of LRRK2 kinase inhibitors are ATPcompetitive, where the molecules compete with ATP for binding to the ATP-binding pocket in the kinase domain (183,184). MLi-2 is a compound that exhibits exceptional potency and specificity both in vitro and in mouse models, where it has been shown to be well-tolerated with no adverse effects on body weight, food intake or behavior (185,186). Although MLi-2 failed to slow or halt the progression of PD in mice and never reached clinical trials, it is an important compound for researchers to study LRRK2 function and pathobiology. PF-06685360, or PFE-360, also shows high potency, kinase selectivity and good brain permeability

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in rats (187). Two inhibitors [DNL-201 and DNL-151 (NCT03710707 and NCT04056689, respectively, https:// clinicaltrials.gov)] are already in clinical trials (188, 189). However, there are several challenges facing therapies targeting LRRK2 kinase. As LRRK2 protein expression is not limited to only the brain, it is crucial to assess any adverse effects on other systems in the body, such as in kidneys, lungs and immune cells. Preclinical toxicology studies show possible kidney and lung pathology as a results of various LRRK2 inhibitors (185, 187,190,191), and both activation and inhibition of LRRK2 kinase in immune cells have been associated with immune function (192). Another challenge is the current lack of biomarkers and scalable assays that can measure LRRK2 activity in patients. To date, the most promising candidate biomarker is phosphorylated . /fneur. . Rab protein (172,193), as well as levels of auto-phosphorylated LRRK2 at Ser1292 (194). For example, phosphorylated RAB10 has been shown to be significantly increased in the brain of idiopathic PD patients (195). The development of reliable biomarkers is critical for early PD diagnosis (193), patient selection for the enrolment to clinical trials, to identify patients in which LRRK2 inhibition may be most effective and allow for personalized dose adjustments (196). Finally, although increased LRRK2 kinase activity is present in other forms of genetic PD and especially sporadic PD (197), further research must be conducted to assess LRRK2 activity and function in these forms of PD order to assess the viability of LRRK2 inhibitors to treat all types of PD. GBA and LRRK interactions Although there are

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many clear differences between GBA1and LRRK2-associated PD, highlighted in Table 2, there is increasing evidence suggesting a possible interaction between GBA1 and LRRK2 in PD (193). Clinical studies show that individuals carrying both the G2019S LRRK2 variant and a GBA1 variant exhibit symptoms that closely mimic G2019S-LRRK2 PD symptoms and are milder than patients carrying only a GBA1 variant. This includes slower rates of cognitive and motor decline and milder olfactory dysfunction (158). Compound variant carriers may have higher risk of developing PD, coupled with a tendency for a slightly earlier age at onset, compared with patients carrying just one variant and sporadic PD patients (158,(198)(199)(200). This suggests that the G2019S LRRK2 variant might be dominant over pathogenic GBA1 variants, although it could also depend on the varying penetrance of the two genes. In addition, it is also possible that the GBA1/LRRK2-PD patients in the study are exhibiting LRRK2-mediated PD and the GBA1 variants act as a bystander in pathology progression. Biochemical studies appear to support a link between LRRK2 kinase activity and GCase activity. For example, G2019S and R1441G/C variants reduce GCase activity (but not GCase protein levels) in dopaminergic neurons through increased RAB10 phosphorylation (200, 201). However, G2019S and the gain-of-function LRRK2 variant M1646T [association with PD risk is unclear (138, 202)] are reported to increase GCase activity in dried blood spots (203,204). Therefore, the influence of LRRK2 variants on GCase activity appears to be inconsistent between the blood and dopaminergic neurons. However, there are currently a lack of studies focussing on GCase activity

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on LRRK2 which warrants further investigation. In addition, progression and onset are very difficult to study in cell models and compound mutant carriers are extremely rare, posing further difficulties in investigating the convergence of the two pathways. Concluding remarks The discovery of the GBA1 and LRRK2 mutations as the most important genetic risk factors for developing PD has led to enhanced understanding of the underlying causes of PD. Understanding the functional consequences associated with individual variants is imperative to develop highly efficacious gene-targeted therapies to halt or restore neurodegeneration. Further evaluation of GBA1 and LRRK2 variants and clinical presentation, as well as investigations into interactions between the two genes, is needed to develop biomarkers for early diagnosis and intervention and treatment of PD. Research University College London Hospitals Biomedical Research Centre. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher's note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

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An effect analysis of changes in the composition of the water ecological footprint in Jiangyin City, China Ecological footprint (EF) analysis is an effective tool to assess ecosystem appropriation, but weaknesses still exist. In order to estimate a city's ecosystem appropriation, we made two modifications to EF: 1) including different types of freshwater area in the calculating framework; and 2) re-adjusting all equivalent factors in terms of different types of freshwater. The modifications are mainly concerned with changes in the composition of freshwater areas in EF analysis; that is, the proportion of aquaculture freshwater and tap freshwater. An assumption of 25% of aquaculture freshwater was used to conduct a sensitivity test. It shows that variation of the proportion leads to different equivalent and yield factors for tap freshwater areas. The variations not only change the EF of freshwater but also the total EF. The modified EF calculation served to assess ecosystem appropriation in Jiangyin city in 1998. By comparing the original and the modified EF, it was found that the ecological deficit was larger (2.19 ha per capita) in the modified calculation than in the original (2.16 ha per capita). The original calculation is an underestimate because direct consumption of freshwater was not fully included in its calculating framework. From the modified calculation, the EF for different categories of ecosystems decreased, from fossil energy land, arable land and pasture to built-up areas, which together occupy about 97.58% of the total EF. Fossil energy land occupied 68.55% of the total, while freshwater areas took only 0.62%. The

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main ecological deficits were from fossil energy land, arable land, pasture and marine areas. Ecological supply is lacking within Jiangyin and relies on ecosystems outside the city to survive. Ecosystem management is indispensable to optimise the efficiency of ecosystem use and to expand the biological capacity of the city. The modified EF could provide a more reasonable instrument in policy-making and implementing sustainable management of freshwater ecosystems. INTRODUCTION Ecosystem appropriation has become an important topic in the last 10 years. Its involves two flows (Vitousek et al. 1986;Postel et al. 1996), and (2) estimating the appropriation of ecosystems at spatial scales, such as the ecological footprint (EF) (Wackernagel and Rees 1996;Wackernagel et al. 1999a,b;Lasson et al. 1994;Hu 2000;Deutsch et al. 2000). The spatial-based approach indicates ecosystem appropriation using the bio-productive land area. This approach has two obvious advantages: (1) simplicity in calculation, and (2) high applicability in human-environmental interaction analysis. The method also has some obvious weaknesses and has received criticism (van den van Kooten and Bulte 2000;Rees 2000;Ayres 2000) and some modifications (Jansson et al. 1999, Rees 2000Simmons and Lewis 2000;Haberl et al. 2001;Lenzen and Murray 2001;Hu 2002). The improvements focus on: (1) including more categories of ecosystems, such as freshwater areas and other ecosystems with low bioproductivity, adding them to component-based or land disturbance-based real areas, or leaving them in individual categories of land but aggregating them by weights of bio-productivity (Folke et al. 1997;Deutsch et al. 2000;Simmons and Lewis 2000;Lenzen and Murray 2001); (2) introducing other techniques of flux analysis for mass and

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energy, such as input-output analysis, into the calculation (Lenzen and Murray 2001); (3) including more greenhouse gas emissions other than CO 2 into EF analysis (Lenzen and Murray 2001); and (4) injecting fisheries and biomass substitution into the calculation (Wackernagel et al. 2005). In this paper, we have modified the calculation of the freshwater EF based on the original calculation framework proposed by Wackernagel and colleagues (Wackernagel and Rees 1996;Wackernagel et al. 1999a,b), making it more applicable for different conditions. The focus of our modification is to include two different types of freshwater areas (areas of aquaculture freshwater and tap freshwater) into the EF calculation. In addition, a case study using the modified method provides a comparison of the difference in results between the original and the modified calculation. Finally, a sensitivity test served to examine how changes in the composition of freshwater areas, expressed as the proportion of the two types of freshwater areas, influences the results of EF calculation. Ecosystem appropriation in Jiangyin in recent decades Jiangyin is on the south bank of the Yangtze Riverthe largest river in China. The high proportion of water areas in its territory plays a crucial role in its development, and water ecosystem appropriation is one of the main factors affecting its natural pattern of ecosystems services ( Figure 1). Jiangyin became an administrative municipality in 1987 and, since then, there has been enormous ecosystem appropriation caused by urban development. Scarcity in ecosystems services has become very severe, creating a special challenge for ecosystems management, where rapid urban

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development has caused an obvious reduction in the size of natural ecosystems in the last 30 years ( The original framework of EF calculation The first step in EF calculation for a given administrative area is to estimate the per capita area of appropriated ecosystems (aa) with the provision of ith item of major products or services by dividing average annual consumption of ith item ('c' in kg per capita) by its average annual productivity or yield ('p' in kg/ha): In practice, it is often only possible to estimate per capita consumption by dividing aggregate consumption by total population (N). Of course, many items of consumption (e.g. clothing and furniture) come from several inputs and it is useful to estimate separately the area appropriated by each significant input. The total per capita EF ('ef') can then be calculated by summing all areas of ecosystems appropriated by individual items in the annual shopping basket of goods and services: Thus, EF for a certain size of population is the per capita footprint multiplied by the population size (N): EF= N(ef). It is useful to separate consumption into five major categories: food, water, energy, other consumer goods and services. These categories can be subdivided as necessary. The 'embodied' energy and raw materials of a commodity is the total quantities of energy and materials used during the life cycle of that commodity, for its manufacture, transport and disposal. 'Energy intensity' refers to the direct embodied energy per unit of a good or service. Similarly, we can speak of the 'embodied

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EF' of a commodity as its contribution to the consumer EF. Modified EF calculation Modification of the original EF calculation is necessary to fully consider EF analysis of freshwater ecosystems. Our proposed modifications are outlined below. EF of freshwater in the calculation framework Inland waters add another 0.3 billion ha, giving 2.3 billion ha of potential fisheries out of the 36.6 billion ha of ocean and inland waters on our planet (FAO 1999). The majority of marine fish catches occur on continental shelves, and excluding inaccessible or unproductive waters leaves 2.0 billion ha. Although a fraction of the ocean's 36.3 billion ha, these 2.0 billion ha provide over 95% of marine fish catches (WRI 2000). The fishing footprint assumes additional by-catch according to the species composition of national or local fish catches. Inland water and continental shelf areas have replaced the EEZ (Exclusive Economic Zone) to give a far more accurate distribution of global fishing capacity (Wackernagel et al. 2005). Water ecological footprint Hu et al. International Journal of Sustainable Development & World Ecology 213 Period 1950-1960 1970-1980 1980-1990 1990-1998 Total urban area ( Freshwater areas are important for human survival. They include inland areas such as rivers, lakes, reservoirs and other types of catchment, and differ from other types of ecosystem such as forest, grassland and arable land. As shortage of freshwater is a worldwide problem, freshwater ecosystem appropriation should not be omitted in any EF analysis. Therefore, two types of freshwater are considered in our modification: 1) areas for aquaculture fishery, and 2) areas

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for water supply to households, industries and capture fishery (abbreviated as tapwater). Agricultural water uses are not included here to avoid double counting, since they are possibly already included in cropland, grassland, forest and so on. The two kinds of freshwater area obviously differ from each other in bioproductivity, and they perform many ecological services for human welfare. Similar to the original EF calculation, the supply and demand of freshwater ecosystems in terms of spatial area may be obtained according to their equivalent and yield factors. In our calculation, each EF (or the demand for aquaculture and tapwater) is calculated by converting the consumption quantity into freshwater ecosystem area as a global unit, and then summing the numbers (see Appendix I). This separate calculation leads to a higher global equivalent amount of freshwater EF, but a lower equivalent amount of freshwater supply in comparison with estimates from the compound calculation using average values of equivalent and yield factors for freshwater. Proportion of the two types of freshwater areas In conducting analysis of water EF, some scholars have included aquaculture freshwater areas; our modifications include both aquaculture and tapwater areas. Additionally, the area of freshwater for aquaculture is assumed to be approximately 25% of the total area of inland freshwater on a global scale; therefore, tapwater area should occupy the other 75%. This hypothesis is applied to calculate global bioproductivities for aquaculture and tapwater. Fundamental ecological water uses and re-uses of wastewater The fundamental ecological water uses of freshwater are the minimum amount of water used to

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sustain the stability and resilience of local freshwater ecosystems (including their biodiversity). Wackernagel et al. (2005) suggested that 12% of the global surface is required for the other 30 million species on Earth, but they did not clearly specify the requirements for freshwater ecosystems maintenance and this is possibly an underestimate of real water demand in freshwater ecosystems. Therefore, the concept of fundamental ecological water uses is introduced to estimate the actual amount required for freshwater ecosystem maintenance including hydro-biodiversity conservation. Our EF calculation assumed that 25% of the available freshwater (not the area of freshwater) is conserved for fundamental ecological water uses. The actual area needed for freshwater ecosystem maintenance can be determined from basic data on local hydrological balance and is approximately 12% of the local freshwater area. In our calculation, the amount of local fundamental ecological water uses is deducted from the total available local water stock. In addition, the reuse ratio of wastewater was determined to be 40% for living and industrial uses, according to statistical data for Jiangyin city. Adjustments to the parameters of equivalence factors for global bioproductive land According to Wackernagel and colleagues (2005), equivalence factors represent the world average potential productivity of a given bioproductive area relative to the world average potential productivity of all bioproductive areas. For example, the equivalence factor for crops describes potential biomass output attainable in an area with an assumed level of inputs, such as water and fertiliser. Cropland is more productive than rangeland or pasture, so it has a larger value for

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the equivalence factor than pasture. The equivalence factors for cropland, forest, pasture and built-up area in Wackernagel's calculation are derived from the suitability index of the Global Agro-Ecological Zones (GAEZ) in 2000, a spatial model of potential agricultural yields (Wackernagel et al. 2005). In this calculation, EF of fisheries was obtained from their capacity to supply animal protein relative to that of grassland. In terms of the inclusion of two freshwater areas, we readjusted the equivalence factors according to differences in bioproductivity between freshwater and other categories of bioproductive areas. In our calculation, different types of ecosystems were calculated according to FAO (1999FAO ( , 2000 and WRI (2000WRI ( , 2001 original data for agriculture, fisheries and freshwaters. The values of equivalence factors from our modified calculation are very similar to those of Wackernagel, except for marine areas, and differences mainly come from differences in data sources ( Table 2). The productivity of global average bioproductive land is lower when freshwater ecosystem appropriation is introduced into the calculating framework (Table 2), since the productivity of freshwater is lower than that of arable land, forest or marine areas. In addition, the equivalence factors for various types of ecosystems are a little higher than those calculated without considering the two types of freshwater areas. RESULTS AND DISCUSSION Rapid development of Jiangyin relies on raw materials and energy that come directly or indirectly from natural ecosystems that release waste substances into the surroundings, causing negative effects or even damage to ecosystem service flows. However, the city often lacks

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necessary economic mechanisms to sustain its ecological service flows, leading to rapid uncontrolled shifts in natural ecosystems to built-up areas that become larger, while natural areas become smaller. Based on statistical data for Jiangyin (Statistical Bureau of Jiangyin, 1998; Jiangyin Land Chorography Compiling Committee 1998), annual ecosystem appropriation for 1998 was estimated by employing modified calculations (available from the authors). The main characteristics of EF in the city are: 1. Main products: including products consumed by city residents, such as beef, poultry, eggs, milk, fruit, aquatic products, sugar, cake, edible oil, fat, silk, tea, tobacco, alcoholic drinks, rubber products and timber. Indigenous natural ecosystems cannot provide enough products and raw materials, so some products are imported. 2. Freshwater: there is 47.34 million m 3 of surface freshwater annually available for the city; the supply of freshwater mainly relies on inflows from the Yangtze River. The annual water imported is 0.83 billion m 3 , and about 0.32 billion m 3 of this freshwater serves households and industries. 3. Energy: total energy consumption was 81.26 Pj in 1998, mainly coal, liquid or gas fossil fuels and hydroelectricity, and was imported from outside the city. Energy consumption of fossil fuels was 70.09 Pj, 86.26% of the total. The embodied energy was 1.93 Pj in imported goods and 15.15 Pj in exported goods, and 68.39 Pj in total net import of various products (including energy). EF of the city from the original calculation The EF of Jiangyin was calculated from the original calculation (Wackernagel et al. 2002(Wackernagel et al.

