Biology subjects

Tran, T.

Publications and source records attributed to Tran, T..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Design of linked-domain protein inhibitors of UBE2D as tools to study cellular ubiquitination

Ubiquitin (Ub) is a post-translational modification that largely controls proteostasis through mechanisms spanning transcription, translation, and notably, protein degradation. Ub conjugation occurs through a hierarchical cascade of three enzyme classes (E1, E2, and E3s) involving >1000 proteins that regulate the ubiquitination of proteins. The E2 Ub-conjugating enzymes are the midpoint, yet their cellular roles remain under-characterized, partly due to a lack of inhibitors. For example, the cellular roles of the promiscuous E2 UBE2D/UBCH5 are not well described. Here, we develop a highly selective, multivalent, engineered protein inhibitor for the UBE2D family that simultaneously targets the RING- and backside-binding sites. In HeLa cells, these inhibitors phenocopy knockdown of UBE2D by reducing the IC50 to cisplatin and whole-cell proteomics reveal an increased abundance of [~]20% of the identified proteins, consistent with reduced Ub degradation and proteotoxic stress. These precision tools will enable new studies probing UBE2Ds central role in proteome management.

biochemistry↗

bioRxiv2024

Patient-derived colon epithelial organoids reveal lipid-related metabolic dysfunction in pediatric ulcerative colitis

Background & AimsUlcerative colitis (UC) is associated with epithelial metabolic derangements which exacerbate gut inflammation. Patient-derived organoids recapitulate complexities of the parent tissue in health and disease; however, whether colon organoids (colonoids) model metabolic impairments in the pediatric UC epithelium is unclear. This study determined the functional metabolic differences in the colon epithelia using epithelial colonoids from pediatric patients. MethodsWe developed biopsy-derived colonoids from pediatric patients with endoscopically active UC, inactive UC, and those without endoscopic or histologic evidence of colon inflammation (non-IBD controls). We extensively interrogated metabolic dysregulation through extracellular flux analyses and tested potential therapies that recapitulate or ameliorate such metabolic dysfunction. ResultsEpithelial colonoids from active UC patients exhibit elevated oxygen consumption and proton leak supported by enhanced glycolytic capacity and dysregulated lipid metabolism. The hypermetabolic features in active UC colonoids were associated with increased cellular stress and chemokine secretion, specifically during differentiation. Transcriptomic and pathway analyses indicated a role for PPAR- in lipid-induced hypermetabolism in active UC colonoids, which was validated by PPAR- activation in non-IBD colonoids. Accordingly, limiting neutral lipid accumulation in active UC colonoids through pharmacological inhibition of PPAR- induced a metabolic shift towards glucose consumption, suppressed hypermetabolism and chemokine secretion, and improved cellular stress markers. Control and inactive UC colonoids had similar metabolic and transcriptomic profiles. ConclusionsOur pediatric colonoids revealed significant lipid-related metabolic dysregulation in the pediatric UC epithelium that may be alleviated by PPAR- inhibition. This study supports the advancement of colonoids as a preclinical human model for testing epithelial-directed therapies against such metabolic dysfunction. What You Need to KnowO_ST_ABSBackground and ContextC_ST_ABSColon mucosa healing in pediatric UC requires reinstating normal epithelial function but a lack of human preclinical models of the diseased epithelium hinders the development of epithelial-directed interventions. New FindingsUsing colon biopsy-derived epithelial organoids, samples from pediatric patients with active UC show hyperactive metabolic function largely driven by enhanced lipid metabolism. Pharmacologic inhibition of lipid metabolism alleviates metabolic dysfunction, cellular stress, and chemokine production. LimitationsThough our epithelial colon organoids from active UC patients show targetable metabolic and molecular features from non-IBD controls, they were cultured under sterile conditions, which may not fully capture any potential real-time contributions of the complex inflammatory milieu typically present in the disease. Clinical Research RelevanceCurrent therapies for pediatric UC mainly target the immune system despite the need for epithelial healing to sustain remission. We identified a pharmacologic target that regulates epithelial metabolism and can be developed for epithelial-directed therapy in UC. Basic Research RelevancePediatric UC patient tissue adult stem cell-derived colon epithelial organoids retain disease-associated metabolic pathology and can serve as preclinical human models of disease. Excess reliance on lipids as an energy source leads to oxidative and inflammatory dysfunction in pediatric UC colon organoids. Preprint: This manuscript is currently on bioRxiv. doi: https://doi.org/10.1101/2024.08.22.609271 Lay Summary: Using patient tissue-derived colon epithelial organoids, the investigators identified epithelial metabolic dysfunction and inflammation in pediatric ulcerative colitis that can be alleviated by PPAR-a inhibition.

