Global identification of mRNA-interacting circular RNAs by CLiPPR-Seq.

Although the functional role of circular RNA (circRNA) interaction with microRNAs and proteins has been studied extensively, circRNA interactions with the protein-coding mRNAs in intact cells remain largely unknown. Here, by employing AMT-mediated proximity ligation of RNA-RNA duplexes followed by circRNA enrichment and deep sequencing, we report a novel Cross-Linking Poly(A) Pulldown RNase R Sequencing (CLiPPR-seq) technology which identified hundreds of mRNA-interacting circRNAs in three different cell types, including ßTC6, C2C12 and HeLa cells. Furthermore, CLiPP-seq without RNase R treatment was also performed to identify the mRNA expression in these cells. BLAST analysis of circRNAs in CLiPPR-seq sample with the mRNAs in CLiPP-seq samples determined their potential complementary sequences for circRNA-mRNA interaction. Pulldown of circRNAs and poly(A) RNAs confirmed the direct interaction of circRNAs with target mRNAs. Silencing of mRNA-interacting circRNAs led to the altered expression of target mRNAs in ßTC6 cells, suggesting the role of direct interaction of circRNAs with mRNAs in gene expression regulation. CLiPPR-seq thus represents a novel method for illuminating the myriad of uncharacterized circRNA-mRNA hybrids that may regulate gene expression.

The thermal niche and phylogenetic assembly of evergreen tree metacommunities in a mid-to-upper tropical montane zone.

Frost and freezing temperatures have posed an obstacle to tropical woody evergreen plants over evolutionary time scales. Thus, along tropical elevation gradients, frost may influence woody plant community structure by filtering out lowland tropical clades and allowing extra-tropical lineages to establish at higher elevations. Here we assess the extent to which frost and freezing temperatures influence the taxonomic and phylogenetic structure of naturally patchy evergreen forests (locally known as shola) along a mid-upper montane elevation gradient in the Western Ghats, India. Specifically, we examine the role of large-scale macroclimate and factors affecting local microclimates, including shola patch size and distance from shola edge, in driving shola metacommunity structure. We find that the shola metacommunity shows phylogenetic overdispersion with elevation, with greater representation of extra-tropical lineages above 2000 m, and marked turnover in taxonomic composition of shola woody communities near the frost-affected forest edge above 2000 m, from those below 2000 m. Both minimum winter temperature and patch size were equally important in determining metacommunity structure, with plots inside very large sholas dominated by older tropical lineages, with many endemics. Phylogenetic overdispersion in the upper montane shola metacommunity thus resulted from tropical lineages persisting in the interiors of large closed frost-free sholas, where their regeneration niche has been preserved over time.

Identification and Characterization of Circular Intronic RNAs Derived from Insulin Gene.

Circular RNAs (circRNAs) are a large family of noncoding RNAs that have emerged as novel regulators of gene expression. However, little is known about the function of circRNAs in pancreatic ß-cells. Here, transcriptomic analysis of mice pancreatic islet RNA-sequencing data identified 77 differentially expressed circRNAs between mice fed with a normal diet and a high-fat diet. Surprisingly, multiple circRNAs were derived from the intron 2 of the preproinsulin 2 (Ins2) gene and are termed as circular intronic (ci)-Ins2. The expression of ci-Ins2 transcripts in mouse pancreatic islets, and ßTC6 cells were confirmed by reverse transcription PCR, DNA sequencing, and RNase R treatment experiments. The level of ci-Ins2 was altered in ßTC6 cells upon exposure to elevated levels of palmitate and glucose. Computational analysis predicted the interaction of several RNA-binding proteins with ci-Ins2 and their flanking region, suggesting their role in the ci-Ins2 function or biogenesis. Additionally, bioinformatics analysis predicted the association of several microRNAs with ci-Ins2. Gene ontology and pathway analysis of genes targeted by miRNAs associated with ci-Ins2 suggested the regulation of several key biological processes. Together, our findings indicate that differential expression of circRNAs, especially ci-Ins2 transcripts, may regulate ß-cell function and may play a critical role in the development of diabetes.

Harnessing the potential of monocytes/macrophages to regenerate tissue-engineered vascular grafts.