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, 2005 and the results are shown in EF of the city from the modified calculation There was a 2.19-ha ecological deficit per capita, larger than that in the original calculation, and the ecological deficit was in fossil energy land, arable land, pasture, forest, marine areas and aquaculture areas (Table 4). Fossil energy land had the greatest deficit, followed by arable land. The four main types of appropriated ecosystem decreased gradually from fossil energy land, arable land and pasture to built-up area, and occupied 97.58% of the total EF ( Comparison of EF analysis between the modified and original calculation The introduction of freshwater, especially tapwater, into the EF calculation framework changed the equivalent factors and yield factors, and thus made the results of EF calculation different ( Sensitivity test for the assumption of 25% included in the modification The modification to the original EF calculation was based on an important assumption: the area of aquaculture freshwater is about 25% of total inland freshwater. By examining variations in the proportion of aquaculture areas from 15 to 35%, should affect the values of equivalence factors and yield factors; therefore, a sensitivity test was conducted to show how deviations in EF calculation occur. Variation in the equivalence factor for aquaculture areas drops from 66.67% (a deviation of −10%) to −28.57% (a deviation of +10%), and that for tapwater areas declines from −11.76% (a deviation of −10%) to 15.38% (a deviation of +10%). Additionally, variation in the yield factor for aquaculture areas drops from −40.00% (a deviation of −10%)

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to 40.00% (a deviation of +10%), and variation in the yield factor for tapwater areas decreases from 13.33% (a deviation of −10%) to −13.33% (a deviation of +10%). This showed that the variation relative to the assumption of 25% has a greater effect on the equivalence and yield factors of aquaculture areas than on tapwater areas. For EF and ecological deficits, variation of per capita EF drops from −0.0036% (a deviation of −10%) to 0.0047% (a deviation of +10%), and variation in per capita supply remains invariant. The variation in per capita deficits drops from −0.0039% (a deviation of −10%) to 0.0051% (a deviation of +10%). This showed that the variation relative to the assumption of 25% has a relatively greater effect on per capita EF or demand and ecological deficits than on per capita supply. However, this effect is quite subtle (less than ± 0.01%). CONCLUSIONS This research proposed modifications to the EF calculation. The modifications mainly dealt with two types of freshwater area into the original calculation. Compared with the original calculation, the modified form produced more appropriation and more ecological deficits, which consequently reflects much more exactly the situation for ecosystem appropriation by the city's development, and so reduces the underestimate. Including two types of freshwater area in the original calculation had an effect on the results of EF calculation. Due to lower productivity of freshwater than some other types of ecosystem, such as arable and forest, the inclusion of two types of freshwater area decreases the productivity of global average bioproductive land;

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thus, for the other types of ecosystem, the equivalence factors are a little higher than those without considering freshwater. The change in the proportion between areas of aquaculture and tapwater areas led to variations in equivalence and yield factors for freshwater areas, and had a greater influence on equivalence and yield factors for aquaculture areas than for tapwater areas. The variations relative to the assumption of 25% as the proportion of aquaculture freshwater would not only change the EF of freshwater areas but also the total EF of the city, although not the supply of freshwater to the city. However, the influences of the variations relative to the assumption of 25% are not great. Jiangyin's development has exerted great pressure on local ecosystems and greater dependence on external resources in maintaining the city's development. Table 5 Deviations of EF between the modified and original calculations ecosystems services, ecosystems management is indispensable for the city to optimise the spatial distribution of ecosystem uses and enhance their efficiency (van den Bergh and Verbruggen 1999, Hu 1998Hu , 2002Wackernagel et al. 1999aWackernagel et al. , 2002. The modified EF calculation offers a better tool to help understand the city's condition in terms of ecosystem appropriation and promotes the implementation of sustainable ecosystems management. 3) Equivalence factor for aquaculture freshwater areas = bioproductivity of global aquaculture freshwater areas (kg/ha/yr)/bioproductivity of global average bioproductive areas (kg/ha/yr); 4) Yield factor for local aquaculture freshwater areas = bioyield of local aquaculture freshwater areas (kg/ha/yr)/bioyield of global aquaculture freshwater areas (kg/ha/yr); 5) Yield factor

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of local tapwater areas = yield of local tapwater areas (kg/ha/yr)/bioyield of global tapwater areas (kg/ha/yr); 6) Average water output for global tapwater areas (kg/ha/yr) = total water amount available to global tapwater areas (kg/yr)/total area of global tapwater areas (ha/yr), this value contains fundamental ecological water uses of tapwater areas. Sensitivity test for main parameters and variables of EF calculation The sensitivity test examines how key parameters and variables of EF calculation, such as equivalence factors, yield factors, demand, supply and deficits change when the assumed value for the area of freshwater for aquaculture is about 25% of the total area of inland freshwater at a global scale vary ± 10%. 1) Variations of equivalence or yield factors relative to the assumed 25% = [equivalence or yield factor of any type of freshwater area at the assumed value of any percentageequivalence or yield factor at the assumed value of 25%]/ equivalence or yield factor at the assumed value of 25% *100%. 2) Variations of per capita demand, supply and ecological deficits relative to the assumption of 25% = [per capita demand, supply and ecological deficits at the assumed value of any percentage -per capita demand, supply and ecological deficits at the assumed value of 25%] / per capita demand, supply and ecological deficits at the assumed value of 25% *100%. 1) Per capita EF of aquaculture freshwater areas = total local consumption of aquatic products (kg/yr)/bioyield of global aquaculture freshwater areas (kg/ha/yr) * equivalence factor for aquaculture freshwater areas/total local population; EF and supply calculation

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for freshwater areas in equivalent units 2) Per capita EF of tapwater areas International Journal of Sustainable Development & World Ecology 221 = total local freshwater consumption (kg/yr) average water output for global tapwater areas (kg/ha/yr) * equivalence factor for tapwater areas/total local population; 3) Total per capita EF of freshwater areas = per capita EF of aquaculture freshwater areas + per capita EF of tapwater areas; 4) Per capita supply of tapwater areas = total local tapwater areas (ha/yr)* equivalence factor for tapwater areas * yield factor for local tapwater areas/total local population. As Wackernagel et al. suggested (1999, 2002, 12% of bioproductive areas is conserved for biodiversity maintenance in EF calculations, but this is not completely applicable in our case study. In our calculation, when the percentage of freshwater areas actually needed for fundamental ecological water uses (hydrobiodiversity maintenance requirement already included) is less than 12%, this assumption is applicable; but when this value is more than 12%, the assumption is not applicable and the percentage actually needed for local fundamental ecological water uses is adopted.

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Automatic extraction of systematic polysemy using tree-cut This paper describes an automatic method for extracting systematic polysemy from a hierarchically organized semantic lexicon (WordNet). Systematic polysemy is a set of word senses that are related in systematic and predictable ways. Our method uses a modification of a tree generalization technique used in (Li and Abe, 1998), and generates a tree-cut, which is a list of clusters that partition a tree. We compare the systematic relations extracted by our automatic method to manually extracted WordNet cousins. Introduction In recent years, several on-line broad-coverage semantic lexicons became available, including LDOCE (Procter, 1978), WordNet (Miller, 1990) and HECTOR . (Kilgarriff, 1998a). These lexicons have been used as a domainindependent semantic resource as well as an evaluation criteria in various Natural Language Processing (NLP) tasks, such as Information Retrieval (IR), Information Extraction (IE) and Word Sense Disambiguation (WSD). However, those lexicons are rather complex. For instance, WordNet (version 1.6) contains a total of over 120,000 words and 170,000 word senses, which are grouped into around 100,000 synsets (synonym sets). In addition to the size, word entries in those lexicon are often polysemous. For instance, 20% of the words in Wordnet have more than one sense, and the average number of senses of those polysemous words is around 3. Also, the distinction between word senses tends to be ambiguous and arbitrary. For example, the following 6 senses are listed in WordNet for the noun "door": 1. door -a swinging or sliding barrier 2. door -the space in a wall 3. door

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-anything providing a means of access (or escape) 4. door -a swinging or sliding barrier that will close off access into a car 5. door -a house that is entered via a door 6. door -a room that is entered via a door Because of the high degree of ambiguity, using such complex semantic lexicons brings some serious problems to the performance of NLP systems. The first, obvious problem is the computational intractability: increased processing time needed to disambiguate multiple possibilities will necessarily slow down the system. Another problem, which has been receiving attention in the past few years, is the inaccuracy: when there is more than one sense applicable in a given context, different systems (or human individuals) may select different senses as the correct sense. Indeed, recent studies in WSD show that, when sense definitions are fine-grained, similar senses become indistinguishable to human annotators and often cause disagreement on the correct tag (Ng et al., 1999;Veronis, 1998;Kilgarriff, 1998b). Also in IR and IE tasks, difference in the correct sense assignment will surely degrade recall and precision of the systems. Thus, it is apparent that, in order for a lexicon to be useful as an evaluation criteria for NLP systems, it must represent word senses at the level of granularity that captures human intuition. In Lexical Semantics, several approaches have been proposed which organize a lexicon based on systematic polysemy: 1 a set of word senses that are related in systematic and predictable ISystematic polysemy (in the sense we use in this paper) is also

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referred to as regular polysemy (Apresjan, 1973) or logical polyseray (Pustejovsky, 1995). ways (e.g. ANIMAL and MEAT meanings of the word "chicken"). 2 In particular, (Buitelaar, 1997(Buitelaar, , 1998 identified systematic relations that exist between abstract semantic concepts in the WordNet noun hierarchy, and defined a set of underspecified semantic classes that represent the relations. Then he extracted all polysemous nouns in WordNet according to those underspecified classes and built a lexicon called CORELEX. For example, a CORELEX class AQU (which represents a relation between ARTIFACT and QUANTITY) contains words such as "bottle", "bucket" and "spoon". Using the abstract semantic classes and organizing a lexicon based on systematic polysemy addresses the two problems mentioned above in the following ways. For the first problem, using the abstract classes can reduce the size of the lexicon by combining several related senses into one sense; thus computation becomes more efficient. For the second problem, systematic polysemy does reflect our general intuitions on word meanings. Although the distinction between systematic vs. non-systematic relations (or related vs. unrelated meanings) is sometimes unclear, systematicity of the related senses among words is quite intuitive and has been well studied in Lexical Semantics (for example, (Apresjan, 1973;Cruse, 1986;Nunberg, 1995;Copestake and Briscoe, 1995)). However, there is one critical issue still to be addressed: the level of granularity at which the abstract classes are defined. The problem is that, when the granularity of the abstract classes is too coarse, systematic relations defined at that level may not hold uniformly at more fine-grained levels (Vossen et al.,

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1999). For instance, the CORELEX class AQU mentioned above also contains a word "dose" .3 Here, the relation between the senses of "dose" is different from that of "bottle", "bucket" and "spoon", which can be labeled as CONTAINER-CONTAINERFUL relation. We argue that human intuitions can distinguish meanings 2Note that systematic polysemy should be contrasted with homonymy which refers to words which have more than one unrelated sense (e.g. FINANCIAL_INSTITUTION and SLOPING_LAND meanings of the word "bank"). 3Senses of "dose" in WordNet are: (1) a measured portion of medicine taken at any one time, and (2) the quantity of an active agent (substance or radiation) taken in or absorbed at any one time. at this level, where differences between the systematic relations are rather clear, and therefore lexicons that encode word senses at this level of granularity have advantages over fine-grained as well as coarse-grained lexicons in various NLP tasks. Another issue we like to address is the ways for extracting systematic polysemy. Most often, this procedure is done manually. For example, the current version of WordNet (1.6) encodes the similarity between word senses (or synsets) by a relation called cousin. But those cousin relations were identified manually by the WordNet lexicographers. A similar effort was also made in the EuroWordnet project (Vossen et al., 1999). However, manually inspecting a large, complex lexicon is very time-consuming and often prone to inconsistencies. In this paper, we propose a method which automatically extracts systematic polysemy from a hierarchically organized semantic lexicon (WordNet). Our method uses a modification of a

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tree generalization technique used in (Li and Abe, 1998), and generates a tree-cut, which is a list of clusters that partition a tree. Then, we compare the systematic relations extracted by our automatic method to the WordNet cousins. Preliminary results show that our method discovered most of the WordNet cousins as well as some more interesting relations. 2 Tree Generalization using Tree-cut and MDL Before we present our method, we first give a brief summary of the tree-cut technique which we adopted from (Li and Abe, 1998). This technique is used to acquire generalized case frame patterns from a corpus using a thesaurus tree. Tree-cut Models A thesaurus tree is a hierarchically organized lexicon where leaf nodes encode lexical data (i.e., words) and internal nodes represent abstract semantic classes. A tree-cut is a partition of a thesaurus tree. It is a list of internal/leaf nodes in the tree, and each node represents a set of all leaf nodes in a subtree rooted by the node. Such set is also considered as a cluster. 4 Clusters in a tree-cut exhaustively cover all leaf nodes of the tree, and they are mutually disjoint. For example, for a thesaurus tree in Figure 1 . Thus, a treecut corresponds to one of the levels of abstraction in the tree. Using a thesaurus tree and the idea of treecut, the problem of acquiring generalized case frame patters (for a fixed verb) from a corpus is to select the best tree-cut that accounts for both observed and unobserved case frame instances. In

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(Li and Abe, 1998), this generalization problem is viewed as a problem of selecting the best model for a tree-cut that estimates the true probability distribution, given a sample corpus data. Formally, a tree-cut model M is a pair consisting of a tree-cut F and a probability parameter vector O of the same length, where F and ® are: (1) F= [Cx,..,Ck], O=[P(C,),..,P(Ck)] (2) words, that is, P(C) = ~=1 P(nj). Here, compared to knowing all P(nj) (where 1 < j < m) individually, knowing one P(C) can only facilitate an estimate of uniform probability distribution among members as the best guess, that is, P(nj) = P(C) for all j. Therefore, in general, m when clusters C1..Cm are merged and generalized to C according to the thesaurus tree, the estimation of a probability model becomes less accurate. The MDL Principle To select the best tree-cut model, (Li and Abe, 1998) uses the Minimal Description Length (MDL) principle (Rissanen, 1978). The MDL is a principle of data compression in Information Theory which states that, for a given dataset, the best model is the one which requires the minimum length (often measured in bits) to encode the model (the model description length) and the data (the data description length). For the problem of case frame generalization, the MDL principle fits very well in that it captures the trade-off between the simplicity of a model, which is measured by the number of clusters in a tree-cut, and the goodness of fit to the data, which is measured by the

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estimation accuracy of the probability distribution. The calculation of the description length for a tree-cut model is as follows. Given a thesaurus tree T and a sample S consisting of the case frame instances, the total description 0.2]). Note that P(C) is the probability of cluster C = {nl, .., nm) as a whole. It is essentially the sum of all (true) probabilities of the member 4A leaf node is also a cluster whose cardinality is 1. L(M,S)=L(F)+L(eT)+L(SJF, e) (3) where L(F) is the model description length, L(OIF) is the parameter description length (explained shortly), and L(SIF , O) is the data description length. Note that L(F) + L(OIF ) essentially corresponds to the usual notion of the model description length. where G is the set of all cuts in T, and IG I denotes the size of G. This value is a constant for • SFor justification and detailed explanation of these formulas, see (Li and Abe, 1998). all models, thus it is omitted in the calculation of the total length. The parameter description length L(OIF ) indicates the complexity of the model. It is the length required to encode the probability distribution of the clusters in the tree-cut F. It is calculated as where k is the length of ®, and IS[ is the size of S. Finally, the data description length L(SIF, O) is the length required to encode the whole sample data. It is calculated as where, for each n E C and each C E F, and P(n)-P(C) ICl P(C)-f(c) (8)