physiology↗

bioRxiv2024

An enhancer-AAV toolbox to target and manipulate distinct interneuron subtypes

In recent years, we and others have identified a number of enhancers that, when incorporated into rAAV vectors, can restrict the transgene expression to particular neuronal populations. Yet, viral tools to access and manipulate specific neuronal subtypes are still limited. Here, we performed systematic analysis of single cell genomic data to identify enhancer candidates for each of the telencephalic interneuron subtypes. We established a set of enhancer-AAV tools that are highly specific for distinct cortical interneuron populations and striatal cholinergic interneurons. These enhancers, when used in the context of different effectors, can target (fluorescent proteins), observe activity (GCaMP) and manipulate (opto-genetics) specific neuronal subtypes. We also validated our enhancer-AAV tools across species. Thus, we provide the field with a powerful set of tools to study neural circuits and functions and to develop precise and targeted therapy.

neuroscience↗

bioRxiv2024

TOMM40 regulates hepatocellular and plasma lipid metabolism via an LXR-dependent pathway

The gene encoding TOMM40 (Transporter of Outer Mitochondrial Membrane 40) is adjacent to that encoding APOE, which has a central role in lipid and lipoprotein metabolism. Human genetic variants near APOE and TOMM40 are strongly associated with plasma lipid levels, but a specific role for TOMM40 in lipid metabolism has not been established. Investigating this, we show that suppression of TOMM40 in human hepatoma cells upregulates expression of APOE and LDLR in part via activation of LXRB (NR1H2) by oxysterols, with consequent increased uptake of VLDL and LDL. This is in part due to disruption of mitochondria-endoplasmic reticulum contact sites, with resulting accrual of reactive oxygen species and non-enzymatically derived oxysterols. With TOMM40 knockdown, cellular triglyceride and lipid droplet content are increased, effects attributable in part to receptor-mediated VLDL uptake, since lipid staining is significantly reduced by concomitant suppression of either LDLR or APOE. In contrast, cellular cholesterol content is reduced due to LXRB-mediated upregulation of the ABCA1 transporter as well as increased production and secretion of oxysterol-derived cholic acid. Consistent with the findings in hepatoma cells, in vivo knockdown of TOMM40 in mice results in significant reductions of plasma triglyceride and cholesterol concentrations, reduced hepatic cholesterol and increased triglyceride content, and accumulation of lipid droplets leading to development of steatosis. These findings demonstrate a role for TOMM40 in regulating hepatic lipid and plasma lipoprotein levels and identify mechanisms linking mitochondrial function with lipid metabolism. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/600910v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@17b0c8forg.highwire.dtl.DTLVardef@17599aaorg.highwire.dtl.DTLVardef@1bb6ab2org.highwire.dtl.DTLVardef@15a035f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

The impact of various microplastics on bacterial community and antimicrobial resistance genes in Norwegian and South African wastewater

Wastewater treatment plants (WWTPs) may serve as hotspots for pathogens and promote antimicrobial resistance (AMR). Plastic debris in wastewater could further contribute to AMR dissemination. The aim of this study is to investigate the impact of various microplastic types on bacterial communities and AMR gene abundance in Norwegian and South African wastewater. Microcosm experiments were designed as follows: Five manufactured microplastic pellet types were used for testing and two rock aggregate types were used as controls. In addition, each material type was subjected to artificial aging treatments using either ultra-violet light or hydrogen peroxide. Each material was incubated in flasks containing inlet/outlet wastewater obtained from Norwegian/South African WWTPs. Nucleic acids were extracted after a one-week incubation period. The detection of the blaFOX and blaMOX genes was performed using quantitative PCR. Extracted DNA was sequenced using a MinION device. Non-metric multi-dimensional scaling plot on full-length 16S sequencing data at the species level showed samples were clustered into distinct material groups. These results were in line with the ANOSIM test showing significant p-values in both Norwegian and South African WWTP settings. Indicator species analysis showed a strong association between many Acinetobacter species with the plastic group than the rock group. Aging treatment using hydrogen peroxide showed some effects on microbial. The abundance of blaFOX and blaMOX genes in the Norwegian wastewater outlet were generally lower compared to those in the inlet, though results were contrary in South African wastewater samples. The relative abundance of AMR genes seemed to be increased on several plastic types (PET, PE, PLA) but decreased on PVC-A. WWTP treatments in this study did not effectively reduce the abundance of AMR genes. In addition, plastic categories were shown to play a pivotal role in developing distinct bacterial communities and AMR profiles.

