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Ravichandran, S.

Publications and source records attributed to Ravichandran, S..

13 recordsLinked to original sources

Latent generative search unlocks de novo design of untapped biomolecular interactions at scale

De novo protein design has advanced rapidly, yet designing binders to polar, solvent-exposed epitopes and small, flexible ligands remains challenging. Such hydrated surfaces and flexible molecules, including carbohydrates, provide few of the hydrophobic contacts favoured by current methods and have largely resisted de novo binders. To address this challenge, here we introduce latent generative search for binder design, a novel framework that uses reward-guided search at inference time to steer the Proteina-Complexa generative model. The model codesigns sequence and structure - generating them together in a continuous latent space - and thereby removes the inverse-folding step on which current methods rely. In a screen of more than one million designs by multiplexed phage display, latent generative search produced more validated binders than every other method tested, its codesigned sequences surpassing post hoc redesign. It delivered high-affinity binders across therapeutic receptors, a viral attachment protein and intracellular signalling targets. Our approach also accessed previously untapped biology, generating the first de novo proteins that bind a free carbohydrate, including one that discriminates between blood-group antigens - a polar, flexible target class beyond the reach of current design methods.

bioengineering↗

Nucleic acid turnover and lipid remodeling distinguish T cell activation and exhaustion states via label-free Raman spectroscopy

T cell exhaustion impairs immune control of chronic diseases including tuberculosis, HIV, malaria, and cancer. Its clinical implications are vast, predicting HIV-associated malignancy and treatment response and limiting the efficacy of cell therapies. Despite the advantages of monitoring and removing exhausted T cells, current detection methods require expensive antibody labeling, destructive workflows, or days-long functional assays. Here, we introduce Raman spectroscopy as a label-free assay for distinguishing T cell states directly from culture while preserving viability for downstream use. We achieve >97% accuracy in discriminating unstimulated, activated, and exhausted T cells across three donors and multiple hardware setups. We identify vibrational modes associated with nucleic acid turnover and lipid remodeling as key features that distinguish T cell activation and exhaustion. In heterogeneous populations, we quantify exhaustion percentage with R2= 1 and strong correlation to adenine (r= -0.91) and amide II protein (r= 0.94) vibrational modes. This work establishes vibrational fingerprinting as a direct measure of T cell exhaustion beyond surface marker expression towards scalable immune diagnostics, in-line monitoring, and selective immunopheresis.

biophysics↗

Lifestyle intervention is associated with attenuation of ER stress/inflammation and enhancement of naive immune cell identity in older adults with metabolic disease

Chronic inflammation and cellular stress are hallmarks of aging, obesity, and type 2 diabetes (T2D), but whether these programs can be modulated by lifestyle intervention in late life, particularly in the presence of established metabolic disease, remain unknown. We profiled circulating immune cells from older adults with obesity and T2D (ages 66-83 years; n = 9) before and after a 6-month lifestyle intervention combining caloric restriction with exercise training. Participants showed substantial weight loss ([~]7%) alongside improvements in glycemic control, insulin sensitivity, and physical performance. Longitudinal single-cell transcriptomic and epigenomic profiling identified two major changes. First, intervention was associated with downregulation of inflammatory and endoplasmic reticulum (ER) stress transcriptional programs, with the most pronounced effects observed in CD14+ monocytes. DDIT3 (CHOP) was transcriptionally and epigenetically downregulated and its inferred regulatory network encompassed multiple inflammatory mediators. Second, naive CD4+ T, naive Treg, and naive B cells exhibited an upregulation of naive cell identity genes, with naiveness scores increasing after intervention, which declines with age in an independent healthy adult cohort. Together, these findings suggest that lifestyle intervention is associated with coordinated remodeling of both innate and adaptive immune compartments in older adults, revealing substantial plasticity of the aging immune system especially targeting ER stress, inflammation, and naive lymphocyte identity programs.

