Expression Of mRNA Transcripts Research Articles

USP34 regulates endothelial PAR1 mRNA transcript expression and cellular signaling.

Signaling by G protein-coupled receptors (GPCRs) is regulated by temporally distinct processes including receptor desensitization, internalization, and lysosomal sorting, and are tightly controlled by post-translational modifications. While the role of phosphorylation in regulating GPCR signaling is well studied and established, the mechanisms by which other post-translational modifications, such as ubiquitination, regulate GPCR signaling are not clearly defined. We hypothesize that GPCR ubiquitination and deubiquitination is critical for proper signaling and cellular responses. In the present study, we show that the deubiquitinase ubiquitin-specific protease-34 (USP34) regulates thrombin-stimulated protease-activated receptor-1 (PAR1)-induced p38 autophosphorylation and activation. The PAR1-stimulated p38 signaling pathway is driven by ubiquitination. Interestingly, small interfering RNA-induced knockdown of USP34 expression markedly increased PAR1 cell surface abundance and protein expression without modulating PAR1 ubiquitination or the ubiquitination status of p38 signaling pathway components. In addition, increased PAR1 expression observed in USP34-depleted cells was not caused by altered PAR1 constitutive internalization, agonist-induced internalization, or receptor degradation. Rather, we report that loss of USP34 expression increased mRNA transcript expression of the PAR1-encoding gene, F2R. This study unexpectedly identified a critical role for USP34 in regulation of F2R mRNA transcript expression, which modulates PAR1 cell surface levels and thrombin-stimulated p38 mitogen-activated protein kinase signaling.

P53 Guselkumab binding to CD64+ IL-23–producing myeloid cells enhances potency for neutralizing IL-23 signalling

Abstract IL-23 is implicated in the pathogenesis of psoriasis (PsO), and myeloid cells expressing FcγRI (CD64) are the primary source of IL-23 in lesional PsO skin tissue. The incidence and prevalence of psoriatic arthritis increases with PsO severity, and joint disease activity is positively correlated with frequency of peripheral CD64+ monocytes. We evaluated: CD64 and IL-23 expression in PsO patient skin biopsies; binding of anti-IL-23p19 subunit IgG1 monoclonal antibodies (mAbs) guselkumab [(GUS) fully human, native Fc-region] and risankizumab [(RZB) humanized, mutated Fc-region] to CD64; and functional consequences of CD64 binding by anti-IL-23p19 subunit mAbs in in vitro assays. CD64, IL-23p19 and IL-23p40 subunits mRNA transcripts were analysed from bulk and single-cell RNAseq datasets. Binding of mAbs to IFNγ-primed human monocytes and IL-23–secreting inflammatory monocytes, and capture of endogenously secreted IL-23, were evaluated by flow cytometry. Internalization of IL-23 and GUS/RZB within CD64+ macrophages was assessed with live-cell confocal imaging. GUS and RZB potency for inhibiting IL-23 was determined in a co-culture of THP-1 cells (CD64+ monocyte cell line activated to produce IL-23) and an IL-23 reporter cell line (measuring biologically active IL-23). IL-23p19 mRNA transcript expression in the co-culture was measured by qPCR. Expression of CD64, IL-23p19, and IL-23p40 mRNA transcripts was increased in lesional vs. non-lesional PsO skin, as were the abundance of myeloid cell types co-expressing CD64/IL-23p19 mRNA transcripts. In in vitro assays, GUS (not RZB) demonstrated Fc-mediated binding to CD64 on IFNγ-primed monocytes. Moreover, CD64-bound GUS simultaneously captured IL-23 secreted from the same cells. GUS (not RZB) bound to the CD64+ macrophages and mediated IL-23 internalization to low pH intracellular compartments. GUS and RZB equipotently inhibited signalling by IL-23 present in THP-1-conditioned medium. However, in a co-culture of IL-23–producing THP-1 cells with an IL-23–responsive reporter cell line, GUS demonstrated enhanced potency vs. RZB for inhibiting IL-23 signalling. GUS did not alter IL-23p19 mRNA transcript expression in the co-culture. These findings were consistent with previous observations of CD64+ myeloid cells as a key source of IL-23 in lesional PsO skin tissue. GUS binding to CD64 on IL-23-producing cells likely contributed to the enhanced potency of GUS vs. RZB in inhibiting IL-23 signalling in the co-culture assay. These in-vitro data support a hypothesis for optimal localization of GUS in inflamed tissues, where CD64+ IL-23-producing myeloid cells are increased and in proximity to IL-23-responsive lymphoid cells, enhancing GUS neutralization of IL-23 at its source of production.

