Biology subjects

Mukund, A.

Publications and source records attributed to Mukund, A..

5 recordsLinked to original sources

bioRxiv2025

Increased versican and fibrosis in mesenteric lymph nodes disrupts immune surveillance and drives systemic bacterial dissemination in cirrhosis

Background and ObjectiveMesenteric lymph nodes (MLN) are immunological barriers against bacterial translocation (BT). Enhanced gut BT through MLN facilitates bacterial spread and higher mortality in cirrhosis. We aimed to elucidate mechanisms underlying MLN failure to effectively contain BT during advanced cirrhosis. DesignBT and immune cells were analyzed in lymphoid organs and circulation of control and CCl4 models, with and without MLN (MLNx). MLN proteomics identified versican (VCAN) as major upregulated protein in cirrhosis, whose immunomodulatory function was examined in vitro and in vivo in CCl and (Bile duct ligation) BDL models. Plasma VCAN were measured in end-stage cirrhosis patients and analyzed as mortality predictor. ResultsIn control rats, bacteria were confined to MLN, whereas cirrhotics showed BT to MLN, lymph, and portal blood. Compared to control, CCl4 rats had increased activated Th-cells in MLN but reduced in circulation. In control-MLNx rats, activated Th-cells were reduced in circulation vs controls. In BDL models, MLN CFU correlated with VCAN level. In vitro, VCAN enhanced T cell suppression and impaired migration which was reversed by CD44 blockade. In vivo VCAN knockdown reduced fibrosis and bacterial burden in MLN, while restoring Th-cell activation locally and systemically. Clinically, plasma VCAN levels were elevated in advanced cirrhosis patients and remained an independent predictor of 28-day sepsis-related mortality. ConclusionIncreased VCAN impairs T cell activation and migration in MLN, fostering immune suppression and bacterial persistence. Plasma VCAN levels serve as promising biomarker for MLN dysfunction and prognostic factor for predicting sepsis-related mortality in end-stage cirrhosis. What is already known on this topic - Enhanced gut bacterial translocation through mes-enteric lymph nodes (MLN) facilitates systemic bacterial spread and increases mortality in cirrhosis. The mechanisms underlying MLN failure to effectively contain bacterial spread during advanced cirrhosis remain largely unknown. What this study adds - The study unveils a critical role of lymph node fibrosis and elevated versican (VCAN) expression in causing deranged immune responses and bacterial clearance in MLN, increasing systemic bacterial load and immunosuppression. Most importantly, high plasma VCAN emerges as a prognostic biomarker for functional failure of MLN and 28-day mortality predictor in critically ill patients with cirrhosis. How this study might affect research, practice or policy - VCAN, representing enhanced MLN fibrosis and dysfunction, emerges as a predictive biomarker of adverse clinical out-comes in patients with advanced cirrhosis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/690350v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@369f4eorg.highwire.dtl.DTLVardef@14c232org.highwire.dtl.DTLVardef@d99b21org.highwire.dtl.DTLVardef@57ac95_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2022

Large-scale mapping and systematic mutagenesis of human transcriptional effector domains

Human gene expression is regulated by over two thousand transcription factors and chromatin regulators1,2. Effector domains within these proteins can activate or repress transcription. However, for many of these regulators we do not know what type of transcriptional effector domains they contain, their location in the protein, their activation and repression strengths, and the amino acids that are necessary for their functions. Here, we systematically measure the transcriptional effector activity of >100,000 protein fragments (each 80 amino acids long) tiling across most chromatin regulators and transcription factors in human cells (2,047 proteins). By testing the effect they have when recruited at reporter genes, we annotate 307 new activation domains and 592 new repression domains, a [~]5-fold increase over the number of previously annotated effectors3,4. Complementary rational mutagenesis and deletion scans across all the effector domains reveal aromatic and/or leucine residues interspersed with acidic, proline, serine, and/or glutamine residues are necessary for activation domain activity. Additionally, the majority of repression domain sequences contain either sites for SUMOylation, short interaction motifs for recruiting co-repressors, or are structured binding domains for recruiting other repressive proteins. Surprisingly, we discover bifunctional domains that can both activate and repress and can dynamically split a cell population into high- and low-expression subpopulations. Our systematic annotation and characterization of transcriptional effector domains provides a rich resource for understanding the function of human transcription factors and chromatin regulators, engineering compact tools for controlling gene expression, and refining predictive computational models of effector domain function.