Cell-free tissue-engineered vascular grafts provide a promising alternative to treat cardiovascular disease, but timely endothelialization is essential for ensuring patency and proper functioning post-implantation. Recent studies from our lab showed that blood cells like monocytes (MCs) and macrophages (Mϕ) may contribute directly to cellularization and regeneration of bioengineered arteries in small and large animal models. While MCs and Mϕ are leucocytes that are part of the innate immune response, they share common developmental origins with endothelial cells (ECs) and are known to play crucial roles during vessel formation (angiogenesis) and vessel repair after inflammation/injury. They are highly plastic cells that polarize into pro-inflammatory and anti-inflammatory phenotypes upon exposure to cytokines and differentiate into other cell types, including EC-like cells, in the presence of appropriate chemical and mechanical stimuli. This review focuses on the developmental origins of MCs and ECs; the role of MCs and Mϕ in vessel repair/regeneration during inflammation/injury; and the role of chemical signalling and mechanical forces in Mϕ inflammation that mediates vascular graft regeneration. We postulate that comprehensive understanding of these mechanisms will better inform the development of strategies to coax MCs/Mϕ into endothelializing the lumen and regenerate the smooth muscle layers of cell-free bioengineered arteries and veins that are designed to treat cardiovascular diseases and perhaps the native vasculature as well.

Patterns of understory invasion in invasive timber stands of a tropical sky island.

Current climate and land cover change threaten global mountaintops with increased spread of invasive species. Long-established plantations of invasive trees on these mountaintops can alter their surroundings, further increasing invader-facilitated invasion. Identifying the ecological conditions promoting such associations can help develop better management interventions. The Western Ghats's Shola Sky Islands (>1400 m MSL) host vast stretches of invasive tree plantations that sustain the colonization of other invasive woody, herbaceous, and fern species in their understories. Here, we analyzed vegetation and landscape variables from 232 systematically placed plots in randomly selected grids using non-metric multidimensional scaling and Phi coefficient approaches to examine patterns of association (positive interactions) between understory invasive species with specific invasive overstory species. We also conducted GLMM with zero inflation to determine the influence of environmental variables where such associations occur. We find that understory invasion of multiple species under the canopy of other invasives is widespread across the Shola Sky Islands. Stands of Eucalyptus host the colonization of 70% of non-native invasive species surveyed across the Shola Sky Islands. In particular, the Lantana camara invasion is strongly associated with Eucalyptus stands. We also found that climatic variables affect the colonization of understory woody invasive species, while invasion by exotic herbaceous species is associated with the density of road networks. Canopy cover impacts all invasives negatively, while fire incidence was negatively associated with invasion by Lantana spp. and the Pteridium spp. While the restoration of natural habitats primarily targets the highly invasive Acacia, less invasive Eucalyptus and Pinus are often not included. Our study suggests that retaining such invasive species in natural habitats, particularly protected areas, can hinder ongoing grassland restoration efforts by facilitating further invasions by multiple woody and herbaceous species.

Quantification of Circular RNAs Using Digital Droplet PCR.

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 water-in-oil droplets, and the PCR occurs in the individual droplets. The dd-PCR has several advantages over conventional real-time qPCR, including increased accuracy in detecting low-abundance targets, omitting reference genes for quantification, eliminating technical replicates for samples, and showing high resilience to inhibitors in the samples. Recently, dd-PCR has become one of the most popular methods for accurately quantifying target DNA or RNA for gene expression analysis and diagnostics. Circular RNAs (circRNAs) are a large family of recently discovered covalently closed RNA molecules lacking 5' and 3' ends. They have been shown to regulate gene expression by acting as sponges for RNA-binding proteins and microRNAs. Furthermore, circRNAs are secreted into body fluids, and their resistance to exonucleases makes them serve as biomarkers for disease diagnosis. This article aims to show how to perform divergent primer design, RNA extraction, cDNA synthesis, and dd-PCR analysis to accurately quantify specific circular RNA (circRNA) levels in cells. In conclusion, we demonstrate the precise quantification of circRNAs using dd-PCR.

Why is calcite a strong phosphorus sink in freshwater? Investigating the adsorption mechanism using batch experiments and surface complexation modeling.

