Scholarly Works, Biomedical Sciences and Pathobiology

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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.
Endothelial deletion of EPH receptor A4 alters single-cell profile and Tie2/Akap12 signaling to preserve blood-brain barrier integrity
Cash, Alison; de Jager, Caroline; Brickler, Thomas; Soliman, Eman; Ladner, Liliana; Kaloss, Alexandra M.; Zhu, Yumeng; Pridham, Kevin J.; Mills, Jatia; Ju, Jing; Basso, Erwin Kristobal Gudenschwager; Chen, Michael; Johnson, Zachary; Sotiropoulos, Yianni; Wang, Xia; Xie, Hehuang; Matson, John B.; Marvin, Eric A.; Theus, Michelle H. (National Academy of Sciences, 2023-10-10)
Neurobiological consequences of traumatic brain injury (TBI) result from a complex interplay of secondary injury responses and sequela that mediates chronic disability. Endothelial cells are important regulators of the cerebrovascular response to TBI. Our work demonstrates that genetic deletion of endothelial cell (EC)-specific EPH receptor A4 (EphA4) using conditional EphA4f/f/Tie2-Cre and EphA4f/f/VE-Cadherin-CreERT2 knockout (KO) mice promotes blood–brain barrier (BBB) integrity and tissue protection, which correlates with improved motor function and cerebral blood flow recovery following controlled cortical impact (CCI) injury. scRNAseq of capillary-derived KO ECs showed increased differential gene expression of BBB-related junctional and actin cytoskeletal regulators, namely, A-kinase anchor protein 12, Akap12, whose presence at Tie2 clustering domains is enhanced in KO microvessels. Transcript and protein analysis of CCI-injured whole cortical tissue or cortical-derived ECs suggests that EphA4 limits the expression of Cldn5, Akt, and Akap12 and promotes Ang2. Blocking Tie2 using sTie2-Fc attenuated protection and reversed Akap12 mRNA and protein levels cortical-derived ECs. Direct stimulation of Tie2 using Vasculotide, angiopoietin-1 memetic peptide, phenocopied the neuroprotection. Finally, we report a noteworthy rise in soluble Ang2 in the sera of individuals with acute TBI, highlighting its promising role as a vascular biomarker for early detection of BBB disruption. These findings describe a contribution of the axon guidance molecule, EphA4, in mediating TBI microvascular dysfunction through negative regulation of Tie2/Akap12 signaling.
Monoubiquitination of histone H2B is a crucial regulator of the transcriptome during memory formation
Navabpour, Shaghayegh; Farrell, Kayla; Kincaid, Shannon E.; Omar, Nour; Musaus, Madeline; Lin, Yu; Xie, Hehuang; Jarome, Timothy J. (Cold Spring Harbor Laboratory Press, 2024-03)
Posttranslational modification of histone proteins is critical for memory formation. Recently, we showed that monoubiquitination of histone H2B at lysine 120 (H2Bub) is critical for memory formation in the hippocampus. However, the transcriptome controlled by H2Bub remains unknown. Here, we found that fear conditioning in male rats increased or decreased the expression of 86 genes in the hippocampus but, surprisingly, siRNA-mediated knockdown of the H2Bub ligase, Rnf20, abolished changes in all but one of these genes. These findings suggest that monoubiquitination of histone H2B is a crucial regulator of the transcriptome during memory formation.
Neurological manifestations of encephalitic alphaviruses, traumatic brain injuries, and organophosphorus nerve agent exposure
VanderGiessen, Morgen; de Jager, Caroline; Leighton, Julia; Xie, Hehuang; Theus, Michelle; Johnson, Erik; Kehn-Hall, Kylene (Frontiers, 2024-12-13)
Encephalitic alphaviruses (EEVs), Traumatic Brain Injuries (TBI), and organophosphorus nerve agents (NAs) are three diverse biological, physical, and chemical injuries that can lead to long-term neurological deficits in humans. EEVs include Venezuelan, eastern, and western equine encephalitis viruses. This review describes the current understanding of neurological pathology during these three conditions, provides a comparative review of case studies vs. animal models, and summarizes current therapeutics. While epidemiological data on clinical and pathological manifestations of these conditions are known in humans, much of our current mechanistic understanding relies upon animal models. Here we review the animal models findings for EEVs, TBIs, and NAs and compare these with what is known from human case studies. Additionally, research on NAs and EEVs is limited due to their classification as high-risk pathogens (BSL-3) and/or select agents; therefore, we leverage commonalities with TBI to develop a further understanding of the mechanisms of neurological damage. Furthermore, we discuss overlapping neurological damage mechanisms between TBI, NAs, and EEVs that highlight novel medical countermeasure opportunities. We describe current treatment methods for reducing neurological damage induced by individual conditions and general neuroprotective treatment options. Finally, we discuss perspectives on the future of neuroprotective drug development against long-term neurological sequelae of EEVs, TBIs, and NAs.
