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Mahajan, G.

Publications and source records attributed to Mahajan, G..

11 recordsLinked to original sources

Genetic Perturbation Prediction Models Fail Out-of-Distribution: A Rigorous Benchmark and Systematic Evaluation

Perturbation prediction models - which forecast transcriptional responses to gene inactivation - are increasingly proposed as in silico surrogates for genome-scale drug discovery screens. Yet, rigorous evaluation of whether these models genuinely generalize beyond their training distribution has been lacking. Existing benchmarks rely predominantly on random train-test splits, obscuring systematic failure modes that only emerge under realistic out-of-distribution (OOD) conditions. Here we introduce EL-PERTURB (Extrapolative Learning for PERTURBation Prediction), a benchmarking framework that stress-tests 20 perturbation prediction models spanning six architecture classes on both essential gene and genome-wide Perturb-seq screens, using a novel stratified OOD splitting strategy that partitions train and test sets into functionally distinct regions of transcriptional space. We find that model performance degrades substantially under OOD conditions, and that architectural complexity - including single-cell foundation model embeddings - confers no reliable advantage over well-tuned simpler baselines. EL-PERTURB establishes a rigorous benchmark for perturbation effect prediction across a gradient of generalization difficulty, providing a guide for building models that can genuinely serve as surrogates for wet-lab screening.

systems biology↗

A genetically-encoded nanobody sensor reveals conformational diversity in beta-arrestins orchestrated by distinct seven transmembrane receptors

Agonist-induced interaction of G protein-coupled receptors (GPCRs) with {beta}-arrestins ({beta}arrs) is a critical mechanism that regulates the spatio-temporal pattern of receptor localization and downstream signaling. While the underlying mechanism governing GPCR-{beta}arr interaction is primarily conserved and involves receptor activation and phosphorylation, there are several examples of receptor-specific fine-tuning of {beta}arr-mediated functional outcomes. Considering the key contribution of conformational plasticity of {beta}arrs in driving receptor-specific functional responses, it is important to develop and characterize novel sensors capable of reporting distinct {beta}arr conformations in cellular context. Here, we design an intrabody version of a {beta}arr-recognizing nanobody (nanobody32), referred to as intrabody32 (Ib32), in NanoLuc enzyme complementation assay format, and measure its ability to recognize {beta}arr1 and 2 in live cells upon activation of a broad set of GPCRs. We discover that Ib32 robustly recognizes activated {beta}arr1 and 2 in the plasma membrane as well as in the endosomes, and effectively mirrors {beta}arr recruitment profile upon stimulation of GPCRs. We also design an Ib32 sensor for single-photon polarization microscopy with a change in linear dichroism as readout and demonstrate its utility for monitoring {beta}arr activation upon stimulation of angiotensin receptor by its natural and biased agonists. Interestingly, when used side-by-side with a previously described sensor of {beta}arr1 conformation known as Ib30, Ib32 uncovers distinct conformational signatures imparted on {beta}arrs by different GPCRs, which is further corroborated using an orthogonal limited proteolysis assay. Taken together, our study presents Ib32 as a novel sensor to monitor {beta}arr activation and leverages it to uncover conformational diversity encoded in the GPCR-{beta}arr system with direct implications for improving the current understanding of GPCR signaling and regulatory paradigms.

biochemistry↗

Molecular insights into atypical modes of beta-arrestin interaction with seven transmembrane receptors

{beta}-arrestins are multifunctional proteins that are critically involved in regulating spatio-temporal aspects of GPCR signaling. The interaction of {beta}-arrestins with GPCRs is typically conceptualized in terms of receptor activation and phosphorylation primarily in the carboxyl-terminus. Interestingly however, there are several GPCRs that harbor majority of phosphorylation sites in their 3rd intracellular loop (ICL3) instead of carboxyl-terminus but still robustly engage {beta}-arrestins. Moreover, there are several 7TMRs that are now characterized as intrinsically-biased, {beta}-arrestin-coupled receptors (ACRs) due to lack of functional G-protein-coupling but robust {beta}-arrestin binding leading to functional outcomes. The molecular basis of {beta}-arrestin interaction and activation upon binding to these types of 7TMRs is currently elusive, and it represents a major knowledge gap in our current understanding of this signaling system. Here, we present seven cryo-EM structures of {beta}-arrestins in basal state, activated by the muscarinic M2 receptor (M2R) through its ICL3, and a {beta}-arrestin-coupled receptor known as decoy D6 receptor (D6R). These structural snapshots combined with biochemical, cellular, and biophysical experiments including HDX-MS and MD simulation provide novel insights into the ability of {beta}-arrestins to preferentially select specific phosphorylation patterns in the receptors, and also illuminate the structural diversity in 7TMR-{beta}-arrestin interaction. Surprisingly, we also observe that the carboxyl-terminus of {beta}-arrestin2 but not {beta}-arrestin1 undergoes structural transition from a {beta}-strand to -helix upon activation by D6R, which may preclude the core-interaction with the activated receptor. Taken together, our study elucidates previously unappreciated aspects of 7TMR-{beta}-arrestin interaction, and provides important mechanistic clues about how the two isoforms of {beta}-arrestins can recognize and regulate a large repertoire of GPCRs.