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ISl Note here that, in (7), the probability of C is divided evenly among all n in C. This way, words that are not observed in the sample receive a non-zero probability, and the data sparseness problem is avoided. Then, the best model is the one which requires the minimum total description length. Figure 2 shows the MDL lengths for all five tree-cut models that can be produced for the thesaurus tree in Figure 1. The best model is the one with the tree-cut [AIRCRAFT, ball, kite, puzzle] indicated by a thick curve in the figure. Generalization Technique Using the generalization technique in (Li and Abe, 1998) described in the previous section, we wish to extract systematic polysemy automatically from WordNet. Our assumption is that, if a semantic concept is systematically related to another concept, words that have one sense under one concept (sub)tree are likely to have another sense under the other concept (sub)tree. To give an example, Figure 3 shows parts of WordNet noun trees for ARTIFACT and MEASURE, where subtrees under CONTAINER and C0NTAINERFUL respectively contain "bottle", "bucket" and "spoon". Note a dashed line in the figure indicates an indirect link for more than one level. Based on this assumption, it seems systematic polysemy in the two trees can be extracted straight-forwardly by clustering each tree according to polysemy as a feature, and by matching of clusters taken from each tree. 6 To this end, the notion of tree-cut and the MDL principle seem to comprise an excellent tool. However, we must be

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careful in adopting Li and Abe's technique directly: since the problem which their technique was applied to is fundamentally different from ours, some procedures used in their problem may not have any interpretation in our problem. Although both problems are essentially a tree generalization problem, their problem estimates the true probability distribution from a random sample of examples (a corpus), whereas our problem does not have any additional data to estimate, since all data (a lexicon) is already known. This difference raises the following issue. In the calculation of the data description length in equation (6), each word in a cluster, observed or unobserved, is assigned an estimated probability, which is a uniform fraction of the probability of the cluster. This procedure does not have interpretation if it is applied to our problem. Instead, we use the distribution of feature frequency proportion of the clusters, and calculate the data description length by the following formula: C1,..,Ck], 0 = [P(C,),.., P(Ck)]. This corresponds to the length required to encode all words in a cluster, for all clusters in a tree-cut, assuming Huffman's algorithm (Huffman, 1952) assigned a codeword of length -log2P(Ci) to each cluster C/ (whose propor-6We could also combine two (or possibly more) trees into one tree and apply clustering over that tree once. In this paper, we describe clustering of two trees for example purpose. tion is P(Ci) = .~_~_d~ Isl J" All other notions and formulas are applicable to our problem without modification. Clustering Method Our clustering method uses the the modified generalization technique

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described in the last section to generate tree-cuts. But before we apply the method, we must transform the data in Wordnet. This is because WordNet differs from a theaurus tree in two ways: it is a graph rather than a tree, and internal nodes as well as leaf nodes carry data, First, we eliminate multiple inheritance by separating shared subtrees. Second, we bring down every internal node to a leaf level by creating a new duplicate node and adding it as a child of the old node (thus making the old node an internal node). After trees are transformed, our method extracts systematic polysemy by the following three steps. In the first step, all leaf nodes of the two trees are marked with either 1 or 0 (1 if a node/word appears in both trees, or 0 otherwise), In the second step, the generalization technique is applied to each tree, and two tree-cuts are obtained. To search for the best tree-cut, instead of computing the description length for M1 possible tree-cuts in a tree, a greedy dynamic programming algorithm is used. This algorithm , called Find-MDL in (Li and Abe, 1998), finds the best tree-cut for a tree by recursively finding the best tree-cuts for all of its subtrees and merging them from bottom up. This algorithm is quite efficient, since it is basically a depth-first search with minor overhead for computing the description length. Finally in the third step, clusters from the two tree-cuts are matched up, and the pairs which have substantial overlap are

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selected as systematic polysemy. Figure 4 shows parts of the final tree-cuts for ARTIFACT and MEASURE obtained by our method. ~ In both trees, most of the clusters in the tree-cuts are from nodes at depth 1 (counting the root as depth 0). That is because the tree-cut technique used in our method is sensitive to the structure of the tree. More specifically, the MDL principle inherently penalizes a complex tree-cut by assigning a long parameter length. Therefore, unless the entropy of the feature distribution is large enough to make the data length overshadow the parameter length, simpler tree-cuts partitioned at abstract levels are preferred. This situation tends to happen often when the tree is bushy and the total feature frequency is low. This was precisely the case with ARTIFACT and MEASURE, where both Tin the figure, bold letters indicate words which are polysemous in the two tree. Related vs. Unrelated Clusters Of the cluster pairs we extracted automatically, not all are systematically related; some are unrelated, homonymous relations. They are essentially false positives for our purposes. Table 1 shows the number of related and unrelated relations in the extracted cluster pairs. Although the results vary among category combinations, the ratio of the related pairs is rather low: less than 60% on average. There are several reasons for this. First, there are some pairs whose relations are spurious. For example, in ARTIFACT-GROUP class, a pair [LUMBER, SOCIAL_GROUP] was extracted. Words which are common in the two clusters are "picket", "board" and "stock". This relation is obviously

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homonymous. Second, some clusters obtained by tree-cut are rather abstract, so that pairing two abstract clusters results in an unrelated pair. For example, in ARTIFACT-MEASURE class, a pair [INSTRUMENTALITY, LINEAR_UNIT] was selected. Words which are common in the two clusters include "yard", "foot" and "knot" (see the previous Figure 4). Here, the concept INSTRUMENTALITY is very general (at depth 1), and it also contains many (polysemous) words. So, matching this cluster with another abstract cluster is likely to yield a pair which has just enough overlapping words but whose relation is not systematic. In the case of [INSTRUMENTALITY, LINEAR_UNIT], the situation is even worse, because the concept of LINEAR_UNIT in MEASURE represents a collection of terms that were chosen arbitrarily in the his- Automatic vs. Manual Clusters To compare the cluster pairs our method extracted automatically to manually extracted clusters, we use WordNet cousins. A cousin relation is relatively new in WordNet, and the coverage is still incomplete. However, it gives us a good measure to see whether our automatic method discovered systematic relations that correspond to human intuitions. A cousin relation in WordNet is defined between two synsets (currently in the noun trees only), and it indicates that senses of a word that appear in both of the (sub)trees rooted by those synsets are related, s The cousins were manuMly extracted by the WordNet lexicographers. Table 2 shows the number of cousins listed for each top relation class and the number of cousins our automatic method recovered (in the 'Auto' column). As you see, the

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total recall ratio is over 80% (27/33~ .82). In the right three columns of Table 2, we also show the breakdown of the recovered cousins, whether each recovered one was an exact match, or it was more general or specific than the corresponding WordNet cousin. From this, we can see that more than half of the recovered cousins were more general than the WordNet cousins. That is partly because some WordNet cousins have only one or two common words. For example, a WordNet cousin [PAINTING, COLORING_MATERIAL] in ARTIFACT-SUBSTANCE has only one common word "watercolor". Such SActually, cousin is one of the three relations which indicate the grouping of related senses of a word. Others are sister and twin. In this paper, we use cousin to refer to all relations listed in "cousin.tps" file (available in a WordNet distribution). 26 a minor relation tends to be lost in our tree generalization procedure. However, the main reason is the difficulty mentioned earlier in the paper: the problem of applying the tree-cut technique to a bushy tree when the data is sparse. In addition to the WordNet cousins, our automatic extraction method discovered several interesting relations. Table 3 shows some examples, 5 Conclusions and Future Work In this paper, we proposed an automatic method for extracting systematic polysemy from WordNet. As we reported, preliminary results show that our method identified almost all WordNet cousins as well as some new ones. One difficulty is that applying the generalization technique using the MDL principle to the bushy WordNet trees seems to

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yield a tree-cut at rather abstract level. For future work, we plan to compare the systematic relations extracted by our automatic method to corpus data. In particular, we like to test whether our method extracts the same groups of senses which human annotators disagreed (Ng et al., 1999). We also like to test whether our method agrees with the finding that multiple senses which occur in a discourse are often systematically polysemous (Krovetz, 1998).

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Development of Verified Innovation Process for Healthcare Solutions (VIPHS): A Stepwise Model for Digital Health . Many digital health projects often stop in the pilot or test phase. Realisation of new digital health services is often challenging due to lack of guidelines for the step-by-step roll-out and implementation of the systems when changing work processes and procedures are needed. This study describes development of the Verified Innovation Process for Healthcare Solutions (VIPHS) – a stepwise model for digital health innovation and utilisation using service design principles. A multiple case study (two cases) involving participant observation, role play, and semi-structured interviews were conducted for the model development in prehospital settings. The model might be helpful to support realisation of innovative digital health projects in a holistic, disciplined, and strategic way. Introduction It is often difficult to go from a project phase to an operational implementation phase.Especially the failure rate of digital transformation projects is huge.The reason why 70 percent of all digital transformations fail is a lack of discipline in defining and executing proper steps for digital transformations to take off and stay ahead [1]. When it comes to innovation in digital health, making the innovation projects successful is even more challenging due to the complexity in healthcare services.Digital technologies can support and improve delivery of health services [2].Improvements often occur by overcoming challenges in communication due to time and place and allowing people and resources to interact in easier ways [3].This means more people and systems can be involved in digital health.Thus, various activities can be

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created due to the increased number of people and systems, and their relationships.Moreover, ethical issues, healthcare policies, and the ecology of health information systems adds to the complexity, and once in operation, support, service and maintenance adds to the picture. To make innovative digital health projects successful, there is a need for guidelines and models that can support utilisation of the projects with a holistic approach.The existing recommendations and approaches are limited.Most of them are either focusing on interoperability issues, related to health information infrastructure, or concerning patients' accessibility to their own health data [4][5][6][7].Two regions in Sweden reported that the most difficult thing in implementation phase of digital health projects is step-bystep roll-out of the systems [8].There is a lack of models and guidelines that can support digital health innovation and utilisation in a holistic, disciplined, and strategic way. Service design is a multidisciplinary, integrative, and holistic way of innovating or improving services to make them efficient for organisations and more usable, useful, and desirable for customers [9].Service design choreographs interactions, processes, and technologies in complex systems to co-create value for relevant stakeholders [10].The aim of this paper is to present a stepwise model for digital health innovation and utilisation following service design principles, and describe its development process. Methods A multiple case study was conducted to develop a stepwise model for digital health innovation and utilisation.Two cases were used for the development process in an iterative manner.For triangulation, participant observation, roleplay, and semistructured interviews were used to collect data in prehospital settings in Sweden.

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Two separate projects allowed to develop the VIPHS model.The development process was conducted in an iterative manner, which means that the results from the second project provided input to improve the first model from the first project.The first project, PrehospIT-Stroke, aimed to create better conditions for more efficient use of digital health within prehospital healthcare.It focused on the foundation for national harmonization of semantic and technical interoperability, with the acute stroke chain as example, aiming to improve IT support and solutions in the acute phase as well as in follow-up, business development and quality assurance at both local and national level.The second project, ViPHS (video support in the prehospital stroke chain) aimed to evaluate whether a collaborative assessment using video involving a neurologist could be effective to support the transportation decision for potential LVO (large vessel occlusion) patients, especially where there is a risk for considerable transport time to a thrombectomy facility due to geographical distance and/or first stop at local hospital. Six principles of service design [11] were applied in the two projects.Table 1 shows the principles, their meanings, and how they were applied in our studies' contexts. Holistic Sustainably addressing the needs of all stakeholders through the entire service Conducting care process analysis and process mapping of the entire service using a holistic approach in the initial stage The first project, PrehospIT-Stroke, carried out in the following three phases: 1. Detailed care process analysis, identification of critical decision-making points, inventory analysis, and recommendation regarding technical and semantic interoperability, 2. Tests in a lab environment

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of technical and semantic recommendation (Connectathon [12]), 3. Simulated full-scale operational field test in an ambulance. The first phases included process mapping of the stroke chain, inventory of process support, standards and de-facto standards around semantics and interoperability, analysis, and preparation of a recommendation for harmonisation.In the second phase, the system structure was tested in the PrehospIT Connectathon in a lab environment, where the project partners' proprietary solutions were tested technically together in a simulated care process utilizing the proposed interoperability standards and methods.This test showed that the different systems could communicate with each other effectively without failures.In the last phase, the system was tested in a full-scale realistic operational simulation (roleplay), where an entire ambulance mission from dispatch to handover was simulated [13].Eleven ambulance teams (22 ambulance clinicians) were recruited to the study.The teams were instructed to either start using a computerized decision support system (CDSS), described in [13], or to work as usual.Two representative patient cases with stroke-like symptoms were created.In one case, the patient had severe stroke symptoms, and in the other case, the patient had moderate stroke symptoms.All simulations were filmed, and all participants were interviewed after the simulations.During the semi-structured interview (average 35 minutes), questions regarding the CDSS and the system were asked; how compatible the CDSS was with their current way of working, what advantages the system brought compared to their current practice, and what problems and challenges they experienced.The interviews and films were then analysed, and the management of the patients with and without CDSS were compared. The second

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project, ViPHS, was conducted in the following four phases: 1. Analysis of process and inventory and recommendation regarding technical and semantic interoperability 2. Tests in a simulated prehospital environment 3. Initial field test in ambulance 4. Field tests in a larger scale In the first phase, the market for mobile video solutions was investigated.Different solutions were tested and evaluated in terms of image quality, function, malfunctions, and so on.An ambulance was equipped with a solution that was considered as the best alternative and tests on transmission capacity were carried out by driving the ambulance around in areas with poor cellular coverage.In the second phase, a simulated test (roleplays) was performed.Four ambulance teams (eight ambulance clinicians) and four neurologists from a regional stroke centre were recruited.The simulation was set up consisting of a video-equipped ambulance standing outside in a parking lot, an office, and a bench outside.Two patient cases were constructed inspired by real patient cases.In one case, a male patient was found by a colleague in an office with severe stroke symptoms, in the other case, a female patient was found outside with moderate stroke symptoms.After the simulations, each participant was asked questions regarding the simulation design and their experience in a semi-structured interview (average 39 minutes).In phase three, the system was tested in the field in a small pilot study.Video equipment was installed in three ambulances in an ambulance organization in Region Västra Götaland in western Sweden.The staff were trained in the system and new guidelines for the care of patients with stroke symptoms were

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written.For each videoequipped ambulance, a person was appointed as responsible for staff training, that guidelines were updated and implemented, and the collection of study protocols.The pilot study lasted one year and all other ambulances in the organization served as a control group.In phase four, the study was expanded both geographically and with a number of ambulances.Twelve ambulances throughout the region were equipped with video.In this phase the technical solution was also adopted to a video-platform supported and maintained by the region.Thereby a potential expansion of the service as well as full-scale operational support and maintenance beyond the project phase should be facilitated.This study has been going on for a year and is expected to finish in year 2023. Figure 1. The VIPHS model for innovation and utilization Figure 1 shows the stepwise model for digital health developed through the two cases.Red texts on the left side shows where the principles of service design are applied.In the VIPHS model, each step leads to a "Blueprint" which is a document with results obtained and input and recommendations to the next step.The model can also be used partially, not including all the four steps.If the project aims to define a prototype, it can stop after blueprint 2. If the project's aim is a clinical validation (e.g., operational feasibility study or clinical proof of concept), it can stop after blueprint 3. The VIPHS model provides an overall idea on what to achieve in implementation in clinical operations and allow to restart a new project from where the previous project ended.