immunology↗

Rapid residual bead quantification for cell therapy manufacturing using Raman spectroscopy

Adoptive cell therapies are transforming the treatment of cancer and autoimmunity by enhancing patients own immune cells to fight disease. In cell therapy manufacturing, immunomagnetic beads are used to isolate and activate target cells for gene transfer but must be removed downstream to [&le;]10 beads per 300,000 cells. Current quantification requires time-intensive and error-prone manual counting using brightfield microscopy, while existing automated approaches struggle with variable bead-cell morphology and tedious sample preparation steps. Raman spectroscopy offers rapid, morphology-independent detection using molecular signatures generated by inelastic light scattering. Here, we leverage immunomagnetic beads strong Raman signatures to quantify them in area scans from dried samples, achieving single bead resolution and accurate counting of bead clusters with and without cells. Using low power ([&le;]7 mW) and exposure times ([&ge;]0.5 s), the average area under 3 signature Raman peaks (1110 cm-1, 1346 cm-1, and 1595 cm-1) are measured and input to a linear regression model, achieving a mean squared error (MSE) of <0.2 beads. Our results show Raman spectroscopy as a robust, automated approach for bead counting in existing pipelines with potential to improve the safety and throughput of cell therapies.

bioengineering↗

mBER: Controllable de novo antibody design with million-scale experimental screening

AO_SCPLOWBSTRACTC_SCPLOWRecent machine learning approaches have achieved high success rates in designing protein binders that demonstrate in vitro binding to their targets. While open models for unconstrained "minibinder" design have shown great experimental promise, methods for designing binders in specific formats, such as antibodies, have lagged behind in experimental success rates. In this work, we present mBER, an open-source protein binder design system capable of designing antibody-format binders with state-of-the-art experimental success rates. mBER builds on the ColabDesign framework, achieving successful antibody design primarily through the inclusion of informative sequence and structure conditioning information. Using mBER, we designed two libraries comprising over 1 million VHH binders against 436 diverse targets. We experimentally screened the two libraries against 145 of these targets, resulting in a dataset of over 100 million binding interactions. We achieved specific and significant design success against 45% of targets. In a filtered set of designs, we detect binding rates to specific epitopes as high as 38%. Through mBER, we demonstrate that format-specific binder design is possible with no additional training of underlying folding and language models. This work represents the largest reported de novo protein design and validation campaign, and one of the first open-source methods to demonstrate double-digit percentage experimental success rates for antibody binder design.

synthetic biology↗

Chaotic Dynamics and Effects of Cognitive State in Human Single Neurons

How the brain balances flexibility and stability during memory-guided behavior remains unclear. While large-scale brain activity exhibits signatures of chaos, it is unknown whether individual neurons also operate in a chaotic regime, and whether such dynamics are modulated by task engagement. Here, we analyzed over 1800 medial temporal lobe (MTL) neurons recorded intracranially during a declarative memory task. Using sample entropy, correlation dimension, and the largest Lyapunov exponent, we found robust signatures of deterministic chaos in a substantial proportion of individual neurons. Crucially, these signatures were not static: during memory task engagement, neurons exhibited greater entropy and dimensionality, and lower divergence, compared to task-irrelevant periods. Chaos was widespread across MTL subregions and independent of age or gender. These results suggest that chaos is an intrinsic, dynamic feature of single-neuron activity which is selectively amplified during cognitive engagement, and may support flexible temporal coding in the human memory system.

