Quantitative Transcriptase-polymerase Chain Reaction Research Articles

Cyclooxygenase upregulation in cytomegalovirus-infected gingival fibroblasts: Implications for periodontal disease

ObjectiveTo assess the changes in the expression of COX-1 and COX-2 in primary gingival fibroblasts upon infection with two strains of human cytomegalovirus (CMV). MethodsPrimary human gingival fibroblasts (HGFs) were cultured and infected with two strains of human CMV the laboratory strain (Towne) and a clinical isolate (B52) at a multiplicity of infection of 0.5. The relative mRNA levels of COX-1 and COX-2 were evaluated using multiplex reverse transcriptase quantitative polymerase chain reaction with specific hydrolysis probes. Additionally, immunofluorescence was performed for COX-2, IE1, and IE2 viral proteins. ResultsAt 24 h post infection, CMV infection of HGFs resulted in a decrease in COX-1 transcripts in cells infected with both the Towne strain and B52 clinical isolate, and in non-infected cells. Conversely, COX-2 transcripts increased in infected cells (Towne and B52); they were significantly higher in B52 infected cells, similar to the immunofluorescence detection results for COX-2. ConclusionsInfection of HGFs with CMV increased in both mRNA and protein expression of COX-2. Furthermore, the B52 isolate induced higher COX-2 expression than the Towne laboratory strain. This study provides a basis for understanding the putative relationships between CMV infection and inflammatory responses in gingival diseases.

The comparison of decay rates of infectious SARS-CoV-2 and viral RNA in environmental waters and wastewater

The comparison of decay rates of infectious SARS-CoV-2 and viral RNA in environmental waters and wastewater

Hypoxia and Giardia duodenalis: The Role of Hypoxia-Inducible Factor in Altered Epithelial Glucose Metabolism

The gastrointestinal system experiences frequent oxygenic fluctuations and must be able to maintain barrier integrity during periods of suboptimal oxygen concentration, or hypoxia. Epithelial cells rely on the Hypoxia-Inducible Factor (HIF) complex to activate genes that combat cellular stress and adapt cellular metabolism during hypoxia. Importantly, protozoan parasites can modulate host tissue oxygen tension and HIF activation, yet little is known regarding the relationship between enteric protozoa and hypoxia. This research aims to uncover the role of HIF upon Giardia duodenalis infection, a top cause of global diarrheal disease and an excellent infection model for the study of GI physiology. We hypothesize that HIF-target genes are activated upon Giardia infection, promoting alternative cellular glucose metabolism to sustain bioenergetic homeostasis. Caco-2 colonic epithelial cells were infected with Giardia isolate GS/M (MOI 10) for 1.5 or 4.5 hours under normoxic (21% O2) or hypoxic (~1%O2, STEMCELL hypoxia incubator) conditions to capture the early or peak activation of the oxygen-dependent HIF subunit (HIF-1α). RNA was extracted for assessment of transcriptional alterations of HIF-target genes via reverse transcriptase quantitative polymerase chain reaction. Investigation of HIF-mediated intracellular metabolic changes was carried out via liquid-chromatography mass-spectrometry analysis (hydrophilic-interaction chromatography method) on cocultures supplemented with a hypoxia mimetic (DMOG) or a HIF inhibitor (PX-478). Metabolomics experiments were repeated using Caco-2 cells transfected with sodium-dependent glucose cotransporter 1 (SGLT1), a key glucose transporter in the small intestine where Giardia localizes which is not reliably expressed in Caco-2 cells. Under normoxic conditions, genes that aid in cell stress responses ( VEGFA, ANKRD37, GADD45A) and glycolysis ( HK2, LDHA) are upregulated in Giardia-infected cells in a time-dependent manner (p<0.05). Fewer HIF-target genes are upregulated under hypoxic conditions (e.g., VEGFA, GADD45A, PGK1; p<0.05), indicating Giardia-infected cells exhibit a transcriptional profile similar to hypoxic uninfected cells. Interestingly, HIF1α was upregulated in Giardia-infected cells under hypoxic conditions at 1.5 hours (p<0.05). Analysis of the Caco-2 intracellular metabolome indicated HIF-dependent changes to nucleic acid (e.g., guanosine, inosine, uracil) and amino acid (e.g., glycine, threonine) metabolism. DMOG treatment increased the abundance of glycolytic intermediates (e.g., PEP, DHAP) in both cell lines and inhibited the depletion of the Kreb’s Cycle intermediate aconitate in the Caco-2-SGLT1 transfected cells, confirming the promotion of glycolytic flux by HIF. Taken together, our findings indicate Giardia-infected cells illustrate a hypoxic signature, a novel metabolic cell rescue mechanism in response to enteropathogens. Elucidating the role of HIF during enteric parasitic infections will aid in our understanding of the cellular adaptations underpinning GI pathophysiology, while also shedding light on the potential of HIF as a therapeutic target. Natural Science and Engineering Research Council of Canada. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Serum miR-124a and miR-34a as Potential Biomarkers for Rheumatoid Arthritis

