Scholarly Works, Biomedical Sciences and Pathobiology

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Dietary prevention of antibiotic-induced dysbiosis and mortality upon aging in mice
Smith, Kelsey M.; Francisco, Sarah G.; Zhu, Ying; Leroith, Tanya; Davis, Meredith L.; Crott, Jimmy W.; Barger, Kathryn; Greenberg, Andrew S.; Smith, Donald E.; Taylor, Allen; Yeruva, Laxmi; Rowan, Sheldon (Wiley, 2024-12-15)
Oral antibiotic use is both widespread and frequent in older adults and has been linked to dysbiosis of the gut microbiota, enteric infection, and chronic diseases. Diet and nutrients, particularly prebiotics, may modify the susceptibility of the gut microbiome to antibiotic-induced dysbiosis. We fed 12-month-old mice a high glycemic (HG) or low glycemic (LG) diet with or without antibiotics (ampicillin and neomycin) for an additional 11 months. The glycemic index was modulated by the ratio of rapidly digested amylopectin starch to slowly digested amylose, a type-2-resistant starch. We observed a significant decrease in survival of mice fed a HG diet containing antibiotics (HGAbx) relative to those fed a LG diet containing antibiotics (LGAbx). HGAbx mice died with an enlarged and hemorrhagic cecum, which is associated with colonic hyperplasia and goblet cell depletion. Gut microbiome analysis revealed a pronounced expansion of Proteobacteria and a near-complete loss of Bacteroidota and Firmicutes commensal bacteria in HGAbx, whereas the LGAbx group maintained a population of Bacteroides and more closely resembled the LG microbiome. The predicted functional capacity for bile salt hydrolase activity was lost in HGAbx mice but retained in LGAbx mice. An LG diet containing amylose may therefore be a potential therapeutic to prevent antibiotic-induced dysbiosis and morbidity.
Degradation of the α-Carboxyl Terminus 11 Peptide: In Vivo and Ex Vivo Impacts of Time, Temperature, Inhibitors, and Gender in Rat
Tasdemiroglu, Yagmur; Council-Troche, McAlister; Chen, Miao; Ledford, Benjamin; Norris, Russell A.; Poelzing, Steven; Gourdie, Robert G.; He, Jia-Qiang (American Chemical Society, 2024-04-22)
In previous research, a synthetic α-carboxyl terminus 1 (αCT1) peptide derived from connexin 43 (Cx43) and its variant (αCT11) showed beneficial effects in an ex vivo ischemia-reperfusion (I/R) heart injury model in mouse. In an in vivo mouse model of cryo-induced ventricular injury, αCT1 released from adhesive cardiac patches reduced Cx43 remodeling and arrhythmias, as well as maintained cardiac conduction. Whether intravenous injection of αCT1 or αCT11 produces similar outcomes has not been investigated. Given the possibility of peptide degradation in plasma, this study utilized in vivo I/R cardiac injury and ex vivo blood plasma models to examine factors that may limit the therapeutic potential of peptide therapeutics in vivo. Following tail vein administration of αCT11 (100 μM) in blood, no effect on I/R infarct size was observed in adult rat hearts on day 1 (D1) and day 28 (D28) after injury (p > 0.05). There was also no difference in the echocardiographic ejection fraction (EF%) between the control and the αCT11 groups (p > 0.05). Surprisingly, αCT11 in blood plasma collected from these rats was undetectable within ∼10 min after tail vein injection. To investigate factors that may modulate αCT11 degradation in blood, αCT11 was directly added to blood plasma isolated from normal rats without I/R and peptide levels were measured under different experimental conditions. Consistent with in vivo observations, significant αCT11 degradation occurred in plasma within 10 min at 22 and 37 °C and was nearly undetectable by 30 min. These responses were reduced by the addition of protease/phosphatase (PTase/PPTase) inhibitors to the isolated plasma. Interestingly, no significant differences in αCT11 degradation in plasma were noted between male and female rats. We conclude that fast degradation of αCT11 is likely the reason that no beneficial effects were observed in the in vivo I/R model and inhibition or shielding from PTase/PPTase activity may be a strategy that will assist with the viability of peptide therapeutics.
