Biology subjects

Miller, R.

Publications and source records attributed to Miller, R..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Intrastriatal delivery of a zinc finger protein targeting the mutant HTT gene obviates lipid phenotypes in zQ175DN HD mice

Reducing the burden of mutant Huntingtin (mHTT) protein in brain cells is a strategy for treating Huntingtons disease (HD). However, it is still unclear what pathological changes can be reproducibly reversed by mHTT lowering and whether these changes can be measured in peripheral biofluids. We previously found that lipid changes that occur in brain with HD progression could be prevented by attenuating HTT transcription of the mutant allele in a genetic mouse model (LacQ140) with inducible whole body lowering. Here, we tested whether intrastriatal injection of a therapeutic capable of repressing the mutant HTT allele with expanded CAG can provide similar protection against lipid changes in HD mice with a deletion of neo cassette (zQ175DN). Wild-type or zQ175DN mice were injected with AAV9 bearing a cDNA for a zinc finger protein (ZFP) which preferentially targets mutant HTT (ZFP-HTT) to repress transcription (1). Proteins from brain tissues were analyzed using western blot, capillary electrophoresis, and nitrocellulose filtration methods. Lipid analyses of brain tissue and plasma collected from the same mice were conducted by liquid chromatography and mass spectrometry (LC-MS). Somatic instability (SI) index was assessed using capillary gel electrophoresis of PCR products and was shown to be impeded by HTT-ZFP. Lowering mHTT levels by 43% for 4 months prevented loss of total lipid content including subclasses sphingomyelin (SM), ceramide, phosphatidylethanolamine (PE) and others of caudate-putamen in zQ175DN mice. Moreover, LC-MS analysis of plasma demonstrated total lipid increases and lipid changes in monogalactosyl monoacylglyceride (MGMG) and certain phosphatidylcholine (PC) species were reversed with the therapy. In summary, our data demonstrate that analyzing lipid signatures of brain tissue and peripheral biofluids are valuable approaches for evaluating potential therapies in a preclinical model of HD. FundingCHDI Foundation, Dake Family Fund Disclosure statementThe authors have nothing to disclose. Author contributionsAI and KS extracted lipids and performed computational and statistical analysis; AB and CS collected plasma and brain tissues; SL and ES performed protein chemistry; KC maintained mouse colonies, RM performed stereotaxic injections, RB subcloned ZFP cDNAs and prepared virus, MSE, NA, MD, and KKG planned experiments and wrote manuscript.

neuroscience↗

bioRxiv2025

Detection of HTTex1p by western blot and immunostaining of HD human and mouse brain using neo-epitope antibody P90 highlights impact of CAG repeat expansion on its size, solubility, and response to MSH3 silencing

HTT1a was identified in human and mouse Huntingtons disease brain as the pathogenic exon 1 mRNA generated from aberrant splicing between exon 1 and 2 of HTT that contributes to aggregate formation and neuronal dysfunction.1 Detection of the huntingtin exon 1 protein (HTT1a) has been accomplished with fluorescence-based reporter assays (Meso Scale Discovery, Homogeneous Time Resolved Fluorescence) and immunoprecipitation assays in Huntingtons disease knock-in mice but direct detection in homogenates by gel electrophoresis and western blot assay has been lacking. Subcellular fractions prepared from mouse and human Huntingtons disease brain were separated by gel electrophoresis and probed by western blot with neo-epitope monoclonal antibodies 1B12 and 11G2 directed to the C-terminal eight residues of HTT1a. In caudate putamen of an allelic series of 6 month old Huntingtons disease knock-in mice (Q50, Q80, Q111, Q140 and Q175) HTT1a migration was inversely correlated with CAG repeat length and appeared as a SDS soluble high molecular mass smear in Q111, Q140 and Q175 mice but weakly in Q80 and not in WT mice or Q50 indicating a CAG repeat size threshold for detecting HTT1a. HTT1a immunoreactivity diminished if 1B12 and 11G2 antibodies were preincubated with an eight amino acid peptide containing the C-terminus of HTT1a but not with unrelated peptide sequence. Migration of HTT1a and its high molecular mass smear changed with age in caudate putamen of Q111, Q175 and YAC128 mice. Treating Q111 mice with siRNA to MSH3, a modifier of CAG repeat expansion, significantly reduced levels of the high molecular mass smear indicating that the effects of curbing CAG repeat expansion were quantifiable. A prominent 56-60 kDa doublet detected by 1B12 and 11G2 antibodies in lysates from human Huntingtons disease brain was not blocked by preincubation with C-terminal HTT1a blocking peptide and also appeared in brains of Parkinsons disease patients. 1B12 and 11G2 antibodies did not immunoprecipitate HTT proteins from either Huntingtons disease mouse or human brain lysates using conditions that pulled down full length HTT with anti-HTT antibody 2B7. Altogether these data show that 11G2 and 1B12 antibodies can be used in western blot assays to track and quantify immunoreactive HTT1a levels, solubility, and subcellular localization in Huntingtons disease mouse brain. Abbreviated SummarySapp et al., report that pathogenic exon 1 protein HTT1a is detected in brain of mouse models of Huntingtons disease by direct western blot assay using monoclonal antibodies 11G2 and 1B12. Lowering MSH3 mRNA in the caudate putamen to prevent CAG repeat expansion reduced levels of HTT1a.