biophysics↗

XPC loss-of-function triggers melanomagenesis through CDKN2A downregulation

We identified a novel XPC variant, c.2420+5G>A (XPCvar), in siblings with multiple melanomas, inherited alongside c.779+1G>T, which results in an absent or disrupted protein. However, they did not exhibit significantly higher nucleotide excision repair (NER) deficits compared to their unaffected parents, suggesting an NER-independent tumor suppressor function for XPC. XPC knockdown increased cell proliferation and tumorigenicity in vitro without affecting NER. Single-cell RNA sequencing revealed lower Cdkn2a in Xpcvar/var mouse melanocytes. Additionally, XPC knockdown downregulated CDKN2A in vitro. Furthermore, patient fibroblasts showed decreased p16INK4a, which was rescued by XPC overexpression. ChIP-PCR and luciferase assays confirmed XPC binding to the CDKN2A promoter, initiating transcription. Premature stop codon read-through, with gentamicin, restored XPC and p16INK4A in patient fibroblasts. These suggest that XPC regulates CDKN2A and XPC loss might promote melanomagenesis by downregulating CDKN2A, independent of its NER function. We provide preclinical evidence for potential preventive and/or therapeutic strategies for similarly affected individuals. STATEMENT OF SIGNIFICANCECharacterizing a novel XPC variant identified in patients with melanoma revealed a non-canonical role for XPC in regulating CDKN2A transcription. XPC binding to the CDKN2A promoter is crucial for CDKN2A expression and might suppress melanomagenesis. This expands our understanding of melanomagenesis and suggests potential therapies targeting XPC-mediated regulation of CDKN2A.

cancer biology↗

Targeting the Search: An Upgraded Structural and Functional Repository of Antimicrobial Peptides for Biofilm Studies (B-AMP v2.0) with a Focus on Biofilm Protein Targets

Bacterial biofilms, often as multispecies communities, are recalcitrant to conventional antibiotics, making the treatment of biofilm infections a challenge. There is a push towards developing novel anti-biofilm approaches, such as antimicrobial peptides (AMPs), with activity against specific biofilm targets. In previous work, we developed Biofilm-AMP, a structural and functional repository of AMPs for biofilm studies (B-AMP v1.0) with more than 5000 structural models of AMPs and a vast library of AMP annotations to existing biofilm literature. In this study, we present an upgraded version of B-AMP, with a focus on existing and novel bacterial biofilm targets. B-AMP v2.0 hosts a curated collection of 2502 biofilm protein targets across 473 bacterial species, with structural protein models and functional annotations from PDB, UniProt, and PubMed databases. The biofilm targets can be searched for using the name of the source organism, and function and type of protein, and results include designated Target IDs (unique to B-AMP v2.0), UniProt IDs, 3D predicted protein structures, PDBQT files, pre-defined protein function, and relevant scientific literature. To present an example of the combined applicability of both, the AMP and biofilm target libraries in the repository, we present two case studies. In the first case study, we expand an in silico pipeline to evaluate AMPs against a single biofilm target in the multidrug resistant, bacterial pathogen Corynebacterium striatum, using 3D protein-peptide docking models from previous work and Molecular Dynamics simulations ([~]1.18 {micro}s). In the second case study, we build an in silico pipeline to identify candidate AMPs (using AMPs with both anti-Gram positive and anti-Gram negative activity) against two biofilm targets with a common functional classification in Pseudomonas aeruginosa and Staphylococcus aureus, widely-encountered bacterial co-pathogens. With its enhanced structural and functional capabilities, B-AMP v2.0 serves as a comprehensive resource for AMP investigations related to biofilm studies. B-AMP v2.0 is freely available at https://b-amp.karishmakaushiklab.com and will be regularly updated with structural models of AMPs and biofilm targets, as well as 3D protein-peptide interaction models for key biofilm-forming pathogens.

bioinformatics↗

Single injection of very mild dose BOTOX in the vastus lateralis improves testicular spermatogenesis and sperm motility in ageing experimental mice