Acute adenoviral infection elicits an arrhythmogenic substrate prior to myocarditis
Padget, Rachel L.; Zeitz, Michael J.; Blair, Grace A.; Wu, Xiaobo; North, Michael D.; Tanenbaum, Mira T.; Stanley, Kari E.; Phillips, Chelsea M.; King, D. Ryan; Lamouille, Samy; Gourdie, Robert G.; Hoeker, Gregory S.; Swanger, Sharon A.; Poelzing, Steven; Smyth, James W. (American Heart Association, 2024-03-29)
BACKGROUND: Viral cardiac infection represents a significant clinical challenge encompassing several etiological agents, disease stages, complex presentation, and a resulting lack of mechanistic understanding. Myocarditis is a major cause of sudden cardiac death in young adults, where current knowledge in the field is dominated by later disease phases and pathological immune responses. However, little is known regarding how infection can acutely induce an arrhythmogenic substrate before significant immune responses. Adenovirus is a leading cause of myocarditis, but due to species specificity, models of infection are lacking, and it is not understood how adenoviral infection may underlie sudden cardiac arrest. Mouse adenovirus type-3 was previously reported as cardiotropic, yet it has not been utilized to understand the mechanisms of cardiac infection and pathology. METHODS: We have developed mouse adenovirus type-3 infection as a model to investigate acute cardiac infection and molecular alterations to the infected heart before an appreciable immune response or gross cardiomyopathy. RESULTS: Optical mapping of infected hearts exposes decreases in conduction velocity concomitant with increased Cx43^Ser368 phosphorylation, a residue known to regulate gap junction function. Hearts from animals harboring a phospho-null mutation at Cx43^Ser368 are protected against mouse adenovirus type-3–induced conduction velocity slowing. Additional to gap junction alterations, patch clamping of mouse adenovirus type-3–infected adult mouse ventricular cardiomyocytes reveals prolonged action potential duration as a result of decreased IK1 and IKs current density. Turning to human systems, we find human adenovirus type-5 increases phosphorylation of Cx43^Ser368 and disrupts synchrony in human induced pluripotent stem cell-derived cardiomyocytes, indicating common mechanisms with our mouse whole heart and adult cardiomyocyte data. CONCLUSIONS: Together, these findings demonstrate that adenoviral infection creates an arrhythmogenic substrate through direct targeting of gap junction and ion channel function in the heart. Such alterations are known to precipitate arrhythmias and likely contribute to sudden cardiac death in acutely infected patients.
Tumor-derived extracellular vesicles disrupt the blood-brain barrier endothelium following high-frequency irreversible electroporation
Murphy, Kelsey R.; Aycock, Kenneth N.; Marsh, Spencer; Hay, Alayna N.; Athanasiadi, Ilektra; Bracha, Shay; Chang, Christine; Gourdie, Robert G.; Davalos, Rafael V.; Rossmeisl, John H. Jr.; Dervisis, Nikolaos G. (Nature Portfolio, 2024-11-18)
High-frequency irreversible electroporation (H-FIRE), a nonthermal brain tumor ablation therapeutic, generates a central tumor ablation zone while transiently disrupting the peritumoral blood–brain barrier (BBB). We hypothesized that bystander effects of H-FIRE tumor cell ablation, mediated by small tumor-derived extracellular vesicles (sTDEV), disrupt the BBB endothelium. Monolayers of bEnd.3 cerebral endothelial cells were exposed to supernatants of H-FIRE or radiation (RT)-treated LL/2 and F98 cancer cells. Endothelial cell response was evaluated microscopically and via flow cytometry for apoptosis. sTDEV were isolated following H-FIRE and RT, characterized via nanoparticle tracking analysis (NTA) and transmission electron microscopy, and applied to a Transwell BBB endothelium model to quantify permeability changes. Supernatants of H-FIRE-treated tumor cells, but not supernatants of sham- or RT-treated cells, disrupted endothelial cell monolayer integrity while maintaining viability. sTDEV released by glioma cells treated with 3000 V/cm H-FIRE increased permeability of the BBB endothelium model compared to sTDEV released after lower H-FIRE doses and RT. NTA revealed significantly decreased sTDEV release after the 3000 V/cm H-FIRE dose. Our results demonstrate that sTDEV increase permeability of the BBB endothelium after H-FIRE ablation in vitro. sTDEV-mediated mechanisms of BBB disruption may be exploited for drug delivery to infiltrative margins following H-FIRE ablation.