neuroscience↗

bioRxiv2024

Divalent siRNA for prion disease

Prion protein (PrP) lowering is effective in animal models of prion disease and is being tested clinically in prion disease patients, but there remains a need for more potent PrP-lowering drug candidates. Inspired by the reported potency and duration of action of divalent short interfering RNA (siRNA), a new oligonucleotide drug modality for the central nervous system, we sought to discover and develop a new PrP-lowering drug candidate. Herein we identify a mouse Prnp-targeting divalent siRNA molecule, 1682-s4, that lowers PrP to 49% residual brain expression in wild-type mice, and, in the context of intracerebral infection with Rocky Mountain Laboratory (RML) prions, achieves a 2.7-fold increase in survival time with pre-symptomatic chronic treatment and 64% increase in survival time with a single dose after symptom onset. We describe the generation of two transgenic mouse lines, Tg25109 and Tg26372, expressing the full human PRNP gene and its non-coding sequence, and demonstrate their utility for in vivo discovery of potent human PRNP-targeting oligonucleotides. We discover siRNA sequence 2439 against human PRNP and compare its potency in different divalent siRNA chemical scaffolds. We determine that both the fixed UU tail and extended nucleic acid linkages of scaffold s4 contribute to superior potency compared to other scaffolds tested, offering 9.4 and 15.9 percentage points respectively of additional PrP knockdown. A single dose of 348 {micro}g of 2439-s4 lowered whole brain hemisphere human PrP in transgenic mice to 17% residual after 30 days, while 52 {micro}g lowered PrP to 49% residual. 1-2% of the dose of 2439-s4 delivered into cerebrospinal fluid is retained in the brain, and the median effective tissue concentration is estimated at 1.2 micrograms per gram of tissue. Good Laboratory Practices toxicology studies identified no significant liabilities, and the U.S. FDA has cleared an Investigational New Drug application to bring 2439-s4 into clinical trials.

neuroscience↗

bioRxiv2024

Normalized Dynamic Time Warping Increases Sensitivity In Differentiating Functional Network Connectivity In Schizophrenia

Our study advances the application of dynamic time warping (DTW) as a functional connectivity measure by introducing a normalization technique which enhances the detection of schizophrenia effects in comparison to both standard DTW and traditional correlation methods. By rigorously examining the statistical validity of DTW and our proposed normalized DTW measure, we show that it effectively captures interdependencies between fMRI signals beyond linear correlation, offering a more robust, complementary and informative approach to functional connectivity analysis. Through comprehensive evaluations, we demonstrate that normalized DTW is more sensitive to differences in functional brain network connections between schizophrenia and controls, highlighting its potential to provide deeper insight into clinical research. Clinical RelevanceThis study enhances our understanding of the functional specificity of schizophrenia by emphasizing the importance of nonlinear relationships through the introduction of a normalization technique for the DTW metric.

neuroscience↗

bioRxiv2024

Identification of potential sepsis therapeutic drugs using a zebrafish rapid screening approach

