Biology subjects

Shrestha, S.

Publications and source records attributed to Shrestha, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Characterization of PROTAC specificity and endogenous protein interactomes using ProtacID

Here we describe ProtacID, a flexible BioID (proximity-dependent biotinylation)-based approach to identify PROTAC-proximal proteins in living cells. ProtacID analysis of VHL- and CRBN-recruiting PROTACs targeting a number of different proteins (localized to chromatin or cellular membranes, and tested across six different human cell lines) demonstrates how this technique can be used to validate PROTAC degradation targets and identify non-productive (i.e. non-degraded) PROTAC-interacting proteins, addressing a critical need in the field of PROTAC development. We also demonstrate that ProtacID can be used to characterize native, endogenous multiprotein complexes without the use of antibodies, or modification of the protein of interest with epitope tags or biotin ligase tagging.

molecular biology↗

bioRxiv2025

Molecular and cellular features of nerve-invaded cancer cells using a newly characterized experimental model

Perineural invasion (PNI) is the invasion of cancer cells into nerves. Although PNI is a risk factor for cancer recurrence and metastasis, the lack of in vitro experimental models representing natural PNI challenges basic studies and therapeutic screening. In this work, we fully characterized a dorsal root ganglia (DRG)-nerve explant model for PNI and demonstrated the characteristic cellular and molecular features of cancer cells undergoing natural PNI. Briefly, thoracic and lumbar DRGs intactly connected to nerves were co-cultured with breast and prostate cancer cells in a 3D matrix for two weeks. Time-dependent brightfield and fluorescence imaging captured the complex interactions of cancer cells, neurons, axons, and Schwann cells within nerves in the DRG-nerve explant, demonstrating the natural invasion of cancer cells. Fundamental investigations showed that the autonomic neurotransmitters norepinephrine and acetylcholine significantly promote PNI. We also demonstrated increased survival of PNI cells against the cytotoxic drug cisplatin. Additionally, we characterized the proteomics profile of PNI cells for future theranostics applications and validated the results using patient breast tumor samples. Overall, this work characterized and established a clinically relevant model for PNI and revealed the cellular crosstalk of PNI cells within nerves. The established model is suitable for fundamental studies and therapeutic screening pertaining to PNI.

cancer biology↗

bioRxiv2024

Penicillin-binding proteins exhibit catalytic redundancy during asymmetric cell division in Clostridioides difficile

Peptidoglycan synthesis is an essential driver of bacterial growth and division. The final steps of this crucial process involve the activity of the SEDS family glycosyltransferases that polymerize glycan strands and the class B penicillin-binding protein (bPBP) transpeptidases that cross-link them. While many bacteria encode multiple bPBPs to perform specialized roles during specific cellular processes, some bPBPs can play redundant roles that are important for resistance against certain cell wall stresses. Our understanding of these compensatory mechanisms, however, remains incomplete. Endospore-forming bacteria typically encode multiple bPBPs that drive morphological changes required for sporulation. The sporulation-specific bPBP, SpoVD, is important for synthesizing the asymmetric division septum and spore cortex peptidoglycan during sporulation in the pathogen Clostridioides difficile. Although SpoVD catalytic activity is essential for cortex synthesis, we show that it is unexpectedly dispensable for SpoVD to mediate asymmetric division. The dispensability of SpoVDs catalytic activity requires the presence of its SEDS partner, SpoVE, and is facilitated by another sporulation-induced bPBP, PBP3. Our data further suggest that PBP3 interacts with components of the asymmetric division machinery, including SpoVD. These findings suggest a possible mechanism by which bPBPs can be functionally redundant in diverse bacteria and facilitate antibiotic resistance. ImportancePeptidoglycan synthesis requires the transpeptidase activity of penicillin-binding proteins (PBPs), which can have specialized functions during cellular growth, division, and differentiation. However, many bacteria produce PBPs with overlapping functions, and this functional redundancy can increase antibiotic resistance. The spore-forming pathogen, Clostridioides difficile, encodes four PBPs: two are essential for growth and division and another, SpoVD, is essential for spore formation. Here, we show that the transpeptidase activity of SpoVD is dispensable during the first morphological step of sporulation, asymmetric division, because a sporulation-induced PBP, PBP3, partially substitutes for SpoVDs function during this early stage. We find that SpoVD and PBP3 interact during sporulation, suggesting a mechanism by which bPBPs can confer functional redundancy and potentially contribute to antibiotic resistance.

