A Proteomic Strategy for Global Analysis of Plant Protein Complexes

Global analyses of protein complex assembly, composition, and location are needed to fully understand how cells coordinate diverse metabolic, mechanical, and developmental activities. The most common methods for proteome-wide analysis of protein complexes rely on affinity purification-mass spectrometry or yeast two-hybrid approaches. These methods are time consuming and are not suitable for many plant species that are refractory to transformation or genome-wide cloning of open reading frames. Here, we describe the proof of concept for a method allowing simultaneous global analysis of endogenous protein complexes that begins with intact leaves and combines chromatographic separation of extracts from subcellular fractions with quantitative label-free protein abundance profiling by liquid chromatography-coupled mass spectrometry. Applying this approach to the crude cytosolic fraction of Arabidopsis thaliana leaves using size exclusion chromatography, we identified hundreds of cytosolic proteins that appeared to exist as components of stable protein complexes. The reliability of the method was validated by protein immunoblot analysis and comparisons with published size exclusion chromatography data and the masses of known complexes. The method can be implemented with appropriate instrumentation, is applicable to any biological system, and has the potential to be further developed to characterize the composition of protein complexes and measure the dynamics of protein complex localization and assembly under different conditions.     

Functional modulation of lysophosphatidic acid type 2 G-protein coupled receptor facilitates alveolar bone formation

Lipid biosynthesis is recently studied its functions in a range of cellular physiology including differentiation and regeneration. However, it still remains to be elucidated in its precise function. To reveal this, we evaluated the roles of lysophosphatidic acid (LPA) signaling in alveolar bone formation using the LPA type 2 receptor (LPAR2) antagonist AMG-35 (Amgen Compound 35) using tooth loss without periodontal disease model which would be caused by trauma and usually requires a dental implant to restore masticatory function. In this study, in vitro cell culture experiments in osteoblasts and periodontal ligament fibroblasts revealed cell type-specific responses, with AMG-35 modulating osteogenic differentiation in osteoblasts in vitro. To confirm the in vivo results, we employed a mouse model of tooth loss without periodontal disease. Five to 10 days after tooth extraction, AMG-35 facilitated bone formation in the tooth root socket as measured by immunohistochemistry for differentiation markers KI67, Osteocalcin, Periostin, RUNX2, transforming growth factor beta 1 (TGF-β1) and SMAD2/3. The increased expression and the localization of these proteins suggest that AMG-35 elicits osteoblast differentiation through TGF-β1 and SMAD2/3 signaling. These results indicate that LPAR2/TGF-β1/SMAD2/3 represents a new signaling pathway in alveolar bone formation and that local application of AMG-35 in traumatic tooth loss can be used to facilitate bone regeneration and healing for further clinical treatment.     

The Burden and Determinants of Non Communicable Diseases Risk Factors in Nepal: Findings from a Nationwide STEPS Survey

Background

World Health Organization (WHO) estimates for deaths attributed to Non Communicable Diseases (NCDs) in Nepal have risen from 51% in 2010 to 60% in 2014. This study assessed the distribution and determinants of NCD risk factors among the Nepalese adult population.

Methods and Findings

A nationally representative cross-sectional survey was conducted from Jan to June 2013 on the prevalence of NCD risk factors using the WHO NCD STEPS instrument. A multistage cluster sampling method was used to randomly select the 4,200 respondents. The adjusted prevalence ratio (APR) was used to assess the determinants of NCD risk factors using a Poisson regression model. The prevalence of current smoking (last 30 days) was 19% (95%CI:16.6-20.6), and harmful alcohol consumption (≥60 g of pure alcohol for men and ≥40 g of pure alcohol for women on an average day) was 2% (95%CI:1.4-2.9). Almost all (99%, 95%CI:98.3-99.3) of the respondents consumed less than five servings of fruits and vegetables combined on an average day and 3% (95%CI:2.7-4.3) had low physical activity. Around 21% (95%CI:19.3-23.7) were overweight or obese (BMI≥25). The prevalence of raised blood pressure (SBP≥140 mm of Hg or DBP≥90 mm of Hg) and raised blood glucose (fasting blood glucose ≥126 mg/dl), including those on medication were 26% (95%CI:23.6-28.0) and 4% (95%CI:2.9-4.5) respectively. Almost one quarter of respondents, 23% (95%CI:20.5-24.9), had raised total cholesterol (total cholesterol ≥190 mg/dl or under current medication for raised cholesterol). he study revealed a lower prevalence of smoking among women than men (APR:0.30; 95%CI:0.25-0.36), and in those who had higher education levels compared to those with no formal education (APR:0.39; 95%CI:0.26-0.58). Harmful alcohol use was also lower in women than men (APR:0.26; 95%CI:0.14-0.48), and in Terai residents compared to hill residents (APR:0.16; 95%CI:0.07-0.36). Physical inactivity was lower among women than men (APR:0.55; 95%CI:0.38-0.80), however women were significantly more overweight and obese (APR:1.19; 95%CI:1.02-1.39). Being overweight or obese was significantly less prevalent in mountain residents than in hill residents (APR:0.41; 95%CI:0.21-0.80), and in rural compared to urban residents (APR:1.39; 95%CI:1.15-1.67). Lower prevalence of raised blood pressure was observed among women than men (APR:0.69; 95%CI: 0.60-0.80). Higher prevalence of raised blood glucose was observed among urban residents compared to rural residents (APR:2.05; 95%CI:1.29-3.25). A higher prevalence of raised total cholesterol was observed among the respondents having higher education levels compared to those respondents having no formal education (APR:1.76; 95%CI:1.35-2.28).