DNA tetrahedron nanoparticles service as a help carrier and adjvant of mRNA vaccine

Aim of the studyTo investigate the potential of DNA nanoparticles (DNPs) as carriers and adjuvants for mRNA vaccines.Materials and methodsCustomized oligonucleotides were assembled into DNA tetrahedra (DNA-TH), which were subsequently complexed with streptavidin and mRNA encoding green fluorescent protein (GFP). Various assays were conducted to evaluat the stability of the DNPs, their cellular uptake, immune activation potential, and GFP mRNA transcription efficiency. P53-mutant HSC-3 cells were used to establish a subcutaneous xenograft tumor model to explore the effects of DNPs as carriers and adjuvants in a disease model.ResultsThe DNPs were remained stable extracellularly and rapidly taken up by antigen-presenting cells. Compared to naked GFP mRNA, DNPs statistically significantly activated immune responses and facilitated GFP mRNA transcription and protein expression both in vitro and in vivo. Immunization with DNP-GFP mRNA complexes induced higher antibody titers compared to naked mRNA. The DNPs demonstrated good biocompatibility. DNP-p53 inhibited the growth of subcutaneous xenograft tumors in mice with p53-mutant HSC-3 cells, outperforming both the naked p53 mRNA and blank control groups, with a statistically significant difference (P < 0.05).ConclusionDNA nanoparticles show promise for improving mRNA vaccine delivery and efficacy. Further optimization of these nanoparticles could lead to highly effective mRNA vaccine carriers with broad applications.

Increased susceptibility to diet-induced obesity in female mice impairs ovarian steroidogenesis: The role of elevated leptin signalling on nodal activity inhibition in theca cells

ObjectivesSusceptibility to obesity in humans is driven by the intricate interplay of genetic, environmental and behavioural factors. Moreover, the mechanisms linking maternal obesity to infertility remain largely understudied. In this study, we investigated how variable susceptibility to obesity in mice affects ovarian steroidogenesis, with a particular focus on the leptin-mediated dysregulation of Nodal signalling pathway in theca cells (TC). MethodsC56BL/6J (B6) and 129S1/SvlmJ (129) mice, models of maternal obesity (MO), were fed a chow diet (CD) and a high fat diet (HFD) for 16 weeks. To investigate the contrasting effects of leptin on ovarian steroidogenesis, B6 mice pharmacologically treated with leptin for 16 days on CD were used to model hyperleptinemia, while homozygous ob/ob (−/−) mice with genetic leptin deficiency, also on a CD, were used to examine the effects of obesity in the absence of leptin. Following the characterisation of the mouse phenotype, gonadal fat (GON), whole ovaries (WO), ovarian TC and granulosa cell (GC) fractions were collected for mRNA transcription and protein expression analysis. Finally, in vitro treated ovarian explants obtained from B6 mice were used to further elucidate the effects of Nodal on steroidogenesis. ResultsThe significant gain in body weight (BW) and fat mass (FM) in HFD-fed B6 mice (p < 0.05), was associated with increased mRNA transcription of the adipose tissue expansion genes Polymerase I and transcript release factor (Cavin), Secreted frizzled-related protein 5 (Sfrp5) and Mesoderm specific transcript (Mest) in GON (p < 0.05). Furthermore, the HFD-fed B6 mice presented also impaired glucose metabolism and insulin sensitivity (p < 0.05). In contrast, the HFD-fed 129 mice exhibited no changes in BW and FM, maintaining glucose and insulin metabolism. At the ovarian level, decreased protein expression of Steroidogenic Acute Regulatory Protein (StAR) in WO obtained from HFD-fed B6 mice (p = 0.05), was followed by reduced transcription of key steroidogenic genes like Star and Cytochrome P450 17a1 (Cyp17a) in TC (p < 0.05). Furthermore, the transcription of Nodal and its receptors was downregulated (p < 0.05), whereas mRNA levels of Suppressor of cytokine signalling 3 (Socs3) and SMAD family member 7 (Smad7) were upregulated in TC obtained from HFD-fed B6 mice (p < 0.05). No changes were seen in the genes regulating steroidogenesis, Nodal signalling, or Socs3 and Smad7 activity in the ovaries of HFD-fed 129 mice. Importantly, the pharmacological treatment of lean mice with leptin, upregulated the ovarian transcription of Socs3 and Smad7, while downregulating Nodal and its receptors (p < 0.05). Finally, in vitro pharmacological inhibition of Nodal signalling pathway in ovarian explants isolated from CD-fed B6 mice decreased the transcription of Star and Cyp17a in TC (p < 0.05), whereas Nodal treatment of explants obtained from HFD-fed B6 mice restored the transcription of both genes (p < 0.05). ConclusionsIncreased susceptibility to obesity in MO is associated with systemic hyperleptinemia and hypoestrogenism due to compromised ovarian steroidogenesis, largely driven by the inhibitory effects of leptin-Smad7 pathway on Nodal signalling activity in the TC compartment of ovarian follicles.