Increasing H2B Monoubiquitination Improves the Transcriptome and Memory in the Aged Hippocampus
Kincaid, Shannon; Setenet, Gueladouan; Preveza, Natalie J.; Arndt, Kaiser C.; Gwin, Phillip; Lin, Yu; Xie, Hehuang; Jarome, Timothy J. (Society for Neuroscience, 2025-04)
A decline in cognitive abilities is associated with the aging process, affecting nearly 33% of US adults over the age of 70, and is a risk factor for the development of dementia and Alzheimer's disease. Several studies have reported age-related alterations in the transcriptome in the hippocampus, a major site of memory storage that is among the first regions impacted with age, dementia, and Alzheimer's disease. However, much remains unknown about why these transcriptional changes exist in the aged hippocampus and how this impacts memory late in life. Here, we show that monoubiquitination of histone H2B (H2Bubi), an epigenetic mechanism recently reported to be major regulator of the epigenome and transcriptome during memory formation in the young adult brain, decreases with age in the hippocampus of male rats. In vivo CRISPR-dCas9-mediated upregulation of Rnf20, the only ubiquitin E3 ligase for H2B, in the hippocampus significantly improved memory retention in aged rats. Remarkably, RNA-seq analysis revealed that in addition to the 18 genes typically upregulated in the aged rat hippocampus following contextual fear conditioning, Rnf20 upregulation caused learning-related increases and decreases in 40 and 11 unique genes, respectively, suggesting that these 51 genes may be among those most critical for improving memory in advanced age. Together, these data suggest that H2B monoubiquitination is a significant regulator of age-related dysregulation of the transcriptome and impairments in memory.
Transmission pathways of Campylobacter jejuni between humans and livestock in rural Ethiopia are highly complex and interdependent
Singh, Nitya; Thystrup, Cecilie A. N.; Hassen, Bahar M.; Bhandari, Menuka; Rajashekara, Gireesh; Hald, Tine M.; Manary, Mark J.; McKune, Sarah L.; Hassen, Jemal Y.; Smith, Helen L.; Marshall, Jonathan C.; French, Nigel P.; Havelaar, Arie H. (2025-05-03)
Background: Campylobacter jejuni and C. coli are the most common causes of bacterial enteritis worldwide whereas symptomatic and asymptomatic infections are associated with stunting in children in low- and middle-income countries. Little is known about their sources and transmission pathways in low- and middle-income countries, and particularly for infants and young children. We assessed the genomic diversity of C. jejuni in Eastern Ethiopia to determine the attribution of infections in infants under 1 year of age to livestock (chickens, cattle, goats and sheep) and other humans (siblings, mothers). Results: Among 287 C. jejuni isolates, 48 seven-gene sequence types (STs), including 11 previously unreported STs were identified. Within an ST, the core genome STs of multiple isolates differed in fewer than five alleles. Many of these isolates do not belong to the most common STs reported in high-resource settings, and of the six most common global STs, only ST50 was found in our study area. Isolates from the same infant sample were closely related, while those from consecutive infant samples often displayed different STs, suggesting rapid clearance and new infection. Four different attribution models using different genomic profiling methods, assumptions and estimation methods predicted that chickens are the primary reservoir for infant infections. Infections from chickens are transmitted with or without other humans (mothers, siblings) as intermediate sources. Model predictions differed in terms of the relative importance of cattle versus small ruminants as additional sources. Conclusions: The transmission pathways of C. jejuni in our study area are highly complex and interdependent. While chickens are the most important reservoir of C. jejuni, ruminant reservoirs also contribute to the infections. The currently nonculturable species Candidatus C. infans is also highly prevalent in infants and is likely anthroponotic. Efforts to reduce the colonization of infants with Campylobacter and ultimately stunting in low-resource settings are best aimed at protecting proximate sources such as caretakers’ hands, food and indoor soil through tight integration of the currently siloed domains of nutrition, food safety and water, sanitation and hygiene.