Establishing Human and Canine Xenograft Murine Osteosarcoma Models for Application of Focused Ultrasound Ablation
Hay, Alayna N.; Simon, Alex; Ruger, Lauren N.; Gannon, Jessica; Coutermarsh-Ott, Sheryl; Vickers, Elliana R.; Eward, William; Neufeld, Nathan J.; Vlaisavljevich, Eli; Tuohy, Joanne (MDPI, 2025-08-30)
Background: Osteosarcoma (OS) is the most commonly occurring type of bone cancer in both humans and canines. The survival outcomes for OS patients have not improved significantly in decades. A novel and innovative treatment option that is currently under investigation for OS in the veterinary field is the focused ultrasound ablation modality, histotripsy. Histotripsy is a non-thermal, non-invasive, non-ionizing ablation modality that destroys tissue through generation of acoustic cavitation. Objective: In the current study, we sought to investigate the utility of an orthotropic OS xenograft murine model for characterization of chronic ablative and clinical outcomes post-histotripsy ablation. Method: Given the high comparative relevance of canine to human OS, histotripsy was delivered to orthotopic OS tumors in both human and canine xenograft murine models. Results: Histotripsy improved limb function in tumor-bearing mice compared to untreated tumor bearing mice. The results of this study demonstrated the utility of the orthotopic OS xenograft murine model for histotripsy-based preclinical studies. Conclusions: The current study is the first published investigation for the use of an orthotopic xenograft murine model for the development of histotripsy ablation for OS. The developmental process of the model, technical limitations, and future directions are discussed.
Precision Adjuvant Strategies in Vaccine Development for Substance Use Disorders: Variability and Mechanistic Insights
Bian, Yuanzhi; Ci, Qiaoqiao; Luo, Xin M.; Zhang, Chenming (MDPI, 2025-09-20)
Substance use disorders (SUDs) remain a major global health challenge with limited treatment options and high relapse rates. Vaccines that induce drug-sequestering antibodies have shown promise, but their efficacy is hindered by the poor immunogenicity of small-molecule haptens. Adjuvants, substances that enhance immune responses, are critical for overcoming this limitation and improving vaccine efficacy. This review synthesizes over two decades of preclinical and clinical research to guide rational adjuvant design for SUD vaccines. Five major adjuvant classes are examined: aluminum-salt adjuvants, emulsion adjuvants, toll-like receptor (TLR) agonists, protein immunopotentiators, and cytokine modulators. Their physicochemical properties, innate immune activation profiles, and applications in nicotine, stimulant, and opioid vaccines are discussed. Comparative analyses reveal pronounced drug-specific and carrier-specific variability. Case studies illustrate the superior performance of a complementary TLR-agonist pair in a nicotine nanovaccine versus its limited effect in oxycodone vaccines. They also reveal the differential efficacy of an oil-in-water emulsion adjuvant across antigen types. Four principles emerge: (i) no adjuvant is universally optimal; (ii) drug pharmacology influences immune signaling; (iii) adjuvant-carrier compatibility is important; (iv) complementary adjuvant pairings often outperform single agents. These insights support a precision-vaccinology paradigm that tailors adjuvant strategies to each drug class and the delivery vehicle, advancing the development of next-generation SUD vaccines.
Double-Outlet Right Ventricular Malformation in a Two-Year-Old Aberdeen Angus Cow
White, Baker; Carvallo-Chaigneau, Francisco R.; Cecere, Thomas E.; Mckenzie, Harold; Menciotti, Giulio; Umaña Sedó, Sebastián G. (MDPI, 2025-08-30)
A 2-year-old Aberdeen Angus cow was presented with lethargy and decreased appetite at the VA-MD College of Veterinary Medicine Large Animal Teaching Hospital. Initial clinical examination revealed cyanosis, tachycardia, polycythemia, and a significant increase in lactate levels. The heifer experienced spontaneous death while hospitalized, prompting a postmortem examination. Gross evaluation demonstrated that the aorta arose entirely from the right ventricle, while the main pulmonary artery maintained its normal position, consistent with a diagnosis of double-outlet right ventricle. Additional cardiac abnormalities were identified, including an atrial septal defect, ventricular septal defect and marked right ventricular hypertrophy. These defects fall under the category of double-outlet right ventricle malformation. While congenital heart defects are a more recognized cause of cardiac failure and mortality in calves, they should remain a consideration in cases of sudden death, even in adult cattle.