Abstract Background: MicroRNAs (miRs) are defined as noncoding small RNAs that are involved in the regulation of various immune functions, indicating they could be possible biomarkers for immune-mediated disorders. We aimed to evaluate miR-124a and miR-34a levels in serum as diagnostic biomarkers for rheumatoid arthritis (RA) and to investigate their correlation with the disease activity in RA patients. Methods: Our study consisted of 40 patients with RA and 40 controls. The disease activity for the RA patients was evaluated using the Disease Activity Score in 28 joints (DAS28). Relative quantification of miR-124a and miR-34a expressions in serum was conducted by reverse transcriptase quantitative real-time polymerase chain reaction. Results: Expression levels of miR-124a and miR-34a in serum were significantly lower in RA patients (median: 0.64 and 0.30, respectively) compared to controls (median: 3.12 and 1.88, respectively), P < 0.001. Their serum levels were negatively associated with disease activity and inversely correlated with DAS28, C-reactive protein, and erythrocyte sedimentation rate (P < 0.001). Conclusion: Serum miR-124a and miR-34a could serve as potential noninvasive biomarkers for RA diagnosis and reflect the disease activity in RA patients.

Phytochemical screening, gas chromatograph/mass spectrometer (GCMS) analysis and molecular toxicological potential of Hunteria umbellata aqueous fruit extract against Staphylococcus aureus in accessory gene regulators (AGRs)

BackgroundFruits of Hunteria umbellata (HU) have been utilized in folk medicine as potent against Staphylococcus aureus (SA) infections, particularly skin and nasal related conditions. However, there is scarcity of literature concerning toxicological evaluation of graded doses of HU fruit, especially at molecular level, specifically targeting the accessory gene regulator (AGR) system to prevent abuse of doses in the treatment of bacterial infections. This research evaluated molecular toxicological property of SA exposed to varied concentrations of aqueous HU fruit extract ranging from 500 to 3.90625 µg/µL using broth microdilution method and quantification of AGR I and II genes’ expression employing two-step reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). We first identified quality and quantity of chemical compounds in aqueous fruit of HU using phytochemistry and hybrid gas chromatograph–mass spectrometer (GC–MS) technique. Additionally, preliminary bactericidal potential of HU was assessed before molecular toxicology.ResultsResults revealed six phytochemicals and twenty analytical grade compounds from a standard library were identified from chromatograms of HU fruit extract. Some main compounds detected are n-Hexadecanoic acid (25.24%), 2-Pentadecanone, 6,10,14-trimethyl (16.08%), Cuparene (16.63%), Tetradecanoic acid (6.21%) and 9-Octadecenoic acid, (E)- (5.70%). Bactericidal activity shows significant (p < 0.05) toxicity in the tested (8) concentrations of HU fruits in a dose-response relationship compared to the controls (positive and negative). The quantified expression of AGR I and II genes in SA was most significantly increased (p < 0.05) at both 250 and 500 µg/µL of HU fruit extract while least significant increase (p < 0.05) was recorded at 125 µg/µL compared to control.ConclusionsNotably, the study highlighted a potential risk of augmented bacterial infection especially with higher doses of HU extracts during boils’ treatment and other epidermal infections instigated by Staph. Expression of both AGR genes at higher doses (250 and 500 µg/µL) is indicative of further expression of several other genes responsible for virulence factors in a variety of human bacterial infections. Thus, consumption of HU fruit to treat boils and blisters should be with great caution especially at high doses.

Titanium Surfaces with a Laser-Produced Microchannel Structure Enhance Pre-Osteoblast Proliferation, Maturation, and Extracellular Mineralization In Vitro.