In the military, combat wound infections can progress rapidly to life-threatening sepsis. Discovery of effective small molecule drugs to prevent and/or treat sepsis is a priority. To identify potential sepsis drug candidates, we used an optimized larval zebrafish model of endotoxicity/sepsis (Philip et al., 2017) to screen commercial libraries of U.S. Food and Drug Administration (FDA)- approved drugs and other active pharmaceutical ingredients (API) known to affect pathways implicated in the initiation and progression of sepsis in humans (i.e., inflammation, mitochondrial dysfunction, coagulation, and apoptosis). We induced endotoxicity in 3- and 5-day post fertilization larval zebrafish (characterized by mortality and tail fin edema (vascular leakage)) by immersion exposure to Pseudomonas aeruginosa 60 g/mL lipopolysaccharide (LPS) for 24 hours, then screened for the rescue potential of 644 selected drugs simultaneously with LPS at 10 M. After LPS exposure, we used a neurobehavioral assay (light-dark test) to further evaluate rescue from endotoxicity and to determine possible off-target drug side effects. We identified 29 drugs with > 60% rescue of tail edema and mortality. Three drugs (Ketanserin, Tegaserod, and Brexpiprazole) produced 100% rescue and did not differ from the controls in the light-dark test, suggesting a lack of off-target neurobehavioral effects. Further testing of these three drugs at a nearly 100% lethal concentration of Klebsiella pneumoniae LPS (45 g/mL) showed 100% rescue from mortality and 88%-100% mitigation against tail edema. The success of the three identified drugs in a zebrafish endotoxicity/sepsis model warrants further evaluation in mammalian sepsis models.

pharmacology and toxicology↗

bioRxiv2024

Extensive longevity and DNA virus-driven adaptation in nearctic Myotis bats

Summary ParagraphThe genus Myotis is one of the largest clades of bats, and exhibits some of the most extreme variation in lifespans among mammals alongside unique adaptations to viral tolerance and immune defense1-3. To study the evolution of these phenotypes we generated cell lines and near-complete genome assemblies for eight closely related Myotis species. Using genome-wide screens of positive selection, analyses of structural variation, and functional experiments in primary cells, we identify patterns of adaptation contributing to longevity, cancer resistance, and viral interactions. We demonstrate distinct modes of adaptation to DNA and RNA viruses compared to all other mammals, with bats exhibiting genome-wide overrepresentation of positive selection for DNA virus-interacting proteins and elevated rates of copy number variation for RNA virus-interacting proteins. Characterization of Myotis-specific duplications of the key immune factor protein kinase R (PKR) reveals multiple ancient segregating trans-species copy number polymorphisms. We show that the recurrent evolution of longevity seen in Myotis is associated with positive selection in cancer pathways, and demonstrate a unique response to DNA damage in primary cells of the long-lived M. lucifugus. Together, our results suggest that bats remarkable longevity and immunity are linked through pleiotropic adaptations to viruses and aging-related disease.

genomics↗

bioRxiv2024

Comparing efficiencies of population control methods for responding to foreign animal disease threats in wild pigs

Introductions of foreign animal diseases (FADs) into free-ranging wildlife can be difficult to control and devastating for domestic livestock trade. Combating a new FAD introduction in wildlife with an emergency response requires quickly limiting spread of the disease by intensely removing wild animals and recovering their carcasses for proper disposal. In the case of African swine fever virus (ASFv) in wild pigs (Sus scrofa), which has been spreading in many regions of the world, there is little information on the time- and cost-efficiency of methods for intensively and consistently removing wild pigs and recovering carcasses in an emergency response scenario. We compared the efficiencies of aerial operations, trapping, an experimental toxic bait, and ground shooting in northcentral Texas, USA during two months in 2023. Removing and recovering carcasses of wild pigs averaged a rate of 0.15 wild pigs/person hour and cost an average of $233.04/wild pig ($USD 2023) across all four methods. Aerial operations required the greatest initial investment but subsequently was the most time- and cost-efficient, costing an average of $7,266 to incrementally reduce the population by 10% including recovering carcasses. Aerial operations required a ground crew of [~]7 people/helicopter to recover carcasses. Costs for reducing the population of wild pigs using trapping were similar, although took 13.5 times longer to accomplish. A benefit of trapping was carcass recovery was incorporated. Toxic baiting was less efficient because carcass recovery required substantial time, and we removed very few wild pigs with ground shooting in this landscape. We recommend combining aerial and trapping methodologies to remove wild pigs and their carcasses efficiently and effectively during a FAD response. Overall, our findings can inform the preparation of resources, personnel needs, and deployment readiness for FAD responses involving wild pigs.