microbiology↗

bioRxiv2024

Enhanced Maturity and Functionality of Vascularized Human Liver Organoids through 3D Bioprinting and Pillar Plate Culture

Liver tissues, composed of hepatocytes, cholangiocytes, stellate cells, Kupffer cells, and sinusoidal endothelial cells, are differentiated from endodermal and mesodermal germ layers. By mimicking the developmental process of the liver, various differentiation protocols have been published to generate human liver organoids (HLOs) in vitro using induced pluripotent stem cells (iPSCs). However, HLOs derived solely from the endodermal germ layer often encounter technical hurdles, such as insufficient maturity and functionality, limiting their utility for disease modeling and hepatotoxicity assays. To overcome this, we separately differentiated EpCAM+ endodermal progenitor cells (EPCs) and mesoderm-derived vascular progenitor cells (VPCs) from the same human iPSC line. These cells were then mixed in BME-2 matrix and concurrently differentiated into vascular human liver organoids (vHLOs). Remarkably, vHLOs exhibited significantly higher maturity than vasculature-free HLOs, as demonstrated by increased coagulation factor secretion, albumin secretion, drug-metabolizing enzyme (DME) expression, and bile acid transportation. To enhance assay throughput and miniaturize vHLO culture, we 3D bioprinted expandable HLOs (eHLOs) in BME-2 matrix on a pillar plate platform derived from EPCs and VPCs and compared with HLOs derived from endoderm alone. Compared to HLOs cultured in a 50 L BME-2 matrix dome in a 24-well plate, vHLOs cultured on the pillar plate exhibited superior maturity, likely due to enhanced nutrient and signaling molecule diffusion. The integration of physiologically relevant patterned liver organoids with the unique pillar plate platform enhanced the capabilities for high-throughput screening and disease modeling.

bioengineering↗

bioRxiv2024

Varietal screening of newly developed blackgram genotypes against Cercospora Leaf Spot (Cercospora canescens) in the Terai region of Nepal.

Screening of 12 blackgram genotypes against Cercospora Leaf Spot (Cercospora canescens) was carried out in RCB design with 3 replications in research plot of Mid-West Academy and Research Institute, Tulsipur, Dang during Bhadra to Mangsir, 2078 B.S. The blackgram genotypes were brought from Grain Legumes Research Program, Khajura, Banke. Disease severity was taken 3 times at 40, 47, and 54 days after sowing. Disease scoring was done as a percentage of leaf area infected on the individual plant at 7-day intervals and disease incidence, disease severity, mean AUDPC, and mean yield was calculated. Disease incidence was not significant among the tested genotypes. Disease severity at 40, 47, and 54 DAS was highly significant among the genotypes. Mean disease score and mean area under disease progressive curve (AUDPC) were also highly significant. Among the genotypes, 10 genotypes were categorized as moderately resistant and 2 genotypes (BLG 0066-1-1 and BLG 0035-1) were categorized as moderately susceptible. The highest Mean AUDPC value (324.1) was possessed by BLG 0035-1 followed by BLG 0066-1-1 (317.6). The lowest mean AUDPC value (175) was possessed by BLG 0069-1. A highly significant difference was found in yield among the black gram genotypes. The highest yield (799 kg/ha) by obtained by BLG 0068-2 followed by Rampur mas (769 kg/ha). The lowest yield (495 kg/ha) was obtained by BLG 0066-1.

pathology↗

bioRxiv2024

Cryopreservation of neuroectoderm on a pillar plate and in situ differentiation into human brain organoids