Transient Lymphatic Remodeling Follows Sub‑Ablative High‑Frequency Irreversible Electroporation Therapy in a 4T1 Murine Model
Esparza, Savieay; Jacobs, Edward; Hammel, Jennifer H.; Michelhaugh, Sharon K.; Alinezhadbalalami, Nastaran; Nagai‑Singer, Margaret; Imran, Khan Mohammad; Davalos, Rafael V.; Allen, Irving C.; Verbridge, Scott S.; Munson, Jennifer M. (Springer, 2025-02-25)
High-frequency irreversible electroporation (H-FIRE) is a minimally invasive local ablation therapy known to activate the adaptive immune system and reprogram the tumor microenvironment. Its predecessor, irreversible electroporation (IRE), transiently increases microvascular density and immune cell infiltration within the surviving non-ablated and non-necrotic tumor region, also known as the viable tumor region. However, the impact of pulse electric field therapies on lymphatic vessels, crucial for T-cell fate and maturation, remains unclear. This study investigates how sub-ablative H-FIRE (SA-HFIRE) affects lymphatic and blood microvascular remodeling in the 4T1 mammary mouse model. We conducted a temporal and spatial analysis to evaluate vascular changes in the viable tumor, peritumoral fat pad, and tumor-draining lymph node posttreatment. Histological examination showed a transient increase in blood vessel density on Day 1 post-treatment, followed by a spike in lymphatic vessel density in the viable tumor region on Day 3 post-treatment, increased lymphatic vessel density in the peripheral fat pad, and minimal remodeling of the tumor-draining lymph node within 3 days following treatment. Gene expression analysis indicated elevated levels of CCL21 and CXCL2 on Day 1 post-treatment, while VEGFA and VEGFC did not appear to contribute to vascular remodeling. Likewise, CCL21 protein content in tumor-draining axillary lymph nodes correlated with gene expression data from the viable tumor region. These findings suggest a dynamic shift in lymphatic and blood microvascular structures post-SA-HFIRE, potentially enhancing the adaptive immune response through CCL21- mediated lymphatic homing and subsequent lymph node microvascular remodeling. Future work will assess the immune and transport function of the microvasculature to inform experiments aimed at the application of adjuvant therapies during scenarios of tumor partial ablation.
Evolution at Spike protein position 519 in SARS-CoV-2 facilitated adaptation to humans
Cereghino, C.; Michalak, K.; DiGiuseppe, S.; Yu, D.; Faraji, A.; Sharp, A.K.; Brown, Anne M.; Kang, L.; Weger-Lucarelli, James; Michalak, P. (Springer Nature, 2024)
As the COVID-19 pandemic enters its fourth year, the pursuit of identifying a progenitor virus to SARSCoV- 2 and understanding the mechanism of its emergence persists, albeit against the backdrop of intensified efforts to monitor the ongoing evolution of the virus and the influx of new mutations. Surprisingly, few residues hypothesized to be essential forSARS-CoV-2 emergence and adaptation to humans have been validated experimentally, despite the importance that these mutations could contribute to the development of effective antivirals. To remedy this,we searched for genomic regions in the SARS-CoV-2 genome that show evidence of past selection around residues unique to SARSCoV- 2 compared with closely related coronaviruses. In doing so, we identified a residue at position 519 in Spike within the receptor binding domain that holds a static histidine in human-derived SARSCoV- 2 sequences but an asparagine in SARS-related coronaviruses from bats and pangolins. In experimental validation, the SARS-CoV-2 Spike protein mutant carrying the putatively ancestral H519N substitution showed reduced replication in human lung cells, suggesting that the histidine residue contributes to viral fitness in the human host. Structural analyses revealed a potential role of Spike residue 519 in mediating conformational transitions necessary for Spike prior to binding with ACE2. Pseudotyped viruses bearing the putatively ancestral N519 also demonstrated significantly reduced infectivity in cells expressing the human ACE2 receptor compared to H519. ELISA data corroborated that H519 enhances Spike binding affinity to the human ACE2 receptor compared to the putatively ancestral N519. Collectively, these findings suggest that the evolutionary transition at position 519 of the Spike protein played a critical role inSARS-CoV-2 emergence and adaptation to the human host. Additionally, this residue presents as a potential drug target for designing small molecule inhibitors tailored to this site.