microbiology↗

bioRxiv2024

Structure-activity relationship of pyrrolidine pentamine derivatives as inhibitors of the aminoglycoside 6'-N-acetyltransferase type Ib

Resistance to amikacin and other major aminoglycosides is commonly due to enzymatic acetylation by aminoglycoside 6'-N -acetyltransferase type I enzyme, of which type Ib [AAC(6')-Ib] is the most widespread among Gram-negative pathogens. Finding enzymatic inhibitors could be an effective way to overcome resistance and extend the useful life of amikacin. Small molecules possess multiple properties that make them attractive compounds to be developed as drugs. Mixture-based combinatorial libraries and positional scanning strategy led to the identification of a chemical scaffold, pyrrolidine pentamine, that, when substituted with the appropriate functionalities at five locations (R1 - R5), inhibits AAC(6')-Ib-mediated inactivation of amikacin. Structure-activity relationship (SAR) studies showed that while truncations to the molecule result in loss of inhibitory activity, modifications of functionalities and stereochemistry have different effects on the inhibitory properties. In this study, we show that alterations at position R1 of the two most active compounds, 2700.001 and 2700.003, reduced inhibition levels, demonstrating the essential nature not only of the presence of an S -phenyl moiety at this location but also the distance to the scaffold. On the other hand, modifications on the R3, R4, and R5 positions have varied effects, demonstrating the potential for optimization. A correlation analysis between molecular docking values ({Delta}G) and the dose required for two-fold potentiation of compounds described in this and the previous studies showed a significant correlation between {Delta}G values and inhibitory activity. HighlightsO_LIAmikacin resistance in Gram-negatives is mostly caused by the AAC(6')-Ib enzyme C_LIO_LIAAC(6')-Ib has been identified in most Gram-negative pathogens C_LIO_LIInhibitors of AAC(6')-Ib could be used to treat resistant infections C_LIO_LICombinatorial libraries and positional scanning identified an inhibitor C_LIO_LIThe lead compound can be optimized by structure activity relationship studies C_LI

biochemistry↗

bioRxiv2024

Differential predictive value of resident memory CD8+T cell subpopulations in non-small-cell lung cancer patients treated by immunotherapy

A high density of resident memory T cells (TRM) in tumors correlates with improved clinical outcomes in immunotherapy-treated patients. However, in preclinical models, only some subpopulations of TRM are associated with cancer vaccine efficacy. We identified two main TRM subpopulations in tumor-infiltrating lymphocytes derived from non-small cell lung cancer (NSCLC) patients: one co-expressing CD103 and CD49a (DP), and the other expressing only CD49a (MP); both exhibiting additional TRM surface markers like CD69. DP TRM exhibited greater functionality compared to MP TRM. Analysis of T-cell receptor (TCR) repertoire and of the stemness marker TCF-1 revealed shared TCRs between populations, with the MP subset appearing more progenitor-like phenotype. In two NSCLC patient cohorts, only DP TRM predicted PD-1 blockade response. Multivariate analysis, including various biomarkers (CD8, TCF1+CD8+T cells, and PD-L1) associated with responses to anti-PD(L)1, showed that only intra-tumoral infiltration by DP TRM remained significant. This study highlights the non-equivalence of TRM populations and emphasizes the importance of distinguishing between them to better define their role in antitumor immunity and as a biomarker of response to immunotherapy.