One of the primary drivers of Phosphorus (P) limitation in aquatic systems is P adsorption to sediments. Sediments adsorb more P in freshwater compared to other natural solutions, but the mechanism driving this difference is poorly understood. To provide insights into the mechanism, we conducted batch experiments of P adsorption to calcite in freshwater and seawater, and used computer software to develop complexation models. Our simulations revealed three main reasons that, combining together, may explain the greater P adsorption to calcite in freshwater vs. seawater. First, aqueous speciation of P makes a difference. The ion pair CaPO4- is much more abundant in freshwater; although seawater has more Ca2+ ions, MgHPO40 and NaHPO40 are more thermodynamically favored. Second, the adsorbing species of P make a difference. The ion pair CaPO4- (the preferred adsorbate in freshwater) is able to access adsorption sites that are not available to HPO42- (the preferred adsorbate in seawater), thereby raising the maximum concentration of P that can adsorb to the calcite surface in freshwater. Third, water chemistry affects the competition among ions for surface sites. Other ions (including P) compete more effectively against CO32- when immersed in freshwater vs. seawater, even when the concentration of HCO3-/CO32- is higher in freshwater vs. seawater. In addition, we found that under oligotrophic conditions, P adsorption is driven by the higher energy adsorption sites, and by the lower energy sites in eutrophic conditions. This study is the first to model P adsorption mechanisms to calcite in freshwater and seawater.

Emerging Role of Circular RNA-Protein Interactions.

Circular RNAs (circRNAs) are emerging as novel regulators of gene expression in various biological processes. CircRNAs regulate gene expression by interacting with cellular regulators such as microRNAs and RNA binding proteins (RBPs) to regulate downstream gene expression. The accumulation of high-throughput RNA-protein interaction data revealed the interaction of RBPs with the coding and noncoding RNAs, including recently discovered circRNAs. RBPs are a large family of proteins known to play a critical role in gene expression by modulating RNA splicing, nuclear export, mRNA stability, localization, and translation. However, the interaction of RBPs with circRNAs and their implications on circRNA biogenesis and function has been emerging in the last few years. Recent studies suggest that circRNA interaction with target proteins modulates the interaction of the protein with downstream target mRNAs or proteins. This review outlines the emerging mechanisms of circRNA-protein interactions and their functional role in cell physiology.

Identification of Potential circRNA-microRNA-mRNA Regulatory Network in Skeletal Muscle.

Circular RNAs (circRNAs) are a newly discovered family of regulatory RNAs generated through backsplicing. Genome-wide profiling of circRNAs found that circRNAs are ubiquitously expressed and regulate gene expression by acting as a sponge for RNA-binding proteins (RBPs) and microRNAs (miRNAs). To identify circRNAs expressed in mouse skeletal muscle, we performed high-throughput RNA-sequencing of circRNA-enriched gastrocnemius muscle RNA samples, which identified more than 1,200 circRNAs. In addition, we have identified more than 14,000 and 15,000 circRNAs in aging human skeletal muscle tissue and satellite cells, respectively. A subset of abundant circRNAs was analyzed by RT-PCR, Sanger sequencing, and RNase R digestion assays to validate their expression in mouse skeletal muscle tissues. Analysis of the circRNA-miRNA-mRNA regulatory network revealed that conserved circNfix might associate with miR-204-5p, a suppressor of myocyte enhancer factor 2c (Mef2c) expression. To support the hypothesis that circNfix might regulate myogenesis by controlling Mef2c expression, silencing circNfix moderately reduced Mef2c mRNA expression and inhibited C2C12 differentiation. We propose that circNfix promotes MEF2C expression during muscle cell differentiation in part by acting as a sponge for miR-204-5p.

Optogenetic-mediated cardiovascular differentiation and patterning of human pluripotent stem cells.

Precise spatial and temporal regulation of dynamic morphogen signals during human development governs the processes of cell proliferation, migration, and differentiation to form organized tissues and organs. Tissue patterns spontaneously emerge in various human pluripotent stem cell (hPSC) models. However, the lack of molecular methods for precise control over signal dynamics limits the reproducible production of tissue patterns and a mechanistic understanding of self-organization. We recently implemented an optogenetic-based OptoWnt platform for light-controllable regulation of Wnt/ß-catenin signaling in hPSCs for in vitro studies. Using engineered illumination devices to generate light patterns and thus precise spatiotemporal control over Wnt activation, here we triggered spatially organized transcriptional changes and mesoderm differentiation of hPSCs. In this way, the OptoWnt system enabled robust endothelial cell differentiation and cardiac tissue patterning in vitro. Our results demonstrate that spatiotemporal regulation of signaling pathways via synthetic OptoWnt enables instructive stem cell fate engineering and tissue patterning.

