Biology subjects

Ramakrishnan, G.

Publications and source records attributed to Ramakrishnan, G..

11 recordsLinked to original sources

bioRxiv2026

Cell-scale autonomous CMOS motes for intracellular bioelectronics

Integrating autonomous electronics within single cells has remained beyond the reach of modern bioelectronics. Miniaturization at this scale could transform our ability to study and actuate biological processes at the cellular level, complementing existing molecular and fluorescent approaches. As these devices approach the sub-100-{micro}m length scale, volumetric constraints demand fundamentally new approaches to power delivery and telemetry. Here, we report an optically powered 10-picoliter complementary metal oxide semiconductor (CMOS) mote that operates with a power density of 1 pW/pL, comparable to the metabolic rate of cellular systems. These fully CMOS motes can be manufactured at scale yielding 1000 motes from a 4-mm2 silicon die. Multiple motes can be simultaneously powered and interrogated within a single optical field of view using epifluorescence microscopy. We demonstrate intracellular implantation of these motes within the single-celled mixotrophic dinoflagellate Noctiluca scintillans with negligible cytoplasmic displacement, pushing the boundaries of active CMOS bioelectronics to the intracellular domain and establishing a next-generation of truly cell-scale bioelectronic interfaces TeaserA 10-pL autonomous CMOS mote with fluorescence-based backscatter communication enables cell-scale sensing.

bioengineering↗

bioRxiv2026

Rhomboid protease Rhbdl2 regulates macrophage recruitment and wound regeneration in zebrafish

Tissue regeneration requires tight control of immune cell behavior, yet the mechanisms that restrain immune-driven regenerative responses remain poorly defined. Here, we identify the rhomboid intramembrane serine protease Rhbdl2 as a critical regulator of regeneration in zebrafish. We generated rhbdl2 mutants by CRISPR Cas9 and found that it does not affect normal development, but triggers enhanced regenerative growth following injury, accompanied by increased macrophage accumulation at the wound site, which is accompanied by increased early apoptosis and proliferation. Proteomic analyses reveal increased Rac2 protein levels in rhbdl2 mutants, indicating dysregulated immune signaling. Functionally, Rac2 morpholino oligonucleotides mediated knockdown in rhbdl2 mutant larvae suppresses the elevated macrophage recruitment and enhanced tissue regenerative phenotype. Together, these findings uncover Rhbdl2 as an immune checkpoint that constrains macrophage driven enhanced regeneration, with vast implications for inflammatory disease, fibrosis, and tumor immune interactions.

immunology↗

bioRxiv2024

IL-33 protects from recurrent C. difficile infection by restoration of humoral immunity

Clostridioides difficile infection (CDI) recurs in one of five patients. Monoclonal antibodies targeting the virulence factor TcdB reduce disease recurrence, suggesting that an inadequate anti-TcdB response to CDI leads to recurrence. In patients with CDI, we discovered that IL-33 measured at diagnosis predicts future recurrence, leading us to test the role of IL-33 signaling in the induction of humoral immunity during CDI. Using a mouse recurrence model, IL-33 was demonstrated to be integral for anti-TcdB antibody production. IL-33 acted via ST2+ ILC2 cells, facilitating germinal center T follicular helper (GC-Tfh) cell generation of antibodies. IL-33 protection from reinfection was antibody-dependent, as MT KO mice and mice treated with anti-CD20 mAb were not protected. These findings demonstrate the critical role of IL-33 in generating humoral immunity to prevent recurrent CDI. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/623943v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@9ec4aeorg.highwire.dtl.DTLVardef@1827bd3org.highwire.dtl.DTLVardef@18f761dorg.highwire.dtl.DTLVardef@19396d4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO IL-33 restoration induces toxin-B-specific antibody production via the ILC2-TFH axis. In the left panel, IL-33 remediation increases ILC2s, subsequently inducing TFH directly or indirectly. TFH cell induction is pivotal for the production of antibodies. IL-33 also downregulates type 1 and type 3 immunity, favoring type 2 immunity to enhance host survival and reduce morbidity. The middle panel illustrates antibiotic-induced dysbiosis, resulting in decreased IL-33 levels and reduced antibody production. The right panel demonstrates the protective effect in reinfection, attributed to toxin-specific antibodie generated by IL-33 remediation. C_FIG

immunology↗

bioRxiv2024

Real time imaging reveals a role for macrophage protrusive motility in melanoma invasion

Macrophages are primary cells of the innate immune system that mediate tumor progression. However, the motile behavior of macrophages and interactions with tumor cells are not well understood. Here, we exploit the optical transparency of larval zebrafish and perform real time imaging of macrophage-melanoma interactions. We found that macrophages are highly motile in the tumor microenvironment. Macrophages extend dynamic projections between tumor cells that precedes invasive melanoma migration. Modulating macrophage motility with a dominant inhibitory mutation in Rac2 inhibits recruitment to the tumor and impairs tumor invasion. However, a hyperactivating mutation in Rac2 does not affect macrophage recruitment but limits macrophage projections into the melanoma mass and reduces invasive melanoma cell migration. Taken together, these findings reveal a role for Rac2-mediated macrophage protrusive motility in melanoma invasion.

