Scholarly Works, Biomedical Sciences and Pathobiology

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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.
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.
Potential of Stem-Cell-Induced Peripheral Nerve Regeneration: From Animal Models to Clinical Trials
Wynne, Taylor M.; Fritz, Virginia Grey; Simmons, Zachary T.; Zahed, Malek; Seth, Ananya; Abbasi, Tamir; Reymundi, Michael J.; Roballo, Kelly C. S. (MDPI, 2024-11-23)
Peripheral nerve injury has become an increasingly prevalent clinical concern, causing great morbidity in the community. Although there have been significant advancements in the treatment of peripheral nerve damage in recent years, the issue of long-term nerve regeneration remains. Furthermore, Wallerian degeneration has created an obstacle to long-term nerve regeneration. For this reason, there has been extensive research on the use of exogenous and endogenous stem cells as an adjunct or even primary treatment option for peripheral nerve injury. The plasticity and inducibility of stem cells make them an enticing option for initiating neuronal cell regrowth and optimal sensory and functional nerve regeneration. Peripheral nerve injury has a broad range of causative factors and etiologies. As such, unique stem cell-induced peripheral nerve treatments are being investigated to ameliorate the damage incited by all causes, including trauma, neuropathy, and systemic neurodegenerative diseases. This review is oriented to outline the potential role of stem cell therapies in peripheral nerve injury versus the current standards of care, compare the benefits and drawbacks of specific stem cell lines under investigation, and highlight the current models of stem cell therapy in the peripheral nervous system, with the ultimate goal of narrowing down and optimizing the role and scope of stem cell therapy in peripheral nerve injury.
Predicting patients with septic shock and sepsis through analyzing whole-blood expression of NK cell-related hub genes using an advanced machine learning framework
Du, Chao; Tan, Stephanie C.; Bu, Heng-Fu; Subramanian, Saravanan; Geng, Hua; Wang, Xiao; Xie, Hehuang; Wu, Xiaowei; Zhou, Tingfa; Liu, Ruijin; Xu, Zhen; Liu, Bing; Tan, Xiao-Di (Frontiers, 2024-11-28)
Background: Sepsis is a life-threatening condition that causes millions of deaths globally each year. The need for biomarkers to predict the progression of sepsis to septic shock remains critical, with rapid, reliable methods still lacking. Transcriptomics data has recently emerged as a valuable resource for disease phenotyping and endotyping, making it a promising tool for predicting disease stages. Therefore, we aimed to establish an advanced machine learning framework to predict sepsis and septic shock using transcriptomics datasets with rapid turnaround methods. Methods: We retrieved four NCBI GEO transcriptomics datasets previously generated from peripheral blood samples of healthy individuals and patients with sepsis and septic shock. The datasets were processed for bioinformatic analysis and supplemented with a series of bench experiments, leading to the identification of a hub gene panel relevant to sepsis and septic shock. The hub gene panel was used to establish a novel prediction model to distinguish sepsis from septic shock through a multistage machine learning pipeline, incorporating linear discriminant analysis, risk score analysis, and ensemble method combined with Least Absolute Shrinkage and Selection Operator analysis. Finally, we validated the prediction model with the hub gene dataset generated by RT-qPCR using peripheral blood samples from newly recruited patients. Results: Our analysis led to identify six hub genes (GZMB, PRF1, KLRD1, SH2D1A, LCK, and CD247) which are related to NK cell cytotoxicity and septic shock, collectively termed 6-HubGss. Using this panel, we created SepxFindeR, a machine learning model that demonstrated high accuracy in predicting sepsis and septic shock and distinguishing septic shock from sepsis in a cross-database context. Remarkably, the SepxFindeR model proved compatible with RT-qPCR datasets based on the 6-HubGss panel, facilitating the identification of newly recruited patients with sepsis and septic shock. Conclusions: Our bioinformatic approach led to the discovery of the 6-HubGss biomarker panel and the development of the SepxFindeR machine learning model, enabling accurate prediction of septic shock and distinction from sepsis with rapid processing capabilities.
