Biology subjects

Dahal, S.

Publications and source records attributed to Dahal, S..

11 recordsLinked to original sources

bioRxiv2026

2,6-diaminopurine enhances Aqp4 stop codon readthrough and AQP4 perivascular localization in the brain

In the brain, the water channel Aquaporin 4 (AQP4) is largely restricted to astrocytes, enriched at perivascular astrocytic processes, and involved in fluid balance and neurological disease. The perivascular pool is functionally unique as it is depolarized in diseases, targeted by neuromyelitis optica autoantibodies, and implicated in the clearance of brain metabolites such as amyloid beta. The perivascular AQP4 was recently shown to be AQP4X, an extended isoform arising from readthrough, where approximately 20% of translating ribosomes continue through the Aqp4 stop codon. Because Aqp4 terminates with a UGA, here we tested whether Aqp4 readthrough can be enhanced by 2,6-diaminopurine, a purine analog known to promote UGA decoding. Using dual luciferase and immunoblotting for AQP4X in cultured cells, we show that the drug promotes Aqp4 readthrough and increases AQP4X levels. For in vivo validation, we use the wild-type mouse as well as a genetically engineered mouse with a stop-to-sense mutation allowing Aqp4 readthrough at 100%. 2,6-diaminopurine elevates perivascular AQP4X levels by approximately 10% after 20 hours of single intracranial injection. It fails to enhance AQP4X levels in animals with stop-to-sense mutation, suggesting its action involves Aqp4 readthrough. Aqp4 readthrough can be a pharmacological target for modulating perivascular AQP4X in neurological disease models.

neuroscience↗

bioRxiv2026

Comparative genomics analysis reveals that antimicrobial activity in Pseudomonas protegens PBL3 is associated with gene clusters participating in multiple cellular functions

The environmental bacterium Pseudomonas protegens PBL3 has antagonistic activity against the bacterial pathogen Burkholderia glumae, the causal agent of the rice disease Bacterial Panicle Blight (BPB). The antimicrobial activity of the P. protegens PBL3 was found in the bacteria-free secreted fraction (secretome), but the specific molecules, as well as the genetic basis conferring activity, have not been identified. To elucidate the genes and biosynthetic pathways governing the antimicrobial activity of the P. protegens PBL3 secretome, we used comparative genomics, by leveraging six Pseudomonas spp. strains, with available genomic sequences and exhibiting contrasting antimicrobial activities against B. glumae. We hypothesized that Pseudomonas spp. strains with antimicrobial activity against B. glumae have conserved genes with P. protegens PBL3 and those genes are absent in the strains lacking activity. To test this hypothesis, we performed comparative genomics analysis across the six strains using two complementary approaches, anvio and progressiveMauve, and using P. protegens PBL3 as the reference genome. This analysis revealed 188 genes uniquely present in antimicrobial-producing strains. Seven of those genes were associated with biosynthetic gene clusters predicted to encode secondary metabolites; additional genes were grouped into twenty-five contiguous clusters with functions associated with secretion, signal transduction, regulation, transport/efflux, carbohydrate metabolism and one with additional uncharacterized function. Altogether, this study provided a complex and multi-functional network of candidate genes in antimicrobial-producing strains, suggesting that P. protegens PBL3 employs not only classical biosynthetic pathways but also integrated regulatory, metabolic, and export modules to synthesize and deploy antimicrobials. ImportanceBacterial plant diseases can be controlled through biological control, a strategy wherein beneficial microorganisms known as biological control agents (BCAs) interfere with the biological activities of pathogens through several mechanisms. One of these mechanisms is antibiosis, by which antimicrobial molecules produced by the BCA prevent pathogen multiplication or actively kill the pathogen. While this mechanism of antibiosis has been widely recognized, the specific molecules associated with the antimicrobial activity are not always identified, given their diverse and complex chemical structures as well as the unique and intricate biosynthetic pathways. This study unraveled different pathways underlying the antimicrobial activity in the environmental bacterium Pseudomonas protegens PBL3 to advance the discovery of effective antimicrobials to control bacterial plant diseases.

microbiology↗

bioRxiv2025

PCSK9 dependent cholesterol acquisition and utilization underlies metastatic organ preference in pancreatic cancer.