Cryopreservation in cryovials extends cell storage at low temperatures, and advances in organoid cryopreservation improve reproducibility and reduce generation time. However, cryopreserving human organoids presents challenges due to the limited diffusion of cryoprotective agents (CPAs) into the organoid core and the potential toxicity of these agents. To overcome these obstacles, we developed a cryopreservation technique using a pillar plate platform. To illustrate cryopreservation application to human brain organoids (HBOs), early-stage HBOs were produced by differentiating induced pluripotent stem cells (iPSCs) into neuroectoderm (NEs) in an ultralow atachement (ULA) 384-well plate. These NEs were transferred and encapsulated in Matrigel on the pillar plate. The early-stage HBOs on the pillar plate were exposed to four commercially available CPAs, including PSC cryopreservation kit, CryoStor CS10, 3dGRO, and 10% DMSO, before being frozen overnight at -80{degrees}C and subsequently stored in a liquid nitrogen dewar. We examined the impact of CPA type, organoid size, and CPA exposure duration on cell viability post-thaw. Additionally, the differentiation of early-stage HBOs on the pillar plate was assessed using RT-qPCR and immunofluorescence staining. The PSC cryopreservation kit proved to be the least toxic for preserving these HBOs on the pillar plate. Notably, smaller HBOs showed higher cell viability post-cryopreservation than larger ones. An incubation period of 80 minutes with the PSC kit was essential to ensure optimal CPA diffusion into HBOs with a diameter of 400 - 600 {micro}m. These cryopreserved early-stage HBOs successfully matured over 30 days, exhibiting gene expression patterns akin to non-cryopreserved HBOs. The cryopreserved early-stage HBOs on the pillar plate maintained high viability after thawing and successfully differentiated into mature HBOs. This on-chip cryopreservation method could extend to other small organoids, by integrating cryopreservation, thawing, culturing, staining, rinsing, and imaging processes within a single system, thereby preserving the 3D structure of the organoids.

bioengineering↗

bioRxiv2024

Genomic sequencing and neutralizing serological profiles during acute dengue infection: A 2017 cohort study in Nepal

Dengue virus (DENV) is a mosquito-borne flavivirus that poses a threat to nearly 50% of the global population. DENV has been endemic in Nepal since 2006; however, little is known about how DENV is evolving or the prevalence of anti-DENV immunity within the Nepalese population. To begin to address these gaps, we performed a serologic and genetic study of 49 patients from across Nepal who presented at central hospitals during the 2017 dengue season with suspected DENV infection. Of the 49 subjects assessed, 21 (43%) were positive for DENV NS1 antigen; of these; 5 were also anti-DENV IgM+ IgG+; 7 were DENV IgM+ IgG-, 2 were IgM- IgG+, and 7 were IgM- IgG- by specific ELISAs. Seven of the 21 NS1+ sera were RNA+ by RT-PCR (six DENV2, one DENV3), suggesting that DENV2 was the dominant serotype in our cohort. Whole-genome sequencing of two DENV2 isolates showed similarity with strains circulating in Singapore in 2016, and the envelope genes were also similar to strains circulating in India in 2017. DENV-neutralizing antibodies (nAbs) were present in 31 of 47 sera tested (66%); among these, 20, 24, 26, and 12 sera contained nAbs against DENV1, 2, 3, and 4 serotypes, respectively. Serology analysis suggested that 12 (26%) and 19 (40%) of the 49 subjects were experiencing primary and secondary DENV infections, respectively. Collectively, our results provide evidence for current and/or past exposure to multiple DENV serotypes in our cohort, and the RNA analyses further indicate that DENV2 was the likely dominant serotype circulating in Nepal in 2017. These data suggest that expanded local surveillance of circulating DENV genotypes and population immunity will be important to effectively manage and mitigate future dengue outbreaks in Nepal.

genomics↗

bioRxiv2024

Dengue Virus Surveillance in Nepal Yields the First On-Site Whole Genome Sequences of Isolates from the 2022 Outbreak

