Chloroquine sensitizes breast cancer cells to chemotherapy independent of autophagy

Chloroquine (CQ) is a 4-aminoquinoline drug used for the treatment of diverse diseases. It inhibits lysosomal acidification and therefore prevents autophagy by blocking autophagosome fusion and degradation. In cancer treatment, CQ is often used in combination with chemotherapeutic drugs and radiation because it has been shown to enhance the efficacy of tumor cell killing. Since CQ and its derivatives are the only inhibitors of autophagy that are available for use in the clinic, multiple ongoing clinical trials are currently using CQ or hydroxychloroquine (HCQ) for this purpose, either alone, or in combination with other anticancer drugs. Here we show that in the mouse breast cancer cell lines, 67NR and 4T1, autophagy is induced by the DNA damaging agent cisplatin or by drugs that selectively target autophagy regulation, the PtdIns3K inhibitor LY294002, and the mTOR inhibitor rapamycin. In combination with these drugs, CQ sensitized to these treatments, though this effect was more evident with LY294002 and rapamycin treatment. Surprisingly, however, in these experiments CQ sensitization occurred independent of autophagy inhibition, since sensitization was not mimicked by Atg12, Beclin 1 knockdown or bafilomycin treatment, and occurred even in the absence of Atg12. We therefore propose that although CQ might be helpful in combination with cancer therapeutic drugs, its sensitizing effects can occur independently of autophagy inhibition. Consequently, this possibility should be considered in the ongoing clinical trials where CQ or HCQ are used in the treatment of cancer, and caution is warranted when CQ treatment is used in cytotoxic assays in autophagy research.     

Dynamic proteome analysis of Cyanothece sp. ATCC 51142 under constant light

Understanding the dynamic nature of protein abundances provides insights into protein turnover not readily apparent from conventional, static mass spectrometry measurements. This level of data is particularly informative when surveying protein abundances in biological systems subjected to large perturbations or alterations in environment such as cyanobacteria. Our current analysis expands upon conventional proteomic approaches in cyanobacteria by measuring dynamic changes of the proteome using a (13)C(15)N-l-leucine metabolic labeling in Cyanothece ATCC51142. Metabolically labeled Cyanothece ATCC51142 cells grown under nitrogen-sufficient conditions in continuous light were monitored longitudinally for isotope incorporation over a 48 h period, revealing 414 proteins with dynamic changes in abundances. In particular, proteins involved in carbon fixation, pentose phosphate pathway, cellular protection, redox regulation, protein folding, assembly, and degradation showed higher levels of isotope incorporation, suggesting that these biochemical pathways are important for growth under continuous light. Calculation of relative isotope abundances (RIA) values allowed the measurement of actual active protein synthesis over time for different biochemical pathways under high light exposure. Overall results demonstrated the utility of "non-steady state" pulsed metabolic labeling for systems-wide dynamic quantification of the proteome in Cyanothece ATCC51142 that can also be applied to other cyanobacteria.     

In silico assessment of potential druggable pockets on the surface of α1-antitrypsin conformers

The search for druggable pockets on the surface of a protein is often performed on a single conformer, treated as a rigid body. Transient druggable pockets may be missed in this approach. Here, we describe a methodology for systematic in silico analysis of surface clefts across multiple conformers of the metastable protein α(1)-antitrypsin (A1AT). Pathological mutations disturb the conformational landscape of A1AT, triggering polymerisation that leads to emphysema and hepatic cirrhosis. Computational screens for small molecule inhibitors of polymerisation have generally focused on one major druggable site visible in all crystal structures of native A1AT. In an alternative approach, we scan all surface clefts observed in crystal structures of A1AT and in 100 computationally produced conformers, mimicking the native solution ensemble. We assess the persistence, variability and druggability of these pockets. Finally, we employ molecular docking using publicly available libraries of small molecules to explore scaffold preferences for each site. Our approach identifies a number of novel target sites for drug design. In particular one transient site shows favourable characteristics for druggability due to high enclosure and hydrophobicity. Hits against this and other druggable sites achieve docking scores corresponding to a K(d) in the μM-nM range, comparing favourably with a recently identified promising lead. Preliminary ThermoFluor studies support the docking predictions. In conclusion, our strategy shows considerable promise compared with the conventional single pocket/single conformer approach to in silico screening. Our best-scoring ligands warrant further experimental investigation.     

The diet of the striped hyena in Nepal's lowland regions

Striped hyenas (Hyaena hyaena) are extremely rare in Nepal, and only a few people have studied them in their natural forest and grassland habitat. Their rarity is due to anthropogenic pressures such as hunting, habitat modification, being killed on roads, and depletion of their natural prey. Here, we studied the feeding ecology of hyenas in lowland, Nepal. We employed an opportunistic sampling to collect hyena scats in a range of habitats and the line transect sampling to identify the prey of the hyena in the study site. We collected 68 hyena scats between 2015 and 2018. Most of the hyena scat (39.7%) was found in the Churia Hill forest followed by riverbed (26.4%), mixed forest (14.7%), Sal (Shorea robusta)‐dominated forest (11.7%), and grassland area (7.3%). We found eleven mammalian prey species, plants, and some unidentified items in the hyena scats. The frequency of occurrence and relative biomass of the medium‐sized wild boar (Sus scrofa) were higher than other smaller prey species such as hare (Lepus nigricollis) and rhesus macaque (Macaca mulatta). Similarly, the proportion of large prey species such as nilgai (Boselaphus tragocamelus) in the hyena diet was lower compared with wild boar, hares, and rhesus macaques indicating medium‐sized wild boar is the most preferred prey species. Livestock contributed 17.3% of the total dietary biomass. Domesticated species such as goats, sheep, cows, and even dogs were found in the diet of hyenas. Predation of livestock by hyenas could cause conflict, especially if this ongoing issue continues in the future. Rather, more conservation effort is required in lowland areas of Nepal to protect the hyenas' natural prey species, particularly in wildlife habitats to reduce the lure of taking domestic livestock. Similarly, conservation education at the local level and active involvement of government authorities in the conservation of this species might be helpful to mitigate human–hyena conflict in the human‐dominated landscape.     