The clinical success of dental titanium implants is profoundly linked to implant stability and osseointegration, which comprises pre-osteoblast proliferation, osteogenic differentiation, and extracellular mineralization. Because of the bio-inert nature of titanium, surface processing using subtractive or additive methods enhances osseointegration ability but limits the benefit due to accompanying surface contamination. By contrast, laser processing methods increase the roughness of the implant surface without contamination. However, the effects of laser-mediated distinct surface structures on the osteointegration level of osteoblasts are controversial. The role of a titanium surface with a laser-mediated microchannel structure in pre-osteoblast maturation remains unclear. This study aimed to elucidate the effect of laser-produced microchannels on pre-osteoblast maturation. Pre-osteoblast human embryonic palatal mesenchymal cells were seeded on a titanium plate treated with grinding (G), sandblasting with large grit and acid etching (SLA), or laser irradiation (L) for 3-18 days. The proliferation and morphology of pre-osteoblasts were evaluated using a Trypan Blue dye exclusion test and fluorescence microscopy. The mRNA expression, protein expression, and protein secretion of osteogenic differentiation markers in pre-osteoblasts were evaluated using reverse transcriptase quantitative polymerase chain reaction, a Western blot assay, and a multiplex assay, respectively. The extracellular calcium precipitation of pre-osteoblast was measured using Alizarin red S staining. Compared to G- and SLA-treated titanium surfaces, the laser-produced microchannel surfaces enhanced pre-osteoblast proliferation, the expression/secretion of osteogenic differentiation markers, and extracellular calcium precipitation. Laser-treated titanium implants may enhance the pre-osteoblast maturation process and provide extra benefits in clinical application.

Influence of storage solution, temperature, assay time and concentration on RT-qPCR nucleic acid detection for SARS-CoV-2 detection of SARS-CoV-2 by the RT-qPCR

Real-time reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is an important method for the early diagnosis of coronavirus disease 2019 (COVID-19). This study investigated the effects of storage solution, temperature and detection time on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid detection by RT-qPCR. Various concentrations of SARS-CoV-2 were added to inactive and non-inactive storage solution and the viral suspensions were stored at various temperatures (room temperature, 4, −20 and −80 °C). Then, at five different detection time points, the Ct values were determined by RT-qPCR. Active and inactive storage solutions and storage temperature have a great impact on the detection of N gene of SARS-CoV-2 at different concentration corridors but have little impact on the ORF gene. The storage time has a greater impact on the N gene and ORF gene at high concentrations but has no effect on the two genes at low concentrations. In conclusion, storage temperature, storage time and storage status (inactivated, non-inactivated) have no effect on the nucleic acid detection of SARS-CoV-2 at the same concentration. For different concentrations of SARS-CoV-2, the detection of N gene is mainly affected.

Identification of reference genes and analysis of heat shock protein gene expression (Hsp90) in arta (Calligonum comosum L.) leaf under heat stress

In southern Tunisia, plants are subjected to high temperatures and disturbances, causing the degradation of soils and plants. Studying wild plants and their response to heat stress can facilitate their conservation and help ecosystem rehabilitation. Understanding heat stress responses has gradually become a hotspot in desert plant research. The adaptive responses of Calligonum comosum species to high temperature were examined in this regard. For C.comosum gene expression studies, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is the best technique, in addition to the use of appropriate reference genes. So, in our research, we analyzed four different housekeeping genes to select the most stable one during heat stress conditions. To analyze the results, we applied various statistical programs, including GeNorm, Normfinder, Bestkeeper, and the comparative ΔCt method, to identify the most appropriate reference genes for normalizing gene expression under heat stress conditions. Ultimately, our analysis revealed that α-tubulin (α-tub) emerged as the most stable reference gene.Additionally, we subjected C. comosum plants to thermal stress to elucidate the expression patterns of heat shock protein (hsp90) genes. Fourteen-day-old plants were exposed to a temperature of 60 °C for a period ranging from 30 to 240 min. The highest level of hsp90 gene expression was realized upon exposure to heat for 180 min, underscoring the upregulation of this protein in response to thermal stress.This study is the first systematic investigation for selecting reference genes for qPCR in C. comosum under heat stress. These findings will substantially benefit gene expression studies conducted under similar challenging conditions, contributing to our understanding of desert plant responses to heat stress.