To grow at distant sites, metastatic cells must overcome major challenges posed by the unique cellular and metabolic composition of secondary organs1. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease that metastasizes to the liver and lungs. Despite evidence of metabolic reprogramming away from the primary site, the key drivers that dictate the ability of PDAC cells to colonize the liver or lungs and survive there are undefined. We identified Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) as predictive of colonization of the liver versus lungs by integrating datasets describing the metastatic tropism of human PDAC cell lines2, with in vivo metastasis modeling in mice and gene expression correlation analysis. PCSK9 is a negative regulator of low density lipoprotein(LDL)-cholesterol import and, accordingly, PCSK9-low PDAC cells exhibit strong preference for colonizing LDL-rich liver tissue. Cholesterol taken up by liveravid PCSK9-low cells is converted into the signaling oxysterol, 24(S)-hydroxycholesterol, which reprograms the surrounding microenvironment to induce nutrient release from neighboring hepatocytes. Conversely, PCSK9-high, lung-avid PDAC cells rely on transcriptional upregulation of the distal cholesterol synthesis pathway to generate intermediates, chiefly, 7dehydrodesmosterol and 7-dehydrocholesterol, with protective action against ferroptosis, a vulnerability in the oxygen-rich microenvironment of the lung. Increasing PCSK9 levels redirected liver-avid cells to the lung whereas its ablation drove lung-avid cells to the liver, thereby establishing PCSK9 as necessary and sufficient for secondary organ site preference. Our studies reveal PCSK9-driven differential utilization of the distal cholesterol synthesis pathway as a key and potentially actionable driver of metastatic growth in PDAC.

cancer biology↗

bioRxiv2024

Exploiting the Achilles' Heel of Viral RNA Processing to Develop Novel Antivirals

Viruses continue to pose a significant health burden to the human population, and recent history has shown a concerning surge in viral threats. Treatment options for viral infections are limited, and viruses have proven adept at evolving resistance to many existing therapies, highlighting a significant vulnerability in our defenses. In response to this challenge, we explored the modulation of cellular RNA metabolic processes as an alternative paradigm to antiviral development. Many viruses depend on the host cells RNA splicing machinery, and small alterations to this host process results in catastrophic changes in viral protein production, ultimately inhibiting virus replication. Previously, the small molecule 5342191 was identified as a potent inhibitor of HIV-1 replication by altering viral RNA accumulation at doses that minimally affect host gene expression. In this report, we document 5342191 as a potent inhibitor of adenovirus, coronavirus, and influenza replication. In each case, 5342191-mediated reduction in virus replication was associated with altered viral RNA accumulation and loss of viral structural protein expression. Interestingly, while resistant viruses were rapidly isolated for compounds targeting either virus-encoded proteases or polymerases, we have not yet isolated 534219-resistant variants of coronavirus or influenza. As with HIV-1, 5342191s inhibition of coronaviruses and influenza is mediated through the activation of specific cell signaling networks, including GPCR and/or MAPK signaling pathways that ultimately affect SR kinase expression. Together, these studies highlight the therapeutic potential of compounds that target cellular processes essential for the replication of multiple viruses. Not only do these compounds hold promise as broad-spectrum antivirals, but they also offer the potential of greater resilience in combating viral infections.

microbiology↗

bioRxiv2024

Integrated forward and reverse degradomics uncovers the proteolytic landscape of aortic aneurysms and the roles of MMP9 and mast cell chymase