8302 A Novel Tool to Map Estrogen Responsiveness Within Estrogen Sensitive Hypothalamic Neurons in Mice

Abstract Disclosure: A.L. Cara: None. A.L. Hall: None. K.K. Soma: None. J. van Veen: None. S.M. Correa: None. Identification of hormone sensitive cells has been traditionally evaluated by hormone receptor expression. Receptor positive cells can be labeled via expression of mRNA transcript via in situ hybridization, presence of protein via immunohistology, or via conditionally-dependent Cre recombinase driven reporter expression. However, these methods used to label and manipulate cells only indicate which cells are capable of sensing hormones, and not necessarily which cells are responding to a hormonal signal in a specific physiologic or behavioral context. Furthermore, broad labeling of all receptor positive cells may not accurately reflect the heterogeneous nature of certain cell populations, particularly in the brain. The hypothalamus is a brain region with well characterized hormone receptor expression, including estrogen receptors, such as ERα (encoded by the Esr1 gene). Estrogens have a broad array of physiologic and behavioral functions which are mediated through hypothalamic nuclei, including thermoregulation, metabolism, and reproduction. Neuronal populations mediating these functions have been characterized, targeted, and manipulated based on ERα expression, but the dynamic response to estrogens in these highly heterogeneous populations has yet to be uncovered. We developed a custom reporter virus that fluorescently labels estrogen-sensitive and responsive cells. The virus contains a flip-excision switch (FLEX) to drive Cre-dependent expression of a FusionRed fluorescent reporter, eight tandem estrogen response elements (ERE), and a destabilized enhanced green fluorescent protein (EGFP) with a 2-hour half-life. The Cre-dependent expression of FusionRed identifies all Esr1-Cre cells at the time of transduction, while the ERE-EGFP identifies a subset of Esr1-Cre+ cells that are actively responding to estrogens via nuclear receptor signaling. We stereotactically injected the ERE-EGFP virus to distinct hypothalamic nuclei of Esr1-Cre female mice. Following transduction, mice were sacrificed during different stages of the estrous cycle. Spatial analysis of ERE-EGFP expression revealed dynamic changes across the estrous cycle, with responses differing based on hypothalamic nucleus. Reporter expression dynamics were compared to estrogen levels measured by LC-MS/MS in ovary, serum, and microdissected brain tissue. A separate group of mice were ovariectomized and given estradiol benzoate (EB) or vehicle control, and neurons were flow sorted for bulk RNA sequencing. We discovered transcriptomic differences in FusionRed+ cells with high or low ERE-EGFP expression, including upregulation of estrogen target genes (e.g., Pgr, encoding progesterone receptor) with EB treatment in ERE-EGFP+ cells. In summary, this new viral reporter reveals divergent responses to estrogens across subsets of estrogen sensitive neurons, brain regions, and hormonal states. Presentation: 6/2/2024

Chromatin Accessibility Plays an Important Epigenetic Role on Antibody Expression From CMV Promoter and DNA Elements Flanking the CHO TI Host Landing-Pad.

Targeted integration (TI) Chinese hamster ovary (CHO) platforms are commonly used for protein expression. However, the impact of epigenetic modifications on protein expression in TI cell lines remains elusive since almost all the epigenetic studies focus on random integration (RI) of the gene of interest and only within the promoter region. To address the impact of epigenetic modifications on TI CHO cells, we utilized a standard mAb-1 to identify and characterize TI clones with the same transgene copy numbers but different levels of transgene transcription and titer. Surprisingly, while CMV promoters were not methylated and histone acetylation/methylation was present, these epigenetic markers did not trend with mRNA transcription and protein expression in our TI model. Instead, ATAC-seq data analysis revealed that differences in chromatin accessibility within the TI site could be a major factor impacting these observed differences. However, neither chromatin accessibility nor histone acetylation/methylation profiles in early cultures were predictive of high-expressing clones early during the CLD process. Finally, modulation of the histone profiles (H3K27ac and H3K4me3) at the CMV promoters within the TI integration site using dCas9 fusion proteins was not effective in further increasing mAb titers which could have been likely due to interference of the dCas9 fusion proteins with transcription from the CMV promoters. Overall, our data suggests increasing chromatin accessibility at the TI site is the most effective way to increase mRNA transcription and hence, productivity in TI cell lines.

Whole-transcriptome analyses of ovine lung microvascular endothelial cells infected with bluetongue virus

Bluetongue virus (BTV) infection induces profound and intricate changes in the transcriptional profile of the host to facilitate its survival and replication. However, there have been no whole-transcriptome studies on ovine lung microvascular endothelial cells (OLMECs) infected with BTV. In this study, we comprehensively analysed the whole-transcriptome sequences of BTV-1 serotype-infected and mock-infected OLMECs and subsequently performed bioinformatics differential analysis. Our analysis revealed 1215 differentially expressed mRNA transcripts, 82 differentially expressed long noncoding RNAs (lncRNAs) transcripts, 63 differentially expressed microRNAs (miRNAs) transcripts, and 42 differentially expressed circular RNAs (circRNAs) transcripts. Annotation from Gene Ontology, enrichment from the Kyoto Encyclopedia of Genes and Genomes, and construction of endogenous competing RNA network analysis revealed that the differentially expressed RNAs primarily participated in viral sensing and signal transduction pathways, antiviral and immune responses, inflammation, and extracellular matrix (ECM)-related pathways. Furthermore, protein‒protein interaction network analysis revealed that BTV may regulate the conformation of ECM receptor proteins and change their biological activity through a series of complex mechanisms. Finally, on the basis of real-time fluorescence quantitative polymerase chain reaction results, the expression trends of the differentially expressed RNA were consistent with the whole-transcriptome sequencing data, such as downregulation of the expression of COL4A1, ITGA8, ITGB5, and TNC and upregulation of the expression of CXCL10, RNASEL, IRF3, IRF7, and IFIHI. This study provides a novel perspective for further investigations of the mechanism of the ECM in the BTV-host interactome and the pathogenesis of lung microvascular endothelial cells.