Digital Surface-Enhanced Raman Spectroscopy-Lateral Flow Test Dipstick: Ultrasensitive, Rapid Virus Quantification in Environmental Dust
Wang, Wei; Srivastava, Sonali; Garg, Aditya; Xiao, Chuan; Hawks, Seth; Pan, Jin; Duggal, Nisha; Isaacman-VanWertz, Gabriel; Zhou, Wei; Marr, Linsey C.; Vikesland, Peter J. (American Chemical Society, 2024-03-07)
This study introduces a novel surface-enhanced Raman spectroscopy (SERS)-based lateral flow test (LFT) dipstick that integrates digital analysis for highly sensitive and rapid viral quantification. The SERS-LFT dipsticks, incorporating gold-silver core-shell nanoparticle probes, enable pixel-based digital analysis of large-area SERS scans. Such an approach enables ultralow-level detection of viruses that readily distinguishes positive signals from background noise at the pixel level. The developed digital SERS-LFTs demonstrate limits of detection (LODs) of 180 fg for SARS-CoV-2 spike protein, 120 fg for nucleocapsid protein, and 7 plaque forming units for intact virus, all within <30 min. Importantly, digital SERS-LFT methods maintain their robustness and their LODs in the presence of indoor dust, thus underscoring their potential for accurate and reliable virus diagnosis and quantification in real-world environmental settings.
Neuropathogenesis of Encephalitic Alphaviruses in Non-Human Primate and Mouse Models of Infection
Woodson, Caitlin M.; Carney, Shannon K.; Kehn-Hall, Kylene (MDPI, 2025-02-14)
Encephalitic alphaviruses, including eastern, Venezuelan, and western equine encephalitis virus (EEEV, VEEV, and WEEV, respectively) are New World alphaviruses primarily transmitted by mosquitos that cause debilitating and lethal central nervous system (CNS) disease in both humans and horses. Despite over one hundred years of research on these viruses, the underpinnings of the molecular mechanisms driving virally induced damage to the CNS remain unresolved. Moreover, virally induced encephalitis following exposure to these viruses causes catastrophic damage to the CNS, and survivors of infection often suffer from permanent neurological sequelae as a result of sustained neuroinflammation and neurological insults encountered. Animal models are undoubtedly invaluable tools in biomedical research, where physiologically relevant models are required to study pathogenesis and host–pathogen interactions. Here, we review the literature to examine nonhuman primate (NHP) and mouse models of infection for EEEV, VEEV, and WEEV. We provide a brief overview of relevant background information for each virus, including geography, epidemiology, and clinical disease. The primary focus of this review is to describe neuropathological features associated with CNS disease in NHP and mouse models of infection and compare CNS invasion and neuropathogenesis for aerosol, intranasal, and subcutaneous routes of exposure to EEEV, VEEV, and WEEV.
The Impact Aerobic and Anaerobic Incubations of Poultry Litter Have on Class 1 Integron Resistome and Microbiome
Maurer, John J.; Hoke, Alexa; Das, Keshav C.; Wu, Jian; Williams, Mark A.; Kinstler, Sydney; Ritz, Casey; Pittman, Gregory P.; Berghaus, Roy; Lee, Margie D. (MDPI, 2025-02-13)
Animal manure is a desirable fertilizer because of its rich nitrogen, but it also contains a large and diverse reservoir of antimicrobial resistance (AMR) genes (ARGs). To reduce this AMR reservoir, five treatments (passive aeration, forced aeration, static or anaerobic incubations, autoclaving) were assessed for their impact on the poultry litter resistome. Bacterial DNA was extracted from the litter and the qPCR-estimated copy number of 16S rrs, class1 integrons (intI1) and associated resistance genes (aadA, sul1). Then, 16S amplicon metagenomic sequencing was used to determine community diversity and composition. Depending on incubation conditions, class 1 integrons and their associated ARGs were reduced by 0.5 to 1.0 Log₁₀/g poultry litter. Only autoclaving reduced integrons and associated AMR genes by three Log₁₀. Changes in AMR abundance reflected fluctuations in litter bacteriome composition at the family, genus, and sequence variant level. There was a negative correlation between class 1 integron and AMR genes, with genera belonging to Actinobacteria, Firmicutes, and Proteobacteria phyla. While these poultry litter treatments failed to reduce AMR abundance, aerobic and anaerobic treatments reduced taxons that contained pathogenic species. The approach to remediating resistance in poultry litter may be more effective if is focused on reducing bacterial pathogens.