BackgroundDysregulated proteolysis is implicated in thoracic (TAA) and abdominal aortic aneurysm (AAA) pathogenesis, but the proteolytic landscapes (degradomes) of aneurysmal and normal aorta, and contributions of individual proteases remain undefined. Here, a proteome-wide approach was used to uncover TAA and AAA degradomes, compare them quantitatively and define the specific role in aortic remodeling of two proteases consistently identified in the aneurysms, mast cell chymase (CMA1) and matrix metalloprotease 9 (MMP9). MethodsThe mass spectrometry-based N-terminomics strategy Terminal Amine Isotopic Labeling of Substrates (TAILS) was applied to Marfan syndrome TAAs (n=5), AAAs (n=16) and corresponding non-diseased aorta (TAs, n=4, and AAs, n=8) as a forward degradomics application, i.e., to define substrate and protease degradomes, and 8-plex iTRAQ-TAILS was used for quantitative comparison. Cleavage sites of CMA1 and MMP9 were sought by reverse degradomics, i.e., digestion of aortic proteins with these proteases, followed by 6-plex iTRAQ-TAILS. CMA1 and MMP9 proteolysis of biglycan was investigated using Amino-Terminal Oriented Mass spectrometry of Substrates (ATOMS). ResultsWe experimentally annotated 16,923 proteolytically derived peptides (substrate degradome) and 90 proteases (protease degradome) in the aorta. Quantitative substrate degradome comparisons identified specific differentially modulated pathways and networks in TAA and AAA. Reverse degradomics elucidated > 300 CMA1 and MMP9 substrate cleavage sites, of which, many, including orthogonally validated biglycan cleavage, occurred in the disease degradomes. ConclusionsUnbiased, proteome-wide forward degradomics of the aortic wall from TAA, AAA and non-diseased tissue generated the first systems biology view of vascular wall breakdown and public resource for the hitherto occult proteolytic landscape, demonstrating widespread extracellular matrix remodeling. The findings provide insights on aortic aneurysm pathways and potential disease biomarkers. Mapping of specific contributions of CMA1 and MMP9 on the aortic forward substrate degradome using reverse degradomics provides a strategy for defining the activities of all proteases involved in aortic disease.

biochemistry↗

bioRxiv2024

Unification of cell-scale metabolic activity with biofilm behavior by integration of advanced flow and reactive-transport modeling and microfluidic experiments

The bacteria Geobacter sulfurreducens (GS) is a promising candidate for broad applications involving bioelectrochemical systems (BES), such as environmental bioremediation and energy production. To date, most GS studies have reported biofilm-scale metrics, which fail to capture the interactions between cells and their local environments via the complex metabolism at the cellular level. Moreover, the dominance of studies considering diffusion-only molecular mass transport models within the biofilm has ignored the role of internal advection though the biofilm in flow BES. Among other things, this incomplete picture of anode-adhered GS biofilms has led to missed opportunities in optimizing the operational parameters for BES. To address these gaps, we have modernized a GS genome-scale metabolic model (GEM) and complemented it with local flow and reactive-transport models (FRTM). We tuned certain interactions within the model that were critical to reproducing the experimental results from a pure-culture GS biofilm in a microfluidic bioelectrochemical cell under precisely controlled conditions. The model provided insights into the role of mass transport in determining the spatial availability of nutrient molecules within the biofilm. Thus, we verified that fluid advection within biofilms was significantly more important and complex than previously thought. Coupling these new transport mechanisms to GEM revealed adjustments in intracellular metabolisms based on cellular position within the biofilm. Three findings require immediate dissemination to the BES community: (i) Michaelis-Menten kinetics overestimate acetate conversion in biofilm positions where acetate concentration is high, whereas Coulombic efficiencies should be nearly 10% lower than is assumed by most authors; (ii) unification of the empirically observed flow sensitivity of biofilm-scale kinetic parameters and cell-scale values are finally achieved; and (iii) accounting for advection leads to estimations of diffusion coefficients which are much lower than proposed elsewhere in the literature. In conclusion, in-depth spatiotemporal understanding of mechanisms within GS biofilm across relevant size scales opens the door to new avenues for BES optimization, from fine-scale processes to large-scale applications, including improved techno-economic analyses.

systems biology↗

bioRxiv2022

Novel Context-Specific Genome-Scale Modelling Explores the Potential of Chlamydomonas reinhardtii for Synthetic Biology Applications