Prevalence and Molecular Analysis of Encephalomyocarditis Virus-2 in the Hazel Dormouse.

The hazel dormouse (Muscardinus avellanarius) population in the UK continues to decline due to habitat loss, despite reintroductions of captive-bred individuals being conducted nationally for over 30years. Disease surveillance of captive-bred and wild dormice is performed to identify novel and existing disease threats which could impact populations. In this study, we firstly investigated cause of death in seven hazel dormice found dead in England, through next-generation sequencing identifying a virus closely related to a wood mouse encephalomyocarditis virus-2 (EMCV-2). Subsequently, lung tissue samples from 35 out of 44 hazel dormice tested positive for EMCV-2 RNA using a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and Sanger sequencing methods developed in this study. Formalin-fixed tissues available for nine hazel dormice which tested positive for EMCV-2 RNA were examined microscopically. Three cases showed moderate interstitial pneumonia with minimal to mild lymphoplasmacytic myocarditis, but no evidence of encephalitis. However, the presence of possible alternative causes of death in these cases means that the lesions cannot be definitively attributed to EMCV-2. Here, we report the first detection of EMCV-2 in hazel dormice and conclude that EMCV-2 is likely to be endemic in the hazel dormouse population in England and may be associated with clinical disease.

Selection, identification and evaluation of optimal reference genes in Chinese sturgeon (Acipenser sinensis) under polypropylene microplastics stress

Polypropylene microplastics (PP-MPs) are emerging environmental contaminants that have the potential to cause adverse effects on aquatic organisms. Reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) is a valuable tool for assessing the gene expression profiles under PP-MPs stress. To obtain an accurate gene expression profile of tissue inflammation and apoptosis that reflects the molecular mechanisms underlying the impact of PP-MPs on Chinese sturgeon, identifying reliable reference genes is crucial for RT-qPCR analysis. In this study, we constructed an experiment model of Chinese sturgeon exposed to PP-MPs, assessed the pathological injury, metabolic profile responses and oxidative stress in liver, evaluated the reliability of 8 reliable reference genes by 4 commonly used algorithms including GeNorm, NormFinder, BeatKeeper, Delta Ct, and then analyzed the performance of inflammatory response genes in liver, spleen and kidney with the best reference gene. HE staining revealed that the cytoplasm full small vacuoles and nucleus diameter increased were occurred in the liver cell of PP-MPs in treatment groups. Additionally, oxidative and biochemical parameters were significantly changes in the liver of treatment groups. For the reference genes in PP-MPs exposure experiments, this study screening the optimal reference genes including: EF1α and GAPDH for liver and spleen, and GAPDH and RPS18 for kidney. Besides, 2 inflammatory response genes (NLRP3, TNF-α) were chosen to assess the optimal reference genes using the least stable reference gene (TUB) as a control, verified the practicality of the select reference genes in different tissues. We also found that the low concentration of PP-MPs could induce the liver tissue damage and inflammatory response in Chinese sturgeon. Our study initially evaluated the impact of short-time exposure with PP-MPs in Chinese sturgeon and provided 3 sets of validated optimal reference genes in Chinese sturgeon exposure to PP-MPs.