Inheritance and ecological effects of exogenous genes from transgenic Brassica napus to Brassica juncea hybrids

●With the rapid development of new breeding techniques, the ecological impacts of transgenic plants receive wide concern again, particularly for potential gene flow from transgenic crops to their relatives. The transgene insertion position, number of gene copies, and flanking sequence of exogenous genes integrated into the recipient genome affect the genetic stability and fitness of offspring.●We employed hybrids F1 and F2, six backcross generations BC1-BC6 and BC1F1 from transgenic Brassica napus with Bacillus thuringiensis (Bt) cry1Ac gene and its wild relative B. juncea through hand pollination. We detected exogenous gene copies, mRNA transcription, and protein expression by ddPCR, qRT-PCR and ELISA, and the fitness of hybrid and backcross generations.●Exogenous genes followed Mendelian segregation expectations in hybrid and backcrossed generations, with stable gene copies, mRNA transcription and protein concentration of exogenous genes in offspring. Exogenous gene copies had no significant effects on plant fitness, but had positive effects on mRNA transcription and protein concentration that varied with growth stages in hybrid and backcross generations.●Our study demonstrated inheritance and ecological effects of exogenous genes from transgenic plants to wild relatives, which will help ecological risk management of biotechnological plants released in the nature.

Abstract We142: Identification of the Molecular Determinants for the Circadian Regulation of the Human KCNH2 promoter

Background: KCNH2 encodes a voltage-gated potassium channel (Kv11.1), which conducts the rapid delayed rectifier potassium current (IKr) critical for ventricular repolarization in the heart. Circadian clock factors regulate the expression of the KCNH2 mRNA transcript in the heart. However, the molecular mechanisms for the circadian expression of KCNH2 transcripts have yet to be defined. Hypothesis: A conserved cis-regulatory element in the core KCNH2 promoter drives circadian regulation of human KCNH2 (hKCNH2) promoter activity. Objective: Identify the molecular determinants of the circadian regulation of hKCNH2 promoter activity and determine the impact of single nucleotide polymorphisms (SNPs) in this region. Methods: Real-time bioluminescence (LumiCycle, Actimetrics) and Dual Glo luciferase assays were employed to identify critical cis elements that regulate the circadian expression of the human KCNH2 promoter using promoter-luciferase reporter constructs. We studied several deletion and mutant hKCNH2 promoter reporter vectors transiently transfected in a myogenic cell line (C2C12 cells). Bioluminescence was measured at 10-minute intervals for 7-10 days. Data were analyzed for circadian characteristics (period, phase and amplitude). Mutant constructs included both engineered and constructs containing naturally occurring rare and common SNPs. Results: Lumicycle data demonstrated that the hKCNH2 promoter reporter showed robust circadian and overall activity in C2C12 cells. Deletion and mutational analysis of the hKCNH2 promoter-reporter construct identified a highly conserved tandem E-box element within 1 Kb of the exon 1 start site that was critical for circadian and overall regulation of hKCNH2 promoter activity. Cells expressing hKCNH2 promoter-reporter constructs with different SNPs in this tandem E-box element showed SNP-specific effects. Surprisingly, a relatively common SNP decreased hKCNH2 circadian promoter activity by 66% (n = 6, p=0.0049). Conclusion: We identified a conserved tandem E-box in the proximal promoter of hKCNH2 Exon1 necessary for circadian and overall hKCNH2 promoter activity. Several SNPs in this region suggested a functional impact that might lead to decreased levels of IKr.

Maintenance of taste receptor cell presynaptic sites requires gustatory nerve fibers.

