Biology subjects

Prakash, A.

Publications and source records attributed to Prakash, A..

6 recordsLinked to original sources

bioRxiv2018

Development and Application of a High-Content Virion Display Human GPCR Array

G protein-coupled receptors (GPCRs) comprise the largest membrane protein family in humans and can respond to a wide variety of ligands and stimuli. Like other multi-pass membrane proteins, the biochemical properties of GPCRs are notoriously difficult to study because they must be embedded in lipid bilayers to maintain their native conformation and function. To enable an unbiased, high-throughput platform to profile biochemical activities of GPCRs in native conformation, we individually displayed 315 human non-odorant GPCRs (>85% coverage) in the envelope of human herpes simplex virus-1 and immobilized on glass to form a high-content Virion Display (VirD) array. Using this array, we found that 50% of the tested commercial anti-GPCR antibodies (mAbs) is ultra-specific, and that the vast majority of those VirD-GPCRs, which failed to be recognized by the commercial mAbs, could bind to their canonical ligands, indicating that they were folded correctly. Next, we used the VirD-GPCR arrays to examine binding specificity of two known peptide ligands and recovered expected interactions, as well as new off-target interactions, three of which were confirmed with real-time kinetics measurements. Finally, we explored the possibility of discovering novel pathogen targets by probing VirD-GPCR arrays with live group B Streptococcus (GBS), a common Gram-positive bacterium causing neonatal meningitis. Using cell invasion assays and a mouse model of hematogenous meningitis, we showed that inhibition of one of the five newly identified GPCRs, CysLTR1, greatly reduced GBS penetration in brain-derived endothelial cells and in mouse brains. Therefore, our work demonstrated that the VirD-GPCR array holds great potential for high-throughput, unbiased screening for small molecule drugs, affinity reagents, and deorphanization.

pharmacology and toxicology↗

bioRxiv2018

The Human RNA-Binding Proteome and Its Dynamics During Arsenite-Induced Translational Arrest

Proteins and RNA functionally and physically intersect in multiple biological processes, however, currently no universal method is available to purify protein-RNA complexes. Here we introduce XRNAX, a method for the generic purification of protein-crosslinked RNA, and demonstrate its versatility to study the composition and dynamics of protein-RNA interactions by various transcriptomic and proteomic approaches. We show that XRNAX captures all RNA biotypes, and use this to characterize the sub-proteomes that interact with coding and non-coding RNAs (ncRNAs), and to identify hundreds of protein-RNA interfaces. Exploiting the quantitative nature of XRNAX, we observe drastic remodeling of the RNA-bound proteome during arsenite-induced stress, distinct from autophagy-induced changes in the total proteome. In addition, we combine XRNAX with CLIP-seq to validate the interaction of ncRNA with Lamin B and EXOSC2. Thus, XRNAX is a resourceful approach to study structural and compositional aspects of protein-RNA interactions to address fundamental questions in RNA-biology.

molecular biology↗

bioRxiv2018

Pathogen susceptibility and fitness costs explain variation in immune priming across natural populations of flour beetles

In many insects, individuals primed with low doses of pathogens live longer after being exposed to the same pathogen later in life. Yet, our understanding of the evolutionary and ecological history of priming of immune response in natural insect populations is limited. Previous work demonstrated population-, sex- and- stage specific variation in the survival benefit of priming response in flour beetles (Tribolium castaneum) infected with their natural pathogen Bacillus thuringiensis. However, the evolutionary forces responsible for this natural variation remained unclear. Here, we tested whether the strength of the priming response (measured as the survival benefit after priming and subsequent infection relative to unprimed controls) was associated with multiple fitness parameters across 10 flour beetle populations. Our results suggest two major selective pressures that may explain the observed inter-population variation in priming: (A) Basal pathogen susceptibility - populations that were more susceptible to infection produced a stronger priming response, and (B) Reproductive success - populations where primed females produced more offspring had lower survival benefit, suggesting a trade-off between priming response and reproduction. Our work is the first empirical demonstration of multiple selective pressures that may govern the adaptive evolution of immune priming in the wild. We hope that this motivates further experiments to establish the role of pathogen-imposed selection and fitness costs in the evolution of priming in natural insect populations.