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The PrehospIT project stopped at step 3 (blueprint 3) since the goal of the project was to demonstrate the benefits for the care process deploying standards etc., enabling multi-system interoperability and improved care processes in a realistic setting.The Connectathon was the prime source for evaluating interoperability.During the user simulations the improved care process was achieved from deploying this interoperability.The ViPHS project is still running in an extended step 4 (blueprint 4) in March 2023.The clinical tests were stopped at the end of 2022.However, the tests resulted in a suggestion of modifying and updating the technical solution before going into more general deployment across the health care region.Planning is currently underway to introduce the ViPHS system in all the region's ambulances. Discussion and Conclusion In digital health, changing systems and work processes, namely digitalisation of services in healthcare, is one of the fundamental challenges.Having models that can guide and support digital health innovation and utilisations would be helpful to solve this issue.We could develop a stepwise model for digital health innovation and utilisation by applying service design principles through empirical settings.The VIPHS model with its clearly defined steps and accompanying "blueprints" could be useful to support the implementation process of digital health projects in a holistic, disciplined, and strategic way.This can contribute to increase the success rate of realizing digital health projects. The ViPHS model's step-by-step method provides valuable data in all steps that contribute to accomplish the next step.For example, step 2 in the ViHPS project revealed that it was necessary to standardise the communication between

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ambulance clinicians and the neurologist.This helped to prepare step 3.In addition, the VIPHS model's stepwise approach made it easier to document and communicate our findings and recommendations to people outside the project group. The VIPHS model was developed in a specific clinical setting, the prehospital stroke care process, which is a limitation of our study.We plan to further improve this model through more case studies in different clinical settings like trauma and fall patient care processes that use advanced technology like artificial intelligence, voice recognition, etc. Table 1 . Principles of service design and application to our studies E. Lee et al. / Development of Verified Innovation Process for Healthcare Solutions (VIPHS)

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Use of the endoscopy global Rating scale by endoscopy services in canada The Canadian Association of Gastroenterology (CAG), through its Quality Program – Endoscopy (QP-E), has been promoting and supporting the use of the Global Rating Scale (GRS) since 2007 (1,2). The CAG recently performed a review of the uptake of the GRS across Canada as well as the anonymous, aggregate results obtained by the participating units since 2007. BACkGrOund The endoscopy Global Rating Scale, or GRS, is a quality improvement tool designed for endoscopy services.It promotes patient-centred standards of quality through the use of an iterative process of measurement, interpretation of observed outcomes, formulation of action plans and ensuring desired outcomes have been achieved.The GRS measures unit performance with regards to 12 domains of quality endoscopy: 1) consent and patient information; 2) safety; 3) patient comfort; 4) quality of the procedure; 5) appropriateness of the procedure; 6) ability to communicate results to referring physicians; 7) equality of access; 8) timeliness of the service; 9) booking procedure; 10) privacy and dignity; 11) aftercare; and 12) ability for patients to provide feedback to the service. The GRS was introduced in Canada by the CAG in 2007 through a pilot project that complemented its use with that of a colonoscopy practice audit.It quickly became apparent to users that the GRS was an excellent tool that provided services with both a measure of the intensity of quality-related processes and monitoring, as well as guidance through interventions that would heighten the quality of that service.It also became apparent that the

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GRS, which was developed in the UK, had some shortcomings as it related to the Canadian model of endoscopy care delivery.This led to the development of an adapted version of the tool, the so-called Canadian GRS (C-GRS) (3), which has now been used across Canada since the fall 2011.Moreover, in the fall 2011, the CAG launched its new GRS website for data entry, which is both user-friendly and accessible. unIT PArTICIPATIOn To date, a total of 39 endoscopy units from across Canada have participated in the QP-E program including the GRS.A total of 27 units participated at least once in the eight cycles of the UK-GRS while a total of 30 sites participated at least once in the four cycles of the C-GRS.Eighteen sites participated in both phases.Figure 1 shows the participation uptake for both phases.As shown, uptake with the UK-GRS was dropping by 2010, with lowest participation in fall 2010.The drop is largely related to concentration of efforts during that time to produce a national consensus on quality and safety indicators in endoscopy (4-6) as well as the fact that the C-GRS was being developed and a shift was being made away from the UK GRS system.Since the release of the C-GRS, uptake has been steadily increasing and overall participation is currently greater than seen before.Of the 22 sites that completed the Spring 2013 GRS cycle, 4 are new units which had not participated before and since the new C-GRS was introduced in 2011, 11 new sites have joined the program. GrS reSulTS Unit performance

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with C-GRS is presented in Figure 2, where, for each item, the percentage of units performing at an "above-D" (either C, B or A) level is shown, along with the percentage of units which, over the past four cycles, have shown some improvement in their GRS score. Results and progress achieved to date show that, for all domains measured by the GRS, the majority of units currently perform at baseline (D level) or below baseline (incomplete D level, so-called D-minus level).This means that, while the quality of care may be excellent, the majority of units have no processes, policies and/or monitoring in place to ensure that quality is uniform and to ensure that deficiencies can be systematically identified and remedied. Results also show that units' performance is best for the domains of "patient comfort", "ability to provide feedback to the service" and "consent and patient information", where 55%, 55% and 43% of units perform above baseline (D) level, respectively.Over these past two years, at least 35% of the units that completed the C-GRS at least twice achieved improvements in scoring in 8 out of 12 domains (Figure 2).Score improvements were seen most commonly for "ability to provide feedback to the service", "comfort"and "privacy" where 50%, 45% and 43%, of units improved, respectively.Conversely, with respect to timeliness of access to the service, where the C-GRS is anchored with the CAG's wait times targets (7), 72% of units are at or below baseline and improvement in scoring was achieved by only 15% of them. The high-achieving units were

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identified as those that achieved levels A and/or B for a given item on at least two consecutive cycles.There were more A/B units when looking at the C-GRS cycle data as compared to the UK-GRS data, especially for the items "providing feedback to the service" (8 A/B units), "aftercare" (4 A/B units), "booking procedure" and "communicating results to the referrer" (3 A/B units in each case).Only 3 units achieved A/B levels on any item of the UK-GRS while a total of nine units achieved A/B levels on any items of the C-GRS. One site has achieved A/B levels in 11 of 12 items of the C-GRS.It is a dedicated screening-related colonoscopy centre that used the UK-GRS as the foundation for many of its operational policies.This unit also uses an electronic reporting system that allows the collection and reporting on colonoscopy quality indicators and performance reports for endoscopists. In summary, these results demonstrate that there is an increasing uptake of the C-GRS amongst Canadian endoscopy units.It also reveals that high-performance, defined as achieving A and or B levels on two consecutive cycles, is possible, and more likely to occur with the C-GRS.In particular, items related to patient feedback, aftercare, communicating results to the referrer, and booking procedure seem to be most achievable initially. In summary, the CAG's QP-E is encouraged by these results and is keen to further promote the use of the GRS, and expanding the GRS Use of the endoscopy global Rating scale by endoscopy services in canada Catherine Dubé MD MSc FRCPC website to

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include electronic resources and to facilitate each unit's review of their achievements.Achieving high scores on the GRS will continue to depend on adequate electronic support in the unit, which is intrinsic to our current understanding of quality in endoscopy. Figure 1 ) Figure 1) Sites completing the GRS

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Targeting the Glutamatergic System for the Treatment of HIV-Associated Neurocognitive Disorders The accumulation of excess glutamate in the extracellular space as a consequence of CNS trauma, neurodegenerative diseases, infection, or deregulation of glutamate clearance results in neuronal damage by excessive excitatory neurotransmission. Glutamate excitotoxicity is thought to be one of several mechanisms by which HIV exerts neurotoxicity that culminates in HIV-associated neurocognitive disorders (HAND). Excess glutamate is released upon HIV infection of macrophage/microglial cells and has been associated with neurotoxicity mediated by gp120, transactivator of transcription (Tat) and other HIV proteins. Several strategies have been used over the years to try to prevent glutamate excitotoxicity. Since the main toxic effects of excess glutamate are thought to be due to excitotoxicity from over activation of glutamate receptors, antagonists of these receptors have been popular therapeutic targets. Early work to ameliorate the effects of excess extracellular glutamate focused on NMDA receptor antagonism, but unfortunately, potent blockade of this receptor has been fraught with side effects. One alternative to direct receptor blockade has been the inhibition of enzymes responsible for the production of glutamate such as glutaminase and glutamate carboxypeptidase II. Another approach has been to regulate the transporters responsible for modulation of extracellular glutamate such as excitatory amino acid transporters and the glutamate-cystine antiporter. There is preliminary experimental evidence that these approaches have potential therapeutic utility for the treatment of HAND. These efforts however, are at an early stage where the next steps are dependent on the identification of drug-like inhibitors as well as the development of predictive

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neuroAIDS animal models. Introduction The introduction of combination antiretroviral therapy (cART) in 1996 brought a dramatic reduction in HIV RNA levels and thus morbidity and mortality rates in human immunodeficiency virus (HIV) infected individuals (Coiras et al. 2009). Although cART has been successful, it has not eradicated the disease as the virus can persist in resting memory CD4 + T cells as well as macrophages and astrocytes (Coiras et al. 2009). These latent reservoirs of HIV are easily and quickly activated if cessation of cART treatment occurs (Lewin et al. 2008). Elimination of these reservoirs is a major goal of current research in the HIV field. Even with continuous cART therapy, significant morbidity persists, in particular in the central nervous system (CNS) (Gorantla et al. 2012) where memory problems and dementia are common. It is thought that HIV-associated neurocognitive disorders (HAND) remain high due to both latent reservoir and reduced penetrance of cART to the brain. HIV initially enters the brain by crossing the blood brain barrier via monocytes and lymphocytes very shortly after infection (Fig. 1). The virus then takes up permanent brain residence mainly in microglia, macrophages and astrocytes (Kaul et al. 2001;Tan and McArthur 2012). HIV does not infect neurons, though neural progenitor cells appear able to take up the virus (Kaul 2008). Neurotoxicity in patients with HIV-1 infection is thought to be mediated by HIV-1 proteins such as gp120 and transactivator of transcription (Tat), as well as other products released from infected cells. The mechanisms of neurotoxicity are thought to be both

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direct and indirect including glutamate excitotoxicity, oxidative stress, increase in apoptosis, altered calcium homeostasis, stimulation of tumor necrosis factor-alpha (TNF-α) and nuclear factor κB (NF-kB) and stimulation of nitric oxide production. These mechanisms are likely acting in concert. Although multiple mechanisms are at play, the objective of this review is to present the evidence which indicates that glutamate excitotoxicity is a factor in HIV neurotoxicity and to focus on how this evidence suggests potential opportunities to ameliorate HAND through pharmacological manipulations of the glutamate system. Mechanisms of glutamate excitotoxicity in HAND The amino acid glutamate is the principal excitatory neurotransmitter in mammalian CNS where it is synthesized and stored in the neuronal cytosol in synaptic vesicles in millimolar concentrations (Nedergaard et al. 2002). Extracellular concentrations of glutamate in the synaptic cleft are kept low (nanomolar ranges) by excitatory amino acid transporters (EAATs). These are glutamate transporters which are located mainly on astrocytes and function in removing excess glutamate from the synaptic cleft after the completion of a signaling event, returning it to homeostatic levels. The accumulation of excess glutamate in the extracellular space as a consequence of CNS trauma, neurodegenerative diseases, infection, or deregulation of glutamate clearance results in excitotoxicity. The presence of excess glutamate in the synaptic clefts activates glutamate gated ion channels and results in high levels of ion influx into neuronal cells allowing the over activation of downstream calcium iondependent effectors and signaling pathways, culminating in neuronal damage. Neuronal damage then causes further release of intracellular glutamate into the extracellular space affecting nearby

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neurons. Most acute and chronic neuronal diseases, including HAND, have implicated this type of bystander pathology of excitotoxicity. HIV infection of macrophages and microglial cells causes excess glutamate release and impaired uptake Macrophages and microglia, the resident macrophages in the CNS, are key cellular components of innate immunity. These cells can release a diversity of trophic factors and cytokines that control the behavior and/or destiny of other cells by promoting cell proliferation, migration, recruitment, and apoptosis. Additionally, macrophages and microglial cells play an important role in the phagocytosis of invading pathogens, tissue repair and the clearance of debris (Liu and Hong 2003). The activity of these cells is highly regulated both spatially and temporally due to the potential deleterious effects of their uncontrolled hyperactivation, which include increase in inflammation and cell death. Microglial cells are particularly susceptible to alterations in their surroundings, becoming easily activated, changing morphology and up-regulating the production of a number of membrane receptors and soluble factors (Kreutzberg 1996;Ransohoff and Perry 2009). Conditions that involve neuronal degeneration like HAND, Alzheimer's disease, cerebral ischemia and multiple sclerosis, have been associated with microglial cells pathological activity (Gao and Hong 2008;Zindler and Zipp 2010). Invasion of the brain seems to occur very early in the progression of the disease. However, there is no convincing evidence of HIV-1 neuronal infection despite the fact that HIV-1 associated neuronal dysfunction is accompanied by substantial neuronal loss in the neocortex, putamen, globus pallidus, substantia nigra and hippocampus (Everall et al. 1991;Masliah et al. 1992). Hence it is widely accepted that

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macrophages and microglial cells are responsible for producing and releasing the neurotoxic factors that cause neuronal death (Gendelman 2012). HIV-1 is capable of infecting CD4 + macrophages and T-cells (Chen et al. 1983;Popovic et al. 1983;Klatzmann et al. 1984a, b). Infected macrophages are thought to cross the blood-brain barrier (BBB), turn into resident CNS macrophages and mediate the spread of the virus in the brain (Fig. 1). There is considerable evidence that supports this hypothesis ("Trojan horse" hypothesis) as the mechanism of brain infection by HIV-1 (Peluso et al. 1985;Budka 1986Budka , 1991Koenig et al. 1986;Kure et al. 1990;Dickson et al. 1993;Fischer-Smith et al. 2001). Upon activation, HIV-1 infected macrophages and microglial cells release chemokines, inflammatory cytokines (TNF-α, IL-1β, IL-6), nitric oxide (NO) and glutamate. Excess glutamate can induce neuronal damage through N-methyl-D-aspartate (NMDA) receptor activation (Cutler and Dudzinski 1974;Fonnum 1984). Indeed, HIV-1 infected patients show elevated CSF glutamate levels that correlate with the severity of the dementia and the degree of brain atrophy (Ferrarese et al. 2001). Several glutamate targets (Fig. 2) have been shown to be affected by HIV infection. HIV-1 infected human macrophages and human primary fetal microglia cells have increased glutaminase mRNA and protein levels resulting in elevated extracellular glutamate levels that cause reduced viability of cortical neurons in co-culture or of neurons incubated with conditioned media from these infected cells (Tian et al. 2008;Erdmann et al. 2009;Huang et al. 2011;Zhao et al. 2012). MK-801 (dizocilpine) a non-competitive antagonist at the NMDA receptor abolished the effects on neuronal viability, suggesting this is

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an NMDA receptor-dependent process (Wong et al. 1986). Treatment of macrophages and microglial cells with proinflammatory factors like lipopolysaccharides (LPS) or HIV-1 Tat protein generates reactive oxygen species (ROS), which seem to contribute to the accumulation of (3) HIV-1 infected perivascular macrophages in the brain parenchyma release viral particles that infect other brain macrophages and microglial cells. (4) Activated macrophages and microglial cells release viral proteins gp120 and Tat, glutamate and other factors such as NO, ROS, cytokines, chemokines and arachidonic acid that can either directly or indirectly affect glutamate metabolism and/or transport. (5) Decrease in glutamate uptake by oligodendrocytes and astrocytes due to increased levels of these toxins released by HIV-1 infected macrophages and microglial cells. These factors also cause an increase in vesicular glutamate release by astrocytes. (6) Viral proteins Tat and gp120 and oxidative stress induced by ROS and NO cause an increase in the activity of xCT in uninfected perivascular macrophages and microglia and as a consequence extracellular levels of glutamate increase. (7) Excessive extracellular glutamate triggers activation of glutamate receptors on neurons causing an increase in the intracellular calcium levels, cell death and neuronal degeneration extracellular glutamate and neuronal cell death in a process involving the glutamate-cystine antiporter (xCT) (Barger et al. 2007;Niki 2009). It has been shown that up to 19 % of astrocytes in the brain of patients with HAND are infected with HIV-1 and that there is a direct correlation between the level of infected astrocytes and the degree of neuropathological changes (Churchill et al. 2009). Unlike macrophages