Design and Evaluation Novel Gene-Specific, Cell-Permeable Antisense Peptide Nucleic Acids to Prevent Staphylococcus Aureus Biofilm Formation
Col, Nihan Akguc; Rao, Jayasimha; Rajagopalan, Govindarajan; Sriranganathan, Nammalwar (Gavin Publishers, 2024-12-27)
Staphylococcus aureus, a Gram-positive bacterium, is a leading cause of various biofilm-associated infections in humans and animals, posing significant economic and healthcare challenges. Biofilms exhibit heightened resistance to antimicrobial agents as well as to immune-mediated clearance, thus persisting for long periods of time. Hence, novel therapeutic approaches are needed to eradicate S. aureus biofilms. Peptide nucleic acids (PNAs), synthetic DNA analogs with a peptide backbone instead of sugar backbone, offer a promising approach. In this study, we designed, synthesized and tested the efficacy of several synthetic antisense PNAs coupled with cell-penetrating peptides (CPPs), targeting essential and biofilm related S. aureus genes to inhibit staphylococcal biofilm growth using standard microtiter plate and tygon catheter biofilm assays. P-PNAs targeting the genes for intercellular adhesion locus, ica, cell wall/membrane/envelope biogenesis, fmhb, accessory regulator, sarA, sensor histidine kinase, saeS, repressor of toxins, rot, response regulator, yycF and histidine kinase, yycG genes were tested. Two scrambled PNAs and CPP alone were used as controls. Only one P-PNA, targeting sarA, showed the strongest biofilm inhibitory activity (up to 40 %) at a concentration of 50 μM or higher. This novel P-PNA could be a useful adjunct for the treatment S. aureus biofilm infections.
Broad Host Range Peptide Nucleic Acids Prevent Gram-Negative Biofilms Implicated in Catheter-Associated Urinary Tract Infections
Karp, Hannah Q.; Nowak, Elizabeth S.; Kropp, Gillian A.; Col, Nihan A.; Schulz, Michael D.; Sriranganathan, Nammalwar; Rao, Jayasimha (MDPI, 2025-08-20)
Biofilms develop in sequential steps resulting in the formation of three-dimensional communities of microorganisms that are encased in self-produced extracellular polymeric substances. Biofilms play a key role in device-associated infections, such as catheter-associated urinary tract infections (CAUTIs), because they protect microorganisms from standard antimicrobial therapies. Current strategies to prevent biofilm formation in catheter-related infections, including prophylactic antibiotics and antibiotic-coated catheters, have been unsuccessful. This finding highlights a need for novel approaches to address this clinical problem. In this study, biofilm-forming phenotypes of common Gram-negative bacteria associated with CAUTIs were treated with antisense peptide nucleic acids (PNAs), and biofilm biomass and bacterial viability were quantified after 24 h of treatment. A cocktail of PNAs targeting the global regulator genes rsmA, amrZ, and rpoS in Pseudomonas aeruginosa significantly reduced viability and thus appropriately eliminated biofilm biomass. Antisense-PNAs against these same gene targets and the motility regulator gene motA inhibited biofilm formation among isolates of Klebsiella pneumoniae, Enterobacter cloacae, and Escherichia coli but did not reduce bacterial viability. These results suggest that antisense-PNAs are a promising new technology in preventing biofilm formation in urinary catheters, especially as a potential complement to conventional antimicrobials.
Elevated EGR1 binding at enhancers in excitatory neurons correlates with neuronal subtype-specific epigenetic regulation
Yin, Liduo; Xu, Xiguang; Conacher, Benjamin; Lin, Yu; Carrillo, Gabriela L.; Cun, Yupeng; Fox, Michael A.; Lu, Xuemei; Xie, Hehuang (2025-08-11)
Background: Brain development and neuronal cell specification are accompanied by epigenetic changes that enable the regulation of diverse gene expression patterns. During these processes, transcription factors interact with cell-type-specific epigenetic marks, binding to unique sets of cis-regulatory elements in different cell types. However, the detailed mechanisms through which cell-type-specific gene regulation is established in neurons remain to be explored. Results: In this study, we conducted a comparative histone modification analysis between excitatory and inhibitory neurons. Our results revealed that neuronal cell-type-specific histone modifications are enriched in super enhancer regions that contain abundant EGR1 motifs. Further CUT&RUN assay confirmed that excitatory neurons exhibit more EGR1 binding sites, primarily located in enhancers. Integrative analysis demonstrated that EGR1 binding is strongly correlated with various epigenetic markers of open chromatin regions and is linked to distinct gene pathways specific to neuronal subtypes. In inhibitory neurons, most genomic regions containing EGR1 binding sites become accessible during early embryonic stages, whereas super enhancers in excitatory neurons, which also host EGR1 binding sites, gain accessibility during postnatal stages. Conclusions: This study highlights the crucial role of transcription factor binding, such as EGR1, to enhancer regions, which may be key to establishing cell-type-specific gene regulation in neurons.