evolutionary biology↗

bioRxiv2017

Experimental Evolution Of Insect Immune Memory Vs. Pathogen Resistance

Under strong pathogen pressure, insects often evolve resistance to infection. Many insects are also protected via immune memory ( immune priming), whereby sub-lethal exposure to a pathogen enhances survival after secondary infection. To understand the evolution and consequences of these immune responses, we imposed strong pathogen selection on flour beetles (Tribolium castaneum), infecting them with Bacillus thuringiensis (Bt) for 11 generations. Populations injected first with heat-killed and then live Bt each generation evolved high basal resistance against multiple Bt strains. In contrast, all populations injected only with a high dose of live Bt evolved less effective but strain-specific priming response. Control populations injected with heat-killed Bt did not evolve priming; and in the ancestor, priming was effective only against a low Bt dose. Thus, pathogens can select for rapid modulation of insect priming ability, leading to divergent immune strategies (generalized resistance vs. specific immune priming) with distinct mechanisms and adaptive benefits.

evolutionary biology↗

bioRxiv2017

apterous A Specifies Dorsal Wing Patterns And Sexual Traits In Butterflies

Butterflies have evolved different color patterns on their dorsal and ventral wing surfaces to serve different signaling functions, yet the developmental mechanisms controlling surface-specific patterning are still unknown. Here, we mutate both copies of the transcription factor apterous in Bicyclus anynana butterflies using CRISPR/Cas9 and show that apterous A functions both as a repressor and modifier of ventral wing color patterns, as well as a promoter of dorsal sexual ornaments in males. We propose that the surface-specific diversification of wing patterns in butterflies proceeded via the co-option of apterous A into various gene regulatory networks involved in the differentiation of discrete wing traits. Further, interactions between apterous and sex-specific factors such as doublesex may have contributed to the origin of sexually dimorphic surface-specific patterns. Finally, we discuss the evolution of eyespot pattern diversity in the family Nymphalidae within the context of developmental constraints due to apterous regulation.\n\nSignificance statementButterflies have evolved different wing patterns on their dorsal and ventral wing surfaces that serve different signaling functions. We identify the transcription factor, apterous A, as a key regulator of this surface-specific differentiation in butterflies. We also show a role for apterous A in restricting the developmental origin of a novel trait, eyespots, to just the ventral wing surface. Dorsal-ventral differentiation of tissues is not just restricted to butterfly wings but occurs in many other organs and organisms from arthropods to humans. Thus, we believe that our work will be of interest to a diverse group of biologists and layman alike interested in the role of development in shaping biodiversity.

developmental biology↗

bioRxiv2017

FEMALE CHEMICAL WARFARE DRIVES FITNESS EFFECTS OF GROUP SEX RATIO

In animals, skewed sex ratios can affect individual fitness either via sexual interactions (e.g. intersexual conflict or intrasexual mate competition) or non-sexual interactions (e.g. sex-specific resource competition). Because most analyses of sex ratio focus on sexual interactions, the relative importance of these mechanisms remains unclear. We addressed this problem using the flour beetle Tribolium castaneum, where male-biased sex ratios increase female fitness relative to unbiased or female-biased groups. Although flour beetles show both sexual and non-sexual (resource) competition, we found that sexual interactions did not explain female fitness. Instead, female fecundity was dramatically reduced even after a brief exposure to flour conditioned by other females. Earlier studies suggested that quinones (secreted toxins) might mediate density-dependent population growth in flour beetles. We identified ethyl- and methyl-benzoquinone (EBQ and MBQ) as the primary components of adult stink glands that regulate female fecundity. In female-biased groups (i.e. at high female density), females upregulated quinones and suppressed each others reproduction. In male-biased groups, low female density lead to low quinone levels, allowing higher fecundity. Thus, quinones serve both as indicators and mediators of female competition, resulting in the observed fitness decline in female-biased groups. Our results underscore the importance of non-sexual interference competition that may often underlie the fitness consequences of skewed sex ratios.

evolutionary biology↗