biochemistry↗

Structure-guided engineering of biased-agonism in the human niacin receptor via single amino acid substitution

The Hydroxycarboxylic acid receptor 2 (HCA2), also known as the niacin receptor or GPR109A, is a prototypical G protein-coupled receptor that plays a central role in the inhibition of lipolytic and atherogenic activities in our body. Interestingly, GPR109A activation also results in vasodilation that is linked to the side-effect of flushing associated with dyslipidemia drugs such as niacin. This receptor continues to be a key target for developing novel pharmacophores and lead compounds as potential therapeutics in dyslipidemia with minimized flushing response, however, the lack of structural insights into agonist-binding and receptor activation has limited the efforts. Here, we present five different cryo-EM structures of the GPR109A-G-protein complexes with the receptor bound to dyslipidemia drugs, niacin or acipimox, non-flushing agonists, MK6892 or GSK256073, and recently approved psoriasis drug, monomethyl fumarate (MMF). These structures allow us to visualize the binding mechanism of agonists with a conserved molecular interaction network, and elucidate the previously lacking molecular basis of receptor activation and transducer-coupling. Importantly, cellular pharmacology experiments, guided by the structural framework determined here, elucidate pathway-selective biased signaling elicited by the non-flushing agonists. Finally, taking lead from the structural insights, we successfully engineered receptor mutants via single amino acid substitutions that either fail to elicit agonist-induced transducer-coupling or exhibits G-protein signaling bias. Taken together, our study provides previously lacking structural framework to understand the agonist-binding and activation of GPR109A, and opens up the possibilities of structure-guided novel drug discovery targeting this therapeutically important receptor.

biochemistry↗

Vaginal microbiome-host interactions modeled in a human vagina-on-a-chip

BackgroundA dominance of non-iners Lactobacillus species in the vaginal microbiome is optimal and strongly associated with gynecological and obstetric health, while the presence of diverse obligate or facultative anaerobic bacteria and a paucity in Lactobacillus species, similar to communities found in bacterial vaginosis (BV), is considered non-optimal and associated with adverse health outcomes. Various therapeutic strategies are being explored to modulate the composition of the vaginal microbiome; however, there is no human model that faithfully reproduces the vaginal epithelial microenvironment for preclinical validation of potential therapeutics or testing hypotheses about vaginal epithelium-microbiome interactions. ResultsHere, we describe an organ-on-a-chip (Organ Chip) microfluidic culture model of the human vaginal mucosa (Vagina Chip) that is lined by hormone-sensitive, primary vaginal epithelium interfaced with underlying stromal fibroblasts, which sustains a low physiological oxygen concentration in the epithelial lumen. We show that the Vagina Chip can be used to assess colonization by optimal L. crispatus consortia as well as non-optimal Gardnerella vaginalis-containing consortia, and to measure associated host innate immune responses. Co-culture of the L. crispatus consortia was accompanied by maintenance of epithelial cell viability, accumulation of D- and L-lactic acid, maintenance of a physiologically relevant low pH, and down regulation of proinflammatory cytokines. In contrast, co-culture of G. vaginalis-containing consortia in the Vagina Chip resulted in epithelial cell injury, a rise in pH, and upregulation of proinflammatory cytokines. ConclusionThis study demonstrates the potential of applying human Organ Chip technology to create a preclinical model of the human vaginal mucosa that can be used to better understand interactions between the vaginal microbiome and host tissues, as well as to evaluate the safety and efficacy of live biotherapeutics products.