The turnover and re-establishment of peripheral taste synapses is vital to maintain connectivity between the primary taste receptor cells and the gustatory neurons which relay taste information from the tongue to the brain. Despite the importance of neuron-taste cell reconnection, mechanisms governing synapse assembly and the specificity of synaptic connections is largely unknown. Here we use the expression of presynaptic proteins, CALHM1 and Bassoon, to probe whether nerve fiber connectivity is an initiating factor for the recruitment of presynaptic machinery in different populations of taste cells. Under homeostatic conditions, the vast majority (>90%) of presynaptic sites are directly adjacent to nerve fibers. In the days immediately following gustatory nerve transection and complete denervation, Bassoon and CALHM1 puncta are markedly reduced. This suggests that nerve fiber innervation is crucial for the recruitment and maintenance of presynaptic sites. In support of this, we find that expression of Bassoon and Calhm1 mRNA transcripts are significantly reduced after denervation. During nerve fiber regeneration into the taste bud, presynaptic sites begin to replenish, but are not as frequently connected to nerve fibers as intact controls (∼50% compared to >90%). This suggests that gustatory neuron proximity, rather than direct contact, likely drives taste receptor cells to express and aggregate presynaptic proteins at the cell membrane. Together, these data support the idea that trophic factors secreted by gustatory nerve fibers prompt taste receptor cells to produce presynaptic specializations at the cell membrane, which in turn may guide neurons to form mature synapses. These findings provide new insights into the mechanisms driving synaptogenesis and synaptic plasticity within the rapidly changing taste bud environment.

NECA alleviates inflammatory responses in diabetic retinopathy through dendritic cell toll-like receptor signaling pathway.

This study examined the impact of 5'-(N- ethylcarboxamido)adenosine (NECA) in the peripheral blood of healthy individuals, those with diabetes mellitus, diabetic retinopathy (DR), and C57BL/6 mice, both in vivo and in vitro. Enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FCM) were used to evaluate the effects of NECA on dendritic cells (DCs) and mouse bone marrow-derived dendritic cells (BMDCs) and the effects of NECA-treated DCs on Treg and Th17 cells. The effect of NECA on the Toll-like receptor (TLR) pathway in DCs was evaluated using polymerase chain reaction (PCR) and western blotting (WB). FCM and ELISA showed that NECA inhibited the expression of surface markers of DCs and BMDCs, increased anti-inflammatory cytokines and decreased proinflammatory cytokines. PCR and WB showed that NCEA decreased mRNA transcription and protein expression in the TLR-4-MyD88-NF-kβ pathway in DCs and BMDCs. The DR severity in streptozocin (STZ) induced diabetic mice was alleviated. NECA-treated DCs and BMDCs were co-cultivated with CD4+T cells, resulting in modulation of Treg and Th17 differentiation, along with cytokine secretion alterations. NECA could impair DCs' ability to present antigens and mitigate the inflammatory response, thereby alleviating the severity of DR.

Placental growth factor promotes neural invasion and predicts disease prognosis in resectable pancreatic cancer

BackgroundSurgery represents the only curative treatment option for pancreatic ductal adenocarcinoma (PDAC), but recurrence in more than 85% of patients limits the success of curative-intent tumor resection. Neural invasion (NI), particularly the spread of tumor cells along nerves into extratumoral regions of the pancreas, constitutes a well-recognized risk factor for recurrence. Hence, monitoring and therapeutic targeting of NI offer the potential to stratify recurrence risk and improve recurrence-free survival. Based on the evolutionary conserved dual function of axon and vessel guidance molecules, we hypothesize that the proangiogenic vessel guidance factor placental growth factor (PlGF) fosters NI. To test this hypothesis, we correlated PlGF with NI in PDAC patient samples and functionally assessed its role for the interaction of tumor cells with nerves.MethodsSerum levels of PlGF and its soluble receptor sFlt1, and expression of PlGF mRNA transcripts in tumor tissues were determined by ELISA or qPCR in a retrospective discovery and a prospective validation cohort. Free circulating PlGF was calculated from the ratio PlGF/sFlt1. Incidence and extent of NI were quantified based on histomorphometric measurements and separately assessed for intratumoral and extratumoral nerves. PlGF function on reciprocal chemoattraction and directed neurite outgrowth was evaluated in co-cultures of PDAC cells with primary dorsal-root-ganglia neurons or Schwann cells using blocking anti-PlGF antibodies.ResultsElevated circulating levels of free PlGF correlated with NI and shorter overall survival in patients with PDAC qualifying for curative-intent surgery. Furthermore, high tissue PlGF mRNA transcript levels in patients undergoing curative-intent surgery correlated with a higher incidence and greater extent of NI spreading to tumor-distant extratumoral nerves. In turn, more abundant extratumoral NI predicted shorter disease-free and overall survival. Experimentally, PlGF facilitated directional and dynamic changes in neurite outgrowth of primary dorsal-root-ganglia neurons upon exposure to PDAC derived guidance and growth factors and supported mutual chemoattraction of tumor cells with neurons and Schwann cells.ConclusionOur translational results highlight PlGF as an axon guidance factor, which fosters neurite outgrowth and attracts tumor cells towards nerves. Hence, PlGF represents a promising circulating biomarker of NI and potential therapeutic target to improve the clinical outcome for patients with resectable PDAC.Graphical

An Evaluation of High-Risk HPV in Squamous Cell Carcinomas of the Lip in a South African Cohort