ecology↗

bioRxiv2024

Two-hit mouse model of heart failure with preserved ejection fraction combining diet-induced obesity and renin-mediated hypertension

Heart failure with preserved ejection fraction (HFpEF) is increasingly common but its pathogenesis is poorly understood. The ability to assess genetic and pharmacologic interventions is hampered by the lack of robust preclinical mouse models of HFpEF. We have developed a novel "2-hit" model, which combines obesity and insulin resistance with chronic pressure overload to recapitulate clinical features of HFpEF. C57BL6/NJ mice fed a high fat diet for >10 weeks were administered an AAV8-driven vector resulting in constitutive overexpression of mouse Renin1d. Control mice, HFD only, Renin only and HFD-Renin (aka "HFpEF") littermates underwent a battery of cardiac and extracardiac phenotyping. HFD-Renin mice demonstrated obesity and insulin resistance, a 2-3-fold increase in circulating renin levels that resulted in 30-40% increase in left ventricular hypertrophy, preserved systolic function, and diastolic dysfunction indicated by altered E/e, IVRT, and strain measurements; increased left atrial mass; elevated natriuretic peptides; and exercise intolerance. Transcriptomic and metabolomic profiling of HFD-Renin myocardium demonstrated upregulation of pro-fibrotic pathways and downregulation of metabolic pathways, in particular branched chain amino acid catabolism, similar to findings in human HFpEF. Treatment of these mice with the sodium-glucose cotransporter 2 inhibitor empagliflozin, an effective but incompletely understood HFpEF therapy, improved exercise tolerance, left heart enlargement, and insulin homeostasis. The HFD-Renin mouse model recapitulates key features of human HFpEF and will enable studies dissecting the contribution of individual pathogenic drivers to this complex syndrome. Addition of HFD-Renin mice to the preclinical HFpEF model platform allows for orthogonal studies to increase validity in assessment of interventions. NEW & NOTEWORTHYHeart failure with preserved ejection fraction (HFpEF) is a complex disease to study due to limited preclinical models. We rigorously characterize a new two-hit HFpEF mouse model, which allows for dissecting individual contributions and synergy of major pathogenic drivers, hypertension and diet-induced obesity. The results are consistent and reproducible in two independent laboratories. This high-fidelity pre-clinical model increases the available, orthogonal models needed to improve our understanding of the causes and assessment treatments for HFpEF.

physiology↗

bioRxiv2024

Preventing acute neurotoxicity of CNS therapeutic oligonucleotides with the addition of Ca2+ and Mg2+ in the formulation

Oligonucleotide therapeutics (ASOs and siRNAs) have been explored for modulation of gene expression in the central nervous system (CNS), with several drugs approved and many in clinical evaluation. Administration of highly concentrated oligonucleotides to the CNS can induce acute neurotoxicity. We demonstrate that delivery of concentrated oligonucleotides to the CSF in awake mice induces acute toxicity, observable within seconds of injection. Electroencephalography (EEG) and electromyography (EMG) in awake mice demonstrated seizures. Using ion chromatography, we show that siRNAs can tightly bind Ca2+ and Mg2+ up to molar equivalents of the phosphodiester (PO)/phosphorothioate (PS) bonds independently of the structure or phosphorothioate content. Optimization of the formulation by adding high concentrations (above biological levels) of divalent cations (Ca2+ alone, Mg2+ alone, or Ca2+ and Mg2+) prevents seizures with no impact on the distribution or efficacy of the oligonucleotide. The data here establishes the importance of adding Ca2+ and Mg2+ to the formulation for the safety of CNS administration of therapeutic oligonucleotides.

neuroscience↗

bioRxiv2024

Clearing-enabled light sheet microscopy as a novel method for three-dimensional mapping of the sensory innervation of the mouse knee