Circular RNAs in myogenesis.

Skeletal muscles have an immense ability to regenerate from the muscle stem cells called satellite cells. The process of skeletal muscle regeneration is called myogenesis, which starts with activation of quiescent satellite cells immediately after muscle injury followed by proliferation and fusion of myoblasts into myotubes. Myogenesis is orchestrated through the expression of a specific set of genes which, at each step regulated by complex gene regulatory networks. Besides the well-established roles of transcription factors, increasing evidence demonstrated that circular (circ)RNAs modulate gene expression during myogenesis and are involved in muscle-related diseases. Here we review the recent findings of circRNAs involved in myogenesis.

'Have we missed reporting adverse drug reactions under Revised National TB Control Programme?' - A mixed method study in Bengaluru, India.

OBJECTIVE: Prompt identification, reporting and management of ADRs during anti tuberculosis treatment can ensure better compliance and treatment outcomes. The study was conducted to identify the gaps and associated factors in reporting of ADRs under RNTCP; assess knowledge, attitude and practice of RNTCP staff regarding pharmacovigilance programme and explore the barriers in reporting of ADRs from provider's perspective. METHODS: Mixed method research with sequential explanatory design was carried out in Tuberculosis Units of RNTCP administrative district of Bangalore city during July to December 2017. Quantitative study was carried out among 222 patients on intensive phase of Category I and Category II DOTS to study the incidence, severity and causality of ADRs; and records of these patients were analysed for gaps in reporting. Knowledge, attitude and practice (KAP) regarding recording and reporting aspect of pharmacovigilance programme was assessed among RNTCP staff. As part of the qualitative study, focus group discussion was carried out among RNTCP staff to study barriers for reporting ADRs from the provider's perspective. RESULTS: Record analysis at the time of recruitment showed documentation of ADRs in only five patients. Subsequent analysis of patient records during the middle and end of the intensive phase (IP) did not show documentation of any ADRs. Simultaneously interviews with patients revealed 116 (52.2%), 72 (32.4%) and 53 (23.8%) patients reported one or more symptoms of ADRs. The commonest ADR symptom reported were fatigability and gastrointestinal symptoms followed by musculoskeletal symptoms. KAP among 25 RNTCP staff showed that 96% of them felt reporting of ADRs was necessary and 92% reported the ADRs to their seniors, however 12% were scared to report. The main reason expressed for non-reporting was 'managing ADRs is more important than reporting' (52%). Also, 32% felt the need for retraining of staff on reporting and documentation. Barriers to reporting of ADRs were both health-system related like insufficient training and inadequate guidelines provided to RNTCP staff and patient-related factors like lack of awareness and reluctance to report ADRs. CONCLUSION: Successful implementation of RNTCP and achievement of TB elimination requires provision of adequate information regarding ADRs to patients and intense follow-up and probing at each contact by programme staff to effectively manage ADRs.

2017-2018 Scientific Advances in Thoracic Oncology: Small Cell Lung Cancer.

SCLC remains an aggressive, deadly cancer with only modest effect on survival from standard chemotherapy. However, with the advent of immunotherapy and comprehensive genomic and transcriptomic profiling, multiple new targets are showing promise in the clinical arena, and just recently programmed death ligand 1 inhibition has been shown to improve the efficacy of standard chemotherapy in extended-disease SCLC. Our increasing understanding of the interactions between different pathways will enable more tailored immunotherapy and targeted therapies based on specific biomarkers and rational combinations. Here we discuss the preclinical and clinical strides in 2017 and 2018 that put us on the threshold of a new era in therapeutics and will, it is hoped, translate into significant improvements in survival.

Proximity to forests drives bird conservation value of coffee plantations: implications for certification.