BackgroundThe 4 serotypes of dengue virus (DENV1-4) can each cause potentially deadly dengue disease, and are spreading globally from tropical and subtropical areas to more temperate ones. Nepal provides a microcosm of this global phenomenon, having met each of these grim benchmarks. To better understand DENV transmission dynamics and spread into new areas, we chose to study dengue in Nepal and, in so doing, to build the onsite infrastructure needed to manage future, larger studies. Methods and ResultsDuring the 2022 dengue season, we enrolled 384 patients presenting at a hospital in Kathmandu with dengue-like symptoms; 79% of the study participants had active or recent DENV infection (NS1 antigen and IgM). To identify circulating serotypes, we screened serum from 50 of the NS1+ participants by RT-PCR and identified DENV1, 2, and 3 - with DENV1 and 3 codominant. We also performed whole-genome sequencing of DENV, for the first time in Nepal, using our new on-site capacity. Sequencing analysis demonstrated the DENV1 and 3 genomes clustered with sequences reported from India in 2019, and the DENV2 genome clustered with a sequence reported from China in 2018. ConclusionThese findings highlight DENVs geographic expansion from neighboring countries, identify China and India as the likely origin of the 2022 DENV cases in Nepal, and demonstrate the feasibility of building onsite capacity for more rapid genomic surveillance of circulating DENV. These ongoing efforts promise to protect populations in Nepal and beyond by informing the development and deployment of DENV drugs and vaccines in real time.

genomics↗

bioRxiv2024

TOR signaling regulates GPCR levels on the plasma membrane and suppresses the Saccharomyces cerevisiae mating pathway

Target of Rapamycin (TOR) complexes and G-protein-coupled receptors (GPCRs) are crucial signaling hubs that coordinate adaptive responses to environmental inputs. While GPCR-mediated regulation of TOR has been extensively studied, little is known about TOR-mediated regulation of GPCRs. Here, we establish TOR as a regulator of GPCR signaling via its control of receptor endocytosis in the yeast mating system. By pairing fluorescence microscopy with yeast genetic approaches, we identify the machinery that bridges TOR nutrient sensing to GPCR internalization. Furthermore, we find that autophagic machinery is employed during mating to deliver active receptor to the vacuole (lysosome), suppressing the mating pathway. These results demonstrate that TOR regulates the localization and signaling of the yeast mating GPCR in both ligand-dependent and -independent contexts. These pathways are highly conserved, suggesting that TOR-regulation of GPCRs may be a broadly conserved mechanism for integrating competing signals involving metabolic state and external communications.

cell biology↗

bioRxiv2024

Regenerative human liver organoids (HLOs) in a pillar/perfusion plate for hepatotoxicity assays

Human liver organoids (HLOs) differentiated from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and adult stem cells (ASCs) can recapitulate the structure and function of human fetal liver tissues, thus being considered as a promising tissue model for liver diseases and predictive compound screening. However, the adoption of HLOs in drug discovery faces several technical challenges, which include the lengthy differentiation process with multiple culture media leading to batch-to-batch variation, short-term maintenance of hepatic functions post-maturation, low assay throughput due to Matrigel dissociation and HLO transfer to a microtiter well plate, and insufficient maturity levels compared to primary hepatocytes. To address these issues, expandable HLOs (Exp-HLOs) derived from human iPSCs were generated by optimizing differentiation protocols, which were rapidly printed on a 144-pillar plate with sidewalls and slits (144PillarPlate) and dynamically cultured for up to 20 days into differentiated HLOs (Diff-HLOs) in a 144-perfusion plate with perfusion wells and reservoirs (144PerfusionPlate) for in situ organoid culture and analysis. The dynamically cultured Diff-HLOs exhibited greater maturity and reproducibility than those cultured statically, especially after a 10-day differentiation period. In addition, Diff-HLOs in the pillar/perfusion plate were tested with acetaminophen and troglitazone for 3 days to assess drug-induced liver injury (DILI) and then incubated in an expansion medium for 10 days to evaluate liver recovery from DILI. The assessment of liver regeneration post-injury is critical to understanding the mechanism of recovery and determining the threshold drug concentration beyond which there will be a sharp decrease in the livers regenerative capacity. We envision that bioprinted Diff-HLOs in the pillar/perfusion plate could be used for high-throughput screening (HTS) of hepatotoxic compounds due to the short-term differentiation of passage-able Exp-HLOs, stable hepatic function post-maturation, high reproducibility, and high throughput with capability of in situ organoid culture, testing, staining, imaging, and analysis. Graphical abstractThe overall process of dynamic liver organoid culture and in situ analysis in the 144PillarPlate/144PerfusionPlate for high-throughput hepatotoxicity assays. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=56 SRC="FIGDIR/small/586638v3_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1f2902aorg.highwire.dtl.DTLVardef@1d6e074org.highwire.dtl.DTLVardef@2a0dc0org.highwire.dtl.DTLVardef@eccde0_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Widespread variation in molecular interactions and regulatory properties among transcription factor isoforms