cell biology↗

bioRxiv2024

SwiftTCR: Efficient Computational Docking protocol of TCRpMHC-I Complexes Using Restricted Rotation Matrices

The T cells ability to discern self and non-self depends on its T cell receptor (TCR), which recognizes peptides presented by MHC molecules. Understanding this TCR-peptide-MHC (TCRpMHC) interaction is important for cancer immunotherapy design, tissue transplantation, pathogen identification, and autoimmune disease treatments. Understanding the intricacies of TCR recognition, encapsulated in TCRpMHC structures, remains challenging due to the immense diversity of TCRs (>108/individual), rendering experimental determination and general-purpose computational docking impractical. Addressing this gap, we have developed a rapid integrative modeling protocol leveraging unique docking patterns in TCRpMHC complexes. Built upon PIPER, our pipeline significantly cuts down FFT rotation sets, exploiting the consistent polarized docking angle of TCRs at pMHC. Additionally, our ultra-fast structure superimposition tool, GradPose, accelerates clustering. It models a case in 3-4 minutes on 12 CPUs, showcasing a speedup of up to 25-40 times compared to the ClusPro webserver. On a benchmark set of 38 TCRpMHC class I (TCRpMHC-I) complexes, our protocol outperforms the state-of-the-art docking tools in model quality. This protocol can potentially provide structural information to TCR repertoires targeting specific peptides. Its computational efficiency can also enrich existing pMHC-specific single-cell sequencing TCR data, facilitating the development of structure-based deep learning (DL) algorithms. These insights are essential for understanding T cell recognition and specificity, advancing the development of therapeutic interventions.

bioinformatics↗

bioRxiv2024

Stable, chronic in-vivo recordings from a fully wireless subdural-contained 65,536-electrode brain-computer interface device

Minimally invasive, high-bandwidth brain-computer-interface (BCI) devices can revolutionize human applications. With orders-of-magnitude improvements in volumetric efficiency over other BCI technologies, we developed a 50-m-thick, mechanically flexible micro-electrocorticography (ECoG) BCI, integrating a 256x256 array of electrodes, signal processing, data telemetry, and wireless powering on a single complementary metal-oxide-semiconductor (CMOS) substrate containing 65,536 recording channels, from which we can simultaneously record a selectable subset of up to 1024 channels at a given time. Fully implanted below the dura, our chip is wirelessly powered, communicating bi-directionally with an external relay station outside the body. We demonstrated chronic, reliable recordings for up to two weeks in pigs and up to two months in behaving non-human primates from somatosensory, motor, and visual cortices, decoding brain signals at high spatiotemporal resolution.

neuroscience↗

bioRxiv2024

Staphylococcus aureus lipid factors modulate melanoma clustering and invasion in zebrafish

The microbiome can influence cancer development and progression. However, less is known about the role of the skin microbiota in melanoma. Here, we take advantage of a zebrafish melanoma model to probe the effects of Staphylococcus aureus on melanoma invasion. We find that S. aureus produces factors that enhance melanoma invasion and dissemination in zebrafish larvae. We used a published in vitro 3D cluster formation assay which correlates increased clustering with tumor invasion. S. aureus supernatant increased clustering of melanoma cells which was abrogated by use of a Rho-Kinase inhibitor, implicating a role for Rho-GTPases. The melanoma clustering response was specific to S. aureus and not other staphylococcal species, including S. epidermidis. Our findings suggest that S. aureus may promote melanoma clustering and invasion via lipids generated by the lipase SAL2 (gehB). Taken together, these findings suggest that specific bacterial products mediate melanoma invasive migration in zebrafish. Summary statementS. aureus supernatant increases melanoma invasion in zebrafish and is likely mediated by production of bacterial lipids.

cancer biology↗

bioRxiv2023

PANDORA v2.0: Benchmarking peptide-MHC II models and software improvements

T-cell specificity to differentiate between self and non-self relies on T-cell receptor (TCR) recognition of peptides presented by the Major Histocompatibility Complex (MHC). Investigations into the three-dimensional (3D) structures of peptide:MHC (pMHC) complexes have provided valuable insights of MHC functions. Given the limited availability of experimental pMHC structures and considerable diversity of peptides and MHC alleles, it calls for the development of efficient and reliable computational approaches for modeling pMHC structures. Here we present an update of PANDORA and the systematic evaluation of its performance in modelling 3D structures of pMHC class II complexes (pMHC-II), which play a key role in the cancer immune response. PANDORA is a modelling software that can build low-energy models in a few minutes by restraining peptide residues inside the MHC-II binding groove. We benchmarked PANDORA on 136 experimentally determined pMHC-II structures covering 44 unique {beta} chain pairs. Our pipeline achieves a median backbone Ligand-Root Mean Squared Deviation (L-RMSD) of 0.42 [A] on the binding core and 0.88 [A] on the whole peptide for the benchmark dataset. We incorporated software improvements to make PANDORA a pan-allele framework and improved the user interface and software quality. Its computational efficiency allows enriching the wealth of pMHC binding affinity and mass spectrometry data with 3D models. These models can be used as a starting point for molecular dynamics simulations or structure-boosted deep learning algorithms to identify MHC-binding peptides. PANDORA is available as a Python package through Conda or as a source installation at https://github.com/X-lab-3D/PANDORA.

