Biology subjects

Shrivastava, R.

Publications and source records attributed to Shrivastava, R..

5 recordsLinked to original sources

bioRxiv2025

C/EBPβ-mediated PARP1 Suppression Drives Tumor Progression of Aneuploid Cells

Aneuploidy--defined as gains and losses of chromosomes--is frequently observed in cancer and has been implicated in promoting tumor progression and metastasis. However, the molecular mechanisms underlying this phenomenon remain poorly understood. By generating new models of aneuploidy, we found that aneuploidy confers remarkable resistance to reactive oxygen species (ROS)-mediated cell death. This resistance is a general consequence of aneuploidy, independent of the specific chromosomes gained or lost. Mechanistically, Poly(ADP-Ribose) Polymerase 1 (PARP1) is suppressed in aneuploid cells, which inhibits PARP1-mediated cell death after ROS (parthanatos). We validated aneuploidy-associated PARP1 suppression across 15 cell models and human tumors, with pronounced effects in metastatic tumors. Importantly, decreased PARP1 levels promote tumor metastasis while increased PARP1 suppresses it. Through a genome-wide CRISPR screen and functional validation, we identified the transcription factor CCAAT/enhancer-binding protein beta (CEBPB) as a critical mediator of PARP1 downregulation and ROS resistance in aneuploid cells. Furthermore, we found that lysosomal dysfunction serves as the upstream mediator of CEBPB activation in aneuploid cells. We propose that aneuploidy-driven CEBPB activation promotes PARP1 suppression, fostering ROS resistance and cancer progression. HighlightsO_LIAneuploidy confers resistance to oxidative stress independent of p53 status, karyotype and cell lineage through inhibition of PARP1 expression and activity C_LIO_LISuppressed PARP1 enhances metastatic potential, while PARP1 restoration suppresses metastatic spread, revealing a novel mechanism linking aneuploidy to cancer progression. C_LIO_LIPARP1 suppression compromises DNA damage repair and cell death to multiple genotoxic stressors, including reactive oxygen species, alkylating agents, and UV radiation. C_LIO_LIA genome-wide CRISPR screen and a CRISPRa screen identifies CEBPB as a critical transcription factor mediating PARP1 regulation and ROS resistance. C_LIO_LINuclear CEBPB increases significantly after aneuploidization in experimental systems and in primary human cancers. C_LI O_FIG O_LINKSMALLFIG WIDTH=195 HEIGHT=200 SRC="FIGDIR/small/670212v2_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@1bf4edforg.highwire.dtl.DTLVardef@d3311eorg.highwire.dtl.DTLVardef@13daf25org.highwire.dtl.DTLVardef@b5e65f_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Universal Digital High Resolution Melt for the detection of pulmonary mold infections

BackgroundInvasive mold infections (IMIs) such as aspergillosis, mucormycosis, fusariosis, and lomentosporiosis are associated with high morbidity and mortality, particularly in immunocompromised patients, with mortality rates as high as 40% to 80%. Outcomes could be substantially improved with early initiation of appropriate antifungal therapy, yet early diagnosis remains difficult to establish and often requires multidisciplinary teams evaluating clinical and radiological findings plus supportive mycological findings. Universal digital high resolution melting analysis (U-dHRM) may enable rapid and robust diagnosis of IMI. This technology aims to accomplish timely pathogen detection at the single genome level by conducting broad-based amplification of microbial barcoding genes in a digital polymerase chain reaction (dPCR) format, followed by high-resolution melting of the DNA amplicons in each digital reaction to generate organism-specific melt curve signatures that are identified by machine learning. MethodsA universal fungal assay was developed for U-dHRM and used to generate a database of melt curve signatures for 19 clinically relevant fungal pathogens. A machine learning algorithm (ML) was trained to automatically classify these 19 fungal melt curves and detect novel melt curves. Performance was assessed on 73 clinical bronchoalveolar lavage (BAL) samples from patients suspected of IMI. Novel curves were identified by micropipetting U-dHRM reactions and Sanger sequencing amplicons. ResultsU-dHRM achieved an average of 97% fungal organism identification accuracy and a turn-around-time of 4hrs. Pathogenic molds (Aspergillus, Mucorales, Lomentospora and Fusarium) were detected by U-dHRM in 73% of BALF samples suspected of IMI. Mixtures of pathogenic molds were detected in 19%. U-dHRM demonstrated good sensitivity for IMI, as defined by current diagnostic criteria, when clinical findings were also considered. ConclusionsU-dHRM showed promising performance as a separate or combination diagnostic approach to standard mycological tests. The speed of U-dHRM and its ability to simultaneously identify and quantify clinically relevant mold pathogens in polymicrobial samples as well as detect emerging opportunistic pathogens may provide information that could aid in treatment decisions and improve patient outcomes.