A181 EXPLORING THE ROLE OF KLEBSIELLA VARIICOLA AND KLEBSIELLA PNEUMONIAE IN PEDIATRIC ULCERATIVE COLITIS PATHOGENESIS

Abstract Background Recent studies indicated that Klebsiella (K) pneumoniae (isolated from the stool of IBD patients) and K. variicola (isolated from the mesenteric tissue of Crohn disease patients) have the potential to induce inflammation in epithelial and preadipocyte cells, exacerbating colitis in murine models. Aims We isolated these strains from pediatric ulcerative colitis (UC) patients' appendix (relevant to UC pathogenesis given the protective effects of appendectomy) and other non-inflamed colon sections. We hypothesized that these isolates are invasive and induce inflammation in UC. These strains have not been previously studied in UC. Methods K. variicola and K. pneumoniae were isolated from two pediatric UC patients' appendices and other non-inflamed colon sections and identified using 16S DNA Sanger sequencing. Virulence of the two strains was determined by infecting Caco2 cell lines and performing adhesion and invasion assays, quantifying biofilm formation, and assessing barrier functions using transepithelial electrical resistance (TEER) measurement. Importantly, we monitored the production of pro-inflammatory (e.g., IL-8, TNF-α) and anti-inflammatory (IL-10) cytokines using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Results K. variicola and K. pneumoniae exhibited high levels of biofilm formation compared to adherent-invasive Escherichia coli (AIEC), which are commonly isolated from IBD patients. Furthermore, the Klebsiella strains adhered to epithelial cells within 2-3 hours post infection. TEER experiments showed compromised barrier integrity after 6 hours and overnight infection. Except for K. pneumoniae isolated from the ascending colon, the tested strains exhibited a significant increase in IL-8 expression. Similarly, K. variicola from the peri-appendicular region demonstrated elevated TNF-α gene expression. Conclusions K. variicola and K. pneumoniae isolated from UC patients have the potential to form biofilms, disrupt barrier integrity, and trigger inflammatory responses. These findings unravel a potential role for pathogenic Klebsiella strains in driving UC pathogenesis. Importantly, these data shed light on the role of appendix-associated bacteria in the development of UC. Future work includes using comparative genomics to map the virulence determinants of these bacteria. Funding Agencies CIHRWomen and Children's Health Research Institute (WCHRI)

Seven-year prospective study on yearly incidence of Orbivirus infection of captive white-tailed deer and potential Culicoides vectors.

Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) are arthropod-borne viruses that are transmitted by biting midges in the genus Culicoides (Diptera: Ceratopogonidae) and can cause hemorrhagic disease in certain ruminants. The objectives of this study were to measure the incidence of BTV and EHDV infections in captive white-tailed deer herd as well as tissues and corresponding presence of Culicoides midges at a location near Clinton, LA. During a 7-yr study with yearly outbreaks of hemorrhagic disease in the deer herd, 15 species of Culicoides were captured using Centers for Disease Control (CDC) black light traps. Reverse transcriptase quantitative polymerase chain reaction (PCR) was performed to screen for BTV and EHDV in pools of midges and tissues of deer. From 2012 to 2018, 1,711 pools of midges representing 24,859 specimens were tested, and specimens from 5 of the 15 collected species (Culicoides debilipalpis, Culicoides stellifer, Culicoides venustus, Culicoides haematopotus, and Culicoides crepuscularis) were found to be PCR positive for BTV and EHDV. Most of the BTV-positive pools of biting midges were from specimens of C. debilipalpis and C. stellifer, and most of the EHDV-positive pools were from specimens of C. venustus and C. stellifer. During the 7-yr period, 112 white-tailed deer that died at the study location were PCR positive for BTV or EHDV: detected BTV serotypes were 10 and 12 and EHDV serotypes were 1, 2, and 6. There was a significant increase in BTV/EHDV antibody prevalence in white-tailed deer during the study; antibody-positive rates increased from 15% to 78% in the deer herd of approximately 100 animals.

Eco-epidemiology of Rickettsia amblyommatis and Rickettsia parkeri in naturally infected ticks (Acari: Ixodida) from South Carolina

BackgroundSpotted fever group Rickettsia (SFGR) is the largest group of Rickettsia species of clinical and veterinary importance emerging worldwide. Historically, SFGR cases were linked to Rickettsia rickettsii, the causal agent of Rocky Mountain spotted fever; however, recently discovered species Rickettsia parkeri and Rickettsia amblyommatis have been shown to cause a wide range of clinical symptoms. The role of R. amblyommatis in SFGR eco-epidemiology and the possible public health implications remain unknown.MethodsThis study evaluated statewide tick surveillance and land-use classification data to define the eco-epidemiological relationships between R. amblyommatis and R. parkeri among questing and feeding ticks collected across South Carolina between 2021 and 2022. Questing ticks from state parks and feeding ticks from animal shelters were evaluated for R. parkeri and R. amblyommatis using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) on pooled samples. A Bayesian multivariable logistic regression model for pool testing data was used to assess associations between R. parkeri or R. amblyommatis infection and land-use classification variables among questing ticks. The Spearman correlation was used to evaluate the relationship between the two tested pathogens.ResultsThe infection prevalence for R. amblyommatis was 24.8% (23.4–26.3%) among questing ticks, and 39.5% (37.4–42.0%) among feeding ticks; conversely, for R. parkeri it was 19.0% (17.6–20.5%) among questing ticks and 22.4% (20.3–24.5%) among feeding ticks. A negative, refractory correlation was found between the species, with ticks significantly more likely to contain one or the other pathogen, but not both simultaneously. The Bayesian analysis revealed that R. amblyommatis infection was positively associated with deciduous, evergreen, and mixed forests, and negatively associated with hay and pasture fields, and emergent herbaceous wetlands. Rickettsia parkeri infection was positively associated with deciduous, mixed, and evergreen forests, herbaceous vegetation, cultivated cropland, woody wetlands, and emergent herbaceous wetlands, and negatively associated with hay and pasture fields.ConclusionsThis is the first study to evaluate the eco-epidemiological factors driving tick pathogenicity in South Carolina. The negative interactions between SFGR species suggest the possible inhibition between the two pathogens tested, which could have important public health implications. Moreover, land-use classification factors revealed environments associated with tick pathogenicity, highlighting the need for tick vector control in these areas.Graphical