immunology↗

bioRxiv2023

Ribose lowers RpoS translation through RbsD mRNA

The Escherichia coli sigma factor RpoS accumulates during starvation, activates transcription of the general stress response, and then rapidly returns to low levels upon nutrient replenishment. Sugar levels modulate RpoS levels by signaling though central metabolism, ATP and the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) transport system, which favors glucose. However, there have been few examples of dedicated control pathways for specific sugars regulating RpoS. We screened an overexpression library with a RpoS- LacZ reporter and found that overexpressing the ribose metabolism rbsD gene reduced RpoS levels. A functional RbsD ribose pyranase protein was not necessary for the effect, suggesting that the RbsD mRNA was responsible for the effect. We used a series of LacZ fusions and RT-qPCR to determine how RbsD mRNA affects RpoS and found that regulation of RpoS occurred at the level of translation. Furthermore, the effect of rbsD overexpression was diminished in strain that did not have the RpoS untranslated hairpin loop or the small RNA chaperone Hfq. RbsD mRNA has previously been shown to bind to the sRNA DsrA. We demonstrated that the effect of RbsD on RpoS was dependent on the sRNAs DsrA and ArcZ and Hfq. Finally, we showed that the sugar ribose lowers RpoS levels in a manner that requires RbsD. In summary, our results demonstrate that RpoS levels respond specifically to ribose though a component of the ribose metabolism pathway and the main player, RbsD, acts as dual function mRNA that has a regulatory role by interacting with the sRNAs that control RpoS.

genetics↗

bioRxiv2023

CCT and Cullin1 regulate the TORC1 pathway to promote dendritic arborization in health and disease.

The development of cell-type-specific dendritic arbors is integral to the proper functioning of neurons within their circuit networks. In this study, we examine the regulatory relationship between the cytosolic chaperonin CCT, key insulin pathway genes, and an E3 ubiquitin ligase (Cullin1) in homeostatic dendritic development. CCT loss of function (LOF) results in dendritic hypotrophy in Drosophila Class IV (CIV) multidendritic larval sensory neurons, and CCT has recently been shown to fold components of the TOR (Target of Rapamycin) complex 1 (TORC1), in vitro. Through targeted genetic manipulations, we have confirmed that LOF of CCT and the TORC1 pathway reduces dendritic complexity, while overexpression of key TORC1 pathway genes increases dendritic complexity in CIV neurons. Both CCT and TORC1 LOF significantly reduce microtubule (MT) stability. CCT has been previously implicated in regulating proteinopathic aggregation, thus we examined CIV dendritic development in disease conditions as well. Expression of mutant Huntingtin leads to dendritic hypotrophy in a repeat-length-dependent manner, which can be rescued by TORC1 disinhibition via Cullin1 LOF. Together, our data suggest that Cullin1 and CCT influence dendritic arborization through regulation of TORC1 in both health and disease. SIGNIFICANCEThe insulin pathway has become an increasingly attractive target for researchers interested in understanding the intersection of metabolism and brain health. We have found connections between the insulin pathway and cytosolic protein maintenance in the development of neuronal dendrites. These pathways converge on the dendritic cytoskeleton, particularly microtubules. Neurons expressing mutant Huntingtin also show defects in dendritic development and the underlying cytoskeleton, and we find that disinhibition of the insulin pathway can rescue dendritic hypotrophy in these neurons. This work advances our understanding of the molecular interactions between the insulin pathway and neuronal development in both health and Huntingtons Disease conditions.

neuroscience↗

bioRxiv2023

Molecular Basis of Cell Membrane Adaptation in Daptomycin-Resistant Enterococcus faecalis

Daptomycin is a last-resort lipopeptide antibiotic that disrupts cell membrane (CM) and peptidoglycan homeostasis. Enterococcus faecalis has developed a sophisticated mechanism to avoid daptomycin killing by re-distributing CM anionic phospholipids away from the septum. The CM changes are orchestrated by a three-component regulatory system, designated LiaFSR, with a possible contribution of cardiolipin synthase (Cls). However, the mechanism by which LiaFSR controls the CM response and the role of Cls are unknown. Here, we show that cardiolipin synthase activity is essential for anionic phospholipid redistribution and daptomycin resistance since deletion of the two genes (cls1 and cls2) encoding Cls abolished CM remodeling. We identified LiaY, a transmembrane protein regulated by LiaFSR, as an important mediator of CM remodeling required for re-distribution of anionic phospholipid microdomains via interactions with Cls1. Together, our insights provide a mechanistic framework on the enterococcal response to cell envelope antibiotics that could be exploited therapeutically.