An in vitro evaluation of intravenous lipid emulsion on three common canine toxicants
Jones, Emery; Walton, Stuart A.; Davis, Jennifer; Council-Troche, McAlister (Frontiers, 2024-09-25)
Objective: To determine whether intravenous lipid emulsion (ILE) therapy significantly reduces the concentration of baclofen, ibuprofen, and/or bromethalin in canine whole blood over time. Animals: Seven 500 mL bags of canine DEA 1.1 negative blood were divided into aliquots of 125 mL and randomly assigned to one of three treatment groups (baclofen, ibuprofen, bromethalin) or four control groups (a positive control for each treatment group and a negative control group). Procedures: Injectable ibuprofen (200 mg/kg), baclofen (8 mg/kg), or bromethalin (3 mg/kg) was apportioned into 125 mL aliquots of canine whole blood and incubated for 30 min at 38.5°C. ILE (12.4 mL, Intralipid®) was added to each sample and the solution vortexed [215 rpm for 15 min at 37°C (98.6°F)]. Samples were obtained at designated time points (0, 15, 30, 60, 180, 360 min), centrifuged, and separated into serum and RBC fractions. Serum samples were ultracentrifuged (22,000 g for 10 min at 37°C) to separate lipid rich and poor fractions. Samples were stored at −80°C prior to analysis. Results: A significant decrease in total drug concentration was established for bromethalin and its metabolite desmethylbromethalin compared to positive controls. ILE significantly reduced desmethylbromethalin at the 30-and 360-min time points. The remainder of the desmethylbromethalin time points did not reach significance. Bromethalin concentration was significantly reduced at all time points compared to positive controls. Neither baclofen nor ibuprofen had significant changes in concentration. Conclusion: ILE therapy was effective at reducing the total drug concentration of bromethalin and its metabolite desmethylbromethalin supporting the lipid sink theory. As a single compartment in vitro study, this study does not evaluate other proposed mechanisms of action of ILE therapy. ILE therapy may have other means of significantly decreasing lipophilic drug concentration in cases of toxicosis.
Case report: Placental chorioadenoma in a primiparous pug dog
Balogh, Orsolya; Diab, Santiago; Parker, Acadia; de Sousa, Gabriela C.; Cecere, Julie T.; McCarter, Samantha J.; Sponenberg, Dan Phillip (Frontiers, 2024-11-27)
A single 1.7 cm × 1.2 cm × 1 cm focal, raised, smooth, round, pink to flesh-colored mass protruding from the chorioallantois of the zonary placenta was found during Cesarean section in a primiparous pug bitch. Microscopically, the non-encapsulated, non-infiltrative, exophytic mass originated from the chorioallantoic stroma overlying the labyrinth and was composed of many arborizing fronds generally lined by 1 to 2 layers of cuboidal, columnar and occasionally polygonal, large trophoblastic cells, but frequently by a larger number of cells that occasionally piled up to form small nests or nodules. The gross and microscopic characteristics of this mass were compatible with a benign neoplastic process arising in the chorioallantois and involving placental trophoblasts, hence the diagnosis of a placental chorioadenoma. Five of the six newborn puppies were viable and of normal birthweight, while one puppy, which had lower birthweight than the others, could not be resuscitated and was humanely euthanized. Although placental tumors in dogs are very rare, this case is compelling evidence in the argument for routine inspection of canine placentas to identify potentially deleterious macro- or microscopic conditions that may be linked to negative fetal outcomes.
Phosphorylation of Ser711 Residue in the Hypervariable Region of Zoonotic Genotype 3 Hepatitis E Virus is Important for Virus Replication
Wang, Bo; Subramaniam, Sakthivel; Tian, Debin; Mahsoub, Hassan M.; Heffron, C. Lynn; Meng, Xiang-Jin (American Society for Microbiology, 2024-10-08)
Hepatitis E virus (HEV) is distinct from other hepatotropic viruses because it is zoonotic. HEV-1 and HEV-2 exclusively infect humans, whereas HEV-3 and HEV-4 are zoonotic. However, the viral and/or host factors responsible for cross-species HEV transmission remain elusive. The hypervariable region (HVR) in HEV is extremely heterogenetic and is implicated in HEV adaptation. Here, we investigated the potential role of Serine phosphorylation in the HVR in HEV replication. We first analyzed HVR sequences across different HEV genotypes and identified a unique region at the N-terminus of the HVR, which is variable in the human-exclusive HEV genotypes but relatively conserved in zoonotic HEV genotypes. Using predictive tools, we identified four potential phosphorylation sites that are highly conserved in zoonotic HEV-3 and HEV-4 genomes but absent in human-exclusive HEV-1 strains. To explore the functional significance of these putative phosphorylation sites, we introduced mutations into the HEV-3 infectious clone and indicator replicon, replacing each Serine residue individually with alanine or aspartic acid, and assessed the impact of these substitutions on HEV-3 replication. We found that the phospho-blatant S711A mutant significantly reduced virus replication, whereas the phospho-mimetic S711D mutant modestly reduced virus replication. Conversely, mutations in the other three Serine residues did not significantly affect HEV-3 replication. Furthermore, we demonstrated that Ser711 phosphorylation did not alter host cell tropism of zoonotic HEV-3. In conclusion, our results showed that potential phosphorylation of the Ser711 residue significantly affects HEV-3 replication in vitro, providing new insights into the potential mechanisms of zoonotic HEV transmission.