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and microglia, astrocytes are latently infected by HIV-1 making these cells incapable of releasing viral particles. Nonetheless, HIV-1 mRNAs (Tat, Rev and Ref) and proteins (Nef) accumulate inside astrocytes affecting their function (Sabri et al. 2003). Changes induced in astrocytes by HIV infection include activation, production and release of inflammatory cytokines and chemokines, and glutamate release ( Fig. 1) (Benveniste 1998;Bajetto et al. 2002;John et al. 2003;Farina et al. 2007). Cytokines such as TNF-α and IL-1β released by activated microglia, macrophages or astrocytes treated with viral proteins like gp120 or Tat cause increased glutamate release and decreased extracellular glutamate uptake by astrocytes due to down regulation of EAAT1 and EAAT2 gene expression (Wang et al. 2004;Lee et al. 2005;Brabers and Nottet 2006;Cheung et al. 2008). Fig. 2 Potential ways to regulate glutamate excitotoxicity for the treatment of HAND (1) Inhibition of glutaminase -glutaminase is a neuronal enzyme that produces glutamate by the deamination of glutamine. During HIV-1 infection, it is increased in glial cells and the synaptic cleft. (2) Inhibition of GCPII -GCPII is an astrocytic enzyme that catalyzes the hydrolysis of NAAG to N-acetyl aspartate (NAA) and glutamate. (3) Blockade of glutamate receptors-glutamate receptors such as NMDA, AMPA, kainate and mGluR are targets for inhibition of glutamate excitotoxicity in HAND. (4) Inhibition of xCT -xCT transports extracellular cys2 into cells and intracellular glutamate into the extracellular space. (5) Activation of glutamate transporters (e.g. EAAT1)glutamate transporters mobilize glutamate away from the synaptic cleft. Glutamate and NAAG are released through intracellular vesicles at the presynaptic terminal during

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neurotransmission. The illustration shows NAAG and glutamate in the same vesicles but it is not known if they are in the same or different vesicles Moreover, excess extracellular glutamate can induce elevation of intracellular calcium levels in astrocytes which in turn increases even more the release of glutamate from those cells in an autocrine manner . Calcium influx in astrocytes may decrease EAAT1 levels in these cells by a CD38-dependent mechanism contributing to the accumulation of extracellular glutamate (Bruzzone et al. 2004;Liu et al. 2010). In short, a major contributor to the neuronal toxicity due to glutamate in HAND seems to be the aberrant glutamate transport and/or metabolism in astrocytes caused by viral toxins, cytokines or glutamate. Glutamate toxicity in HIV-1 infected patients could also depend on altered glutamate transport in oligodendrocytes (Domercq et al. 2007). Co-culture of LPS-activated microglia and oligodendrocytes or incubation of oligodendrocytes with conditioned media from LPS-activated microglia inhibited glutamate uptake by oligodendrocytes resulting in extracellular glutamate accumulation and cell death (Pang et al. 2010). Moreover, peroxynitrite, the product of the reaction between superoxide and NO, is a potent inhibitor of EAAT glutamate transporters GLAST, GLT-1 and EAAC1 (Trotti et al. 1996). Taken together, compromised clearance of extracellular glutamate from the synaptic cleft by oligondendrocytes, allows levels of glutamate to increase and cause toxicity in neurons. GP120 neurotoxicity and glutamate Gp120 is the main HIV envelope glycoprotein that, along with gp41, allows the entry of HIV-1 into cells via the CD4 receptor along with CCR5 and CXCR4 receptors. The amino acid sequence

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of this protein consists of five variable regions (V1-V5) and five constant domains (C1-C5) (Checkley et al. 2011). There is both in vitro and in vivo evidence indicating that gp120 triggers neurotoxicity (Doble 1999). Gp120 is toxic to cultured rat hippocampal neurons in vitro (Brenneman et al. 1988;Dreyer and Lipton 1995), produces cognitive deficits in rats (Glowa et al. 1992) and causes impaired neuronal development in rat neonates (Hill et al. 1993;Bagetta et al. 1994). Further, transgenic mice expressing gp120 in astrocytes develop neurodegeneration (Toggas et al. 1994). Gp120 neurotoxicity is thought to contribute to cortical atrophy (Lipton 1992a), a condition associated with cognitive impairment observed in some AIDS patients (Power and Johnson 1995). NMDA receptor antagonists, but not CNQX, an AMPA receptor antagonist, prevent in vitro gp120 neurotoxicity in rodent cultures suggesting that gp120 neurotoxicity is mediated, at least in part, by the NMDA receptor (Lipton et al. 1991;Lipton 1992a, b;Muller et al. 1992;Savio and Levi 1993). Interestingly, if glutamate is removed enzymatically from the cell culture milieu, gp120 excitotoxicity is lost (Lipton et al. 1991). Furthermore, neurodegeneration in gp120-expressing transgenic mice can be ameliorated by the NMDA antagonist memantine (Toggas et al. 1996) and the glutamate release inhibitor riluzole (Sindou et al. 1994). Gp120 is also known to trigger NMDA receptormediated cell death in human neurons (Corasaniti et al. 1995;Lannuzel et al. 1995;Wu et al. 1996). However, gp120 receptors (CD4 receptors) are not found in neurons so that gp120 neurotoxicity mediated by NMDA receptors is likely through an indirect mechanism (Lipton et al. 1991)

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involving glutamate release from infected macrophage and microglia. Macrophages are essential in order to see the neurotoxic effect of gp120 (Lipton 1992c). Unlike neurons, macrophages express CD4 receptors that recognize gp120. In addition, stimulation of macrophages with gp120 releases arachidonic acid which impairs glutamate uptake by astrocytes (Dreyer and Lipton 1995) thus providing an explanation for the build-up of glutamate in the synapse. Tat neurotoxicity and glutamate HIV-1 Tat is a small basic protein (86-101 residues, depending on the viral isolate) encoded by the HIV-1 genome that plays an important role enhancing the efficiency of transcription from viral dsDNA (Debaisieux et al. 2012). Tat protein is encoded by two exons, the first one corresponds to the 72 N-terminal residues and the second one the remaining 14-32 residues (Cheng et al. 1998). In addition to its role in viral replication, this protein is secreted from intact HIV-1 infected cells at nanomolar levels and is found in serum, CSF and brain of HIV-1 infectedpatients (Xiao et al. 2000). Fragments of Tat have been shown to cause apoptosis in human peripheral blood mononuclear cells, T-cells, neuroblastoma, rat cortical neurons and human fetal primary neuronal cells (New et al. 1998). Moreover, injection of HIV-1 Tat in mice caused neurotoxicity, seizures, death, neuronal degeneration, astrocytosis and microglia activation (Sabatier et al. 1991;Philippon et al. 1994). Tat peptides were shown to be neuroexcitatory and neurotoxic in cultured human fetal neurons triggering the release of calcium ion from intracellular stores (Haughey et al. 1999(Haughey et al. , 2001Holden et al. 1999). This calcium

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release causes membrane depolarization, activation of metabolic pathways, ROS generation and apoptosis (Nath et al. 1996;Kaul et al. 2001). Release of Tat-mediated calcium ion seems dependent on NMDA receptor activation since NMDA receptor antagonists MK-801 and D-2-amino-5phosphonovalerate (AP-5), significantly decrease cell death induced in neurons and astrocytes by Tat . It is thought that Tat causes the release of Zn 2+ from its binding site on the NMDA receptor, causing activation and increasing its capacity to allow calcium ion influx (Chandra et al. 2005). Tat can bind to lipoprotein related protein (LRP) receptor and form a complex with postsynaptic density protein-95 (PSD-95), NMDA receptor and neuronal nitric oxide synthase (nNOS) at the cell membrane in neurons ). By a mechanism not fully understood this complex can cause apoptosis in both NMDA receptor positive and negative neurons. Although most studies implicate NMDA receptors, some evidence suggests that the toxic effects of the Tat protein are mediated through non-NMDA receptors. In fetal neurons the non-NMDA receptor antagonists kynurenate, CNQX and NBQX significantly decreased Tat-induced cell death while there was no significant effect of MK-801 or AP5 (Nath et al. 1996;Cheng et al. 1998). Tat has also been reported to cause an increase in expression levels and activity of xCT in rat primary microglia resulting in increased glutamate release (Gupta et al. 2010). Also, Tat decreases the expression of manganese superoxide dismutase, which could lead to lower capacity for anti-oxidant response in cells and ultimately induce oxidative stress (Flores et al. 1993). Finally, Tat seems to have synergistic effects

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on other toxins like glutamate and HIV-1 gp120 causing a significant increase in their neurotoxic potency (Wang et al. 1999;Nath et al. 2000). Brief exposure of hippocampal neurons in neonatal rats to Tat and physiological levels of NMDA caused marked cell loss supporting the idea that locally released Tat could enhance NMDA receptor activation-dependent neurotoxic effects (Wang et al. 1999). Accessory and regulatory HIV proteins neurotoxicity and glutamate In addition to gp120 and Tat, other less well studied HIV proteins have been identified and have been shown to contribute to glutamate-related toxicity. These include gp160, gp41 and viral protein R (Vpr) (Hussain et al. 2008;Gorantla et al. 2012). Gp41 facilitates the release of glutamate from glial cells in vitro suggesting that this protein may contribute to the excitotoxic effects of HIV infection (Kort 1998). Gp41 was shown to be more effective than gp120 at releasing glutamate in rat parietal cortical slices (Wang and White 2000). Another study showed that both gp120 and its precursor gp160, can both alter NMDAinduced intracellular free calcium levels leading to neurotoxicity (Lannuzel et al. 1995). Vpr transgenic mice displayed higher levels of glutamate in the cortex and basal ganglia along with lower levels of glutamate transporters, EAAT1 and EAAT2 (Noorbakhsh et al. 2010;Power et al. 2012). These findings correlated to disturbances in both motor and cognitive behaviors (Noorbakhsh et al. 2010). Reduction of glutamate receptor signaling Since the main toxic effects of excess glutamate are thought to be due to excitotoxicity from over activation of glutamate receptors, antagonists of these receptors

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have been popular therapeutic targets for treatment of HAND (Fig. 2). Early work to ameliorate the effects of excess extracellular glutamate focused on NMDA receptor antagonism, in particular with the use of 1-amino-3, 5-dimethyl-adamantane (memantine) (Lipton 2004). Memantine is an uncompetitive low affinity antagonist of the NMDA receptor that is approved to treat the symptoms of Alzheimer's disease. Memantine can block excessive glutamate activity without interfering with the normal functioning of the receptor (Lipton 2004). Preclinical research mainly focused on the effect of memantine on gp120 induced neuronal damage. Memantine can prevent gp120 toxicity (Lipton 1992b;Muller et al. 1992;Muller et al. 1996) as well as the combined toxicity of gp120 and Tat in neuronal cultures (Nath et al. 2000). The first in vivo evidence of memantine's neuroprotective effects was established in the gp120 transgenic mice with a significant enhancement of dendritic and presynaptic terminal densities after treatment (Toggas et al. 1996). Impaired synaptic transmission and long-term potentiation (LTP) have been reported in SCID mice injected with human macrophages infected with HIV-1 ( Anderson et al. 2004). In this same study, memantine was shown to attenuate these deficits. Based on this preclinical evidence, a 20-week, randomized, doubleblind, placebo-controlled trial involving HIV-infected participants with mild to severe cognitive impairment was carried out. Memantine showed good tolerability but no improvement in cognitive deficits; a longer follow-up is underway (Schifitto et al. 2007;Zhao et al. 2010). Other NMDA receptor antagonists such as MK-801, AP-5 and 7-chloro kynurenic acid have also been shown to prevent gp120 induced neurotoxicity in vitro (Lipton

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et al. 1991;Lipton 1992a, b, c;Corasaniti et al. 1995). In contrast, the non-NMDA receptor antagonist, CNQX failed to show any protection (Lipton et al. 1991;Corasaniti et al. 1995). In a recent study in rat hippocampal neurons, several NMDA receptor antagonists were screened for their effectiveness to prevent Tat-induced cell death and synapse loss. MK-801, memantine and ifenprodil but not the GluN2A-selective NMDA receptor antagonist TCN201 were neuroprotective. Memantine and ifenprodil protected against Tat-induced cell death but had no effect on synapse loss. MK-801 and TCN201 had the opposite effects (Shin et al. 2012). In general, however, the use of glutamate receptor antagonists in patients has been fraught with side effects and few potent glutamate receptor antagonists have made it through advanced clinical trials. Inhibition of enzymes responsible for the formation of glutamate Given the side effects observed in the clinic while trying to block postsynaptic glutamate receptors directly, one alternative is to try to reduce the presynaptic generation and release of glutamate. In this regard, two enzymes thought to contribute to increased levels of glutamate in the synapse are glutamate carboxypeptidase II (GCPII) and glutaminase. Inhibition of these two enzymes could help abrogate the effects of glutamate excitotoxicity (Fig. 2). GCPII is a membrane-bound glial enzyme that catalyzes the hydrolysis of N-acetyl-aspartyl-glutamate (NAAG) to N-acetyl aspartate (NAA) and glutamate. NAAG is an abundant peptide neurotransmitter in mammalian brain that is thought to act as an agonist at group II metabotropic glutamate receptors and a mixed agonist at the NMDA receptor (Westbrook et al. 1986;Neale et al.

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2000) , although some controversy exists regarding these activities (Fricker et al. 2009). GCPII-catalyzed hydrolysis of NAAG is believed to function both to terminate NAAG mediated neurotransmission and to liberate glutamate which then acts at various glutamate receptors. Consequently, GCPII inhibitors could help lower glutamate concentration at the synapse and alleviate glutamate excitotoxicity. This hypothesis has been substantiated by numerous reports where GCPII inhibitors have shown to increase extracellular NAAG and decrease glutamate in the brain measured by microdialysis (Slusher et al. 1999;Nagel et al. 2006) and provide neuroprotective activity in over twenty animal models of disease (Barinka et al. 2012) including inflammatory and neuropathic pain (Chen et al. 2002;Carpenter et al. 2003;Yamamoto et al. 2004), brain ischemia (Slusher et al. 1999), motor neuron disease (Ghadge et al. 2003), spinal cord and traumatic brain injury (Long et al. 2005;Zhong et al. 2005), peripheral neuropathy (Zhang et al. 2006;Carozzi et al. 2009), epilepsy (Witkin et al. 2002) and drug abuse Xi et al. 2010). Further, a GCPII inhibitor, 2-MPPA (2-(3-mercaptopropyl) pentanedioic acid), was used in humans in an exploratory study to assess safety and tolerability of GCPII inhibition. 2-MPPA did not provoke any adverse CNS effects and was well tolerated (van der Post et al. 2005). The potential therapeutic utility of GCPII inhibition in AIDS-related neurotoxicity was recently evaluated in an in vitro model using rat embryonic hippocampal cultures and gp120IIIB. Gp120IIIB exhibits specificity for CXCR4, the receptor known to induce neuronal apoptosis. 2-PMPA, a potent and selective GCPII inhibitor (Jackson et al. 1996) prevented gp120III-induced apoptosis

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in a dose-dependent manner. Apoptosis could be resumed in the presence of mGlu3 receptor antagonists or in the presence of antibodies to transforming growth factor (TGF)-β. The results suggest neuroprotection can be mediated through increases in NAAG and subsequent action at the mGlu3 receptors and TGF-β release. Consistent with the localization of GCPII in astrocytic cells, 2-PMPA failed to provide neuroprotection in the absence of glia (Thomas et al. 2009). Evaluation of GCPII inhibitors in animal models of HAND is underway. Glutaminase catalyzes the hydrolysis of glutamine to glutamate and it is thought to be a major source of glutamate production in the CNS. HIV-1 infected human macrophages and human primary fetal microglia have increased glutaminase mRNA and protein levels (Tian et al. 2008;Erdmann et al. 2009;Huang et al. 2011;Zhao et al. 2012). Macrophages infected with various HIV-1 strains were reported to release high levels of glutamate in the presence of glutamine and this release was inhibited by glutaminase siRNA as well as several structurally diverse glutaminase inhibitors including 6-diazo-5-oxo-L-norleucine (DON), BPTES and its analogs (Zhao et al. 2004;Erdmann et al. 2007). Additional mechanistic studies of glutamate generation in HIV-1 infected macrophages revealed up-regulation of the glutaminase isoform GAC. Glutaminase is normally found in mitochondria but upon infection it is released into cytosol and extracellular space where high levels of glutamine could be rapidly converted to glutamate (Erdmann et al. 2009). Unfortunately, current prototype glutaminase inhibitors are non-specific and reactive (Zhao et al. 2004) or exhibit poor solubility (Wang et al. 2010;Hartwick and Curthoys 2011). Consequently,