Gene expression data of cell cultures is commonly measured in biological and medical studies to understand cellular decision-making in various conditions. Metabolism, affected but not solely determined by the expression, is much more difficult to measure experimentally. Thus, finding a reliable method to predict cell metabolism for given expression data will greatly benefit model-aided metabolic engineering. We have developed such a pipeline that can explore cellular fluxomics from expression data, using only a high-quality genome-scale metabolic model. This is done through two main steps: first, construct a protein-constrained metabolic model by integrating protein and enzyme information into the metabolic model. Secondly, overlay the expression data onto the modified model using a new two-step non-convex and convex optimization formulation, resulting in context-specific models with optionally calibrated rate constants. The resulting model computes proteomes and intracellular flux states that are consistent with the measured transcriptomes. Therefore, it provides detailed cellular insights that are difficult to glean individually from the omic data or metabolic models alone. As a case study, we apply the pipeline to interpret triacylglycerol (TAG) overproduction by Chlamydomonas reinhardtii, using time-course RNA-Seq data. The pipeline allows us to compute C. reinhardtii metabolism under nitrogen deprivation and metabolic shifts after an acetate boost. We also suggest a list of possible bottlenecking proteins that need to be overexpressed to increase the TAG accumulation rate, as well as discussing other TAG-overproduction strategies.

systems biology↗

bioRxiv2022

L-norepinephrine induces ROS formation but alters microbial community composition by altering cellular metabolism

Neuroendocrine compounds discharged into wastewater systems represent an emerging challenge at the intersection of human physiology and environmental microbiology. L-norepinephrine (L-NE) L-NE, which has been recognized to potentiate growth of human and animal bacterial pathogens, is discharged in sewage through urine and feces. While extensive pure culture studies have established L-NEs capacity to modulate bacterial virulence through iron acquisition and quorum sensing pathways, its impact on complex microbial communities, where intricate metabolic networks and interspecies interactions dominate, remains largely unexplored. This knowledge gap is particularly critical as urbanization drives increasing neuroendocrine compound loads in wastewater influents in metropolitan areas. Through parallel treatments of L-NE (1x10{square}{square} to 1x10{square}{square} M), dextrose, and H{square}O{square} in municipal and agricultural wastewater communities, we uncovered sophisticated metabolic and regulatory mechanisms that challenge the conventional understanding of microbial substrate utilization. Despite containing 10-fold less carbon, L-NE treatments achieved superior growth (10{square} CFU mL{square}{superscript 1}) while maintaining Pseudomonadaceae-dominated communities. Targeted metaproteomics revealed coordinated upregulation of oxidative stress genes (oxyR, soxRS) and antioxidant enzymes, while proteome-constrained metabolic modeling demonstrated distinct pathway modulation in central carbon and nitrogen metabolism. Notably, when compared to dextrose-supplemented controls representing typical carbon substrate utilization, L-NE treatments showed similar taxonomic profiles without preferential enrichment of known pathogenic families. However, L-NE significantly enhanced autoinducer gene (luxS, qseC) expression, suggesting increased virulence potential through community-level metabolic reprogramming. These findings reveal L-NE as a potent modulator of microbial community dynamics in engineered ecosystems, with important implications for treatment process stability and downstream environmental impacts. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/482069v2_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1d80968org.highwire.dtl.DTLVardef@51cb63org.highwire.dtl.DTLVardef@b37org.highwire.dtl.DTLVardef@e07503_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOTOC FigureC_FLOATNO C_FIG

microbiology↗

bioRxiv2021

Unleashing a Novel Function of Endonuclease G in Mitochondrial Genome Instability

Having its own genome makes mitochondria a unique and semiautonomous organelle within cells. Mammalian mitochondrial DNA (mtDNA) is double-stranded closed circular molecule of about 16 kb coding 37 genes. Mutations, including deletions in the mitochondrial genome can culminate in different human diseases. Mapping of the deletion junctions suggests that the breakpoints are generally seen at hotspots. 9-bp deletion (8271-8281), seen in the intergenic region of cytochrome c oxidase II/tRNALys, is the most common mitochondrial deletion. While it is associated with several diseases like myopathy, dystonia, and hepatocellular carcinoma, it has also been used as an evolutionary marker. However, the mechanism responsible for its fragility is unclear. In the current study, we show that Endonuclease G, a mitochondrial nuclease responsible for nonspecific cleavage of nuclear DNA during apoptosis, can induce breaks at sequences associated with 9-bp deletion, when it is present on a plasmid or in the mitochondrial genome. Through a series of in vitro and intracellular studies, we show that Endonuclease G binds to G-quadruplex structures formed at the hotspot and induces DNA breaks. Besides, we reconstitute the whole process of 9-bp deletion using purified Endonuclease G to induce breaks in mtDNA, followed by mitochondrial extract-mediated DSB repair and establish that microhomology-mediated end joining is responsible for the generation of mtDNA deletion. Finally, we show that the whole process is regulated by different stress conditions, which may modulate release of Endonuclease G to the mitochondrial matrix. Therefore, we uncover a new role for Endonuclease G in generating deletions, which is dependent on the formation of G4 DNA within the mitochondrial genome. Thus, in this study we identify a novel property of Endonuclease G, besides its role in apoptosis, and the recently described elimination of paternal mitochondria during fertilization.