Most human Transcription factors (TFs) genes encode multiple protein isoforms differing in DNA binding domains, effector domains, or other protein regions. The global extent to which this results in functional differences between isoforms remains unknown. Here, we systematically compared 693 isoforms of 246 TF genes, assessing DNA binding, protein binding, transcriptional activation, subcellular localization, and condensate formation. Relative to reference isoforms, two-thirds of alternative TF isoforms exhibit differences in one or more molecular activities, which often could not be predicted from sequence. We observed two primary categories of alternative TF isoforms: "rewirers" and "negative regulators", both of which were associated with differentiation and cancer. Our results support a model wherein the relative expression levels of, and interactions involving, TF isoforms add an understudied layer of complexity to gene regulatory networks, demonstrating the importance of isoform-aware characterization of TF functions and providing a rich resource for further studies.

systems biology↗

bioRxiv2024

Reproducible generation of human liver organoids (HLOs) on a pillar plate platform via microarray 3D bioprinting

Human liver organoids (HLOs) hold significant potential for recapitulating the architecture and function of liver tissues in vivo. However, conventional culture methods of HLOs, forming Matrigel domes in 6-/24-well plates, have technical limitations such as high cost and low throughput in organoid-based assays for predictive assessment of compounds in clinical and pharmacological lab settings. To address these issues, we have developed a unique microarray 3D bioprinting protocol of progenitor cells in biomimetic hydrogels on a pillar plate with sidewalls and slits, coupled with a clear bottom, 384-deep well plate for scale-up production of HLOs. Microarray 3D bioprinting, a droplet-based printing technology, was used to generate a large number of small organoids on the pillar plate for predictive hepatotoxicity assays. Foregut cells, differentiated from human iPSCs, were mixed with Matrigel and then printed on the pillar plate rapidly and uniformly, resulting in coefficient of variation (CV) values in the range of 15 - 18%, without any detrimental effect on cell viability. Despite utilizing 10 - 50-fold smaller cell culture volume compared to their counterparts in Matrigel domes in 6-/24-well plates, HLOs differentiated on the pillar plate exhibited similar morphology and superior function, potentially due to rapid diffusion of nutrients and oxygen at the small scale. Day 25 HLOs were robust and functional on the pillar plate in terms of their viability, albumin secretion, CYP3A4 activity, and drug toxicity testing, all with low CV values. From three independent trials of in situ assessment, the IC50 values calculated for sorafenib and tamoxifen were 6.2 {+/-} 1.6 {micro}M and 25.4 {+/-} 8.3 {micro}M, respectively. Therefore, our unique 3D bioprinting and miniature organoid culture on the pillar plate could be used for scale-up, reproducible generation of HLOs with minimal manual intervention for high-throughput assessment of compound hepatotoxicity.

bioengineering↗

bioRxiv2024

Dynamic culture of cerebral organoids using a pillar/perfusion plate for the assessment of developmental neurotoxicity