systems biology↗

bioRxiv2021

Dynamic spreading of chromatin-mediated gene silencing and reactivation between neighboring genes in single cells

In mammalian cells genes that are in close proximity are coupled transcriptionally: silencing or activating one gene can affect its neighbors. Understanding these dynamics is important for natural processes, such as heterochromatin spreading during development and aging, and when designing synthetic gene regulation. Here, we systematically dissect this process in single cells by recruiting and releasing repressive chromatin regulators at dual-gene synthetic reporters, and measuring how fast gene silencing and reactivation spread as a function of intergenic distance and configuration of insulator elements. We find that silencing by KRAB, associated with histone methylation, spreads between two genes within hours, with a time delay that increases with distance. This fast KRAB-mediated spreading is not blocked by the classical cHS4 insulators. Silencing by histone deacetylase HDAC4 of the upstream gene can also lead to downstream gene silencing, but with a days-long delay that does not change with distance. This slower silencing can sometimes be stopped by insulators. Gene reactivation of neighboring genes is also coupled, with strong promoters and insulators determining the order of reactivation. We propose a new model of multi-gene regulation, where both gene silencing and gene reactivation can act at a distance, allowing for coordinated dynamics via chromatin regulator recruitment.

synthetic biology↗

bioRxiv2020

High-throughput discovery and characterization of human transcriptional effectors

Thousands of proteins localize to the nucleus; however, it remains unclear which contain transcriptional effectors. Here, we develop HT-recruit - a pooled assay where protein libraries are recruited to a reporter, and their transcriptional effects are measured by sequencing. Using this approach, we measure gene silencing and activation for thousands of domains. We find a relationship between repressor function and evolutionary age for the KRAB domains, discover Homeodomain repressor strength is collinear with Hox genetic organization, and identify activities for several Domains of Unknown Function. Deep mutational scanning of the CRISPRi KRAB maps the co-repressor binding surface and identifies substitutions that improve stability/silencing. By tiling 238 proteins, we find repressors as short as 10 amino acids. Finally, we report new activator domains, including a divergent KRAB. Together, these results provide a resource of 600 human proteins containing effectors and demonstrate a scalable strategy for assigning functions to protein domains.

genetics↗

bioRxiv2018

OSPREY 3.0: Open-Source Protein Redesign for You, with Powerful New Features

We present O_SCPCAPOSPREYC_SCPCAP 3.0, a new and greatly improved release of the O_SCPLOWOSPREYC_SCPLOW protein design software. O_SCPLOWOSPREYC_SCPLOW 3.0 features a convenient new Python interface, which greatly improves its ease of use. It is over two orders of magnitude faster than previous versions of O_SCPLOWOSPREYC_SCPLOW when running the same algorithms on the same hardware. Moreover, O_SCPLOWOSPREYC_SCPLOW 3.0 includes several new algorithms, which introduce substantial speedups as well as improved biophysical modeling. It also includes GPU support, which provides an additional speedup of over an order of magnitude. Like previous versions of O_SCPLOWOSPREYC_SCPLOWO_SCPCAP, C_SCPCAPO_SCPLOWOSPREYC_SCPLOW 3.0 offers a unique package of advantages over other design software, including provable design algorithms that account for continuous flexibility during design and model conformational entropy. Finally, we show here empirically that O_SCPLOWOSPREYC_SCPLOW 3.0 accurately predicts the effect of mutations on protein-protein binding. O_SCPLOWOSPREYC_SCPLOW 3.0 is available at http://www.cs.duke.edu/donaldlab/osprey.php as free and open-source software.\n\nO_TEXTBOX\n\nWe present the third major release of the OSPREY protein design software, along with comparisons to experimental data that confirm its ability to optimize protein mutants for desired functions. osprey 3.0 has significant efficiency, ease-of-use, and algorithmic improvements over previous versions, including GPU acceleration and a new Python interface.\n\nC_TEXTBOX\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC=\"FIGDIR/small/306324v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (54K):\norg.highwire.dtl.DTLVardef@f08b0dorg.highwire.dtl.DTLVardef@393b61org.highwire.dtl.DTLVardef@16997ddorg.highwire.dtl.DTLVardef@1713405_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