microbiology↗

bioRxiv2023

A synthetic delivery vector for mucosal vaccination

The success of mRNA-based vaccines during the Covid-19 pandemic has highlighted the value of this new platform for vaccine development against infectious disease. However, the CD8+ T cell response remains modest with mRNA vaccines, and these do not induce mucosal immunity, which would be needed to prevent viral spread in the healthy population. To address this drawback, we developed a dendritic cell targeting mucosal vaccination vector, the homopentameric STxB. Here, we describe the highly efficient chemical synthesis of the protein, and its in vitro folding. This straightforward preparation led to a synthetic delivery tool whose biophysical and intracellular trafficking characteristics were largely indistinguishable from recombinant STxB. The chemical approach allowed for the generation of new variants with bioorthogonal handles. Selected variants were chemically coupled to several types of antigens derived from the mucosal viruses SARS-CoV-2 and type 16 human papillomavirus. Upon intranasal administration in mice, mucosal immunity, including resident memory CD8+ T cells and IgA antibodies was induced against these antigens. Our study thereby identifies a novel synthetic antigen delivery tool for mucosal vaccination with an unmatched potential to respond to an urgent medical need.

biochemistry↗

bioRxiv2023

Anaplerotic nutrient stress drives synergy of angiogenesis inhibitors with therapeutics targeting tumor metabolism

Tumor angiogenesis is a cancer hallmark, and its therapeutic inhibition has provided meaningful, albeit limited, clinical benefit. While anti-angiogenesis inhibitors deprive the tumor of oxygen and essential nutrients, cancer cells activate metabolic adaptations to diminish therapeutic response. Despite these adaptations, angiogenesis inhibition incurs extensive metabolic stress, prompting us to consider such metabolic stress as an induced vulnerability to therapies targeting cancer metabolism. Metabolomic profiling of angiogenesis-inhibited intracranial xenografts showed universal decrease in tricarboxylic acid cycle intermediates, corroborating a state of anaplerotic nutrient deficit or stress. Accordingly, we show strong synergy between angiogenesis inhibitors (Avastin, Tivozanib) and inhibitors of glycolysis or oxidative phosphorylation through exacerbation of anaplerotic nutrient stress in intracranial orthotopic xenografted gliomas. Our findings were recapitulated in GBM xenografts that do not have genetically predisposed metabolic vulnerabilities at baseline. Thus, our findings cement the central importance of the tricarboxylic acid cycle as the nexus of metabolic vulnerabilities and suggest clinical path hypothesis combining angiogenesis inhibitors with pharmacological cancer interventions targeting tumor metabolism for GBM tumors.

cancer biology↗

bioRxiv2023

A spatially resolved single cell genomic atlas of the adult human breast

The adult human breast comprises an intricate network of epithelial ducts and lobules that are embedded in connective and adipose tissue. While previous studies have mainly focused on the breast epithelial system, many of the non-epithelial cell types remain understudied. Here, we constructed a comprehensive Human Breast Cell Atlas (HBCA) at single-cell and spatial resolution. Our single-cell transcriptomics data profiled 535,941 cells from 62 women, and 120,024 nuclei from 20 women, identifying 11 major cell types and 53 cell states. These data revealed abundant pericyte, endothelial and immune cell populations, and highly diverse luminal epithelial cell states. Our spatial mapping using three technologies revealed an unexpectedly rich ecosystem of tissue-resident immune cells in the ducts and lobules, as well as distinct molecular differences between ductal and lobular regions. Collectively, these data provide an unprecedented reference of adult normal breast tissue for studying mammary biology and disease states such as breast cancer.

genomics↗

bioRxiv2023

Potential selection and maintenance of manure-originated multi-drug resistant plasmids at sub-clinical antibiotic concentrations