Despite the potential toxicity of commercial chemicals to the development of the nervous system (known as developmental neurotoxicity or DNT), conventional in vitro cell models have primarily been employed for the assessment of acute neuronal toxicity. On the other hand, animal models used for the assessment of DNT are not physiologically relevant due to the heterogenic difference between humans and animals. In addition, animal models are low-throughput, time-consuming, expensive, and ethically questionable. Recently, human brain organoids have emerged as a promising alternative to assess the detrimental effects of chemicals on the developing brain. However, conventional organoid culture systems have several technical limitations including low throughput, lack of reproducibility, insufficient maturity of organoids, and the formation of the necrotic core due to limited diffusion of nutrients and oxygen. To address these issues and establish predictive DNT models, cerebral organoids were differentiated in a dynamic condition in a unique pillar/perfusion plate, which were exposed to test compounds to evaluate DNT potential. The pillar/perfusion plate facilitated uniform, dynamic culture of cerebral organoids with improved proliferation and maturity by rapid, bidirectional flow generated on a digital rocker. Day 9 cerebral organoids in the pillar/perfusion plate were exposed to ascorbic acid (DNT negative) and methylmercury (DNT positive) in a dynamic condition for 1 and 3 weeks, and changes in organoid morphology and neural gene expression were measured to determine DNT potential. As expected, ascorbic acid didnt induce any changes in organoid morphology and neural gene expression. However, exposure of day 9 cerebral organoids to methylmercury resulted in significant changes in organoid morphology and neural gene expression. Interestingly, methylmercury did not induce adverse changes in cerebral organoids in a static condition, thus highlighting the importance of dynamic organoid culture in DNT assessment.

bioengineering↗

bioRxiv2024

A resource to enable chemical biology and drug discovery of WDR Proteins

Protein class-focused drug discovery has a long and successful history in pharmaceutical research, yet most members of druggable protein families remain unliganded, often for practical reasons. Here we combined experiment and computation to enable discovery of ligands for WD40 repeat (WDR) proteins, one of the largest human protein families. This resource includes expression clones, purification protocols, and a comprehensive assessment of the druggability for hundreds of WDR proteins. We solved 21 high resolution crystal structures, and have made available a suite of biophysical, biochemical, and cellular assays to facilitate the discovery and characterization of small molecule ligands. To this end, we use the resource in a hit-finding pilot involving DNA-encoded library (DEL) selection followed by machine learning (ML). This led to the discovery of first-in-class, drug-like ligands for 9 of 20 targets. This result demonstrates the broad ligandability of WDRs. This extensive resource of reagents and knowledge will enable further discovery of chemical tools and potential therapeutics for this important class of proteins.

biochemistry↗

bioRxiv2024

Same Sentences, Different Grammars, Different Brain Responses: An MEG study on Case and Agreement Encoding in Hindi and Nepali Split-Ergative Structures

At first glance, the brains language network appears to be universal, but languages clearly differ. How does the brain adapt to the specific details of individual grammatical systems? Here, we present an MEG study on case and agreement in Hindi and Nepali. Both languages use split-ergative case systems. However, these systems interact with verb agreement differently - in Hindi, case features conspire to determine which noun phrase (NP) the verb agrees with (subject, object, or neither), but in Nepali the verb always agrees with the subject NP. We found that left inferior frontal and left anterior temporal regions are sensitive to case features in both languages. Across case configurations, these same brain areas in Hindi participants show different patterns of activity for sentences that require masculine vs. feminine marking on the verb, before the comprehenders encounter it. Additionally, the left temporoparietal junction in Hindi shows different activity for subject and object agreement configurations. Both findings are not observed in Nepali participants. We suggest that this brain response demonstrates a unique-to-Hindi selection of an agreement controller and pre-encoding of the verbs morphological features. This shows that brain activity reflects psycholinguistic processes that are intimately tied to grammatical features. HighlightsO_LIThe left inferior frontal lobe and the left anterior temporal lobe distinguish accusative objects versus bare object NPs in Hindi and Nepali, and pre-emptively encode gender agreement features in Hindi. C_LIO_LIThe left inferior parietal lobe shows a differential sensitivity to object-agreement and subject-agreement constructions in Hindi that is absent in Nepali C_LIO_LIMEG can reveal differences in neural activity that reflect specific requirements of different grammatical systems C_LI

neuroscience↗

bioRxiv2024

A hierarchical Bayesian interaction model to estimate cell-type-specific methylation quantitative trait loci incorporating priors from cell-sorted bisulfite sequencing data