P1238 Exploring the Role of Klebsiella variicola and Klebsiella pneumoniae in Pediatric Ulcerative Colitis Pathogenesis

Abstract Background Recent studies indicated that Klebsiella (K) pneumoniae (isolated from the stool of IBD patients) and K. variicola (isolated from the mesenteric tissue of Crohn disease patients) have the potential to induce inflammation in epithelial and preadipocyte cells, exacerbating colitis in murine models. We isolated these strains from pediatric ulcerative colitis (UC) patients' appendix (relevant to UC pathogenesis given the protective effects of appendectomy) and other non-inflamed colon sections. We hypothesized that these isolates are invasive and induce inflammation in UC. These strains have not been previously studied in UC. Methods K. variicola and K. pneumoniae were isolated from two pediatric UC patients' appendices and other non-inflamed colon sections and identified using 16S DNA Sanger sequencing. Virulence of the two strains was determined by infecting Caco2 cell lines and performing adhesion and invasion assays, quantifying biofilm formation, and assessing barrier functions using transepithelial electrical resistance (TEER) measurement. Importantly, we monitored the production of pro-inflammatory (e.g., IL-8, TNF-α) and anti-inflammatory (IL-10) cytokines using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Results K. variicola and K. pneumoniae exhibited high levels of biofilm formation compared to adherent-invasive Escherichia coli (AIEC), which are commonly isolated from IBD patients. Furthermore, the Klebsiella strains adhered to epithelial cells within 2-3 hours post infection. TEER experiments showed compromised barrier integrity after 6 hours and overnight infection. Except for K. pneumoniae isolated from the ascending colon, the tested strains exhibited a significant increase in IL-8 expression. Similarly, K. variicola from the peri-appendicular region demonstrated elevated TNF-α gene expression. Conclusion K. variicola and K. pneumoniae isolated from UC patients have the potential to form biofilms, disrupt barrier integrity, and trigger inflammatory responses. These findings unravel a potential role for pathogenic Klebsiella strains in driving UC pathogenesis. Importantly, these data shed light on the role of appendix-associated bacteria in the development of UC. Future work includes using comparative genomics to map the virulence determinants of these bacteria.

Utilization of temperature-mediated activation of thyroid hormone-induced molecular memory to evaluate early signaling events in the olfactory epithelium of Rana [Lithobates] catesbeiana tadpoles

The amphibian olfactory system is highly distinct between aquatic tadpole and terrestrial frog life stages and therefore must remodel extensively during thyroid hormone (TH)-dependent metamorphosis. Developmentally appropriate functioning of the olfactory epithelium is critical for survival. Previous studies in other Rana [Lithobates] catesbeiana premetamorphic tadpole tissues showed that initiation of TH-induced metamorphosis can be uncoupled from execution of TH-dependent programs by holding tadpoles in the cold rather than at warmer permissive temperatures. TH-exposed tadpoles at the nonpermissive (5 °C) temperature do not undergo metamorphosis but retain a “molecular memory” of TH exposure that is activated upon shift to a permissive warm temperature. Herein, premetamorphic tadpoles were held at permissive (24 °C) or nonpermissive (5 °C) temperatures and injected with 10 pmoles/g body weight 3,5,3′-triiodothyronine (T3) or solvent control. Olfactory epithelium was collected at 48 h post-injection. RNA-sequencing (RNA-Seq) and reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) analyses generated differentially expressed transcript profiles of 4328 and 54 contigs for permissive and nonpermissive temperatures, respectively. Translation, rRNA, spliceosome, and proteolytic processes gene ontologies were enriched by T3 treatment at 24 °C while negative regulation of cell proliferation was enriched by T3 at 5 °C. Of note, as found in other tissues, TH-induced basic leucine zipper-containing protein-encoding transcript, thibz, was significantly induced by T3 at both temperatures, suggesting a role in the establishment of molecular memory in the olfactory epithelium. The current study provides critical insights by deconstructing early TH-induced induction of postembryonic processes that may be targets for disruption by environmental contaminants.