microbiology↗

bioRxiv2023

Targeting the deNEDDylating enzyme NEDP1 to ameliorate ALS phenotypes through Stress Granules dissolution

In Amyotrophic Lateral Sclerosis (ALS) motor neuron disease, mutations in proteins that upon stress localize within cytoplasmic protein inclusions called Stress Granules (SGs), are linked to the formation of aberrant inclusions, which are related to neuronal cell death. By combining studies in human cells and C. elegans including the use of Nanobodies, we found that inhibition of NEDP1, the enzyme responsible for the processing and deconjugation of the Ubiquitin-like molecule NEDD8 from substrates, promotes the elimination both of physiological and pathological SGs. The hyper-NEDDylation of Poly-(ADP-ribose) polymerase-1 enzyme upon NEDP1 inhibition compromises PAR production and is a key mechanism for the observed SG phenotype. Importantly, the above-described effects are related to improved cell survival in human cells, and in C. elegans, NEDP1 deletion ameliorates ALS-phenotypes related to animal motility. Our studies reveal NEDP1 as potential therapeutic target for ALS, based on the elimination of aberrant SGs.

cell biology↗

bioRxiv2018

Biosynthetic intermediates of the enterobacterial common antigen overcome outer membrane lipid dyshomeostasis in Escherichia coli

The outer membrane (OM) is an essential component of the Gram-negative bacterial envelope that protects cells against external threats. To maintain a functional OM, cells require distinct mechanisms to ensure balance of proteins and lipids in the membrane. Mutations in OM biogenesis and/or homeostasis pathways often result in permeability defects, but how molecular changes in the OM affect barrier function is unclear. Here, we seek potential mechanism(s) that can alleviate permeability defects in Escherichia coli cells lacking the Tol-Pal complex, which accumulate excess PLs in the OM. We identify mutations in enterobacterial common antigen (ECA) biosynthesis that re-establish OM barrier function against large hydrophilic molecules, yet did not restore lipid homeostasis. Furthermore, we demonstrate that build-up of biosynthetic intermediates, but not loss of ECA itself, contributes to the rescue. This suppression of OM phenotypes is unrelated to known effects that accumulation of ECA intermediates have on the cell wall. Finally, we reveal that an unusual diacylglycerol pyrophosphoryl-linked lipid species also accumulates in ECA mutants, and might play a role in the rescue phenotype. Our work provides insights into how OM barrier function can be restored independent of lipid homeostasis, and highlights previously unappreciated effects of ECA-related species in OM biology.

microbiology↗

bioRxiv2017

Bacterial outer membrane homeostasis via retrograde phospholipid transport

Biogenesis of the outer membrane (OM) in Gram-negative bacteria, which is essential for viability, requires the coordinated transport and assembly of proteins and lipids, including lipopolysaccharides (LPS) and phospholipids (PLs), into the membrane. While pathways for LPS and OM protein assembly are well-studied, how PLs are transported to and from the OM is not clear. Mechanisms that ensure OM stability and homeostasis are also unknown. The trans-envelope Tol-Pal complex, whose physiological role has remained elusive, is important for OM stability. Here, we establish that the Tol-Pal complex is required for PL transport and OM lipid homeostasis in Escherichia coli. Cells lacking the complex exhibit defects in lipid asymmetry and accumulate excess phospholipids (PLs) in the OM. This imbalance in OM lipids is due to defective retrograde PL transport in the absence of a functional Tol-Pal complex. Thus, cells ensure the assembly of a stable OM by maintaining an excess flux of PLs to the OM only to return the surplus to the inner membrane. Our findings also provide insights into the mechanism by which the Tol-Pal complex may promote OM invagination during cell division.

microbiology↗