molecular biology↗

bioRxiv2021

Genome-scale modeling of Pseudomonas aeruginosa PA14 unveils its broad metabolic capabilities and role of metabolism in drug potentiation

P. aeruginosa is an opportunistic human pathogen that is one of the leading causes of hospital-acquired infections. We have developed an updated genome-scale model (GEM) of Pseudomonas aeruginosa PA14 for systems-study of the pathogen. We used both automated and semi-manual approaches to reconstruct and curate the model. After an extensive literature research, we added organism-specific reactions (e.g., phenazine transport and redox metabolism, cofactor metabolism, carnitine metabolism, oxalate production, etc.) to the model. This effort led to a highly curated, three-compartment, and mass-and-charge balanced BiGG model of PA14 that contains 1509 genes, 1779 metabolic reactions and 1151 unique metabolites. The model (iSD1509) has the largest genome coverage of P. aeruginosa PA14 to date with 424 more genes than the previous model (iPau1129). It is also the most accurate with prediction accuracies as high as 92.4% (for gene essentiality) and 93.5% (for substrate utilization). The model simulates growth in both aerobic and anaerobic conditions. It predicts the biosynthesis of the virulence factor phenazine as a process for the pathogen to grow in low-oxygen environment. Further, a mechanism for the overproduction of a drug susceptibility biomarker (gluconate) can be elucidated by the principles of optimal growth. Finally, the model also simulates drug activity potentiation and protection by fumarate and glyoxylate, respectively, and provides mechanistic explanations for these processes. Overall, iSD1509 can be utilized to decipher the metabolic mechanisms associated with virulence and antibiotic susceptibility of P. aeruginosa PA14 to aid in the development of effective intervention strategies.

systems biology↗

bioRxiv2018

The genetics and genome-wide screening of perennialism loci in Zea diploperennis

Perennialism is common among the higher plants, yet we know little about its inheritance. To address this, six hybrids were made by reciprocally crossing perennial Zea diploperennis Iltis, Doebley & R. Guzman with three varieties/inbred lines of annual maize (Z. mays L. spp. mays). We specifically focused on the plants ability to regrow after flowering and senescence. All the F1 plants demonstrated senescence and regrowth for several cycles, indicating a dominant effect of the Z. diploperennis alleles. The regrowth ability was stably transmitted to progeny of the hybrids in segregation ratios that suggested the trait was controlled by two dominant, complementary loci. Genome-wide screening with genotyping-by-sequencing (GBS) identified two major regrowth loci reg1 and reg2 on chromosomes 2 and 7, respectively. GBS results were validated using a larger F2 population and PCR markers derived from the single nucleotide polymorphisms within the locus intervals. These markers will be employed to select near-isogenic lines for the two loci and to identify candidate genes in the loci in Z. diploperennis.\n\nSignificance StatementOur study contributes to our general understanding of inheritance of perennialism in the higher plants. Previous genetic studies of the perennialism in Zea have yielded contradictory results. We take a reductionist approach by specifically focusing on the plants ability to regenerate new shoots after senescence without regard to associated traits, such as rhizome formation, tillering or environmental impacts. Using this criterion, inheritance of perennialism in Zea appears to be dominantly and qualitatively inherited. Importantly, our data indicate that there is no major barrier to transferring this trait into maize or other grass crops for perennial crop development, which enhances sustainability of grain crop production in an environmentally friendly way.

plant biology↗