BackgroundTo determine the prevalence of HR-HPV in a series of lip SCC from South African patients, using currently accepted HPV-testing methodologies and to define the clinical and histomorphologic features of HPV-associated lip SCC.MethodsFifty SCC of lip and 50 control cases were tested for HR-HPV using p16 and HR-HPV DNA PCR. p16-equivocal/positive and HPV DNA PCR-positive SCC were further evaluated for the expression of HPV-16 and HPV-18 mRNA transcripts using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to confirm transcriptionally active HPV.Resultsp16 was positive in 22% (n = 11) and equivocal in 4% (n = 2) of the SCC. One p16-positive case showed positivity for both HPV-16 DNA and HPV-16 E6/E7 mRNA transcripts (HPV prevalence rate of 2%). The HPV-positive case was non-keratinizing and occurred in an 80-year-old female. The two p16-equivocal cases were HR-HPV DNA positive and mRNA PCR negative. p16 was found to have a positive predictive value of 9%.ConclusionFindings from our cohort of lip SCC suggest that HR-HPV may have an insignificant role in the pathogenesis of SCC at this site. Due to its low ppv, p16 is insufficient to establish HR-HPV infection in SCC of the lip. The combination of p16 and DNA PCR appears to correlate with the presence of transcriptionally active virus. HPV E6/E7 mRNA detection is the gold standard for identifying HR-HPV. mRNA testing is not widely available in sub-Saharan Africa due to technical and financial constraints; however, the test appears to be of great value in p16-equivocal lip SCC.

Deciphering Microbiome, Transcriptome, and Metabolic Interactions in the Presence of Probiotic Lactobacillus acidophilus against Salmonella Typhimurium in a Murine Model.

Salmonella enterica serovar Typhimurium (S. Typhimurium), a foodborne pathogen that poses significant public health risks to humans and animals, presents a formidable challenge due to its antibiotic resistance. This study explores the potential of Lactobacillus acidophilus (L. acidophilus 1.3251) probiotics as an alternative strategy to combat antibiotic resistance associated with S. Typhimurium infection. In this investigation, twenty-four BALB/c mice were assigned to four groups: a non-infected, non-treated group (CNG); an infected, non-treated group (CPG); a group fed with L. acidophilus but not infected (LAG); and a group fed with L. acidophilus and challenged with Salmonella (LAST). The results revealed a reduction in Salmonella levels in the feces of mice, along with restored weight and improved overall health in the LAST compared to the CPG. The feeding of L. acidophilus was found to downregulate pro-inflammatory cytokine mRNA induced by Salmonella while upregulating anti-inflammatory cytokines. Additionally, it influenced the expression of mRNA transcript, encoding tight junction protein, oxidative stress-induced enzymes, and apoptosis-related mRNA expression. Furthermore, the LEfSe analysis demonstrated a significant shift in the abundance of critical commensal genera in the LAST, essential for maintaining gut homeostasis, metabolic reactions, anti-inflammatory responses, and butyrate production. Transcriptomic analysis revealed 2173 upregulated and 506 downregulated differentially expressed genes (DEGs) in the LAST vs. the CPG. Functional analysis of these DEGs highlighted their involvement in immunity, metabolism, and cellular development. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis indicated their role in tumor necrosis factor (TNF), mitogen-activated protein kinase (MAPK), chemokine, Forkhead box O (FOXO), and transforming growth factor (TGF-β) signaling pathway. Moreover, the fecal metabolomic analysis identified 929 differential metabolites, with enrichment observed in valine, leucine, isoleucine, taurine, glycine, and other metabolites. These findings suggest that supplementation with L. acidophilus promotes the growth of beneficial commensal genera while mitigating Salmonella-induced intestinal disruption by modulating immunity, gut homeostasis, gut barrier integrity, and metabolism.

Panax notoginseng saponins prevent dementia and oxidative stress in brains of SAMP8 mice by enhancing mitophagy

BackgroundMitochondrial dysfunction is one of the distinctive features of neurons in patients with Alzheimer’s disease (AD). Intraneuronal autophagosomes selectively phagocytose and degrade the damaged mitochondria, mitigating neuronal damage in AD. Panax notoginseng saponins (PNS) can effectively reduce oxidative stress and mitochondrial damage in the brain of animals with AD, but their exact mechanism of action is unknown.MethodsSenescence-accelerated mouse prone 8 (SAMP8) mice with age-related AD were treated with PNS for 8 weeks. The effects of PNS on learning and memory abilities, cerebral oxidative stress status, and hippocampus ultrastructure of mice were observed. Moreover, changes of the PTEN-induced putative kinase 1 (PINK1)-Parkin, which regulates ubiquitin-dependent mitophagy, and the recruit of downstream autophagy receptors were investigated.ResultsPNS attenuated cognitive dysfunction in SAMP8 mice in the Morris water maze test. PNS also enhanced glutathione peroxidase and superoxide dismutase activities, and increased glutathione levels by 25.92% and 45.55% while inhibiting 8-hydroxydeoxyguanosine by 27.74% and the malondialdehyde production by 34.02% in the brains of SAMP8 mice. Our observation revealed the promotion of mitophagy, which was accompanied by an increase in microtubule-associated protein 1 light chain 3 (LC3) mRNA and 70.00% increase of LC3-II/I protein ratio in the brain tissues of PNS-treated mice. PNS treatment increased Parkin mRNA and protein expression by 62.80% and 43.80%, while increasing the mRNA transcription and protein expression of mitophagic receptors such as optineurin, and nuclear dot protein 52.ConclusionPNS enhanced the PINK1/Parkin pathway and facilitated mitophagy in the hippocampus, thereby preventing cerebral oxidative stress in SAMP8 mice. This may be a mechanism contributing to the cognition-improvement effect of PNS.