Phenelzine-based probes reveal Secernin-3 is involved in thermal nociception
Bustin, Katelyn A.; Shishikura, Kyosuke; Chen, Irene; Lin, Zongtao; McKnight, Nate; Chang, Yuxuan; Wang, Xie; Li, Jing Jing; Arellano, Eric; Pei, Liming; Morton, Paul D.; Gregus, Ann M.; Buczynski, Matthew W.; Matthews, Megan L. (Academic Press - Elsevier, 2023-03-15)
Chemical platforms that facilitate both the identification and elucidation of new areas for therapeutic development are necessary but lacking. Activity-based protein profiling (ABPP) leverages active site-directed chemical probes as target discovery tools that resolve activity from expression and immediately marry the targets identified with lead compounds for drug design. However, this approach has traditionally focused on predictable and intrinsic enzyme functionality. Here, we applied our activity-based proteomics discovery platform to map non-encoded and post-translationally acquired enzyme functionalities (e.g. cofactors) in vivo using chemical probes that exploit the nucleophilic hydrazine pharmacophores found in a classic antidepressant drug (e.g. phenelzine, Nardil®). We show the probes are in vivo active and can map proteome-wide tissue-specific target engagement of the drug. In addition to engaging targets (flavoenzymes monoamine oxidase A/B) that are associated with the known therapeutic mechanism as well as several other members of the flavoenzyme family, the probes captured the previously discovered N-terminal glyoxylyl (Glox) group of Secernin-3 (SCRN3) in vivo through a divergent mechanism, indicating this functional feature has biochemical activity in the brain. SCRN3 protein is ubiquitously expressed in the brain, yet gene expression is regulated by inflammatory stimuli. In an inflammatory pain mouse model, behavioral assessment of nociception showed Scrn3 male knockout mice selectively exhibited impaired thermal nociceptive sensitivity. Our study provides a guided workflow to entangle molecular (off)targets and pharmacological mechanisms for therapeutic development.
MCU expression in hippocampal CA2 neurons modulates dendritic mitochondrial morphology and synaptic plasticity
Pannoni, Katy E.; Fischer, Quentin S.; Tarannum, Renesa; Cawley, Mikel L.; Alsalman, Mayd M.; Acosta, Nicole; Ezigbo, Chisom; Gil, Daniela V.; Campbell, Logan A.; Farris, Shannon (Nature Research, 2025-02-06)
Neuronal mitochondria are diverse across cell types and subcellular compartments in order to meet unique energy demands. While mitochondria are essential for synaptic transmission and synaptic plasticity, the mechanisms regulating mitochondria to support normal synapse function are incompletely understood. The mitochondrial calcium uniporter (MCU) is proposed to couple neuronal activity to mitochondrial ATP production, which would allow neurons to rapidly adapt to changing energy demands. MCU is uniquely enriched in hippocampal CA2 distal dendrites compared to proximal dendrites, however, the functional significance of this layer-specific enrichment is not clear. Synapses onto CA2 distal dendrites readily express plasticity, unlike the plasticity-resistant synapses onto CA2 proximal dendrites, but the mechanisms underlying these different plasticity profiles are unknown. Using a CA2-specific MCU knockout (cKO) mouse, we found that MCU deletion impairs plasticity at distal dendrite synapses. However, mitochondria were more fragmented and spine head area was diminished throughout the dendritic layers of MCU cKO mice versus control mice. Fragmented mitochondria might have functional changes, such as altered ATP production, that could explain the structural and functional deficits at cKO synapses. Differences in MCU expression across cell types and circuits might be a general mechanism to tune mitochondrial function to meet distinct synaptic demands.