The role of gut microbiota in different murine models of systemic lupus erythematosus
Lu, Ran; Luo, Xin M. (Taylor & Francis, 2024-07-16)
Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by immune system dysfunction that can lead to serious health issues and mortality. Recent investigations highlight the role of gut microbiota alterations in modulating inflammation and disease severity in SLE. This review specifically summaries the variations in gut microbiota composition across various murine models of lupus. By focusing on these differences, we aim to elucidate the intricate relationship between gut microbiota dysbiosis and the development and progression of SLE in preclinical settings.
TCDD and CH223191 alter T cell balance but fail to induce anti-inflammatory response in adult lupus mice
Gutierrez, Fernando; Murphy, Quiyana M.; Swartwout, Brianna K.; Read, Kaitlin A.; Edwards, Michael R.; Abdelhamid, Leila; Cabana-Puig, Xavier; Testerman, James C.; Xu, Tian; Lu, Ran; Amin, Pavly; Cecere, Thomas E.; Reilly, Christopher M.; Oestreich, Kenneth J.; Ciupe, Stanca M.; Luo, Xin M. (The American Association of Immunologists, 2024-02-14)
Aryl hydrocarbon receptor (AhR) responds to endogenous and exogenous ligands as a cytosolic receptor, transcription factor, and E3 ubiquitin ligase. Several studies support an anti-inflammatory effect of AhR activation. However, exposure to the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during early stages of development results in an autoimmune phenotype and exacerbates lupus. The effects of TCDD on lupus in adults with pre-existing autoimmunity have not been described. We present novel evidence that AhR stimulation by TCDD alters T cell responses but fails to impact lupus-like disease using an adult mouse model. Interestingly, AhR antagonist CH223191 also changed T cell balance in our model. We next developed a conceptual framework for identifying cellular and molecular factors that contribute to physiological outcomes in lupus and created models that describe cytokine dynamics that were fed into a system of differential equations to predict the kinetics of T follicular helper (Tfh) and regulatory T (Treg) cell populations. The model predicted that Tfh cells expanded to larger values following TCDD exposure compared with vehicle and CH223191. Following the initial elevation, both Tfh and Treg cell populations continuously decayed over time. A function based on the ratio of predicted Treg/Tfh cells showed that Treg cells exceed Tfh cells in all groups, with TCDD and CH223191 showing lower Treg/Tfh cell ratios than the vehicle and that the ratio is relatively constant over time. We conclude that AhR ligands did not induce an anti-inflammatory response to attenuate autoimmunity in adult lupus mice. This study challenges the dogma that TCDD supports an immunosuppressive phenotype.
Single nucleotide polymorphisms are associated with strain-specific virulence differences among clinical isolates of Cryptococcus neoformans
Jackson, Katrina M.; Kono, Thomas; Betancourt, Jovany J.; Wang, Yina; Kabbale, Kisakye D.; Ding, Minna; Kezh, Perry; Ha, Grace; Yoder, J. Marina; Fulton, Sophie R.; Mukaremera, Liliane; Tiffin, Peter; Gusa, Asiya; Meya, David B.; Billmyre, R. Blake; Xue, Chaoyang; Nielsen, Kirsten (Nature Research, 2024-12-02)
Studies across various pathogens highlight the importance of pathogen genetic differences in disease manifestation. In the human fungal pathogen Cryptococcus neoformans, sequence type (ST) associates with patient outcome. We performed a meta-analysis of four genomic studies and identified overlapping gene regions associated with virulence, suggesting the importance of these gene regions in cryptococcal disease in diverse clinical isolates. We explored the relationship between virulence and strain genetic differences using the cryptococcosis mouse model and a closely related library of ST93 clinical isolates. We identified four in vivo virulence phenotypes: hypervirulence, typical virulence with CNS disease, typical virulence with non-CNS disease, and latent disease. Hypervirulent isolates were clade specific and associated with an interferon gamma (IFNγ) dominated immune response. Using a genome wide association study (GWAS), we identified nine genes with polymorphisms associated with IFNγ production, including the inositol sensor ITR4. The itr4Δ mutant recapitulated the hypervirulence phenotype and ITR4 affects expression of two IFNγ associated genes. Finally, we showed that IFNγ production is associated with SNPs that downregulate ITR4 and with SNP accumulation in other IFNγ associated genes. These data highlight the complex role of pathogen genetics in virulence and identify genes associated with hypervirulence and IFNγ in Cryptococcus neoformans.