Low genetic diversity in the endangered great Indian bustard (Ardeotis nigriceps) across India and implications for conservation

The great Indian bustard (Ardeotis nigriceps) is an endemic endangered bird of the Indian subcontinent with a declining population, as a result of hunting and continuing habitat loss. In this first genetic study of this little-known species, we investigate the diversity of the mitochondrial DNA (hypervariable control region II and cytochrome b gene) among samples (n = 63) from five states within the current distribution range of great Indian bustards in India. We find just three haplotypes defined by three variable sites, a comparatively low genetic diversity of π = 0.0021 ± 0.0012 for cytochrome b, 0.0008 ± 0.0007 for the control region (CR), and 0.0017 ± 0.0069 for combined regions and no phylogeographic structure between populations. We provide evidence for a bottleneck event, estimate an effective population size (Ne) that is roughly concordant with recent population size estimates based on field surveys (~200 to 400), but extremely low for a widely distributed species. We also discuss the conservation implications. Based on our findings, we strongly recommend upgrading the IUCN threat status from Endangered to Critically Endangered.     

Sex-dimorphic moderate hypoglycemia preconditioning effects on Hippocampal CA1 neuron bio-energetic and anti-oxidant function

Molecular and Cellular Biochemistry - Hypoglycemia is a detrimental complication of rigorous management of type 1 diabetes mellitus. Moderate hypoglycemia (MH) preconditioning of male rats...     

Socioeconomic and cultural differentials in age at marriage and the effect on fertility in Nepal

Age at marriage is one of the factors that influence the fertility behaviour of women, particularly in a society like Nepal where contraceptive use is low. Socioeconomic and cultural factors, particularly religion and ethnicity, are important variables in determining age at marriage in Nepal. Fertility was negatively related with age at marriage. Marriage duration had a greater influence on fertility than age at marriage, although these were strongly correlated.     

Actin re-distribution in response to hydrogen peroxide in airway epithelial cells

Reactive oxygen species (ROS) disrupt the barrier function of airway epithelial cells through a mechanism that appears to involve remodeling of the actin cytoskeleton. Similarly, keratinocyte growth factor (KGF) has been shown to protect against ROS-induced loss of barrier function through a mechanism that may also involve the actin cytoskeleton. To further determine the role of the actin cytoskeleton in ROS-induced barrier injury, we quantified the relative amount of total actin associated with the cytoskeleton following exposure to hydrogen peroxide (H(2)O(2)) and pretreatment with KGF. We also determined the role of the actin-myosin contractile mechanism in the process by quantifying the relative amount of myosin heavy chain (MHC) associated with the cytoskeleton. While the transepithelial resistance (TER) of a monolayer of airway epithelial cells (Calu-3) decreased after 2 h of continuous exposure to 0.5 mM H(2)O(2), actin and MHC, both dissociated from the cytoskeleton within 15 min of H(2)O(2) exposure. The TER of the monolayers remained depressed although both actin and myosin returned to the cytoskeleton by 4 h after the initiation of H(2)O(2) exposure. Filamentous actin (f-actin) staining suggested that the re-associating actin took the form of short fibers associated with cortical actin rather than long stress fibers. Furthermore, pretreatment with KGF prevented the loss of actin and MHC from the actin cytoskeleton but did not prevent the decrease in TER. These studies suggest that actin disassembly from the cytoskeleton is important in the loss of barrier function, but that it is not the overall amount of actin that is associated with the cytoskeleton that is important, rather it is the contribution this actin makes to the architectural cohesiveness of the cell that contributes to the barrier function.     

Effect of Urbanization and Seasonality in Bird Communities of Kathmandu Valley,Nepal

Kathmandu Valley, Nepal is undergoing rapid urbanization but its effects on the bird communities have not been reported till date. Kathmandu Valley was categorized into urban, sub-urban and rural to study the impact of urbanization in bird communities. By mobilizing volunteers, we monitored 24 transects each with one km long in summer and winter seasons of 2016. A total of 13,749 individuals of birds belonging to 102 species were recorded. Species richness and diversity of all birds declined from rural to urban areas and showed significant variation along urban–rural gradients. Insectivore was the most species-rich guild while nectarivore the least. The richness of insectivore, frugivore and carnivore guilds showed significant variations along the urban–rural gradients and higher preference towards the rural areas. Similarly, species richness of all birds and richness of insectivore and carnivore guilds showed significant seasonal variation and were higher in the winter season. Our results indicate that richness, diversity and feeding guilds of birds show different response towards the urbanization gradients and seasons. Sub-urban areas can function as bird refugia, however, habitat enrichments (like increasing green spaces, setting up new parks and gardens, plantation of native fruiting trees etc.) are utmost necessary to support the bird communities in urban areas of Kathmandu Valley.