microbiology↗

Predicting drug concentrations in PDMS microfluidic organ chips

Microfluidic organ-on-a-chip (Organ Chip) cell culture devices are often fabricated using polydimethylsiloxane (PDMS) because it is biocompatible, transparent, elastomeric, and oxygen permeable; however, hydrophobic small molecules can absorb to PDMS, which makes it challenging to predict drug responses. Here, we describe a combined simulation and experimental approach to predict the spatial and temporal concentration profile of a drug under continuous dosing in a PDMS Organ Chip containing two parallel channels separated by a porous membrane that is lined with cultured cells, without prior knowledge of its log P value. First, a three-dimensional finite element simulation of drug loss into the chip was developed that incorporates absorption, adsorption, convection, and diffusion, which predicts changes in drug levels over time and space as a function of potential PDMS diffusion coefficients and log P values. By then experimentally measuring the diffusivity of the compound in PDMS and determining its partition coefficient through mass spectrometric analysis of the drug concentration in the channel outflow, it is possible to estimate the effective log P range of the compound. The diffusion and partition coefficients were experimentally derived for the antimalarial drug and potential SARS-CoV-2 therapeutic, amodiaquine, and incorporated into the model to quantitatively predict the drug-specific concentration profile over time measured in human Lung Airway Chips lined with bronchial epithelium interfaced with pulmonary microvascular endothelium. The same strategy can be applied to any device geometry, surface treatment, or in vitro microfluidic model to predict the spatial and temporal gradient of a drug in 3D without prior knowledge of the partition coefficient or the rate of diffusion in PDMS. Thus, this approach may expand the use of PDMS Organ Chip devices for various forms of drug testing.

bioengineering↗

Venlafaxine stimulates an MMP-9-dependent increase in excitatory/inhibitory balance in a stress model of depression

Emerging evidence suggests that there is a reduction in overall cortical excitatory to inhibitory balance in major depressive disorder (MDD), which afflicts approximately 14-20% of individuals. Reduced pyramidal cell arborization occurs with stress and MDD, and may diminish excitatory neurotransmission. Enhanced deposition of perineuronal net (PNN) components also occurs with stress. Since parvalbumin-expressing interneurons are the predominant cell population that is enveloped by PNNs, which enhance their ability to release GABA, excess PNN deposition likely increases pyramidal cell inhibition. In the present study we investigate the potential for matrix metalloprotease-9 (MMP-9), an endopeptidase secreted in response to neuronal activity, to contribute to the antidepressant efficacy of venlafaxine, a serotonin/norepinephrine reuptake inhibitor. Chronic venlafaxine increases MMP-9 levels in murine cortex, and increases both pyramidal cell arborization and PSD-95 expression in the cortex of wild-type but not MMP-9 null mice. We have previously shown that venlafaxine reduces PNN deposition and increases the power of ex vivo gamma oscillations in conventionally-housed mice. Gamma power is increased with pyramidal cell disinhibition and with remission from MDD. Herein we observe that PNN expression is increased in a corticosterone-induced stress model of disease and reduced by venlafaxine. As compared to mice that receive concurrent venlafaxine, corticosterone treated mice also display reduced ex vivo gamma power and impaired working memory. Autopsy-derived prefrontal cortex samples show elevated MMP-9 levels in anti-depressant treated MDD patients as compared to controls. These preclinical and postmortem findings highlight a link between extracellular matrix regulation and MDD.

neuroscience↗

Local design principles at hippocampal synapsesrevealed by an energy-information trade-off

Synapses across different brain regions display distinct structure-function relationships. We investigate the interplay of fundamental design principles that shape the transmission properties of the excitatory CA3-CA1 pyramidal cell connection, a prototypic synapse for studying the mechanisms of learning in the hippocampus. This small synapse is characterized by probabilistic release of transmitter, which is markedly facilitated in response to naturally occurring trains of action potentials. Based on a physiologically realistic computational model of the CA3 presynaptic terminal, we show how unreliability and short-term dynamics of vesicle release work together to regulate the trade-off of information transfer versus energy use. We propose that individual CA3-CA1 synapses are designed to operate at close to maximum possible capacity of information transfer in an efficient manner. Experimental measurements reveal a wide range of vesicle release probabilities at hippocampal synapses, which may be a necessary consequence of long-term plasticity and homeostatic mechanisms that manifest as presynaptic modifications of release probability. We show that the timescales and magnitude of short-term plasticity render synaptic information transfer nearly independent of differences in release probability. Thus, individual synapses transmit optimally while maintaining a heterogeneous distribution of presynaptic strengths indicative of synaptically-encoded memory representations. Our results support the view that organizing principles that are evident on higher scales of neural organization percolate down to the design of an individual synapse.

neuroscience↗

Growth behavior and glyphosate resistance level in 10 biotypes of Echinochloa colona in Australia