Urinary Mitochondrial DNA Induces an Inflammatory Response in Peripheral Blood Mononuclear Cells

Background: Mitochondria are semiautonomous cell organelles having its own nucleic acid. Mitochondrial DNA (Mt-DNA) remain in hypomethylated (CpG) state and impose an immunogenic response by binding to the toll-like receptor (TLR-9) through the NF-kB pathway. Innate immune cells recognize the hypomethylated pattern of mt-DNA and quickly trigger the innate immune response. The immunomodulatory effects of urinary mt-DNA derived from renal transplant recipients with COVID-19-associated acute kidney injury (AKI) have not been studied. Materials and Methods: Healthy donor peripheral blood mononuclear cell (PBMC) was cultured with the urinary Mt-DNA derived from the renal transplant recipients, who previously developed SARS-CoV-2 infection associated AKI. Cell activation was measured by the flow cytometry. In cell pellets, interleukin IL-6, IL-10, and Myd88, TLR-9 mRNA transcript expression was measured by the reverse transcription polymerase chain reaction. The IL-6 and IL-10 cytokine levels were measured by the enzyme-linked immunosorbent assay in culture supernatants. Results: The urinary mitochondrial DNA (umt-DNA) significantly induces the activation of &gt; 75% of PBMCs. The m-RNA transcript expression of the inflammatory gene in control versus umt-DNA treated PBMCs was for IL-6 (0.99 ± 0.05 vs. 2.18 ± 1.15 au; P = 0.004), MYD88 was (1.00 ± 0.05 vs. 1.55 ± 0.31; P &lt; 0.001), TLR-9 (1.00 ± 0.05 vs. 3.33 ± 1.37 au; P &lt; 0.001) was upregulated, and the IL-10 (1.00 ± 0.13 vs. −1.73 ± 0.58; P &lt; 0.001) level was downregulated. However, in PBMC culture supernatants, IL-6 level in control versus umt-DNA-treated groups were (37.50 ± 13.79 vs. 186.9 ± 15.50 pg/mL; P &lt; 0.001), which was significantly higher in umt-DNA-treated groups and the IL-10 (8.80 ± 2.16 vs. 7.60 ± 3.12 pg/mL; P = 0.32) level was similar between the control- and umt-DNA-treated groups. Conclusions: Urinary Mt-DNA significantly induces the inflammatory cytokine IL-6 secretion from the PBMCs through the Myd88-dependent pathway.

Association of HLA-E single nucleotide polymorphisms with human myeloid leukemia.

Single nucleotide polymorphisms (SNPs) of HLA-E are related to the occurrence of many diseases, but their functions remain unclear. In this study, the function of SNPs at HLA-E rs76971248 and rs1264457 on the myeloid leukemia cells was analyzed by a progressive procedure, included genotyping, mRNA transcription, regulatory element, protein expression, and anti-tumor effect. The frequencies of rs76971248 G and rs1264457 G were found higher in myeloid leukemia patients than those in healthy blood donors (p < 0.05). For myeloid leukemia, rs76971248 T was protective, while rs1264457 G was susceptible. We also found that rs76971248 affected HLA-E mRNA transcription and membrane HLA-E (mHLA-E) expression in K562 cells through differently binding to transcription factor HOXA5 (p < 0.0001), while rs1264457 affected mHLA-E expression by changing mRNA transcription and an encoding amino acid (p < 0.01). In contrast, the expression of soluble HLA-E (sHLA-E) was not influenced by both rs1264457 and rs76971248. The higher HLA-E expression was detected among myeloid leukemia patients, and the K562 cells with higher HLA-E molecules played a significant inhibitory effect on the killing activity of NK-92MI cells (p < 0.05). In conclusion, the higher HLA-E expression of myeloid leukemia cells is promoted by rs76971248 G and rs1264457 G, which helps escape from NK-92MI cells' killing.

Effects of Viable and Heat-Inactivated Bifidobacterium longum D42 on Proliferation and Apoptosis of HT-29 Human Colon Cancer Cells.