The goal of this study was to determine minimum selection concentrations of various antibiotics using four manure-originated multi-drug resistant plasmids in a surrogate Escherichia coli host. These plasmids carried genes conferring resistance phenotypes to several antibiotic classes including {beta}-lactams, lincosamides, phenicols, macrolides, sulfonamides and tetracyclines. The minimum selection concentrations of antibiotics tested in nutrient-rich medium were determined: 14.1-28.2 mg/L for penicillin G, 0.1 mg/L for oxytetracycline, 0.45 mg/L for chlortetracycline, 2 mg/L for lincomycin, 1 mg/L for florfenicol, 1.3-4 mg/L for azithromycin, 0.13-0.25 mg/L for tetracycline, 0.004-0.01 mg/L for cefotaxime. Penicillin G, oxytetracycline, chlortetracycline, lincomycin and florfenicol had minimum selection concentrations in nutrient-defined medium slightly changed within 3.5-fold range compared to those in nutrient-rich medium. The minimum selection concentrations of antibiotics interfering folic acid synthesis in bacteria were also determined: 63 mg/L for sulfamethoxazole, 11.2 mg/L for sulfisoxazole and 0.06 mg/L for trimethoprim. Mixing two antibiotics changed minimum selection concentrations within 3.7-fold range compared to those in single antibiotic tests. Relatively high plasmid loss rates (> 90%) were observed when culturing plasmid-bearing strains in antibiotic-free nutrient-rich and nutrient-defined media. Overall results suggested that these plasmids can be maintained at concentrations environmentally relevant in waste water treatment plants, sewage, manure and manured soil although they are not stable in antibiotic-free environments. IMPORTANCEAntibiotic resistance crisis is a grave concern in healthcare systems around the world. To combat this crisis, we sought to find out how likely manure-originated multi-drug resistant plasmids are to be selected and maintained in different environment matrices. Our study showed that these plasmids conferring resistance to {beta}-lactams, lincosamides, phenicols, macrolides, sulfonamides and tetracyclines can be selected at minimum selection concentrations which are lower than minimum inhibition concentrations of the E. coli host strain. Lincomycin, oxytetracycline, chlortetracycline, tetracycline, chloramphenicol, trimethoprim had minimum selection concentrations lower than the antibiotic concentrations in several environment matrices reported previously. Our findings suggest that despite the burden and the high rate of plasmid loss, these plasmids can still be selected, maintained and circulated well in some polluted environments.

microbiology↗

bioRxiv2023

Genetic characterization of extended-beta-lactamase (ESBL) plasmids captured from dairy manures

This study was to assess the gene diversity and characterize a large set of plasmids harboring extended {beta}-lactamase (ESBL) genes from raw and digested dairy manure. A total of eighty-four plasmids that were captured in this E. coli recipient were sequenced using Illumina MiSeq sequencing technology. Twenty-four plasmids of interest were subsequently sequenced using MinION technology in order that a hybrid assembly could be performed on short-and long-read sequences to circularize and complete these plasmids. The size of sequenced plasmids ranged between 40 and 260 kb with various incompatibility groups: IncC, IncI1, IncN, IncY, IncB/O/K/Z, IncX1, IncHI2, IncHI2A, IncFIB(K), IncFII. A variety of extended {beta}-lactamase genes were identified: blaCTXM -1, blaCTXM -14, blaCTXM -15, blaCTXM-27, blaCTXM-55, blaCTXM-61, blaPER-1, blaIMP-27. Interestingly, the blaIMP-27 gene, a novel metallo-{beta}-lactamase discovered in the last decade, was found located on an integrated region in the host chromosome. And one plasmid carrying the blaCMY-2 gene, an AmpC gene, also expressed ESBL phenotype. Four virulence factors, including cia, cib, traT and terC, were detected on some of these plasmids. In addition, six type-2 toxin-antitoxin systems were detected: MazF/E, PemK/I, HipA/B, YdcE/D, RelB/E and HigB/A. Twenty-two out of twenty-four complete plasmids carried putative prophage regions; and most of prophage hits were marked as incomplete, except that the largest plasmid pT525A and the IncY plasmid pT415A had prophage hits with higher scores. IMPORTANCEThe widespread of antibiotic resistant bacteria is largely due to the exchange of mobile genetic elements such as plasmids. Plasmids harboring extended {beta}-lactamase (ESBL) genes originated from dairy manure potentially become entrained in manured soil, which subsequently enter the human food chain. Currently there is a lack of detailed information on these plasmids in the environment, specifically in dairy manure. This study unveils the abundance and diversity of ESBL-carrying plasmids from both raw and digested manures which were captured in gfp-labelled E. coli CV601. In addition, the study provides insightful information of plasmid characteristics including incompatibility groups, ESBL genes combined with other resistance genes, mobile genetic elements (transposons, insertion sequence), toxin-antitoxin systems, virulence factors and prophage sequences.