Effects of subclinical Theileria orientalis Ikeda genotype infection on average daily gain ratios and a satisfactory rating in the breeding soundness exam in bull test stations in Virginia
Guynn, Sierra R.; Currin, John F.; Todd, S. Michelle; Greiner, Scott P.; Lahmers, Kevin K. (Texas A&M University Libraries, 2023-07-17)
Theileria orientalis Ikeda genotype is a tick borne hemoprotozoan that typically causes economic losses in dairy and beef cattle in Australia, New Zealand and Japan. Acute clinical infections from T. orientalis Ikeda include anemia, icterus, ill-thrift and death. The acute phase of the infection has been associated with decreased libido in dairy bulls, decreased live weight gain in beef bulls and increased mortality in naïve adults and calves. A sequela to acute infections within a herd is persistent subclinical infections, which have been associated with decreased mean daily gain in suckling beef calves. In late 2017, T. orientalis Ikeda was detected in beef cattle from multiple counties in Virginia and was associated with anemia, weakness, late term abortions and death. As of 2022, T. orientalis Ikeda has been identified in beef cattle in 31 of 95 Virginia counties. Beef production, typically in naturally bred cow-calf operations, is the second largest agricultural commodity in Virginia. Central bull testing programs for performance evaluation and marketing of beef bulls has existed for over 60 years in Virginia. T. orientalis Ikeda was first detected at the Southwest bull test station in 2020 when screened at conclusion of the test. The objective of this study was to determine if subclinical infection with T. orientalis Ikeda affected the average daily gain (ADG) ratios of all bulls on test and the achievement of a satisfactory rating of the breeding soundness exam (BSE) for senior bulls.
Theileria orientalis Ikeda infection does not negatively impact growth performance or breeding soundness exam results in young beef bulls at bull test stations
Guynn, Sierra R.; Greiner, Scott P.; Currin, John F.; Todd, S. Michelle; Assenga, Alphonce; Hungerford, Laura L.; Lahmers, Kevin K. (Frontiers, 2024-07-18)
Introduction: Theileria orientalis Ikeda genotype is an emerging cattle disease in the US. Since 2017, when T. orientalis Ikeda was discovered in beef cattle in two counties in Virginia, cattle infections have risen to include ~67% of Virginia counties and 14 states. Consistent with New Zealand studies, many infected herds in Virginia were >90% positive upon initial testing without overt evidence of infection. Central bull tests present a unique opportunity to study the effects of T. orientalis Ikeda infections, as bulls from multiple source herds are consolidated. The objective of this study was to determine if infection with T. orientalis Ikeda affected the average daily gain (ADG), adjusted yearling weight (AYW) and breeding soundness of bulls at two test stations in Virginia over a period of years. Materials and methods: The bulls were fed and housed similarly to compare their growth performance and breeding soundness. For T. orientalis Ikeda testing, DNA was extracted from whole blood for quantitative polymerase chain reaction. Results: The number of bulls infected with T. orientalis Ikeda at initial delivery to the stations increased significantly over the years studied. Multivariable linear regression models, using Angus bulls from Virginia test stations, indicated no significant effect on ADG or AYW in bulls that became test positive during the test or were positive for the duration, compared to Angus bulls that were negative for the duration. At LOC A, the odds of passing a breeding soundness exam (BSE) were not significantly different for bulls that turned positive during the test or were positive for the duration, compared to bulls that were negative for the duration of the test. At LOC B, bulls that became positive during the test were 2.4 times more likely (95% CI: 1.165–4.995, p = 0.016) to pass their BSE compared to bulls that remained negative throughout the test. Discussion: We do not suppose that an obscured infection of T. orientalis Ikeda is protective for bulls to pass a BSE. However, this study demonstrates an obscured infection of T. orientalis Ikeda does not negatively affect weight gain or achievement of a satisfactory BSE rating at the central bull test stations in Virginia.