A major barrier that hampers our understanding of the precise anatomic distribution of pain sensing nerves in and around the joint is the limited view obtained from traditional two dimensional (D) histological approaches. Therefore, our objective was to develop a workflow that allows examination of the innervation of the intact mouse knee joint in 3D by employing clearing-enabled light sheet microscopy. We first surveyed existing clearing protocols (SUMIC, PEGASOS, and DISCO) to determine their ability to clear the whole mouse knee joint, and discovered that a DISCO protocol provided the most optimal transparency for light sheet microscopy imaging. We then modified the DISCO protocol to enhance binding and penetration of antibodies used for labeling nerves. Using the pan-neuronal PGP9.5 antibody, our protocol allowed 3D visualization of innervation in and around the mouse knee joint. We then implemented the workflow in mice intra-articularly injected with nerve growth factor (NGF) to determine whether changes in the nerve density can be observed. Both 3D and 2D analytical approaches of the light sheet microscopy images demonstrated quantifiable changes in midjoint nerve density following 4 weeks of NGF injection in the medial but not in the lateral joint compartment. We provide, for the first time, a comprehensive workflow that allows detailed and quantifiable examination of mouse knee joint innervation in 3D.

neuroscience↗

bioRxiv2024

mRNA nuclear clustering leads to a difference in mutant huntingtin mRNA and protein silencing by siRNAs in vivo

Huntingtons disease (HD) is an autosomal dominant neurodegenerative disease caused by CAG repeat expansion in the first exon of the huntingtin gene (HTT). Oligonucleotide therapeutics, such as short interfering RNA (siRNA), reduce levels of huntingtin mRNA and protein in vivo and are considered a viable therapeutic strategy. However, the extent to which they silence HTT mRNA in the nucleus is not established. We synthesized siRNA cross-reactive to mouse (wild-type) Htt and human (mutant) HTT in a di-valent scaffold and delivered to two mouse models of HD. In both models, di-valent siRNA sustained lowering of wild-type Htt, but not mutant HTT mRNA expression in striatum and cortex. Near-complete silencing of both mutant HTT protein and wild-type Htt protein was observed in both models. Subsequent fluorescent in situ hybridization (FISH) analysis shows that di-valent siRNA acts predominantly on cytoplasmic mutant HTT transcripts, leaving clustered mutant HTT transcripts in the nucleus largely intact in treated HD mouse brains. The observed differences between mRNA and protein levels, exaggerated in the case of extended repeats, might apply to other repeat-associated neurological disorders.

neuroscience↗

bioRxiv2024

Rapid profiling of transcription factor-cofactor interaction networks reveals principles of epigenetic regulation

Transcription factor (TF)-cofactor (COF) interactions define dynamic, cell-specific networks that govern gene expression; however, these networks are understudied due to a lack of methods for high-throughput profiling of DNA-bound TF-COF complexes. Here we describe the Cofactor Recruitment (CoRec) method for rapid profiling of cell-specific TF-COF complexes. We define a lysine acetyltransferase (KAT)-TF network in resting and stimulated T cells. We find promiscuous recruitment of KATs for many TFs and that 35% of KAT-TF interactions are condition specific. KAT-TF interactions identify NF-{kappa}B as a primary regulator of acutely induced H3K27ac. Finally, we find that heterotypic clustering of CBP/P300-recruiting TFs is a strong predictor of total promoter H3K27ac. Our data supports clustering of TF sites that broadly recruit KATs as a mechanism for widespread co-occurring histone acetylation marks. CoRec can be readily applied to different cell systems and provides a powerful approach to define TF-COF networks impacting chromatin state and gene regulation.