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a meaningful evaluation of the potential of glutaminase inhibition to prevent glutamate excitotoxicity in animal models of neuroAIDS awaits the identification of better drug-like glutaminase inhibitors Regulation of glutamate transporters Oxidative stress in macrophages and microglia is also thought to contribute to increased extracellular glutamate through xCT. Treatment of macrophages and microglial cells with pro-inflammatory factors like lipopolysaccharides (LPS) or HIV-1 Tat protein causes lipid peroxidation due to the ROS that are generated. Cells affected by oxidative stress show alterations in cell signaling, membrane organization, and protein and DNA modification (Niki 2009). In order to neutralize lipid peroxidation, glutathione-Stransferase (GST) catalyzes reactions between reduced glutathione (GSH) and oxidized lipids to form products like 4hydroxynonenal (4-HNE) and acrolein. This detoxification process irreversibly consumes the GSH available in the cell decreasing the anti-oxidant protective power. Replenishment of intracellular GSH is thought to occur through xCT which shuttles cystine (cys2) inside the cell in exchange for glutamate that is released from the cell. Cys2 is the main source of cysteine, required for GSH synthesis. This enhanced cys2 uptake to replenish GSH causes an increase in extracellular glutamate that in turn can contribute to neurotoxicity (Barger et al. 2007). The antiporter also supports a redox cycle over the plasma membrane where cys2 uptake is followed by intracellular reduction to cysteine and secretion of surplus cysteine into the extracellular space (Banjac et al. 2008;Conrad and Sato 2011). Overall, xCT appears to support an endogenous antioxidant response (through the uptake of cys2 as well as the release of cysteine) with the concomitant

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increase of extracellular glutamate. However, the antioxidant response can become neurotoxic when it is activated during inflammation associated with neurodegeneration. When macrophages are stimulated by LPS or TNFα, they import cys2 and release cysteine into the medium resulting in a reduced milieu conducive to T cell activation; the accumulation of extracellular thiols is inhibited by glutamate suggesting the involvement of xCT (Angelini et al. 2002). Exposure of cultured primary microglia to HIV Tat causes dose-dependent increases in extracellular glutamate; these increases become higher in the presence of morphine in accordance with the immunomodulatory properties of this opiate agonist. Tat-induced glutamate release was associated with increased expression of the xCT antiporter and was inhibited by the xCT prototype inhibitors DLaminoadipic acid and 4-carboxyphenylglycine (Gupta et al. 2010). These findings suggest that Tat-mediated activation of xCT could be playing a role in HIV-related pathology and that xCT inhibitors have potential for the treatment of HAND. HIV-mediated neurotoxicity can also result in inhibition of glutamate transporters GLT1, GLAST, and EAAC1 (Trotti et al. 1996) or in reduction of their expression (Noorbakhsh et al. 2010;Power et al. 2012). Since these transporters are involved in the reuptake of glutamate by the cell after glutamate neurotransmission, inhibition or reduction of expression of these transporters results in aberrant activation of glutamate receptors (Sheldon and Robinson 2007). Consequently, one potential approach is to search for transport activators that could increase either their activity or their expression. This approach has shown promise in other glutamate -related disorders such as ALS (Rothstein et al. 2005). Recent

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work reports on the identification of pyridazine derivatives that increase the protein levels of the glutamate transporter EAAT2 in astrocytes. (Xing et al. 2011). The effect of these activators in models of neurodegenerative disease including HAND awaits investigation. Animal models to investigate effects of glutamate regulation in HAND In order to investigate if glutamatergic-based therapeutics can be effective in eliminating the symptoms of HAND, one needs an appropriate preclinical animal model. The generation of animal models of HIV has proven to be very challenging owing to the fact that HIV itself is not infectious to rodents. In order to create an accurate animal model for HAND several criteria need to be met. The animal model needs to possess target CNS cells that are permissive to virus infection, have a chronic infection period, display altered blood-brain barrier function to permit transmigration of infected cells and have the ability to maintain viral reservoirs (Gorantla et al. 2012). It would also need to feature the generation of viral proteins such as Tat and gp120 as well as the release of neurotoxic products such as proinflammatory cytokines, chemokines, quinolinic acid, glutamate, arachidonic acid and nitric acid among others. Since the discovery of HIV, several animal models of HAND have been created (Table 1), but like many models of neurodegenerative diseases, no one current model recapitulates the exact characteristics of HAND or HIV-1 associated dementia (HAD) (Gorantla et al. 2012;Jaeger and Nath 2012). Some of the first and most logical models to be generated were where the full-length HIV-1 DNA was

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inserted into the mouse genome under the control of various promoters (Santoro et al. 1994;Thomas et al. 1994;Gorantla et al. 2012). A HIV-1 transgenic rat was also developed and cognitive deficits such as impairments in spatial learning as well as evidence of other clinical manifestations of the disease have been reported making it a suitable model for testing therapeutics in rats (Reid et al. 2001;Vigorito et al. 2007;Lashomb et al. 2009). The most commonly used mouse models for HIV are those that express some of the viral proteins generated upon HIV infection such as Tat and gp120 (Toggas et al. 1994;Kim et al. 2003;Gorantla et al. 2012). GFAP-Tat Tg mice possess doxycycline-inducible expression of the Tat protein under control of GFAP promoter while GFAP-HIVgp-120 Tg mice exhibit expression of gp120 protein driven by GFAP promoter that is not inducible (Toggas et al. 1994;Kim et al. 2003;Bruce-Keller et al. 2008). Spatial learning on the Morris water maze was shown to be impaired in the gp-120 mice (D'Hooge et al. 1999). This is thought to be due to excitotoxic mechanisms as a result of increased NMDA receptor signaling and impaired hippocampal long-term potentiation (LTP) which is believed to be the NMDA receptor-dependent biological correlate of learning and memory (Lipton 1994;Toggas et al. 1996). Indeed, as mentioned previously, the first in vivo evidence of the NMDA receptor antagonist, memantine's neuroprotective effects was established in these gp120 transgenic mice (Toggas et al. 1996). Like the gp120 mice, the Tat transgenic mice also exhibit memory deficits as demonstrated by diminished performance

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in hippocampal-dependent memory tasks such as the Barnes maze, Morris water maze, fear conditioning and novel object recognition (Carey et al. 2012;Fitting et al. 2012). Interestingly, Tat transgenic mice display an increase in expression of the xCT antiporter which could be the response to increased oxidative stress and excitotoxicity (Bridges et al. 2004). Like gp-120, the Tat protein has been shown to interfere with LTP (Li et al. 2004;Fitting et al. 2012). Since the gp120 and Tat proteins both induce impairments to the glutamate system, these models can be appropriately used to test glutamatergic therapeutics. Direct injection of these proteins into brain areas has also been used to model HAND and have shown cognitive and sensorimotor gating impairments as well as interference in LTP (Glowa et al. 1992;Pugh et al. 2000;Sanchez-Alavez et al. 2000;Li et al. 2004;Fitting et al. 2006;Fernandes et al. 2007). As mentioned above, injection of HIV-1 Tat in mice caused neurotoxicity, seizures, death, neuronal degeneration, astrocytosis and microglia activation (Sabatier et al. 1991;Philippon et al. 1994). Future generation of double or triple transgenic lines combined with the introduction of some neurotoxic products or supernatants from HIV-infected macrophages might be needed to convey the collective effects of the various viral proteins and other HIV-generated toxins in the CNS. To overcome the fact that HIV does not infect mice, two approaches were undertaken to circumvent the restriction of HIV-1 entry to rodent species. The first approach was on the host side with the generation of various types of humanized mouse models that incorporated a functional

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human immune system (HIS) into severe combined immunodeficient (SCID) mice and thus permitting HIV infection (Jaeger and Nath 2012). HIV-1 infected monocyte derived macrophages (MDM) were also injected into these SCID mice to create HIV encephalitic (HIVE) mice and many of the pathological features of HIVE as well as cognitive and plasticity deficits were reproduced in these mice which were attenuated with memantine (Tyor et al. 1993;Avgeropoulos et al. 1998;Zink et al. 2002;Anderson et al. 2004;Sas et al. 2007). These mice have been widely used for therapeutic testing but biosafety requirements make them difficult to work with (Gorantla et al. 2012). The other approach to overcome the issue of species recognition was on the side of the virus itself. This was accomplished by replacing the coding region of HIV-1 gp120 with that of gp80 from a rodent-infectious retrovirus called ectotropic murine leukemia virus resulting in the EcoHIV construct (Potash et al. 2005). Cognitive testing has not been carried out in these mice nor have any defects in LTP or the glutamate system been reported to date. These mice have been successfully used for the preclinical evaluation of antiretroviral drugs and vaccines Saini et al. 2007). All of these models have potential to be used to evaluate new therapeutics but each have their individual pros and cons and none of them encompass the entire HAND symptom repertoire. It is clear that the glutamate system is affected following HIV infection of the CNS and therapeutics aimed at ameliorating these deficits may be beneficial for the symptoms of HAND.

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In summary, glutamate excitotoxicity is one of several mechanisms by which HIV exerts neurotoxicity that culminates in HAND. Glutamate is released upon HIV infection of macrophage/microglial cells and has been associated with neurotoxicity mediated by gp120, Tat and other HIV proteins. Several strategies have been used over the years to try to prevent glutamate excitotoxicity, including direct blockade of glutamate receptors. Unfortunately, this approach is fraught with side effects. Another approach has been to inhibit enzymes responsible for the production of glutamate such as glutaminase and GCPII or to regulate the transporters responsible for modulation of extracellular glutamate such as EAAT and xCT. These efforts however, are at an early stage where next steps are dependent on the identification of drug-like inhibitors as well as the development of predictive neuroAIDS animal models.

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Activation energies of water molecular dynamics and Stokes-Einstein equation An analysis of the values and signs of the activation energies of temperature dependences (TDs) of the self-diffusion coefficient (D) and dynamic viscosity ({\eta}) in the range from 0 {\deg}C to 100 {\deg}C confirmed that the molecular dynamics of water is based on the synergism of endo and exo reactions of rearrangement of hydrogen bonds in the supramolecular structure water. The ratio of linear approximation coefficients TD of the complex characteristic D{\eta} before and after 25 {\deg}C correlates with the ratio of cluster sizes prevailing in the water structure in these temperature ranges. This result is consistent with the Stokes-Einstein equation and the hypothesis that the decomposition of hexagonal ice-like clusters is completed in the vicinity of 25 {\deg}C. A reliable approximation of the TD complex D{\eta} by a bimodal function of type Texp (E/RT) was used to refine the Stokes-Einstein equation. Introduction In [1], it was established that the molecular physics of the anomalous temperature (kBthe Boltzmann constant, r is the particle radius) for D and T1 in [1,2] took β = 1, and for η β = 0. EA values for these characteristics were comparable with the hydrogen bond energy and varied greatly in the intervals 0 -25 o C and 26-100 o C. Moreover, in the case of D and T1, the EA values were negative (endo reaction), and for η, positive (exo reaction). In [1,2,3] it was suggested that, in the vicinity of 25 o C, the transformation of the ice-like supramolecular structure

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is completed by the decomposition of tetrahedral hexagonal clusters (W6) into dimers and smaller clusters. As a rule, the value D is calculated from T1 values, which are determined by the spin-lattice relaxation of protons, measured by the NMR. A confirmation of the adequacy of the D values is usually the relation (2) [4,5,6]. By definition, D should characterize the displacement, that is, the translational mobility of the center of gravity of the water molecules located next to the oxygen atom. The dynamics of proton spins in the general case includes rotational motions of molecules, tunneling jumps of a proton along HBs chains and differs for spin ortho and para isomers of water [7]. The ambiguity of the correspondence between the dynamics of proton spins and the mobility of oxygen atoms is expressed in the qualitative nature of the correlations between the values of D and T1. Equation (2) is also evaluative, since it was derived using a statistical model of thermodynamics that does not take into account the TDs features of the dynamic characteristics of water. The bimodal function (1) (see Figure 1a). Since in accordance with (2) these coefficients are kB/6πr, the parameter r at T < 25 °C should be 2.7 times larger than at T > 25 °C. This result is consistent with the hypothesis of a sharp transformation of SMS in the vicinity of 25 °C and the decay of W6 into dimers and clusters that are 2-3 times smaller than the size of W6 [1,4]. Transformation also corresponds to the

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transition between two metastable phases of SMS waterlow density liquid (LDL) and high density liquid (HDL) [1]. The prevailing loose tetrahedral configuration of HBs is characteristic of LDL, and the denser configuration of disturbed HBs is predominant for HDL [1]. Table Temperature interval, parameter β and activation energy of Arrhenius approximations of the temperature dependences of the water characteristics. The values of EA and ER for D and T1 practically coincide in the range of 0-25 °C, but in the range of 26-100 °C their values for D are 10-12% higher than the values for T1. Moreover, the difference in the values obtained by different authors does not exceed ~5% (see Table). space. The first stage is responsible for the physics of viscosity, and the physics of the second stage is related to the physics of the process of water crystallization [1]. The kinetics of both stages depends on the instantaneous structure of the hydrogen bond network in SMS, which in turn depends on T. TDs by the activation mechanism of the endothermic stage of the self-diffusion process. The kinetics of this mechanism is determined by the activation energy , which is 2.8 times greater in the range of 0-25 °C than in the range of 25-100 °C (see Table). This result can be associated with a corresponding increase in the average number of HBs per water molecule in the range of 0-25 °C due to the predominance of W6 clusters with tetrahedral HBs in SMS [1]. Within the bounds of reliability of the approximation of

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TDs of the complex Dη by the function ( ) (Figure 1c), the Stokes-Einstein equation can be represented as:

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Transition metals doped CuAlSe2 for promising intermediate band materials Introducing an isolated intermediate band (IB) into a wide band gap semiconductor can potentially improve the optical absorption of the material beyond the Shockley-Queisser limitation for solar cells. Here, we present a systematic study of the thermodynamic stability, electronic structures, and optical properties of transition metals (M = Ti, V, and Fe) doped CuAlSe2 for potential IB thin film solar cells, by adopting the first-principles calculation based on the hybrid functional method. We found from chemical potential analysis that for all dopants considered, the stable doped phase only exits when the Al atom is substituted. More importantly, with this substitution, the IB feature is determined by $3d$ electronic nature of M^{3+} ion, and the electronic configuration of 3d^1 can drive a optimum IB that possesses half-filled character and suitable subbandgap from valence band or conduction band. We further show that Ti-doped CuAlSe2 is the more promising candidate for IB materials since the resulted IB in it is half filled and extra absorption peaks occurs in the optical spectrum accompanied with a largely enhanced light absorption intensity. The result offers a understanding for IB induced by transition metals into CuAlSe2 and is significant to fabricate the related IB materials. Introduction The conversion efficiency is one of the most important factors to estimate the performance of photovoltaic solar cells. At present, many ideas to increase the efficiency of solar cell have been developed including the third generation solar cells. As one of the third generation solar cells, intermediate band

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solar cell (IBSC) is believed to have bright prospects. For traditional photovoltaic solar cells, electrons are directly excited from the valence band (VB) to the conduction band (CB) by absorbing photons. For IBSC, the concept is to introduce an isolated intermediate band (IB) in the main band gap of the host semiconductor to build a three-photon absorption process such that electrons can be excited not only from the VB to the CB but also from the VB to the IB and from the IB to the CB. As a result, the upper limiting efficiency of IBSC was predicted to be as high as 63.1%, greater than the single-junction solar cell (40.7%). [1,2] By further increasing the number of IBs, the efficiency would reach 86.8%. [3,4] Theoretical and experimental reports have verified that IB materials could effectively increase the optical absorption. [5,6,7] Numerous efforts have been made to implement IBSCs, including through quantum dots and the insertion of appropriate impurities into the bulk host semiconductor. [5,6,7,8,9,10,11,12,13,14,15,16,17,18] From previous studies, the bulk IB material is easier to fabricate than quantum dots and has a stronger absorption because of the higher density of the IB states. [18] Among bulk IB host materials, Cu-based chalcopyrite compounds are the important candidate host semiconductors for IBSCs with the high conversion efficiency of 46.7%. [19] When the value of band gap is in the optimum region of 2.2 − 2.8 eV with the IB located in the region of 0.8 − 1.1 eV from VB, the photovoltaic energy conversion is becoming stronger. Hence,