Conclusion

The prevalence of low fruit and vegetable consumption, overweight and obesity, raised blood pressure and raised total cholesterol is markedly high among the Nepalese population, with variation by demographic and ecological factors and urbanization. Prevention, treatment and control of NCDs and their risk factors in Nepal is an emerging public health problem in the country, and targeted interventions with a multi-sectoral approach need to be urgently implemented.     

Influence of lipids on the hydrophobic barrier within the pore of the TWIK-1 K2P channel

Several recent ion channel structures have revealed large side portals, or ‘fenestrations’ at the interface between their transmembrane helices that potentially expose the ion conduction pathway to the lipid core of the bilayer. In a recent study we demonstrated that functional activity of the TWIK-1 K2P channel is influenced by the presence of hydrophobic residues deep within the inner pore. These residues are located near the fenestrations in the TWIK-1 structure and promote dewetting of the pore by forming a hydrophobic barrier to ion conduction. During our previous MD simulations, lipid tails were observed to enter these fenestrations. In this addendum to that study, we investigate lipid contribution to the dewetting process. Our results demonstrate that lipid tails from both the upper and lower leaflets can occupy the fenestrations and partially penetrate into the pore. The lipid tails do not sterically occlude the pore, but there is an inverse correlation between the presence of water within the hydrophobic barrier and the number of lipids tails within the lining of the pore. However, dewetting still occurs in the absence of lipids tails, and pore hydration appears to be determined primarily by those side-chains lining the narrowest part of the pore cavity.     

Foods,macronutrients and fibre in the diet of blue sheep (Psuedois nayaur) in the Annapurna Conservation Area of Nepal

Food resources are often critical regulating factors affecting individual fitness and population densities. In the Himalayan Mountains, Bharal “blue sheep” (Pseudois nayaur) are the main food resource for the endangered snow leopard (Panthera uncia), as well as being preyed upon by other predators. Blue sheep, however, may face a number of challenges including food resource competition with other wild and domestic ungulates, and hunting pressure. Here, we characterized the diet of blue sheep in the Annapurna Conservation Area (ACA) of Nepal and conducted proximate nutritional analysis on a limited number of plants identified as foods. Furthermore, we investigated the macronutrient and fiber balance of these plants using nutritional geometry which is a state‐space approach to modeling multidimensional and interactive nutritional aspects of foraging. A total of 19 plant species/genera were identified in blue sheep pellets using microhistological analysis. On average, across seasons and regions of the study area, the two most frequently occurring plants in pellets were graminoids: Kobressia sp. and Carex spp. The macronutrient balance of Kobresia sp. was relatively high in carbohydrate and low in protein, while other plants in the diet were generally higher in protein and lipid content. Analysis of fiber balance showed that the two most consumed plants of blue sheep (i.e., Kobresia spp. and Carex spp.) contained the highest concentration of hemicellulose, which is likely digestible by blue sheep. The hemicellulose and lignin balance of plants ranged relatively widely, yet their cellulose contents showed less variation. Foraging by blue sheep may therefore be a balance between consuming highly digestible high‐carbohydrate plants and plants less‐digestible but higher in protein and/or lipid.     

In the shadows of snow leopards and the Himalayas: density and habitat selection of blue sheep in Manang,Nepal

There is a growing agreement that conservation needs to be proactive and pay increased attention to common species and to the threats they face. The blue sheep (Pseudois nayaur) plays a key ecological role in sensitive high‐altitude ecosystems of Central Asia and is among the main prey species for the globally vulnerable snow leopard (Panthera uncia). As the blue sheep has been increasingly exposed to human pressures, it is vital to estimate its population dynamics, protect the key populations, identify important habitats, and secure a balance between conservation and local livelihoods. We conducted a study in Manang, Annapurna Conservation Area (Nepal), to survey blue sheep on 60 transects in spring (127.9 km) and 61 transects in autumn (134.7 km) of 2019, estimate their minimum densities from total counts, compare these densities with previous estimates, and assess blue sheep habitat selection by the application of generalized additive models (GAMs). Total counts yielded minimum density estimates of 6.0–7.7 and 6.9–7.8 individuals/km² in spring and autumn, respectively, which are relatively high compared to other areas. Elevation and, to a lesser extent, land cover indicated by the normalized difference vegetation index (NDVI) strongly affected habitat selection by blue sheep, whereas the effects of anthropogenic variables were insignificant. Animals were found mainly in habitats associated with grasslands and shrublands at elevations between 4,200 and 4,700 m. We show that the blue sheep population size in Manang has been largely maintained over the past three decades, indicating the success of the integrated conservation and development efforts in this area. Considering a strong dependence of snow leopards on blue sheep, these findings give hope for the long‐term conservation of this big cat in Manang. We suggest that long‐term population monitoring and a better understanding of blue sheep–livestock interactions are crucial to maintain healthy populations of blue sheep and, as a consequence, of snow leopards.     