Insights from Mass Spectrometry-Based Proteomics on Cryptococcus neoformans
Betancourt, Jovany Jordan; Nielsen, Kirsten (MDPI, 2025-07-17)
Cryptococcus neoformans is an opportunistic fungal pathogen and causative agent of cryptococcosis and cryptococcal meningitis (CM). Cryptococcal disease accounts for up to 19% of AIDS-related mortalities globally, warranting its label as a pathogen of critical priority by the World Health Organization. Standard treatments for CM rely heavily on high doses of antifungal agents for long periods of time, contributing to the growing issue of antifungal resistance. Moreover, mortality rates for CM are still incredibly high (13–78%). Attempts to create new and effective treatments have been slow due to the complex and diverse set of immune-evasive and survival-enhancing virulence factors that C. neoformans employs. To bolster the development of better clinical tools, deeper study into host–Cryptococcus proteomes is needed to identify clinically relevant proteins, pathways, antigens, and beneficial host response mechanisms. Mass spectrometry-based proteomics approaches serve as invaluable tools for investigating these complex questions. Here, we discuss some of the insights into cryptococcal disease and biology learned using proteomics, including target proteins and pathways regulating Cryptococcus virulence factors, metabolism, and host defense responses. By utilizing proteomics to probe deeper into these protein interaction networks, new clinical tools for detecting, diagnosing, and treating C. neoformans can be developed.
Larval environment reshapes mosquito disease risk via phenotypic and molecular plasticity
Chandrasegaran, Karthikeyan; Walker, Melody; Marano, Jeffrey M.; Rami, Spruha; Bisese, Adaline; Weger-Lucarelli, James; Lahondère, Chloé; Robert, Michael A.; Childs, Lauren M.; Vinauger, Clément (2025-06-21)
Early-life environmental conditions can exert profound, lasting effects on adult phenotypes, with major consequences for fitness and disease transmission, especially in holometabolous insects like mosquitoes, which are a key vector species. Yet, the molecular mechanisms through which juvenile environments shape adult physiology and behavior via transstadial effects remain largely unresolved. Here, we demonstrate that larval competition, a key ecological stressor, profoundly alters adult body size, survival, reproductive output, host-seeking behavior, olfactory neurophysiology, and vector competence in the mosquito Aedes aegypti. Crucially, using transcriptomic profiling and integrative network analyses, we identify seven regulatory hub genes whose expression is strongly associated with size-dependent variation in olfactory behavior, reproductive investment, and Zika virus transmission potential. These hub genes belong to gene modules enriched for functions in chemosensory processing, metabolic regulation, and signal transduction, revealing a molecular framework mediating environmentally induced plasticity across metamorphosis. Integrating these empirical findings into a transmission model, we show that incomplete larval control can inadvertently increase outbreak risk by producing larger, longer-lived, and more competent vectors. Our results uncover molecular mechanisms underpinning phenotypic plasticity in disease vectors and highlight the critical need to account for transstadial effects in models of vector-borne disease transmission.
Development of a Selective Agar for the Detection of Probiotic Strain Ligilactobacillus animalis NP51 and Other Lactic Acid Bacteria in Cattle Feed
Thompson, Kasey; Akter, Shamima; Ferguson-Noel, Naola; Maurer, John J.; Lee, Margie D. (MDPI, 2025-06-13)
The enormous potential of bacteriotherapy in disease treatment and prevention has created a large probiotic market. Significant challenges exist in assessing probiotic quality, efficacy and viability. Lactic acid bacteria (LAB) are commonly used probiotics and the most abundant of the vertebrate microbiota. The goal of this study was to make MRS agar specific for probiotic Ligilactobacillus animalis NP51, since the current formulation is not sufficiently selective. Here, 53 chemicals were screened to identify compound(s) that reduced the growth of non-LAB and fungi on de Mann, Rogosa, and Sharpe (MRS) agar, and which were selective for LAB and specifically the probiotic strain NP51. Cattle feed was selected as the sample type, as it is commonly amended with Lactobacillus or yeast probiotics and often includes silage, a diverse microbial consortium of fungi and LAB. Modified MRS was evaluated for its effectiveness in determining probiotic viability and the detection of L. animalis NP51 in cattle feed, amended with this probiotic. qPCR was used to specifically detect and enumerate NP51 in commercial and experimental feed samples. For four selective agents, nystatin, guanidine hydrochloride, CuSO₄, and ZnCl, it was identified that when used together, they reduced the growth of bacteria and fungi, but did not inhibit the Lactobacillus probiotic NP51 and other LAB. Metagenomic analysis revealed LAB as the major group cultivated on modified MRS agar from the plating of cattle feed amended with silage. As an enrichment, modified MRS broth improved the qPCR detection of probiotic strain NP51. This study illustrated that improvements can be made to existing bacteriological media for enumerating probiotic NP51 and determining the product’s viability.