molecular biology↗

bioRxiv2024

LOCAL SPATIAL FLOWS AND PROPAGATIVE ATTRACTORS: A NOVEL 'FLOWNECTOME' FRAMEWORK FOR ANALYZING BOLD FMRI DYNAMICS

Although the analysis of temporal signal fluctuations and co-fluctuations has long been a fixture of blood oxygenation-level dependent (BOLD) functional magnetic resonance imaging (fMRI) research, the role of local directional flows in both signal propagation and healthy functional integration remains almost entirely neglected. We are introducing an extensible framework, based on localized directional signal flows, to capture and analyze spatial signal propagation and propagative attractor patterns in BOLD fMRI. Novel features derived from this approach are validated in a large resting-state fMRI schizophrenia study where they reveal significant relationships between spatially directional flows, propagative attractor patterns and subject diagnostic status. Plausibly, we find that spatial signal inflow to functional regions tends to positively correlate with net gain/loss in the region's temporal contribution BOLD signal reconstruction. We also find that group ICA (pdGICA) performed on time-varying propagative density maps, which are whole-brain maps of spatial signal inflow to each voxel on successive 30 second windows (ie. propagative attractor maps), produce components that tend to concentrate predominantly in at most six or seven functional regions, in some cases focusing on as few as two. The relationship between the propagative attractor group ICA component maps and functional regions is not sharp, but is focused enough to render the pdGICA component maps functionally tractable. Temporal correlations between pdGICA component timeseries and net gain/loss functional region timeseries on corresponding windows echoes those traditionally observed between functional region timeseries when aligning each pdGICA component with the functional region with which it has greatest spatial overlap. Schizophrenia strongly disrupts the average correlative relationship between pdGICA components and certain functional regions, some of which tend to be implicated in schizophrenia e.g. the thalamus and the anterior cingulate cortex. Schizophrenia also strongly and pervasively linked to the importance of specific pdGICA components in reconstructing subjects' observed time-varying propagative density maps. Over half of the 35 pdGICA component make significantly different average contributions to patient propagative density maps than to those of controls, with the functional footprints of impacted pdGICA components spreading over diverse functional domains. Finally, the magnitudes of local directional flows that carry propagation have spatially structured averages and structured, pervasive schizophrenia effects. The framework introduced here follows a new and fundamentally different data-driven approach to the BOLD fMRI signal. We believe that the empirical measurement of local directional flows and wider spatial signal propagation opens a plethora of new avenues through which to investigate healthy and disordered brain function using BOLD fMRI.

neuroscience↗

bioRxiv2023

Development of primary osteoarthritis during aging in genetically diverse UM-HET3 mice

This study investigated the prevalence and progression of primary osteoarthritis (OA) in aged UM-HET3 mice. Using the Osteoarthritis Research Society International (OARSI) scoring system, we assessed articular cartilage (AC) integrity in 182 knee joints of 22-25 months old mice. Aged UM-HET3 mice showed a high prevalence of primary OA in both sexes. Significant positive correlations were found between cumulative AC (cAC) scores and synovitis in both sexes, and osteophyte formation in female mice. Ectopic chondrogenesis did not show significant correlations with cAC scores. Significant direct correlations were found between AC scores and inflammatory markers in chondrocytes, including matrix metalloproteinase-13 (MMP-13), inducible nitric oxide synthase (iNOS), and the NLR family pyrin domain containing-3 (NLRP3) inflammasome in both sexes, indicating a link between OA severity and inflammation. Additionally, markers of cell cycle arrest, such as p16 and {beta}-galactosidase, also correlated with AC scores. Using micro-CT, we examined the correlations between subchondral bone (SCB) morphology traits and AC scores. In male mice, no significant correlations were found between SCB morphology traits and cAC scores, while in female mice, significant correlations were found between cAC scores and tibial SCB plate bone mineral density. Finally, we explored the effects of methylene blue (MB) and mitoquinone (MitoQ), two agents that affect mitochondrial function, on the prevalence and progression of OA during aging. Notably, MB and MitoQ treatments influenced the diseases progression in a sex-specific manner. MB treatment significantly reduced cAC scores at the medial knee joint, while MitoQ treatment reduced cAC scores, but these did not reach significance. In conclusion, our study provides comprehensive insights into the prevalence and progression of primary OA in aged UM-HET3 mice, highlighting the sex-specific effects of MB and MitoQ treatments. The correlations between AC scores and various pathological factors underscore the multifaceted nature of OA and its association with inflammation and subchondral bone changes.

pathology↗

bioRxiv2023

Bacterial diversity and composition on the rinds of specific melon cultivars and hybrids from across different growing regions in the United States

The goal of this study was to characterize the bacterial diversity on different melon varieties grown in different regions of the US, and determine the influence that region, rind netting, and variety of melon has on the composition of the melon microbiome. Assessing the bacterial diversity of the microbiome on the melon rind can identify antagonistic and protagonistic bacteria for foodborne pathogens and spoilage organisms to improve melon safety, prolong shelf-life, and/or improve overall plant health. Bacterial community composition of melons (n = 603) grown in seven locations over a four-year period were used for 16S rRNA gene amplicon sequencing and analysis to identify bacterial diversity and constituents. Statistically significant differences in alpha diversity based on the rind netting and growing region (p < 0.01) were found among the melon samples. Principal Coordinate Analysis based on the Bray-Curtis dissimilarity distance matrix found that the melon bacterial communities clustered more by region rather than melon variety (R2 value: 0.09 & R2 value: 0.02 respectively). Taxonomic profiling among the growing regions found Enterobacteriaceae, Bacillaceae, Microbacteriaceae, and Pseudomonadaceae present on the different melon rinds at an abundance of [≥] 0.1%, but no specific core microbiome was found for netted melons. However, a core of Pseudomonadaceae, Bacillaceae, and Exiguobacteraceae were found for non-netted melons. The results of this study indicate that bacterial diversity is driven more by the region that the melons were grown in compared to rind netting or melon type. Establishing the foundation for regional differences could improve melon safety, shelf-life, and quality as well as the consumers health.