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CuGaS 2 and CuAlSe 2 are viewed as the most promising IB host materials because their band gaps are 2.45 eV and 2.67 eV, respectively. In the past decade, many theoretical studies, based on the first-principles calculations, have been made to design and understand the bulk CuGaS 2 IB materials. [20,21] Elements of the 3d transition metals and group IVA have been identified as viable candidates to act as substitutes in the cation sites of the CuGaS 2 chalcopyrite hosts. [14,20,21,22,23,24,25,26,27,28] 2 In particular, Ti [29], Sn [30], and Fe [31] doped CuGaS 2 IB semiconductors have been successfully synthesized and display the enhancement of absorption coefficient. However, the photovoltaic energy conversion does not meet the expectations of higher efficiency. More efforts are needed in the future work. With respect to CuAlSe 2 IB materials, the band gap of the host semiconductor is about 2.6 eV. After introducing a IB located at 1.01 eV from VB, the photovoltaic energy conversion will realize 46%. [19] Comparing to CuGaS 2 , perfect CuAlSe 2 has much higher adsorption coefficient in the visible region. [28,32] Nevertheless, few studies on CuAlSe 2 IB materials are reported. For transition metals, we know that introducing local 3d electrons into the wide band gap semiconductor often induces the impurity bands in the main band gap of the host. Considering the advantage of 3d electrons of transition metals and selecting three typical elements Ti, V, and Fe, in this work, we therefore employ density functional theory based on the hybrid density functional method to

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investigate the transition metals M -doped CuAlSe 2 . The effects of doping concentrations and positions are examined. It is our aim to explore the thermodynamic stability induced by doping, understand the forming mechanism of IB, and predict the promising candidates materials for IBSC. This is significant to fabricate the CuAlSe 2 IB materials and develop the related IBSCs. Computational Details Density functional theory (DFT) calculations have been carried out on a representative structure which consists on a supercell derived from the parent body-centered tetragonal CuAlSe 2 structure as shown Fig. 1(a). Different concentrations of the proposed dopants inside the host semiconductor were computed. In order to obtain the expectative doping concentration, calculations for supercells containing 16, 32, 64, and 128 atoms were carried out and compared. The desired structures were achieved by substituting M atom for Cu, for Al, and for Se atom respectively inside a CuAlSe 2 supercell. The configurations were fully optimized using a conjugate-gradient algorithm. The Monkhorst-Pack k-point grids are generated according to the specified kpoint separation 0.02Å −1 and the convergence thresholds are set as 10 −6 eV in energy and 0.005 eV/Å in force. All calculations are based on the projector augmented wave method (PAW) [33] with a cutoff energy of 400 eV as implemented in the Vienna ab initio simulation package (VASP) [34]. In the standard DFT, the generalized gradi-ent approximation (GGA) of Perdew-Burke-Ernzerhof (PBE) version [35] is adopted to describe the electronic exchange-correlation (XC) interactions. However, the standard DFT usually leads to erroneous descriptions for some real systems

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such as underestimating the band gap. One way of overcoming this deficiency is to use Heyd-Scuseria-Ernzerhof (HSE) hybrid functional, where a part of the nonlocal Hartree-Fock (HF) type exchange is admixed with a semilocal XC functional, to give the following expression [36]: where α is the mixing coefficient and ν is the screening parameter that controls the decomposition of the Coulomb kernel into short-range (SR) and long-range (LR) exchange contributions. In this calculation, the HSE exchange-correlation functional is set as 35% mixing of screened HF exchange to PBE functional, namely α = 0.35, which can reproduce the experimental band gap of CuAlSe 2 . Noticeably, Spin polarization is studied due to the existence of a magnetic M atom. Results and discussion CuAlSe 2 crystallizes in a chalcopyrite structure with a space-group of I42d. As shown in Fig. 1(a), each atom in this structure is fourfold coordinated. Namely, each Se atom is coordinated with two Al and two Cu atoms, and each cation is coordinated with four Se ions. The optimized lattice constants of CuAlSe 2 are a = 5.654Å and c = 11.147Å according with the experimental values. The calculated band gap is only 0.86 eV within the GGA method, while it reaches 2.6 eV based on HSE functional (ν = 0.35), which is in good agreement with the experimental value of 2.67 eV. [37] From the electronic characteristics near the Fermi level shown in Fig. 1(b), the VB is mainly formed by Cu-3d and Se-4p electronic states, while the CB mainly results from Al-3s and

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Se-4s4p states. To examine the probability of the formation of doped compounds, we have calculated the formation energy of doped system using the following as 4 where E SC (doped) and E SC (host) is the total energy of the doped and host perfect supercells, respectively. E i is the total energy of the component element i in its pure phase, which has been shown in Table I. ∆n i is the number of exchanged atom i between the host and the doped system (∆n i means that the atom i moves into the host). µ i is the chemical potential referenced to the total energy E i of the pure element phase for the i species, satisfying µ i ≤ 0. µ i = 0 represents the limit where the element is so rich that its pure phase can form. µ i < 0 means that the formation of the compound is favorable rather than the pure elemental phase. ∆H f is the formation enthalpy of doped system related to formation energy. E f represents the chemical reaction ongoing, while E f indicates that the thermodynamic balance is achieved between the host and doping compounds. To analyze the thermodynamic stability of doped systems, we firstly start from the growth of the host CuAlSe 2 . To obtain the CuAlSe 2 phase under a certain chemical environment, we have a balanced system with E f = 0 , and the chemical potentials of Cu, Al, and Se satisfy the equation where the formation enthalpy ∆H f

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(CuAlSe 2 ) can be obtained from our firstprinciples calculations. At the same time, to avoid the secondary phases Cu 2 Se and Al 2 Se 3 , the chemical potentials satisfy the following relations and 2µ Al + 3µ Se < ∆H f (Al 2 Se 3 ) = −6.79 eV. By considering the above constraints Eqs. (3)(4)(5), we can draw the chemical potential range of stable CuAlSe 2 phase excluding the secondary phases of Cu 2 Se and Al 2 Se 3 . Based on the analysis, as long as we know the formation enthalpy of doped systems and the chemical potential of different species i, the formation energy of the doped systems can be evaluated from Eq. (2). The chemical potential of different species i has been listed Table I. We have also calculated the formation enthalpy of different doped systems and show in Table II. Considering the doped system with the transition metal M occupying on the Cu site, namely M Cu , we should control the chemical potentials to satisfy Similarly, for the M respectively occupying on the Al and Se site, M Al and M Se , the chemical potentials must respectively satisfy and By applying the above constraints in Eqs. (3)(4)(5)(6)(7)(8), we can obtain the chemical potential range for the doped CuAlSe 2 system at µ M = 0 (namely under M -rich condition). As shown in Fig. 2, the direction faced the origin satisfies the chemical potential constraints when substituting for Cu atom (plotted with solid line), while

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the direction deviated from the origin satisfies the chemical potential constraints when substituting for Al or Se atom (plotted with dashed line). When µ M < 0 (namely under M -poor condition), the solid line will further shift toward to the origin, while the dashed lines further deviate from the origin. From the chemical potential range shown in Fig. 2(a), therefore, the doped systems of Ti Cu and Ti Se is difficult to grow. The grey region shown in Fig. 2(a) implies the chemical potential range to be able to obtain the doped system of Ti Al . Lowering the chemical potential of Ti, namely representing the Ti-poor condition, the line determined by Ti Al will shift toward to the left, which results in the area of the grey region in Fig. 2(a) reducing. As a result, the Ti Al can be obtained when ∆H f (Ti Al ) − 3.85 ≤ µ T i ≤ 0 eV, where ∆H f (Ti Al ) can be obtained from our first-principles calculations, such as the values shown in Table II. The doping concentration is just corresponding to the variation of µ T i . For our considered several doping concentrations, as shown in Fig. 2(a), the phase boundary of Ti Se is sensitive to the doping content. With regarded to the substitution V atom for Cu or Se atom, as shown in Fig. 2(b), the doped systems of both V Cu and V Se are difficult to grow from the chemical potential constrains. For the substitution V

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atom for Al atom, the line determined by V Al shifts toward to the left comparing with Ti Al . The grey region shown in Fig. 2(b) marks out the range of chemical potential to grow the V Al system. However, the area of grey region in Fig. 2(b) slightly reduces comparing with that in Fig. 2(a). The V Al can be stabilized in the range of ∆H f (V Al ) − 3.85 ≤ µ V ≤ 0 eV. For the Fe doping situation, the Fe Se is impracticable, while the Fe Cu can be obtained in a very small chemical potential region. When substituting Fe atom for Al atom, the Fe Al can be grown in a region of chemical potential as the grey area shown in Fig. 2(c). Namely, the doping at Al site is available when the range of Fe chemical potential satisfies ∆H f (Fe Al ) − 3.85 ≤ µ F e ≤ 0 eV. Thus far, we have cleared that the stable doped phase can be obtained only when substituting M for Al atom, which is according with transition metal doped CuGaS 2 . Additionally, when presenting the chemical potential range of achieving in growth, we assume the constraint of Cu-rich (µ Cu = 0). However, the experimental fabrication often achieves under the Cu-poor situation (µ Cu < 0). For µ Cu < 0, the area of the grey region in Fig. 2 will decrease. Combining with Eqs. (3)(4)(5)(6)(7)(8), to obtain the doped phase, we know that the chemical

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potential of Cu must satisfy −0.85 < µ Cu ≤ 0 eV. After obtaining the stable M -doped CuAlSe 2 , we further investigate the electronic structures to evaluate the feasibility using as IB materials. View from the previous study [19], to achieve the high conversion efficiency, the subbangap formed by IB from VB or CB should be in the range of 0.7 − 1.2 eV, or near to this range. A narrow IB also leads to a high effective mass and a low carrier mobility that makes carrier transport within the band difficult. Thus IB with the finite width is perfect [38]. Importantly, the IB need be half-filled with electrons since both electrons and empty states are required to supply electrons to the CB as well as accepting them from the VB [18]. To examine whether the impurity bands induced by M doping satisfy these requirements above, we have calculated the electronic density of states (DOS) of M -doped CuAlSe 2 for different doping situations. Figure 3(a) shows the DOSs for Ti doping concentrations of 25% and 6.25%, respectively. Clear IB localized around the Fermi level is observed in these two doped CuAlSe 2 systems with substituting Ti atom for Al atom. As the dopant content reaching 25%, the IB is 1.4 eV from the VB maximum (VBM) and 0.7 eV from the CB minimum (CBM). The width of IB is about 0.5 eV. Lowering the doping concentration to 6.25%, the subbandgaps correspondingly become to 1.6 eV and 1.0 eV. The IB position in the

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Ti-doped system is suitable for promising IB materials as proposed by Martí et al [19]. With the increase of doping content as shown in Fig. 3(a), the electronic states to form IB increase and result in the extending of IB, which will suppress the Shockley-Read-Hall recombination [28,39]. Seen from the DOSs of V-doped CuAlSe 2 at two concentration levels of 25% and 6.25% shown in Fig. 3(b), however, no half-filled IB is observed around the Fermi level. The impurity bands are far away from the Fermi level and close to the VBM or CBM which indicates the subbandgap is very small. Furthermore, we can not observe the half-filled IB in Fe-doped CuAlSe 2 systems either. As the DOSs of two dop-ing concentrations shown in Fig. 3(c), the IB is completely empty though it has the isolated feature. As a result, from the aspect of electronic characteristics of IBs, both V-doping and Fe-doping are not good candidates, though the substitutions of V or Fe atom for Al atom satisfy the conditions of thermodynamic stability. The IB feature is related to the electronic configuration of transition metallic ion. In chalcopyrite CuAlSe 2 , four nearest neighboring Se atoms around a Al atom form a tetrahedral crystal field. The M ion substitutes the Al atom in CualSe 2 and therefore is located in a tetrahedral crystal field environment. Based on the crystal field theory, the fivefold degenerated 3d orbital of M will split into two main manifolds: lower twofold degenerate e g states and upper threefold degenerate t 2g

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states. Considering the electronic spin polarization on 3d orbitals, e 2↑ g t 3↑ 2g , namely d 5↑ , represents the five 3d electrons with the majority-spin, while e 2↓ g t 3↓ 2g (d 5↓ ) corresponding to the minority-spin situation. Substituting for Al atom in CuAlSe 2 , Ti, V, and Fe ions exhibit the valence states of Ti 3+ , V 3+ , and Fe 3+ , respectively. The orbital splitting in the tetrahedral crystal field combined with the DOS of M -3d leads to the electronic configurations of Ti 3+ -3d 1 -e 1↑ g , V 3+ -3d 2 -e 2↑ g , and Fe 3+ -3d 5 -e 2↑ g t 3↑ 2g . M 3+ ion is at high-spin state with all 3d electrons being spin up. For the substitution Ti atom for Al atom shown in Fig. 3(a), the IB nature origins from the half filling of e ↑ g band, due to the single 3d electron in Ti 3+ . The coupling between e ↑ g band and VBs is very weak. So there is visible gap between them. For the substitution V atom for Al atom, the number of 3d electrons increases to two. However, as shown in Fig. 3(b), these two 3d electrons full fill the e ↑ g band and lower its energy, which results that the full-filled e ↑ g band strongly couples with VB. The empty 3d bands made up of t ↑ 2g and e ↓ g t ↓ 2g

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are pushed up to the CB. This is reason that the favorable IB can not be observed in V-doped CuAlSe 2 . In Fe doping case, all of five 3d electrons exhibit the majority spin nature, namely e 2↑ g t 3↑ 2g . The full-filled e ↑ g t ↑ 2g bands strongly hybridize with the VBs of the host below the Fermi level. The e ↓ g t ↓ 2g bands are completely empty and form the isolated IBs in the main band gap of CuAlSe 2 . This is just the origin that no half-filled IBs is observed in Fe-doped CuAlSe 2 . Along the analysis for three doping elements above and extending other 3d transition metals such as Cr, Mn, Co, and Ni, the 3d electronic configuration is respectively Cr and Ni 3+ -3d 7 -e 2↑ g t 3↑ 2g e 2↓ g under substituting for Al atom in CuAlSe 2 . For Ni 3+ , we find that the orbitals are all full occupied without the half-filled band around the Fermi level. Seen from the electronic configurations, the halffilled or unfilled band is existent, t 1↑ 2g band for Cr 3+ , t 2↑ 2g band for Mn 3+ , and e 1↓ g band for Co 3+ , which can be viewed as IBs. However, the full-occupied band of e 2↑ g or t 3↑ 2g that exists will strongly couple or hybridize with the VBs of the host. As a result, the subbandgap between VB and IB will

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reduce or disappear. Combining this analysis with three typical doping elements of Ti, V, and Fe, therefore, we conclude that the IB characteristic is determined by the electronic nature of M 3+ ion. We further infer that M 3+ ion with the electronic configuration of d 1 can induce the optimum IB in wide band gap semiconductors. Just this, Ti is the most promising candidate. Adopted the method of Gajdoš et al. [40], the calculated optical properties further confirm the promising of Ti-doped CuAlSe 2 IB materials. Figure 4 shows the absorption coefficient α of Ti-doped CuAlSe 2 in visible light region, comparing with the host. After doping, we can observe a greatly enhanced light absorption intensity and additional absorption peaks in the range of 0.7 − 2.2 eV, which is induced by the IB. Especially, the 25% doping concentration case (Cu 4 Al 3 TiSe 8 ) visibly displays the three-photon absorption process. As shown in Fig. 4, peak 1 implies the electronic transition from IB to CB, and peak 2 represents the electronic transition from VB to IB, while peak 3 results from the electronic transition from VB to CB. The optical result further supports that Ti is the promising candidate to achieving the IB material. Conclusions To explore and design novel structural solar cell materials with higher conversion efficiency, we have investigated the feasibility of M (Ti, V, and Fe) doped CuAlSe 2 as IBSC material from thermodynamic stability, electronic structures, and optical properties, using the first-principles calculation based on HSE hybrid functional.