Spatial Transcriptomics Reveals Regional and Temporal Dynamics of Gene Expression in the Mouse Brain Across Development and Aging
Conacher, Benjamin; Moore, Amanda; Yin, Liduo; Lin, Yu; Xu, Xiguang; Mao, Qinwen; Xie, Hehuang (MDPI, 2025-06-18)
Investigating transcriptomic changes during healthy development and aging provides insights into the molecular mechanisms that regulate the maturation of brain functions and drive age-related decline. Although it has been speculated that aging may represent a reversal of late-stage brain development, direct molecular comparisons between these two processes have remained limited. This study employs spatial transcriptomics to analyze the mouse brain at three key timepoints: postnatal day 21 (P21), 3 months (adult), and 28 months (aged), to identify region-specific differential gene expression dynamics. We identify widespread transcriptional changes across both brain development and aging, with all brain regions exhibiting distinct, region-specific gene expression dynamics that reflect divergent regulatory trajectories across the lifespan. During development, gene expression patterns were strongly enriched for neurogenesis, synaptic plasticity, and myelination, reflecting active circuit formation and white matter maturation. In contrast, aging was characterized by a decline in myelination-related gene expression and a pronounced increase in inflammatory and glial activation pathways, particularly within the hippocampus. While both development and aging involved changes in myelination-associated genes, the underlying mechanisms appear distinct: developmental upregulation supports circuit establishment and refinement, whereas aging-related downregulation may reflect secondary consequences of neuroinflammation and reactive gliosis. These findings underscore that, despite some overlap in affected pathways, neural maturation and age-related decline are driven by fundamentally different regulatory programs. These findings establish a novel spatial transcriptomic reference for brain development and aging, offering a valuable data resource for investigating neurodevelopmental and neurodegenerative mechanisms.
Glucosylceramide Synthase, a Key Enzyme in Sphingolipid Metabolism, Regulates Expression of Genes Accounting for Cancer Drug Resistance
Mostaq, Md Saqline; Kang, Lin; Patwardhan, Gauri A.; Zhao, Yunfeng; Shi, Runhua; Liu, Yong-Yu (MDPI, 2025-05-26)
Emergent cancer drug resistance and further metastasis can mainly be attributed to altered expression levels and functional activities of multiple genes of cancer cells under chemotherapy. In response to challenge with anticancer drugs, enhanced ceramide glycosylation catalyzed by glucosylceramide synthase (GCS) confers drug resistance and enrichment with cancer stem cells. p53 mutations, which gain function in tumor progression, are prevalently extant in ovarian cancers. Via integrated gene expression assessments, we characterized GCS-responsive genes in ovarian cancer cells treated with dactinomycin. NCI/ADR-RES cells dominantly expressed a p53 mutant (7 aa deleted in exon-5) and displayed anti-apoptosis; however, silencing GCS expression rendered these cells sensitive to dactinomycin-induced apoptosis. Microarray analyses of NCI/ADR-RES and its GCS transfected sublines found that elevated GCS expression or ceramide glycosylation was associated with altered expression of 41 genes, notably coding for ABCB1, FGF2, ALDH1A3, apolipoprotein E, laminin 2, chemokine ligands, and IL6, with cellular resistance to induced apoptosis and enrichment with cancer stem cells, promoting cancer progression. These findings were further corroborated through integrated genomic analyses of ovarian cancer from The Cancer Genome Atlas (TCGA) and cancer resistance to platinum-based chemotherapy. Altogether, our present study indicates that altered ceramide glycosylation can modulate expression of these GCS-responsive genes and alter cancer cell attributes under chemotherapy.