Bifidobacterium longum is a common probiotic; both viable and heat-inactivated Bifidobacterium longum have many probiotic effects, such as anticancer effects. But some mechanisms of anticancer effects are still unclear, especially for heat-inactivated probiotics. In this study, we analyzed the effects of viable and heat-inactivated Bifidobacterium longum D42 on human colon cancer cells (HT-29). Cell proliferation, membrane permeability and apoptosis were detected by using the CCK-8 method, LDH method and Annexin V-FITC/PI kits. The ROS level and mitochondrial membrane potential were examined using the fluorescent probes DCFH-DA and JC-1. Real-time fluorescence quantitative PCR (RT-qPCR) and Western blot were used to detect the expression of mitochondrial apoptosis pathway genes and proteins. The results showed that viable and heat-inactivated Bifidobacterium longum D42 at concentrations of 1 × 106 CFU/mL significantly inhibited the proliferation of and increased the level of LDH release of HT-29 colon cancer cells. We found that they could increase the apoptosis rate of HT-29 cells. Moreover, they could also induce apoptosis by inducing cells to produce ROS and destroying the mitochondrial membrane potential of cells. Further studies found that they could increase the mRNA transcription and protein expression levels of the Caspase-3, Caspase-9 and Bax genes in cells, and reduce the mRNA transcription and protein expression levels of the Bcl-2 gene. In summary, our findings revealed that viable and heat-inactivated Bifidobacterium longum D42 have inhibitory effects on proliferation and promote the apoptosis of human colon cancer cells, and also have certain adjuvant drug therapeutic effects and have potential application value in the adjuvant treatment of colon cancer.

Non-homologous End-joining Genotype, mRNA Expression, and DNA Repair Capacity in Childhood Acute Lymphocytic Leukemia.

The capacity for non-homologous end-joining (NHEJ) repair plays a pivotal role in maintaining genome stability and in carcinogenesis. However, there is little literature on the involvement of NHEJ-related genes in childhood acute lymphocytic leukemia (ALL). Our study aimed to elucidate the impact of polymorphisms of X-ray repair cross-complementing group 4 (XRCC4) (rs6869366, rs2075685, rs2075686, rs28360071, rs3734091, rs28360317, rs1805377), XRCC5 (rs828907, rs11685387, rs9288518), XRCC6 (rs5751129, rs2267437, rs132770, rs132774), XRCC7 rs7003908, and DNA ligase IV (LIG4) rs1805388, on the odds of childhood ALL. Genotypes NHEJ-related genes of 266 cases and 266 controls were determined, and the genotype-phenotype correlation was investigated by examining mRNA transcript expression and the capacity for overall and precise NHEJ repair. The variant genotypes of XRCC4 rs3734091, rs28360071, XRCC5 rs828907, and XRCC6 rs5751129 were significantly associated with increased odds of childhood ALL. Further analysis based on susceptibility genotypes showed no significant differences in mRNA transcript expression levels among childhood ALL cases with various putative high-risk genotypes, except XRCC6 rs5751129. Moreover, the overall NHEJ repair capacity was similar among carriers of different XRCC4, XRCC5, and XRCC6 genotypes. However, it is worth noting that individuals carrying the variant C allele at XRCC6 rs5751129 exhibited lower precise NHEJ repair capacity compared to those with the wild-type T allele. Our study identified significant associations between XRCC4 rs3734091, rs28360071, XRCC5 rs828907, and XRCC6 rs5751129 genotypes and childhood ALL. Notably, lower transcriptional expression and reduced precise NHEJ repair capacity were observed in patients carrying the C allele of XRCC6 rs5751129. Further investigations are required to gain deeper insights into childhood ALL development.

Ningxin-Tongyu-Zishen formula alleviates the senescence of granulosa cells on D-galactose-induced premature ovarian insufficiency mice.

Ningxin-Tongyu-Zishen formula (NTZF) is a clinical experience formula for the treatment of premature ovarian insufficiency (POI) in traditional Chinese medicine (TCM), and the potential mechanism is unknown. For in vivo experiments, POI mouse models (C57BL/6 mice), were constructed by subcutaneous injection of D-galactose (D-gal, 200 mg/kg). After treatment of NTZF (10.14, 20.27, 40.54 g/kg;) or estradiol valerate (0.15 mg/kg), ovarian function, oxidative stress (OS) and protein expression of Sirt1/p53 were evaluated. For in vitro experiments, H2O2 (200 μM) was used to treat KGN to construct ovarian granulosa cells (OGCs) cell senescence model. Pretreatment with NTZF (1.06 mg/mL) or p53 inhibitor (Pifithrin-α, 1 μM) was performed before induction of senescence, and further evaluated the cell senescence, OS, mRNA and protein expression of Sirt1/p53. In vivo, NTZF improved ovarian function, alleviated OS and Sirt1/p53 signaling abnormalities in POI mice. In vitro experiments showed that NTZF reduced the level of OS and alleviated the senescence of H2O2-induced KGN. In addition, NTZF activated the protein expression of Sirt1, inhibited the mRNA transcription and protein expression of p53 and p21. Alleviating OGCs senescence and protecting ovarian function through Sirt1/p53 is one of the potential mechanisms of NTZF in the treatment of POI.