MiR-335 Improves Functional Recovery in Rats After Spinal Cord Injury and Protects PC12 Cells Against Injury Via the SPI-Bax/Caspase-3 Axis.

Animal and cellular models of spinal cord injury (SCI) were used to explore the role of miR-335 in regulating cell viability and apoptosis. To investigate the role and the target of miR-335 in SCI. Based on analysis of the GSE19890 data set, miR-335 was identified as a downregulated microRNA (miRNAs) following SCI. Thus, this study investigated whether downregulation of miR-335 is important in the pathological process of SCI. The GSE19890 data set investigating the expression profiles of miRNAs after SCI was downloaded from the GEO database. GSE45006 and GSE4550 data sets were used to identify differentially expressed genes between normal samples and SCI samples. The targets of rno-miR-335 were predicted using the TargetScan database.An experimental model of SCI was established, and agomir-miR-335 was intrathecally injected into rats with SCI. Functional recovery was evaluated by assessment of Basso-Beattie-Bresnahan scores and spinal cord water content and performing hematoxylin-eosin staining. Apoptosis was estimated by TUNEL staining. The H 2 O 2 -treated PC12 cells were used as in vitro models of SCI. Cell viability and apoptosis were examined by cell counting kit-8 and flow cytometry. Caspase-3 expression was evaluated by immunofluorescence staining. Levels of miRNAs and mRNAs were measured by reverse transcriptase quantitative polymerase chain reaction. Western blotting was performed to measure Bcl-2, Bax, cleaved caspase-3, and specificity protein 1 (SP1) protein levels. For in vivo studies, miR-335 was downregulated following SCI, and agomir-miR-335 delivery improved functional recovery through suppressing neuronal apoptosis by inactivating the SP1-Bax/Bcl-2/caspase-3 signaling. During in vitro analysis, miR-335 protected PC12 cells against H 2 O 2 -induced damage by negatively regulating the SP1-Bax/Bcl-2/caspase-3 signaling axis. Moreover, upregulation of SP1 abolished the apoptosis suppressive effects of miR-335 upregulation. MiR-335 ameliorates locomotor impairment in rats with SCI through the suppression of neuronal apoptosis by inactivating SP1-Bax/Bcl-2/caspase-3 signaling.

The pyroptosis mediated biomarker pattern: an emerging diagnostic approach for Parkinson’s disease

BackgroundParkinson’s disease (PD) affects 1% of people over 60, and long-term levodopa treatment can cause side effects. Early diagnosis is of great significance in slowing down the pathological process of PD. Multiple pieces of evidence showed that non-coding RNAs (ncRNAs) could participate in the progression of PD pathology. Pyroptosis is known to be regulated by ncRNAs as a key pathological feature of PD. Therefore, evaluating ncRNAs and pyroptosis-related proteins in serum could be worthy biomarkers for early diagnosis of PD.MethodsNcRNAs and pyroptosis/inflammation mRNA levels were measured with reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Luciferase assays were performed to confirm GSDME as a target of miR-675-5p and HMGB1 as a target of miR-1247-5p. In the serum of healthy controls (n = 106) and PD patients (n = 104), RT-qPCR was utilized to assess miR-675-5p, miR-1247-5p, and two related ncRNAs (circSLC8A1and lncH19) levels. The enzyme-linked immunosorbent assay measured serum levels of pyroptosis-related proteins in controls (n = 54) and PD patients (n = 70).ResultsOur data demonstrated that miR-675-5p and miR-1247-5p significantly changed in PD neuron and animal models. Overexpressed miR-675-5p or downregulated miR-1247-5p could regulate pyroptosis and inflammation in PD neuron models. Using the random forest algorithm, we constructed a classifier based on PD neuron-pyroptosis pathology (four ncRNAs and six proteins) having better predictive power than single biomarkers (AUC = 92%). Additionally, we verified the performance of the classifier in early-stage PD patients (AUC ≥ 88%).ConclusionSerum pyroptosis-related ncRNAs and proteins could serve as reliable, inexpensive, and non-invasive diagnostic biomarkers for PD.LimitationsAll participants were from the same region. Additionally, longitudinal studies in the aged population are required to explore the practical application value of the classifier.