Acetylcholine (ACh), a key neurochemical messenger that plays key roles in neuroplasticity and muscle contraction. While ACh is important for the physiological function of the testis, abnormal levels of ACh cause testicular atrophy and male infertility. BOTOX is a therapeutic form of the botulinum neurotoxin that blocks the excessive release of ACh at the neuromuscular junction. Previously, repeated intracremasteric injections and slight overdose of BOTOX have been reported to induce adverse effects in the testicular parameter of experimental rodents. However, a mild dose of BOTOX is highly beneficial against skin ageing, neurological deficits, overactive urinary bladder problems, testicular pain and erectile dysfunctions. Considering the facts, the possible therapeutic benefit of BOTOX on the testis might be achieved via its minimal dose and indirect mode of action rather than repeated high quantity in the local supply. Therefore, we revisited the effect of BOTOX but with a trace amount injected into the vastus lateralis of the thigh muscle, and analyzed histological parameters of testis and quality of semen in ageing experimental mice. Experimental animals injected with 1 U/kg bodyweight of BOTOX showed enhanced spermatogenesis in associations with increased activities of key antioxidants in the testis, leading to increased total sperm count and motility. This study signifies that a mild intramuscular dose of BOTOX can be considered as a potential treatment strategy to manage and prevent male infertility.

pharmacology and toxicology↗

Combinatorial transcriptional codes specify macrophage polarization destinations

Macrophages are driven to form distinct functional phenotypes in response to different immunological stimuli, in a process widely referred to as macrophage polarization. Transcriptional regulators that guide macrophage polarization in response to a given trigger remain largely unknown. In this study, we interrogate the programmable landscape in macrophages to find regulatory panels that determine the precise polarization state that a macrophage is driven to. Towards this, we configure an integrative network analysis pipeline that utilizes macrophage transcriptomes in response to 28 distinct stimuli and reconstructs contextualized human gene regulatory networks, and identifies epicentres of perturbations in each case. We find that these contextualized regulatory networks form a spectrum of thirteen distinct clusters with M1 and M2 at the two ends. Using our computational pipeline, we identify combinatorial panels of epicentric regulatory factors (RFs) for each polarization state. We demonstrate that a set of three RFs i.e., CEBPB, NFE2L2 and BCL3, is sufficient to change the polarization destination from M1 to M2. siRNA knockdown of the 3-RF set in THP1 derived M0 cells, despite exposure to an M1 stimulant, significantly attenuated the shift to M1 phenotype, and instead increased the expression of M2 markers. Single knockdown of each RF also showed a similar trend. The siRNA-mediated knockdown of the 3-RF set rendered the macrophages hyper-susceptible to Staphylococcus aureus infection, demonstrating the importance of these factors in modulating immune responses. Overall, our results provide insights into the transcriptional mechanisms underlying macrophage polarization and identify key regulatory factors that may be targeted to modulate immune responses.

systems biology↗

Reduced neutralization of SARS-CoV-2 variants by convalescent plasma and hyperimmune intravenous immunoglobulins for treatment of COVID-19

Hyperimmune immunoglobulin (hCoV-2IG) preparations generated from SARS-CoV-2 convalescent plasma (CP) are under evaluation in several clinical trials of hospitalized COVID-19 patients. Here we explored the antibody epitope repertoire, antibody binding and virus neutralizing capacity of six hCoV-2IG batches as well as nine convalescent plasma (CP) lots against SARS-CoV-2 and emerging variants of concern (VOC). The Gene-Fragment Phage display library spanning the SARS-CoV-2 spike demonstrated broad recognition of multiple antigenic sites spanning the entire spike including NTD, RBD, S1/S2 cleavage site, S2-fusion peptide and S2-heptad repeat regions. Antibody binding to the immunodominant epitopes was higher for hCoV-2IG than CP, with predominant binding to the fusion peptide. In the pseudovirus neutralization assay (PsVNA) and in the wild-type SARS-CoV-2 PRNT assay, hCoV-2IG lots showed higher titers against the WA-1 strain compared with CP. Neutralization of SARS-CoV-2 VOCs from around the globe were reduced to different levels by hCoV-2IG lots. The most significant loss of neutralizing activity was seen against the B.1.351 (9-fold) followed by P.1 (3.5-fold), with minimal loss of activity against the B.1.17 and B.1.429 ([&le;]2-fold). Again, the CP showed more pronounced loss of cross-neutralization against the VOCs compared with hCoV-2IG. Significant reduction of hCoV-2IG binding was observed to the RBD-E484K followed by RBD-N501Y and minimal loss of binding to RBD-K417N compared with unmutated RBD. This study suggests that post-exposure treatment with hCoV-2IG is preferable to CP. In countries with co-circulating SARS-CoV-2 variants, identifying the infecting virus strain could inform optimal treatments, but would likely require administration of higher volumes or repeated infusions of hCOV-2IG or CP, in patients infected with the emerging SARS-CoV-2 variants.