nMOWChIP-seq: low-input genome-wide mapping of non-histone targets
Liu, Zhengzhi; Naler, Lynette B.; Zhu, Yan; Deng, Chengyu; Zhang, Qiang; Zhu, Bohan; Zhou, Zirui; Sarma, Mimosa; Murray, Alexander; Xie, Hehuang; Lu, Chang (Oxford University Press, 2022-03-31)
Genome-wide profiling of interactions between genome and various functional proteins is critical for understanding regulatory processes involved in development and diseases. Conventional assays require a large number of cells and high-quality data on tissue samples are scarce. Here we optimized a low-input chromatin immunoprecipitation followed by sequencing (ChIP-seq) technology for profiling RNA polymerase II (Pol II), transcription factor (TF), and enzyme binding at the genome scale. The new approach produces high-quality binding profiles using 1,000-50,000 cells. We used the approach to examine the binding of Pol II and two TFs (EGR1 and MEF2C) in cerebellum and prefrontal cortex of mouse brain and found that their binding profiles are highly reflective of the functional differences between the two brain regions. Our analysis reveals the potential for linking genome-wide TF or Pol II profiles with neuroanatomical origins of brain cells.
Systematic evaluation of parameters in RNA bisulfite sequencing data generation and analysis
Johnson, Zachary; Xu, Xiguang; Pacholec, Christina; Xie, Hehuang (Oxford University Press, 2022-03-31)
The presence of 5-methylcytosine (m5C) in RNA molecules has been known for decades and its importance in regulating RNA metabolism has gradually become appreciated. Despite recent advances made in the functional and mechanistic understanding of RNA m5C modifications, the detection and quantification of methylated RNA remains a challenge. In this study, we compared four library construction procedures for RNA bisulfite sequencing and implemented an analytical pipeline to assess the key parameters in the process of m5C calling. We found that RNA fragmentation after bisulfite conversion increased the yield significantly, and an additional high temperature treatment improved bisulfite conversion efficiency especially for sequence reads mapped to the mitochondrial transcriptome. Using Unique Molecular Identifiers (UMIs), we observed that PCR favors the amplification of unmethylated templates. The low sequencing quality of bisulfite-converted bases is a major contributor to the methylation artifacts. In addition, we found that mitochondrial transcripts are frequently resistant to bisulfite conversion and no p-m5C sites with high confidence could be identified on mitochondrial mRNAs. Taken together, this study reveals the various sources of artifacts in RNA bisulfite sequencing data and provides an improved experimental procedure together with analytical methodology.
Egr2 Deletion in Autoimmune-Prone C57BL6/lpr Mice Suppresses the Expression of Methylation-Sensitive Dlk1-Dio3 Cluster MicroRNAs
Wang, Zuhang; Heid, Bettina; He, Jianlin; Xie, Hehuang; Reilly, Christopher M.; Dai, Rujuan; Ahmed, S. Ansar (Oxford University Press, 2023-12)
We previously demonstrated that the upregulation of microRNAs (miRNAs) at the genomic imprinted Dlk1-Dio3 locus in murine lupus is correlated with global DNA hypomethylation. We now report that the Dlk1-Dio3 genomic region in CD4+ T cells of MRL/lpr mice is hypomethylated, linking it to increased Dlk1-Dio3 miRNA expression. We evaluated the gene expression of methylating enzymes, DNA methyltransferases (DNMTs), and demethylating ten-eleven translocation proteins (TETs) to elucidate the molecular basis of DNA hypomethylation in lupus CD4+ T cells. There was a significantly elevated expression of Dnmt1 and Dnmt3b, as well as Tet1 and Tet2, in CD4+ T cells of three different lupus-prone mouse strains compared to controls. These findings suggest that the hypomethylation of murine lupus CD4+ T cells is likely attributed to a TET-mediated active demethylation pathway. Moreover, we found that deletion of early growth response 2 (Egr2), a transcription factor gene in B6/lpr mice markedly reduced maternally expressed miRNA genes but not paternally expressed protein-coding genes at the Dlk1-Dio3 locus in CD4+ T cells. EGR2 has been shown to induce DNA demethylation by recruiting TETs. Surprisingly, we found that deleting Egr2 in B6/lpr mice induced more hypomethylated differentially methylated regions at either the whole-genome level or the Dlk1-Dio3 locus in CD4+ T cells. Although the role of methylation in EGR2-mediated regulation of Dlk1-Dio3 miRNAs is not readily apparent, these are the first data to show that in lupus, Egr2 regulates Dlk1-Dio3 miRNAs, which target major signaling pathways in autoimmunity. These data provide a new perspective on the role of upregulated EGR2 in lupus pathogenesis.