microbiology↗

bioRxiv2023

Modeling Tick Populations: An Ecological Test Case forGradient Boosted Trees

General linear models have been the foundational statistical framework used to discover the ecological processes that explain the distribution and abundance of natural populations. Analyses of the rapidly expanding cache of environmental and ecological data, however, require advanced statistical methods to contend with complexities inherent to extremely large natural data sets. Modern machine learning frameworks such as gradient boosted trees efficiently identify complex ecological relationships in massive data sets, which are expected to result in accurate predictions of the distribution and abundance of organisms in nature. However, rigorous assessments of the theoretical advantages of these methodologies on natural data sets are rare. Here we compare the abilities of gradient boosted and linear models to identify environmental features that explain observed variations in the distribution and abundance of blacklegged tick (Ixodes scapularis) populations in a data set collected across New York State over a ten-year period. The gradient boosted and linear models use similar environmental features to explain tick demography, although the gradient boosted models found non-linear relationships and interactions that are difficult to anticipate and often impractical to identify with a linear modeling framework. Further, the gradient boosted models predicted the distribution and abundance of ticks in years and areas beyond the training data with much greater accuracy than their linear model counterparts. The flexible gradient boosting framework also permitted additional model types that provide practical advantages for tick surveillance and public health. The results highlight the potential of gradient boosted models to discover novel ecological phenomena affecting pathogen demography and as a powerful public health tool to mitigate disease risks.

ecology↗

bioRxiv2023

Dimerization and autophosphorylation of the MST family of kinases are controlled by the same set of residues

The Hippo pathway controls tissue growth and regulates stem cell fate through the activities of core kinase cassette that begins with the Sterile 20-like kinase MST1/2. Activation of MST1/2 relies on trans-autophosphorylation but the details of the mechanisms regulating that reaction are not fully elucidated. Proposals include dimerization as a first step and include multiple models for potential kinase-domain dimers. Efforts to verify and link these dimers to trans-autophosphorylation were unsuccessful. We explored the link between dimerization and trans-autophosphorylation for MST2 and the entire family of MST kinases. We analyzed crystal lattice contacts of structures of MST kinases and identified an ensemble of kinase-domain dimers compatible with trans-autophosphorylation. These dimers share a common dimerization interface comprised of the activation loop and G-helix while the arrangements of the kinase-domains within the dimer varied depending on their activation state. We then verified the dimerization interface and determined its function using MST2. Variants bearing alanine substitutions of the G-helix prevented dimerization of the MST2 kinase domain both in solution and in cells. These substitutions also blocked autophosphorylation of full-length MST2 and its Drosophila homolog Hippo in cells. These variants retain the same secondary structure as wild-type and capacity to phosphorylate a protein substrate, indicating the loss of MST2 activation can be directly attributed to a loss of dimerization rather than loss of either fold or catalytic function. Together this data functionally links dimerization and autophosphorylation for MST2 and suggests this activation mechanism is conserved across both species and the entire MST family.

biochemistry↗

bioRxiv2023

Size-Dependent Expression of the Fission Yeast Cdc13 Cyclin is Conferred by Translational Regulation

The major fission yeast cyclin, Cdc13, has been shown to increase in concentration in correlation with cell size, and has been proposed to thereby regulate cell size at division. However, the mechanism of its cell-size regulation has been unknown. Here, we show that Cdc13 is regulated post-transcriptionally. Its transcript is not expressed in a size-dependent manner, rather a size-dependent concentration of protein is expressed from a size-independent concentration of mRNA. Moreover, we show that the expression of Cdc13 is, in fact, size dependent, as opposed to simply size-correlated due to time-dependent expression. We identify a 20-amino-acid motif, which includes the APC D-box degron, as necessary and sufficient for size-dependent expression, which allowed us to construct a size-independent allele of cdc13. Using this allele, we show that size-dependent expression of Cdc13 is not required for size control in fission yeast cells.

cell biology↗