Widespread loss of primary habitat in the tropics has led to increased interest in production landscapes for biodiversity conservation. In the Western Ghats biodiversity hotspot in India, shade coffee plantations are located in close proximity to sites of high conservation value: protected and unprotected forests. Coffee is grown here under a tree canopy that may be dominated by native tree species or by nonnative species, particularly silver oak (Grevillea robusta). We investigated the influence of properties at the local scale and the landscape scale in determining bird communities in coffee plantations, with particular emphasis on species of conservation priority. We used systematic point counts in 11 coffee plantation sites and analyzed data in a randomized linear modeling framework that addressed spatial autocorrelation. Greater proportion of silver oak at the local scale and distance to contiguous forests at the landscape scale were implicated as factors most strongly driving declines in bird species richness and abundance, while increased basal area of native tree species, a local-scale variable, was frequently related to increased bird species richness and abundance. The influence of local-scale variables increased at greater distances from the forest. Distance to forests emerged as the strongest predictor of declines in restricted-range species, with 92% reduction in the abundance of two commonly encountered restricted-range species (Pompadour Green Pigeon and Yellow-browed Bulbul) and a 43% reduction in richness of bird species restricted to Indian hill forests within 8 km of forests. Increase in proportion of silver oak from 33% to 55% was associated with 91% reduction in the abundance of one commonly encountered restricted-range species (Crimson-fronted Barbet). One conservation strategy is providing incentives to grow coffee in a biodiversity-friendly manner. One implication of our study is that plantations located at varying distances to the forest cannot be compared fairly for biodiversity friendliness by existing certification methodology. Another is that conservation of existing forests at the landscape scale is essential for maintaining higher biodiversity in coffee plantations. Incentive schemes that promote conservation of remnant forests at the landscape scale and biodiversity-friendly practices locally and that relate to coffee communities as a whole rather than individual planters are likely to be more effective.

Not seeing the grass for the trees: Timber plantations and agriculture shrink tropical montane grassland by two-thirds over four decades in the Palani Hills, a Western Ghats Sky Island.

Tropical montane habitats, grasslands, in particular, merit urgent conservation attention owing to the disproportionate levels of endemic biodiversity they harbour, the ecosystem services they provide, and the fact that they are among the most threatened habitats globally. The Shola Sky Islands in the Western Ghats host a matrix of native forest-grassland matrix that has been planted over the last century, with exotic timber plantations. The popular discourse on the landscape change is that mainly forests have been lost to the timber plantations and recent court directives are to restore Shola forest trees. In this study, we examine spatiotemporal patterns of landscape change over the last 40 years in the Palani Hills, a significant part of the montane habitat in the Western Ghats. Using satellite imagery and field surveys, we find that 66% of native grasslands and 31% of native forests have been lost over the last 40 years. Grasslands have gone from being the dominant, most contiguous land cover to one of the rarest and most fragmented. They have been replaced by timber plantations and, to a lesser extent, expanding agriculture. We find that the spatial pattern of grassland loss to plantations differs from the loss to agriculture, likely driven by the invasion of plantation species into grasslands. We identify remnant grasslands that should be prioritised for conservation and make specific recommendations for conservation and restoration of grasslands in light of current management policy in the Palani Hills, which favours large-scale removal of plantations and emphasises the restoration of native forests.

Novel antidotes for target specific oral anticoagulants.

Target specific oral anticoagulants (dabigatran, rivaroxaban, apixaban, and edoxaban) are changing the landscape of anticoagulation. The major drawback is the absence of an effective antidote for severe bleedings and/or prior to procedures. Currently there are a few promising antidotes undergoing clinical trials. This review summarized the latest development in idarucizumab, andexanet alpha and PER977.

Topographic and Bioclimatic Determinants of the Occurrence of Forest and Grassland in Tropical Montane Forest-Grassland Mosaics of the Western Ghats, India.