Dynamics of RNA m⁵C modification during brain development
Johnson, Zachary; Xu, Xiguang; Lin, Yu; Xie, Hehuang (Elsevier, 2023-05)
Post-transcriptional RNA modifications have been recognized as key regulators of neuronal differentiation and synapse development in the mammalian brain. While distinct sets of 5-methylcytosine (m5C) modified mRNAs have been detected in neuronal cells and brain tissues, no study has been performed to characterize methylated mRNA profiles in the developing brain. Here, together with regular RNA-seq, we performed transcriptome-wide bisulfite sequencing to compare RNA cytosine methylation patterns in neural stem cells (NSCs), cortical neuronal cultures, and brain tissues at three postnatal stages. Among 501 m5C sites identified, approximately 6% are consistently methylated across all five conditions. Compared to m5C sites identified in NSCs, 96% of them were hypermethylated in neurons and enriched for genes involved in positive transcriptional regulation and axon extension. In addition, brains at the early postnatal stage demonstrated substantial changes in both RNA cytosine methylation and gene expression of RNA cytosine methylation readers, writers, and erasers. Furthermore, differentially methylated transcripts were significantly enriched for genes regulating synaptic plasticity. Altogether, this study provides a brain epitranscriptomic dataset as a new resource and lays the foundation for further investigations into the role of RNA cytosine methylation during brain development.
Independent and joint association of cord plasma pantothenate and cysteine levels with autism spectrum disorders and other neurodevelopmental disabilities in children born term and preterm
Raghavan, Ramkripa; Wang, Guoying; Hong, Xiumei; Pearson, Colleen; Xie, Hehuang; Adams, William G.; Augustyn, Marilyn; Wang, Xiaobin (2023-05-11)
Background: Pantothenate (vitamin B5) is a precursor for coenzyme A (CoA) synthesis, which serves as a cofactor for hundreds of metabolic reactions. Cysteine is an amino acid in the CoA synthesis pathway. To date, research on the combined role of early life pantothenate and cysteine levels in childhood neurodevelopmental disabilities is scarce. Objective: To study the association between cord pantothenate and cysteine levels and risk of autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD) and other developmental disabilities (DD) in children born term and preterm. Methods: The study sample (n = 996, 177 born preterm) derived from the Boston Birth Cohort included 416 neurotypical children, 87 ASD, 269 ADHD, and 224 other DD children, who were mutually exclusive. Participants were enrolled at birth and were followed up prospectively (from October 1, 1998, to June 30, 2018) at the Boston Medical Center. Cord blood sample was collected at birth. Plasma pantothenate and cysteine levels were measured using liquid chromatography-tandem mass spectrometry. Results: Higher cord pantothenate (≥50th percentile vs. <50th percentile) was associated with a greater risk of ASD (adjusted odds ratio [aOR]: 1.94, 95% confidence interval [CI]: 1.06, 3.55) and ADHD (aOR: 1.66, 95% CI: 1.14, 2.40), after adjusting for potential confounders. However, cord cysteine alone was not associated with risk of ASD, ADHD, or other DD. When considering the joint association, greater ASD risk was noted when both cord pantothenate and cysteine levels were elevated (≥50th percentile) (aOR: 3.11, 95% CI: 1.24, 7.79), when compared to children with low cord pantothenate (<50th percentile) and high cysteine. Even though preterm and higher pantothenate independently increased the ASD risk, the greatest risk was found in preterm children who also had elevated pantothenate (≥50th percentile), which was true for all three outcomes: ASD (aOR: 5.36, 95% CI: 2.09, 13.75), ADHD (aOR: 3.31, 95% CI: 1.78, 6.16), and other DD (aOR: 3.39, 95% CI: 1.85, 6.24). Conclusions: In this prospective birth cohort, we showed that higher cord pantothenate individually and in combination with higher cysteine or preterm birth were associated with increased risk of ASD and ADHD. More study is needed to explore this biologically plausible pathway.

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