Recently, poor control of Echinochloa colona with glyphosate has been reported in no-till agriculture systems of the northern grain region (NGR) of Australia. Two experiments were conducted using 10 biotypes of E. colona selected from the NGR of Australia to understand differences in their growth behavior and resistance pattern. Growth studies revealed that these biotypes differed in plant height (53-70 cm plant-1), tiller production (30-52 tillers plant-1), leaf production (124-186 leaves plant-1) and seed head production (37-65 seed heads plant-1). Days taken to seed heads and shoot biomass in these biotypes ranged between 40-48 d and 21-27 g plant-1, respectively. Seed production in these biotypes ranged between 5380 and 10244 seeds plant-1; lowest for biotype B17/25 and highest for biotype B17/13. Correlation studies revealed that seed number plant-1 had a positive correlation with plant height (r = 0.67), tiller number plant-1 (r = 0.89), leaf number plant-1 (r = 0.73), seed heads plant-1 (r = 0.78), seed head weight (r = 0.79), shoot biomass (r = 0.77) and root biomass (r = 0.46). The glyphosate dose-response study showed a wide range of responses in these biotypes and the glyphosate dose required to reduce 50% biomass (GR50 values) was estimated between 217 to 2159 g a.e. glyphosate ha-1. GR50 values of biotypes B17/16, B 17/34 and B17/35 were 719, 2159 and 884 g ha-1, respectively, making them 3, 10 and 4-fold resistant to glyphosate compared with the susceptible biotype B17/37. Growth behavior and seed production potential in these biotypes had no correlation with the resistance index. These results suggest that some biotypes of E. colona are highly problematic; for example, biotype B17/34 was not only highly glyphosate-resistant, but also produced a high seed number (9300 seeds plant-1). This study demonstrated that there is a possibility of great risk with the increased use of glyphosate for managing E. colona in the NGR of Australia. The results warrant integrated weed management strategies and improved stewardship guidelines are required for managing glyphosate-resistant biotypes of E. colona and to restrict further movement of resistant biotypes to other regions of Australia.

ecology↗

Atrial fibrillation associated common risk variants in SYNE2 lead to lower expression of nesprin-2α1 and increased nuclear stiffness

RationaleAtrial fibrillation (AF) genome-wide association studies (GWAS) identified significant associations for rs1152591 and linked variants in the SYNE2 gene encoding the nesprin-2 protein that connects the nuclear membrane with the cytoskeleton\n\nObjectiveDetermine the effects of the AF-associated rs1152591 and rs1152595, two linked intronic single nucleotide polymorphisms (SNPs), on SYNE2 expression and investigate the mechanisms for their association with AF.\n\nMethods and ResultsRNA sequencing of human left atrial appendage (LAA) tissues indicated that rs1152591 and rs1152595 were significantly associated with the expressions of SYNE21, a short mRNA isoform, without an effect on the expression of the full-length SYNE2 mRNA. SYNE21 mRNA uses an alternative transcription start site and encodes an N-terminal deleted 62 kDa nesprin-21 isoform, which can act as a dominant-negative on nuclear-cytoskeleton connectivity. Western blot and qPCR assays confirmed that AF risk alleles of both SNPs were associated with lower expression of nesprin-21 in human LAA tissues. Reporter gene transfections demonstrated that the risk vs. reference alleles of rs1152591 and rs1152595 had decreased enhancer activity. SYNE2 siRNA knockdown (KD) or nesprin-21 overexpression studies in human stem cell-derived induced cardiomyocytes (iCMs) resulted in ~12.5 % increases in the nuclear area compared to controls (p<0.001). Atomic force microscopy demonstrated that SYNE2 KD or nesprin-21 overexpression led to 57.5% or 33.2% decreases, respectively, in nuclear stiffness compared to controls (p< 0.0001).\n\nConclusionsAF-associated SNPs rs1152591 and rs1152595 downregulate the expression of SYNE21, increasing nuclear-cytoskeletal connectivity and nuclear stiffness. The resulting increase in mechanical stress may play a role in the development of AF.

genomics↗

Intracellular calcium stores mediate a synapse-specific form of metaplasticity at hippocampal dendritic spines

Long-term plasticity mediated by NMDA receptors supports input-specific, Hebbian forms of learning at excitatory CA3-CA1 connections in the hippocampus. An additional layer of stabilizing mechanisms that act globally as well as locally over multiple time scales may be in place to ensure that plasticity occurs in a constrained manner. Here, we investigate the potential role of calcium (Ca2+) stores associated with the endoplasmic reticulum (ER) in the local regulation of plasticity dynamics at individual CA1 synapses. Our study is spurred by (1) the curious observation that ER is sparsely distributed in dendritic spines, but over-represented in large spines that are likely to have undergone activity-dependent strengthening, and (2) evidence suggesting that ER motility within synapses can be rapid, and accompany activity-regulated spine remodeling. Based on a physiologically realistic computational model for ER-bearing CA1 spines, we characterize the contribution of IP3-sensitive Ca2+ stores to spine Ca2+ dynamics during activity patterns mimicking the induction of long-term potentiation (LTP) and depression (LTD). Our results suggest graded modulation of the NMDA receptor-dependent plasticity profile by ER, which selectively enhances LTD induction. We propose that spine ER can locally tune Ca2+-based plasticity on an as-needed basis, providing a braking mechanism to mitigate runaway strengthening at potentiated synapses. Our model suggests that the presence of ER in the CA1 spine may promote re-use of synapses with saturated strengths.

neuroscience↗