immunology↗

Antibody repertoire induced by SARS-CoV-2 spike protein immunogens

Multiple vaccine candidates against SARS-CoV-2 based on viral spike protein are under development. However, there is limited information on the quality of antibody response generated following vaccination by these vaccine modalities. To better understand antibody response induced by spike protein-based vaccines, we immunized rabbits with various SARS-CoV-2 spike protein antigens: S-ectodomain (S1+S2) (aa 16-1213), which lacks the cytoplasmic and transmembrane domains (CT-TM), the S1 domain (aa 16-685), the receptor-binding domain (RBD) (aa 319-541), and the S2 domain (aa 686-1213 as control). Antibody response was analyzed by ELISA, Surface Plasmon Resonance (SPR) against different Spike proteins in native conformation, and a pseudovirion neutralization assay to measure the quality and function of the antibodies elicited by the different Spike antigens. All three antigens (S1+S2 ectodomain, S1 domain, and RBD) generated strong neutralizing antibodies against SARS-CoV-2. Vaccination induced antibody repertoire was analyzed by SARS-CoV-2 spike Genome Fragment Phage Display Libraries (SARS-CoV-2 GFPDL), which identified immunodominant epitopes in the S1, S1-RBD and S2 domains. Furthermore, these analyses demonstrated that surprisingly the RBD immunogen elicited a higher antibody titer with 5-fold higher affinity antibodies to native spike antigens compared with other spike antigens. These findings may help guide rational vaccine design and facilitate development and evaluation of effective therapeutics and vaccines against COVID-19 disease. One Sentence SummarySARS-CoV-2 Spike induced immune response

microbiology↗

Single-cell transcriptome analysis during cardiogenesis reveals basis for organ level developmental anomalies

Organogenesis involves integration of myriad cell types with reciprocal interactions, each progressing through successive stages of lineage specification and differentiation. Establishment of unique gene networks within each cell dictates fate determination, and mutations of transcription factors that drive such networks can result in birth defects. Congenital heart defects are the most common malformations and are caused by disruption of discrete subsets of progenitors1-3, however, determining the transcriptional changes in individual cells that lead to organ-level defects in the heart, or other organs, has not been tractable. Here, we employed single-cell RNA sequencing to interrogate early cardiac progenitor cells as they become specified during normal and abnormal cardiogenesis, revealing how dysregulation of specific cellular sub-populations can have catastrophic consequences. A network-based computational method for single-cell RNA-sequencing that predicts lineage specifying transcription factors4,5 identified Hand2 as a specifier of outflow tract cells but not right ventricular cells, despite failure of right ventricular formation in Hand2-null mice6. Temporal single-cell transcriptome analysis of Hand2-null embryos revealed failure of outflow tract myocardium specification, whereas right ventricular myocardium differentiated but failed to migrate into the anterior pole of the developing heart. Dysregulation of retinoic acid signaling, responsible for anterior-posterior patterning7, was associated with posteriorization of anterior cardiac progenitors in Hand2-null mutant hearts and ectopic atrial gene expression in outflow tract and right ventricle precursors. This work reveals transcriptional determinants in individual cells that specify cardiac progenitor cell fate and differentiation and exposes mechanisms of disrupted cardiac development at single-cell resolution, providing a framework to investigate congenital heart defects.

developmental biology↗