BackgroundMethylation Quantitative Trait Loci (meQTLs) are chromosomal regions that harbor genetic variants affecting DNA methylation levels. The identification of meQTLs can be accomplished through quantifying the effects of single nucleotide polymorphisms (SNPs) on DNA methylation levels, and these inferred meQTLs can shed light on the complex interplay between the genome and methylome. However, most meQTL studies to date utilize bulk methylation datasets composed of different cell types that may have distinct methylation patterns in each cell type. Current technological challenges hinder the comprehensive collection of large-scale, cell-type-specific (CTS) methylation data, which limits our understanding of CTS methylation regulation. To address this challenge, we propose a hierarchical Bayesian interaction model (HBI) to infer CTS meQTLs from bulk methylation data. ResultsOur HBI method integrates bulk methylations data from a large number of samples and CTS methylation data from a small number of samples to estimate CTS meQTLs. Through simulations, we show that HBI improves the estimation (accuracy and power) of CTS genetic effects on DNA methylation. To systematically characterize genome-wide SNP-methylation level associations in multiple cell types, we apply HBI to bulk methylation data measured in peripheral blood mononuclear cells (PBMC) from a cohort of 431 individuals together with flow-sorted cell-derived methylation sequencing (MC-seq) data measured in isolated white blood cells (CD4+ T-cells, CD8+ T-cells, CD16+ monocytes) for 47 individuals. We demonstrate that HBI can identify CTS meQTLs and improve the functional annotation of SNPs. ConclusionsHBI can incorporate strong and robust signals from MC-seq data to improve the estimation of CTS meQTLs. Applying HBI to link the methylome and genome data helps to identify biologically relevant cell types for complex traits.

genetics↗

bioRxiv2024

Identification, antimicrobial resistance and molecular detection of Shiga toxin-producing E. coli (STEC) isolated from raw milk of cattle in Chitwan, Nepal

Shiga toxin-producing Escherichia coli (STEC) is one of the most common foodborne pathogens leading to gastroenteritis with symptoms of mild diarrhoea to the potential life-threatening haemolytic uremic syndrome. Serotype O157:H7 has been the most predominant type worldwide but there are an increasing number of outbreaks caused by non-O157:H7 STEC serotypes. This is the first study about STEC in milk used in Nepalese communities. A total of 267 raw milk samples were taken from different sites in Chitwan District, one of the major milk-producing districts of Nepal, using multi-stage sampling. The samples underwent microbiological and molecular analysis. The overall prevalence of E. coli was found to be 29.6% (79/267) in raw cattle milk. Z3276 gene, a specific genetic marker of E. coli O157:H7 was detected only in three isolates which also carried stx genes. The overall prevalence of STEC in raw cattle milk was 11.6% (31/267) whereas the prevalence of STEC among E. coli isolates was 39.2% (31/79). The prevalence of O157:H7 STEC is 1.1% (3/267) and of non-O157:H7 STEC is 10.5% (28/267). Out of 12 antibiotics tested, the sensitivity of STEC to an antibiotic was highest with azithromycin, and levofloxacin, and the most resistant was with tetracycline, and doxycycline. The findings revealed that raw cattle milk was contaminated with STEC, posing a potential public health concern. The discovery of a greater occurrence of stx2 genes in this study highlights the possibility of more severe complications in humans. Molecular-based STEC surveillance is essential to minimize the risk of its transmission.

microbiology↗

bioRxiv2023

Recruitment of FBXO22 for Targeted Degradation of NSD2

Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here, we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain of function mutation p.E1099K, resulting in growth suppression, apoptosis, and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 in a covalent and reversible manner to recruit the SCFFBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCFFBXO22. Overall, we present a highly potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a novel FBXO22-dependent TPD strategy.

biochemistry↗