Pulmonary granuloma formation during latent Cryptococcus neoformans infection in C3HeB/FeJ mice involves progression through three immunological phases
Betancourt, Jovany J.; Ding, Minna; Yoder, J. Marina; Mutyaba, Issa; Atkins, Hannah M.; De la Cruz, Gabriela; Meya, David B.; Nielsen, Kirsten (American Society for Microbiology, 2025-01-14)
Cryptococcus neoformans is a fungal pathogen that can cause lethal disease in immunocompromised patients. Immunocompetent host immune responses, such as formation of pulmonary granulomas, control the infection and prevent disseminated disease. Little is known about the immunological conditions establishing the latent infection granuloma in the lungs. To investigate this, we performed an analysis of pulmonary immune cell populations, cytokine changes, and granuloma formation during infection with a latent disease-causing clinical isolate in C3HeB/FeJ mice over 360 days. We found that latently infected mice progress through three phases of granuloma formation where different immune profiles dominate: an early phase characterized by eosinophilia, high IL-4/IL-13, and C. neoformans proliferation in the lungs; an intermediate phase characterized by multinucleated giant cell formation, high IL-1α/IFNγ, granuloma expansion, and increased blood antigen levels; and a late phase characterized by a significant expansion of T cells, granuloma condensation, and decreases in lung fungal burden and blood antigen levels. These findings highlight a complex series of immune changes that occur during the establishment of granulomas that control C. neoformans in the lungs and lay the foundation for studies to identify critical beneficial immune responses to Cryptococcus infections.
Analysis of the core bacterial community associated with consumer-ready Eastern oysters (Crassostrea virginica)
Hines, Ian S.; Markov Madanick, Justin; Smith, Stephen A.; Kuhn, David D.; Stevens, Ann M. (Public Library of Science, 2023-02-22)
Shellfish, such as the Eastern oyster (Crassostrea virginica), are an important agricultural commodity. Previous research has demonstrated the importance of the native microbiome of oysters against exogenous challenges by non-native pathogens. However, the taxonomic makeup of the oyster microbiome and the impact of environmental factors on it are understudied. Research was conducted quarterly over a calendar year (February 2020 through February 2021) to analyze the taxonomic diversity of bacteria present within the microbiome of consumer-ready-to-eat live Eastern oysters. It was hypothesized that a core group of bacterial species would be present in the microbiome regardless of external factors such as the water temperature at the time of harvest or post-harvesting processing. At each time point, 18 Chesapeake Bay (eastern United States) watershed aquacultured oysters were acquired from a local grocery store, genomic DNA was extracted from the homogenized whole oyster tissues, and the bacterial 16S rRNA gene hypervariable V4 region was PCR-amplified using barcoded primers prior to sequencing via Illumina MiSeq and bioinformatic analysis of the data. A core group of bacteria were identified to be consistently associated with the Eastern oyster, including members of the phyla Firmicutes and Spirochaetota, represented by the families Mycoplasmataceae and Spirochaetaceae, respectively. The phyla Cyanobacterota and Campliobacterota became more predominant in relation to warmer or colder water column temperature, respectively, at the time of oyster harvest.
The effect of model structure and data availability on Usutu virus dynamics at three biological scales
Heitzman-Breen, Nora; Liyanage, Yuganthi R.; Duggal, Nisha; Tuncer, Necibe; Ciupe, Stanca M. (Royal Society, 2024-02-07)
Understanding the epidemiology of emerging pathogens, such as Usutu virus (USUV) infections, requires systems investigation at each scale involved in the host-virus transmission cycle, from individual bird infections, to bird-to-vector transmissions, and to USUV incidence in bird and vector populations. For new pathogens field data are sparse, and predictions can be aided by the use of laboratory-type inoculation and transmission experiments combined with dynamical mathematical modelling. In this study, we investigated the dynamics of two strains of USUV by constructing mathematical models for the within-host scale, bird-to-vector transmission scale and vector-borne epidemiological scale. We used individual within-host infectious virus data and per cent mosquito infection data to predict USUV incidence in birds and mosquitoes. We addressed the dependence of predictions on model structure, data uncertainty and experimental design. We found that uncertainty in predictions at one scale change predicted results at another scale. We proposed in silico experiments that showed that sampling every 12 hours ensures practical identifiability of the within-host scale model. At the same time, we showed that practical identifiability of the transmission scale functions can only be improved under unrealistically high sampling regimes. Instead, we proposed optimal experimental designs and suggested the types of experiments that can ensure identifiability at the transmission scale and, hence, induce robustness in predictions at the epidemiological scale.

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