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Based on the chemical potential analysis, we point out that the stable M -doped CuAlSe 2 only exists when substituting for Al atom, and present the chemical potential conditions to growth. Seen from electronic structures, the Ti substitution results in the optimum IB feature, such as half-filled, favorable subbandgaps of 1.4 eV from VB to IB and 0.7 eV from IB to CB, as well limit width of IB for 25% doping concentration. However, the impurity band induced by the V doping adjoins the VB or CB, while the IB formed by the Fe doping is completely empty. Stable V-and Fe-doped phase can not result in the half-filled IB. We therefore conclude that the M 3+ with the electronic configuration of 3d 1 can drive the optimum IB in the wide bandgap semiconductor, namely the IB feature origins from the 3d electronic nature into the host. Combining the analysis above, Ti is considered to be the more promising candidate. The calculated absorption spectra of Ti-doped CuAlSe 2 illustrate three-photons absorption process. Two additional absorption peaks are formed in the band gap region of the host. And the absorption coefficient of doped systems in visible light region is obviously stronger than that of the host material. This study is helpful for the experimental research of IBSC materials from the doping stability and the forming mechanism of IB.

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The effect of COVID-19 on poor treatment control among ambulatory Hypertensive and/or Diabetic patients in Northwest Ethiopia Background Diabetes and hypertension have emerged as important clinical and public health problems in Ethiopia. The need to have long-term sustainable healthcare services for patients with diabetes and hypertension is essential to enhance good treatment control among those patients and subsequently delay or prevent complications. A collective shift towards acute care for COVID-19 patients combined with different measures to contain the pandemic had disrupted ambulatory care. Hence, it is expected to have a significant impact on treatment control of hypertensive and diabetic patients. However, there is limited evidence on the effect of the pandemic on treatment control and its determinants. Therefore, this study aimed to assess the effect of COVID-19 pandemic on treatment control of ambulatory Hypertensive and Diabetic patients and identify the factors for poor treatment control in North West Ethiopia. Methods A retrospective chart review and cross-sectional survey design were conducted between December 2020 and February 2021. Using a stratified systematic random sampling technique, 836 diabetic and/or hypertensive patients were included in the study. Web-based data collection was done using Kobo collect. The changes in the proportion of poor treatment control among ambulatory Hypertensive and/or Diabetic patients during the COVID-19 pandemic period were assessed. A multivariable binary logistic regression mixed model was fitted to identify the determinants of poor treatment control. The odds ratios were reported in both crude and adjusted form, together with their 95% confidence intervals and p-values. Result Poor treatment control increased significantly from

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24.81% (21.95, 27.92) prior to the COVID-19 pandemic to 30.33% (27.01, 33.88), 35.66% (32.26, 39.20), 36.69% (33.40, 40.12), and 34.18% (3102, 37.49) in the first, second, third, and fourth months following the date of the first COVID-19 case detection in Ethiopia, respectively. Marital status (AOR = 0.56, 95%CI; 0.41, 0.74), regimen of medication administration (AOR = 1.30, 95%CI; 1.02, 166), daily (AOR = 0.12, 95%CI; 0.08, 0.20), twice (AOR = 0.42, 95%CI; 0.30. 0.59), and three times (AOR = 0.31, 95%CI; 0.21, 0.47) frequency of medication, number medications taken per day (AOR = 0.79, 95%CI;0.73, 0.87), patients habits like hazardous alcohol use (AOR = 1.29, 95%CI; 1.02, 1.65) and sedentary lifestyle (AOR = 1.72,95%CI;1.46, 2.02), missed appointment during the COVID-19 pandemic (AOR = 2.09, 95%CI; 1.79, 2.45), and presence of disease related complication (AOR = 1.11, 95%CI; 0.93, 1.34) were significantly associated with poor treatment control among Diabetic and/or hypertensive patients during the COVID-19 pandemic. Conclusion The COVID-19 pandemic had a substantial impact on ambulatory Diabetic and/or Hypertensive patients’ treatment control. Being married, as well as the frequency and types of medicines taken per day were all found to be negatively associated with poor treatment control. During the COVID -19 pandemic, patients’ habits such as hazardous alcohol use and sedentary lifestyle, longer follow-up time, having disease-related complication (s), patients taking injectable medication, number of medications per day, and missed appointments were positively associated with poor treatment control in ambulatory diabetic and hypertensive patients. Therefore, it is better to consider the risk factors of poor treatment control

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while designing and implementing policies and strategies for chronic disease control. Conclusion The COVID-19 pandemic had a substantial impact on ambulatory Diabetic and/or Hypertensive patients' treatment control. Being married, as well as the frequency and types of medicines taken per day were all found to be negatively associated with poor treatment control. During the COVID -19 pandemic, patients' habits such as hazardous alcohol use and sedentary lifestyle, longer follow-up time, having disease-related complication (s), patients taking injectable medication, number of medications per day, and missed appointments were positively associated with poor treatment control in ambulatory diabetic and hypertensive patients. Therefore, it is better to consider the risk factors of poor treatment control while designing and implementing policies and strategies for chronic disease control. Background The highly contagious SARS-CoV-2 virus has killed over 4 million people globally since its detection in Wuhan, China, in December 2019 [1]. The COVID-19 epidemic has far-reaching consequences for the healthcare system. The COVID-19 pandemic has a substantial impact on ambulatory follow-up care in Africa's, particularly Ethiopia's, underdeveloped healthcare system. Governments from many nations have been obliged to enact legislative measures to stem the spread of the infection, which include complete lockdown, social isolation, confinement at home, and the suspension of all business activities varying in duration and extent [2,3]. In Ethiopia, diabetes and hypertension have emerged as major clinical and public health challenges. Both are significant causes of premature death and morbidity. To avoid poor glycaemic or blood pressure control, and hence delay or prevent disease progression and accompanying consequences, individuals

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with diabetes and hypertension require long-term sustainable healthcare services. Clinical services have been hampered by the necessity for contact precautions to stop the spread of the COVID-19 pandemic [3,4]. People with chronic medical conditions, such as diabetes and/or hypertension, are particularly prone to infection and have a higher rate of morbidity and mortality as a result of infection. COVID-19 infection was two to three times more likely in people with diabetes or hypertension, and it was associated with worse outcomes and a higher mortality rate. Diabetic individuals, for example, have twice the chance of being admitted to the intensive care unit for COVID-19 infection as non-diabetic patients. Diabetic and hypertensive patients are more susceptible to COVID-19 infection due to a higher risk of infection as a result of leukocyte malfunction, a pro-inflammatory profile, and micro-angiopathic alterations affecting the lungs or Angiotensin converting enzyme -2 receptors [5,6]. The COVID-19 pandemic is expected to have a substantial impact on hypertensive and diabetic patients' treatment control. Because it allows for the cancellation of non-emergent treatments and clinical appointments regularly, which will have an impact on the clinical outcomes for patients with chronic medical illnesses [7][8][9]. Meanwhile, appropriate treatment controls assist delay or preventing problems, as well as reducing COVID-19 infection morbidity and mortality. As a result of the pandemic's impact, some centres have made rapid and urgent shifts toward alternative patient care methodologies, such as virtual encounters (via video or phone) and medication delivery via mail, which are thought to reduce the risk of infection transmission and the burden

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on the health-care system. The number of ambulatory visits has decreased by 30% in some locations compared to pre-COVID-19 period [2,5,10,11] Patients' adherence to healthy lifestyles and medications has been influenced by various measures attempted to manage the pandemic, such as lockdowns. Age, sex, non-adherence to drugs, non-adherence to dietary restrictions, physical inactivity, the number of medications taken, and the existence of co-morbid disease are all known to play a role in diabetes and hypertension control. Factors associated with the COVID-19-related economic and social crisis may have a substantial indirect impact on the care [5,11,12].The pandemic had a huge impact, especially on these patients, where the goal isn't to cure them completely, but rather to slow down the disease's progression and prevent complications. However, there is no evidence of the pandemic's impact on medication adherence and its determinants in diabetic and hypertensive patients. Therefore, this study aimed at assessing treatment control during COVID-19 pandemic periods and its factors. Thus, assessing the treatment control of patients with diabetes and/ or hypertension during the pandemic might have paramount importance for designing and implementing intervention measures. Study design and setting Retrospective chart review and facility-based cross-sectional study design were employed in public Hospitals that are giving chronic care in Northwest Ethiopia, from January to March 2021. Northwest Ethiopia includes Amhara regional state which has 15 Zones and 180 whereas (139 rural and 41 urban). The first COVID-19 case was confirmed on March 30, 2020. Treatment centres, isolation, and quarantine centres were established in the region as the COVID 19

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prevention and treatment strategies. According to the regional health COVID 19 command team report currently, 291,148 susceptible individuals were tested for COVID 19. Of these 11727 cases were detected, 2862 recovered, and 293 death recorded in the region. The region has 80 hospitals (6 referrals, 2 generals, and 72 primaries), 847 health centres, and 3,342 health posts. The study was incorporating all hospitals (referral, district) in the Amhara region with chronic care centres. Source and study population The source population consisted of all patients with diabetes and/or hypertension who had follow-ups at hospitals in the Amhara regional state. The study population consisted of patients who had chronic care appointments and follow-up during the data collection period. Patients with common chronic disease conditions who were at least 18 years old and had been on medication for at least 2 years were included in the study. Participants who returned within the data collection period were not included in the study. Sample size and sampling procedures With the key objectives in mind, the sample size was estimated using the single population proportion formula. A design effect of 2 and a 10% non-response rate were also taken into account. As a result, the ultimate sample size was estimated to be 845 patients. To recruit study participants, all referral and selected district hospitals in the region were included in the sampling procedure. First, stratification was done based on the status of the hospital (referral or district). Hospitals were then chosen from each stratum. Finally, study participants were chosen using a systematic

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random sampling technique in the specified hospitals based on disease type. The total sample was proportionally allocated across illness types. Data collection methods and measurements To collect the required data on the variables of interest, primary and secondary sources were used. The baseline measurement was taken from the most recent measurement before the first date of COVID-19 case detection, and one year following the COVID-19 case detection was divided into four periods, with the first three months, second three months, third three months, and fourth three months being the first, second, third, and fourth periods, respectively. Charts were retrieved from the treatment centres in the selected Hospitals. During data collection, it has been about a year since the emergence of the COVID-19 pandemic. One-year retrospective data was extracted for the same patients before the COVID-19 pandemic. All available epidemiological information was collected including, socio-demographic variables, clinical factors, and patient treatment control. Treatment control: was ascertained by the treating physician working in the respective follow-up clinics as poor or good. Poor treatment control was considered when the treatment target was not achieved on that specific follow-up date. The treating physician used glycaemic target or blood pressure target coupled with other clinical parameters to ascertain treatment control. Missed appointments: when a patient did not attend the follow-up according to the physicians' schedule. Health management information systems and patient charts were used to extract the data using chart extraction form. Patients' interview was made after the appointment logbook and patient chart retrieval. Quality assurance mechanism Data collectors and supervisors

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were provided training to maintain the data's quality. Medical physicians and other trained health professionals who work in treatment centres were recruited. The questionnaire was translated into Amharic, the native language, and then returned to English to ensure consistency. After the questionnaire was converted to electronic data from using Kobo-collect, web-based data collection was done. A pre-test was conducted, and possible adjustments were made, as well as an internal consistency reliability test. The collected data were checked for completeness and consistency daily at the server. Data management and analysis Following completion of data collection, the web-based data was exported to STATA and R for management and analysis. Cleaning, coding, categorization, and error inception were made by the research team. Results were explored using descriptive statistical techniques and prevalence, mean, median, inter-quartile range, and standard deviations were computed. Since the data had hierarchical nature, it could violate the independence of observations and equal variance assumption of the ordinary logistic regression model. Hence, measurements are nested within an individual; we expect that measurements within the same individuals are more likely to be related to each other than the other individuals. To assess the nested effect, intra-class correlation coefficient was computed as; , where: the ordinary logit distribution has variance of p 2 � 3 , σ μ 2 indicates the cluster variance [28]. The calculated ICC was 11.56% in the null model while σ μ 2 was 0.43. This implies that there is a need to take into account the between individual variability by using advanced models. Therefore,

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for the associated factors, we used the binary logistic mixed-effect regression model. Likelihood Ratio (LR) test and Deviance (-2LLR) was used for model comparison. Accordingly, a mixed effect logistic regression model (both fixed and random effect) was the best-fitted model since it had the lowest deviance value. Both bi-variables and multivariable binary logistic regression models were considered. Variables with a pvalue < 0.2 in the bi-variable analysis were considered in the multivariable. Since the mixed effect model was estimating subject-specific estimates, we covert to population average by a conversion factor: 1 ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ð1þ0:346d b 2 Þ p b (where; β = fixed effect and δ b 2 = random effect estimate) for the interpretation purpose. Finally, both crude and adjusted odds ratios with a 95% Confidence Interval (CI) of the selected model were reported. P-value � 0.05 in the multivariable model were used to declare significant factors associated with poor treatment outcomes. Ethical consideration The University of Gondar's institutional review board provided ethical approval. The Amhara public health institution sent a letter of support, and the medical directors of each hospital approved. The goal, objectives, and right to participate or not engage in the study were all explained to the participants. Participants' permission to withdraw from the study at any time and without explanation was clearly stated. Before data collection, each subject gave their written consent. Furthermore, rather than using personal identifiers, code numbers were utilized to ensure confidentiality. Background

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characteristics A total of 836Diabetic and/ or Hypertensive patients were included in the study with a response rate of 99%. The man diagnosis was Diabetes in 410 (49%) patients. The median age of the study participants was 52 years with Inter Quartile Range (IQR) of 18 (43-61). Nearly two-third of the study participants, 543 (65%) were urban dwellers. Besides, the median duration on follow-up was 5 years with an IQR of 5 (3-8) years. A quarter of the patients, 209 (25%), were housewife followed by a government employee (22%) in their employment. Although more than half (54%) of patients were covered by health insurance, 351 (42%) participants paid their medical expenses from their pocket. Three hundred thirty-six (40%) of patients had one or more identified co-morbidities. While109 (13%) of patients have one or more chronic complications. Five hundred thirty-five (64%) of patients take multiple prescribed medications. Related to the habit of the study participants, 84 (10%) of our patients have a history of hazardous alcohol use. Moreover, three hundred eighteen (38%) of the patients didn't meet the WHO recommendation on physical activity for health. RelatedCOVID-19 pandemic, 334 (40%) of patients had at least one emergency visit during the pandemic period. One in four, (23.02%), of the participants, had COVID-19 like symptom (s) during the study period but more than half of the study participants,453 (54.988%), perceived that they had COVID-19 infection. Of those who had symptoms consistent with COVID-19, only 9 were tested positive for COVID-19 infection. Hypertensive and/or Diabetic patients missed their medical appointment(s) during

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the COVID-19 pandemic. Eighty-four patients (10%) missed appointment before COVID-19 but 205 (31%) of them were missed their appointment due to the pandemic. Nearly half of the patients, 117(45%), who missed their appointments ascribed the missed visit for fear of acquiring COVID-19 infection from Hospital (Table 1). (Fig 1). Factors associated with poor treatment control After identifying variables significantly associated with poor treatment control in the bi-variable analysis at a p-value less than 0.2.Variables such as marital status, duration of follow up, regimen of medications, frequency of drug use, number of medications, hazardous alcohol use, sedentary lifestyle, presence of complication, and missed appointment during the pandemic were significantly associated with poor treatment outcome among diabetic and/or hypertensive patients during COVID-19 pandemic at a 5% level of significance. This study revealed that the odds of poor treatment control among married participants were 44% (AOR = 0.56, 95%CI; 0.41, 0.74) lower as compared with unmarried participants. The likelihood of having poor treatment control for patients who take injectable medication was 1.30 (AOR = 1.30, 95%CI; 1.02, 166), times higher as compared with those who take oral medication. Besides, the frequency of drug use per day was significantly associated with poor treatment control. As compared with patients taking drug five or more times per day, the chance of poor treatment control was 88% (AOR = 0.12, 95%CI;0.08, 0.20), 58% (AOR = 0.42, 95%CI;0.30. 0.59), and 69% (AOR = 0.31, 95%CI; 0.21, 0.47) lower for patients taking one, two, and three times per a day, respectively. As the number of

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