The objective of this analysis was to identify topographic and bioclimatic factors that predict occurrence of forest and grassland patches within tropical montane forest-grassland mosaics. We further investigated whether interactions between topography and bioclimate are important in determining vegetation pattern, and assessed the role of spatial scale in determining the relative importance of specific topographic features. Finally, we assessed the role of elevation in determining the relative importance of diverse explanatory factors. The study area consists of the central and southern regions of the Western Ghats of Southern India, a global biodiversity hotspot. Random forests were used to assess prediction accuracy and predictor importance. Conditional inference classification trees were used to interpret predictor effects and examine potential interactions between predictors. GLMs were used to confirm predictor importance and assess the strength of interaction terms. Overall, topographic and bioclimatic predictors classified vegetation pattern with approximately 70% accuracy. Prediction accuracy was higher for grassland than forest, and for mosaics at higher elevations. Elevation was the most important predictor, with mosaics above 2000 m dominated largely by grassland. Relative topographic position measured at a local scale (within a 300 m neighbourhood) was another important predictor of vegetation pattern. In high elevation mosaics, northness and concave land surface curvature were important predictors of forest occurrence. Important bioclimatic predictors were: dry quarter precipitation, annual temperature range and the interaction between the two. The results indicate complex interactions between topography and bioclimate and among topographic variables. Elevation and topography have a strong influence on vegetation pattern in these mosaics. There were marked regional differences in the roles of various topographic and bioclimatic predictors across the range of study mosaics, indicating that the same pattern of grass and forest seems to be generated by different sets of mechanisms across the region, depending on spatial scale and elevation.

Selective inhibitors of nuclear export (SINE) in hematological malignancies.

Regulated nucleo-cytoplasmic transport plays a major role in maintaining cellular homeostasis. CRM1 (chromosome region maintenance 1 or exportin 1 or XPO 1) is responsible for the nucleo-cytoplasmic transport of more than 200 proteins, including most of the tumor suppressor proteins (TSP). CRM1 is overexpressed in pancreatic cancer, osteosarcoma, glioma, cervical and hematological malignancies. This inspired the development of novel agents that selectively inhibit nuclear exportins (SINEs). In this review we focus on the significance of CRM1 in carcinogenesis and review the new development of SINE inhibitiors in hematological malignancies. Selinexor (KPT-330) as the first-in-human SINE agent represents this novel class of anti-cancer agents.

CEPP regimen (cyclophosphamide, etoposide, procarbazine and prednisone) as initial treatment for Hodgkin lymphoma patients presenting with severe abnormal liver function.

ABVD regimen (doxorubicin, bleomycin, vinblastine and dacarbazine) remains the most commonly used front-line therapy for Hodgkin lymphoma. However, atypical and extranodal presentations present challenges to initial therapy, especially in the presence of renal and liver failure. We hereby present two cases of young male patients with atypical presentation of Hodgkin lymphoma with severe abnormal liver function. Patients showed excellent response to cyclophosphamide, etoposide, procarbazine and prednisone (CEPP regimen).

Ecology driving genetic variation: a comparative phylogeography of jungle cat (Felis chaus) and leopard cat (Prionailurus bengalensis) in India.

BACKGROUND: Comparative phylogeography links historical population processes to current/ecological processes through congruent/incongruent patterns of genetic variation among species/lineages. Despite high biodiversity, India lacks a phylogeographic paradigm due to limited comparative studies. We compared the phylogenetic patterns of Indian populations of jungle cat (Felis chaus) and leopard cat (Prionailurus bengalensis). Given similarities in their distribution within India, evolutionary histories, body size and habits, congruent patterns of genetic variation were expected. METHODOLOGY/PRINCIPAL FINDINGS: We collected scats from various biogeographic zones in India and analyzed mtDNA from 55 jungle cats (460 bp NADH5, 141 bp cytochrome b) and 40 leopard cats (362 bp NADH5, 202 bp cytochrome b). Jungle cats revealed high genetic variation, relatively low population structure and demographic expansion around the mid-Pleistocene. In contrast, leopard cats revealed lower genetic variation and high population structure with a F(ST) of 0.86 between North and South Indian populations. Niche-model analyses using two approaches (BIOCLIM and MaxEnt) support absence of leopard cats from Central India, indicating a climate associated barrier. We hypothesize that high summer temperatures limit leopard cat distribution and that a rise in temperature in the peninsular region of India during the LGM caused the split in leopard cat population in India. CONCLUSIONS/SIGNIFICANCE: Our results indicate that ecological variables describing a species range can predict genetic patterns. Our study has also resolved the confusion over the distribution of the leopard cat in India. The reciprocally monophyletic island population in the South mandates conservation attention.