Molecular Basis for Benzodiazepine Agonist Action at the Type 1 Cholecystokinin Receptor

Understanding the molecular basis of drug action can facilitate development of more potent and selective drugs. Here, we explore the molecular basis for action of a unique small molecule ligand that is a type 1 cholecystokinin (CCK) receptor agonist and type 2 CCK receptor antagonist, GI181771X. We characterize its binding utilizing structurally related radioiodinated ligands selective for CCK receptor subtypes that utilize the same allosteric ligand-binding pocket, using wild-type receptors and chimeric constructs exchanging the distinct residues lining this pocket. Intracellular calcium assays were performed to determine biological activity. Molecular models for docking small molecule agonists to the type 1 CCK receptor were developed using a ligand-guided refinement approach. The optimal model was distinct from the previous antagonist model for the same receptor and was mechanistically consistent with the current mutagenesis data. This study revealed a key role for Leu^7.39 that was predicted to interact with the isopropyl group in the N1 position of the benzodiazepine that acts as a “trigger” for biological activity. The molecular model was predictive of binding of other small molecule agonists, effectively distinguishing these from 1065 approved drug decoys with an area under curve value of 99%. The model also selectively enriched for agonist compounds, with 130 agonists identified by ROC analysis when seeded in 2175 non-agonist ligands of the type 1 CCK receptor (area under curve 78%). Benzodiazepine agonists in this series docked in consistent pose within this pocket, with a key role played by Leu^7.39, whereas the role of this residue was less clear for chemically distinct agonists.     

Feeding preferences and nutritional niche of wild water buffalo (Bubalus arnee) in Koshi Tappu Wildlife Reserve,Nepal

The nutritional characteristics of food resources play an important role in the foraging behavior of animals and can provide information valuable to their conservation and management. We examined the nutritional ecology of wild water buffalo (Bubalus arnee; hereafter “buffalo”) in the Koshi Tappu Wildlife Reserve of Nepal during autumn using a multidimensional nutritional niche framework. We identified 54 plant species as being foraged by buffalo. We found that buffalo consumed graminoids and forbs 2–3 times more frequently than browse items. Proximate analyses of the 16 most frequently foraged plants indicated that buffalo diets were highest in carbohydrate (40.41% ± 1.82%) followed by crude protein (10.52% ± 0.93%) and crude fat (1.68% ± 0.23%). The estimated macronutrient balance (i.e., realized nutrient niche) of the buffalo diet (20.5% protein: 72.8% carbohydrate: 6.7% lipid) was not significantly different than the average balance of all analyzed food items based on 95% confidence regions. Our study suggests that buffalo are likely macronutrient specialists, yet may be generalists in the sense that they feed on a wide range of food items to achieve a nutrient balance similar to that available in forage items. However, the four most frequently consumed items tended to be higher in protein energy than less frequently consumed foods, suggesting some preference for higher protein forage relative to relatively abundant carbohydrates. Although limited in scope, our study provides important information on the nutritional ecology of buffalo, which may be useful for the conservation and management of this endangered species.     

Retinol-binding protein 4 inhibits insulin signaling in adipocytes by inducing proinflammatory cytokines in macrophages through a c-Jun N-terminal kinase- and toll-like receptor 4-dependent and retinol-independent mechanism

Retinol-binding protein 4 (RBP4), the sole retinol transporter in blood, is secreted from adipocytes and liver. Serum RBP4 levels correlate highly with insulin resistance, other metabolic syndrome factors, and cardiovascular disease. Elevated serum RBP4 causes insulin resistance, but the molecular mechanisms are unknown. Here we show that RBP4 induces expression of proinflammatory cytokines in mouse and human macrophages and thereby indirectly inhibits insulin signaling in cocultured adipocytes. This occurs through activation of c-Jun N-terminal protein kinase (JNK) and Toll-like receptor 4 (TLR4) pathways independent of the RBP4 receptor, STRA6. RBP4 effects are markedly attenuated in JNK1-/- JNK2-/- macrophages and TLR4-/- macrophages. Because RBP4 is a retinol-binding protein, we investigated whether these effects are retinol dependent. Unexpectedly, retinol-free RBP4 (apo-RBP4) is as potent as retinol-bound RBP4 (holo-RBP4) in inducing proinflammatory cytokines in macrophages. Apo-RBP4 is likely to be physiologically significant since RBP4/retinol ratios are increased in serum of lean and obese insulin-resistant humans compared to ratios in insulin-sensitive humans, indicating that higher apo-RBP4 is associated with insulin resistance independent of obesity. Thus, RBP4 may cause insulin resistance by contributing to the development of an inflammatory state in adipose tissue through activation of proinflammatory cytokines in macrophages. This process reveals a novel JNK- and TLR4-dependent and retinol- and STRA6-independent mechanism of action for RBP4.