microbiology↗

bioRxiv2023

Trans-Lesion Synthesis and Mismatch Repair Pathway Crosstalk Defines Chemoresistance and Hypermutation Mechanisms in Glioblastoma

Almost all Glioblastoma (GBM) are either intrinsically resistant to the chemotherapeutical drug temozolomide (TMZ) or acquire therapy-induced mutations that cause chemoresistance and recurrence. The genome maintenance mechanisms responsible for GBM chemoresistance and hypermutation are unknown. We show that the E3 ubiquitin ligase RAD18 (a proximal regulator of TLS) is activated in a Mismatch repair (MMR)-dependent manner in TMZ-treated GBM cells, promoting post-replicative gap-filling and survival. An unbiased CRISPR screen provides a new aerial map of RAD18-interacting DNA damage response (DDR) pathways deployed by GBM to tolerate TMZ genotoxicity. Analysis of mutation signatures from TMZ-treated GBM reveals a role for RAD18 in error-free bypass of O6mG (the most toxic TMZ-induced lesion), and error-prone bypass of other TMZ-induced lesions. Our analyses of recurrent GBM patient samples establishes a correlation between low RAD18 expression and hypermutation. Taken together we define novel molecular underpinnings for the hallmark tumorigenic phenotypes of TMZ-treated GBM.

cancer biology↗

bioRxiv2023

Targeting Acute Myeloid Leukemia Stem Cells Through Perturbation of Mitochondrial Calcium

We previously reported that acute myeloid leukemia stem cells (LSCs) are uniquely reliant on oxidative phosphorylation (OXPHOS) for survival. Moreover, maintenance of OXPHOS is dependent on BCL2, creating a therapeutic opportunity to target LSCs using the BCL2 inhibitor drug venetoclax. While venetoclax-based regimens have indeed shown promising clinical activity, the emergence of drug resistance is prevalent. Thus, in the present study, we investigated how mitochondrial properties may influence mechanisms that dictate venetoclax responsiveness. Our data show that utilization of mitochondrial calcium is fundamentally different between drug responsive and non-responsive LSCs. By comparison, venetoclax-resistant LSCs demonstrate a more active metabolic (i.e., OXPHOS) status with relatively high steady-state levels of calcium. Consequently, we tested genetic and pharmacological approaches to target the mitochondrial calcium uniporter, MCU. We demonstrate that inhibition of calcium uptake sharply reduces OXPHOS and leads to eradication of venetoclax-resistant LSCs. These findings demonstrate a central role for calcium signaling in the biology of LSCs and provide a therapeutic avenue for clinical management of venetoclax resistance. SignificanceWe identify increased utilization of mitochondrial calcium as distinct metabolic requirement of venetoclax-resistant LSCs and demonstrate the potential of targeting mitochondrial calcium uptake as a therapeutic strategy.

cancer biology↗

bioRxiv2023

Proportionality-based association metrics in count compositional data

MotivationCompositional data comprise vectors that describe the constituent parts of a whole. Data arising from various -omics platforms such as 16S and RNA-sequencing are compositional in nature. However, correlations between features on raw counts have no meaningful interpretation. Metrics of proportionality were formulated to address this problem. However, there is an inherent bias that arises when calculating these metrics empirically on count-based measures due to variability in read depths. ResultsWe quantify the bias introduced by empirically calculating proportionality-based association metrics in count data. Additionally, we propose a means of estimating these metrics within a logit-normal multinomial model in pursuit of more accurate estimates. The model-based estimates are shown to outperform empirical estimates in simulated data, and are additionally applied to a mouse embryonic stem-cell single-cell sequencing dataset as well as a pediatric-onset multiple sclerosis metagenomic dataset. Availability and ImplementationAn R package is available at https://CRAN.R-project.org/package=countprop. Supplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