bioinformatics↗

bioRxiv2023

Parallel evolution and enhanced virulence upon in vivo passage of an RNA virus in Drosophila melanogaster

Virus evolution is strongly affected by antagonistic co-evolution of virus and host. Host immunity positively selects for viruses that evade the immune response, which in turn may drive counter-adaptations in host immune genes. We investigated how host immune pressure shapes virus populations, using the fruit fly Drosophila melanogaster and its natural pathogen Drosophila C virus (DCV), as a model. We performed an experimental evolution study in which DCV was serially passaged for ten generations in three fly genotypes differing in their antiviral RNAi response: wild-type flies and flies in which the endonuclease gene Dicer-2 was either overexpressed or inactivated. All evolved virus populations replicated more efficiently in vivo and were more virulent than the parental stock. The number of polymorphisms increased in all three host genotypes with passage number, which was most pronounced in Dicer-2 knockout flies. Mutational analysis showed strong parallel evolution, as mutations accumulated in a specific region of the VP3 capsid protein in every lineage in a host genotype-independent manner. The parental tyrosine at position 95 of VP3 was substituted with either one of five different amino acids in 14 out of 15 lineages. However, no consistent amino acid changes were observed in the viral RNAi suppressor gene 1A, nor elsewhere in the genome in any of the host backgrounds. Our study indicates that the RNAi response restricts the sequence space that can be explored by viral populations. Moreover, our study illustrates how evolution towards higher virulence can be a highly reproducible, yet unpredictable process.

microbiology↗

bioRxiv2020

Low-frequency vibrational modes in G-quadruplexes reveal the mechanical properties of nucleic acids

Low-frequency vibrations play an essential role in biomolecular processes involving DNA such as gene expression, charge transfer, drug intercalation, and DNA-protein recognition. However, understanding of the vibrational basis of these mechanisms relies on theoretical models due to the lack of experimental evidence. Here we present the low-frequency vibrational spectra of G-quadruplexes (structures formed by four strands of DNA) and B-DNA characterized using femtosecond optical Kerr-effect spectroscopy. Contrary to expectation, we found that G-quadruplexes show several strongly underdamped delocalized phonon-like modes that have the potential to contribute to the biology of the DNA at the atomic level. In addition, G-quadruplexes present modes at a higher frequency than B-DNA demonstrating that changes in the stiffness of the molecule alter its gigahertz to terahertz vibrational profile. These results demonstrate that current theoretical models fail to predict basic properties of the vibrational modes of DNA. Statement of significanceA number of recent studies have identified thermally excited low-frequency vibrational modes as a key deciding factor in the biological function of DNA. However, the nature of these vibrational modes has never been established. Here, vibrational spectroscopy with unrivalled signal-to-noise in the gigahertz to terahertz range is used to determine the low-frequency Raman spectra of nucleotides and oligomeric DNAs carefully chosen to form G-quadruplexes, structures formed by four strands of DNA common in the genome. These G-quadruplexes exhibit an unusual group of highly-underdamped delocalized vibrational modes--not reproduced by any of the theoretical models in use--which are expected to be the thermally excited. This provides a new perspective on the role of low-frequency vibrational modes in protein interactions and allostery.

biophysics↗

bioRxiv2020

Cell autonomous versus systemic Akt isoform deletions uncovered new roles for Akt1 and Akt2 in breast cancer

Studies in three mouse models of breast cancer identified profound discrepancies between cell autonomous and systemic Akt1 or Akt2 deletion on breast cancer tumorigenesis and metastasis. First, unlike systemic Akt1 deletion, which inhibits metastasis, cell autonomous Akt1 deletion does not. Second, systemic Akt2 deletion does not inhibit mammary tumorigenesis and metastasis, but cell autonomous Akt2 deletion eliminates ErbB2 expressing cells in the mammary gland and prevents tumorigenesis. However, the elevation in insulin by Akt2 systemic deletion hyperactivates tumor Akt, enabling ErbB2 expression, and exacerbates mammary tumorigenesis. Decreasing insulin level inhibits accelerated tumorigenesis by systemic Akt2 deletion. Single cell mRNA sequencing revealed that systemic Akt1 deletion maintains the pro-metastatic cluster within primary tumors but ablates pro-metastatic neutrophils. Systemic Akt1 deletion inhibits metastasis by impairing the survival and mobilization of tumor-associated neutrophils. Importantly, neutrophil-specific deletion of Akt1 is sufficient to exert resistance to metastasis. The results underscore the importance of determining systemic effects rather than cell autonomous effects as a proof of concept for cancer therapy.

cancer biology↗