NS2B-D55E and NS2B-E65D Variations Are Responsible for Differences in NS2B-NS3 Protease Activities Between Japanese Encephalitis Virus Genotype I and III in Fluorogenic Peptide Model
Wahaab, Abdul; Zhang, Yan; Liu, Ke; Rasgon, Jason L.; Kang, Lei; Hameed, Muddassar; Li, Chenxi; Anwar, Muhammad Naveed; Zhang, Yanbing; Shoaib, Anam; Li, Beibei; Qiu, Yafeng; Wei, Jianchao; Ma, Zhiyong (MDPI, 2024-11-26)
Japanese encephalitis virus (JEV) NS2B-NS3 is a protein complex composed of NS3 proteases and an NS2B co-factor. The N-terminal protease domain (180 residues) of NS3 (NS3(pro)) interacts directly with a central 40-amino acid hydrophilic domain of NS2B (NS2B(H)) to form an active serine protease. In this study, the recombinant NS2B(H)-NS3(pro) proteases were prepared in E. coli and used to compare the enzymatic activity between genotype I (GI) and III (GIII) NS2B-NS3 proteases. The GI NS2B(H)-NS3(pro) was able to cleave the sites at the internal C, NS2A/NS2B, NS2B/NS3, and NS3/NS4A junctions that were identical to the sites proteolytically processed by GIII NS2B(H)-NS3(pro). Analysis of the enzymatic activity of recombinant NS2B(H)-NS3(pro) proteases using a model of fluorogenic peptide substrate revealed that the proteolytical processing activity of GIII NS2B(H)-NS3(pro) was significantly higher than that of GI NS2B(H)-NS3(pro). There were eight amino acid variations between GI and GIII NS2B(H)-NS3(pro), which may be responsible for the difference in enzymatic activities between GI and GIII proteases. Therefore, recombinant mutants were generated by exchanging the NS2B(H) and NS3(pro) domains between GI and GIII NS2B(H)-NS3(pro) and subjected to protease activity analysis. Substitution of NS2B(H) significantly altered the protease activities, as compared to the parental NS2B(H)-NS3(pro), suggesting that NS2B(H) played an essential role in the regulation of NS3(pro) protease activity. To further identify the amino acids responsible for the difference in protease activities, multiple substitution mutants including the individual and combined mutations at the variant residues 55 and 65 of NS2B(H) were generated and subjected to protease activity analysis. Replacement of NS2B-55 and NS2B-65 of GI to GIII significantly increased the enzymatic activity of GI NS2B(H)-NS3(pro) protease, whereas mutation of NS2B-55 and NS2B-65 of GIII to GI remarkably reduced the enzymatic activity of GIII NS2B(H)-NS3(pro) protease. Overall, these data demonstrated that NS2B-55 and NS2B-65 variations in the hydrophilic domain of NS2B co-contributed to the difference in NS2B(H)-NS3(pro) protease activities between GI and GIII. However, it will be crucial to explore these mutations in other in vivo and/or in vitro models. Collectively, these observations will be useful for understanding the replication of JEV GI and GIII viruses.
Spatial Transcriptomics and Single-Nucleus Multi-Omics Analysis Revealing the Impact of High Maternal Folic Acid Supplementation on Offspring Brain Development
Xu, Xiguang; Lin, Yu; Yin, Liduo; Serpa, Priscila da Silva; Conacher, Benjamin; Pacholec, Christina; Carvallo, Francisco; Hrubec, Terry; Farris, Shannon; Zimmerman, Kurt; Wang, Xiaobin; Xie, Hehuang (MDPI, 2024-11-07)
Background: Folate, an essential vitamin B9, is crucial for diverse biological processes, including neurogenesis. Folic acid (FA) supplementation during pregnancy is a standard practice for preventing neural tube defects (NTDs). However, concerns are growing over the potential risks of excessive maternal FA intake. Objectives/Methods: Here, we employed a mouse model and spatial transcriptomic and single-nucleus multi-omics approaches to investigate the impact of high maternal FA supplementation during the periconceptional period on offspring brain development. Results: Maternal high FA supplementation affected gene pathways linked to neurogenesis and neuronal axon myelination across multiple brain regions, as well as gene expression alterations related to learning and memory in thalamic and ventricular regions. Single-nucleus multi-omics analysis revealed that maturing excitatory neurons in the dentate gyrus (DG) are particularly vulnerable to high maternal FA intake, leading to aberrant gene expressions and chromatin accessibility in pathways governing ribosomal biogenesis critical for synaptic formation. Conclusions: Our findings provide new insights into specific brain regions, cell types, gene expressions and pathways that can be affected by maternal high FA supplementation.

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