Apple cider vinegar exhibits promising antibiofilm activity against multidrug-resistant Bacillus cereus isolated from meat and their products.

Bacillus cereus (B. cereus) biofilm is grown not only on medical devices but also on different substrata and is considered a potential hazard in the food industry. Quorum sensing plays a serious role in the synthesis of biofilm with its surrounding extracellular matrix enabling irreversible connection of the bacteria. The goal of the current investigation was to ascertain the prevalence, patterns of antimicrobial resistance, and capacity for B. cereus biofilm formation in meat and meat products in Egypt. In all, 150 meat and meat product samples were used in this study. For additional bacteriological analysis, the samples were moved to the Bacteriology Laboratory. Thereafter, the antimicrobial, antiquorum sensing, and antibiofilm potential of apple cider vinegar (ACV) on B. cereus were evaluated. Out of 150 samples, 34 (22.67%) tested positive for B. cereus. According to tests for antimicrobial susceptibility, every B. cereus isolates tested positive for colistin and ampicillin but negative for ciprofloxacin and imipenem. The ability to form biofilms was present in all 12 multidrug-resistant B. cereus isolates (n = 12); of these, 6 (50%), 3 (25%), and 3 (25%) isolates were weak, moderate, and strong biofilm producers, respectively. It is noteworthy that the ACV demonstrated significant inhibitory effects on B. cereus isolates, with minimum inhibitory concentrations varying between 2 and 8 μg/ml. Furthermore, after exposing biofilm-producing B. cereus isolates to the minimum biofilm inhibitory concentrations 50 of 4 μg/ml, it demonstrated good antibiofilm activity (>50% reduction of biofilm formation). Strong biofilm producers had down-regulated biofilm genes (tasA and sipW) and their regulator (plcR) compared to the control group, according to reverse transcriptase quantitative polymerase chain reaction analysis. Our study is the first report, that spotlights the ACV activity against B. cereus biofilm and its consequence as a strong antibacterial and antibiofilm agent in the food industry and human health risk.

Amelioration of Oxidative Stress in Rats with Chronic Obstructive Pulmonary Disease through Shenqi Huatan Decoction Activation of Peroxisome Proliferator-Activated Receptor Gamma-Mediated Activated Protein Kinase/Forkhead Transcription Factor O3a Signaling Pathway.

Chronic obstructive pulmonary disease (COPD) is a common respiratory disease. Currently, no specific treatment strategy has been established; therefore, finding new treatment methods is essential. Clinically, Shenqi Huatan Decoction (SQHT) is a traditional Chinese medicinal formula for COPD treatment; however, its mechanism of action in treatment needs to be clarified. The COPD rat model was replicated by cigarette smoking and tracheal injection using the LPS method. The control group and the SQHT groups were treated with dexamethasone and SQHT by gavage, respectively. After treatment, superoxide dismutase (SOD) serum levels, total antioxidant capacity (TAOC), lipid peroxidation, and malondialdehyde (MDA) were detected by enzyme-linked immunosorbent assay (ELISA). Activated protein kinase alpha (AMPK-α), forkhead transcription factor O3a (FOXO3a), manganese SOD (MnSOD), and peroxisome proliferator-activated receptor gamma (PPARγ) were detected using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and Western blot. Microribonucleic acid and protein expression levels were measured, and pathological changes in lung tissue were observed using hematoxylin and eosin staining. The pathological findings suggested that SQHT substantially affects COPD treatment by enhancing alveolar fusion and reducing emphysema. ELISA results showed that SQHT could lower the blood levels of MDA and lipid peroxide and raise SOD and TAOC levels, suggesting that it could lessen oxidative stress. In the lung tissue of rats with COPD, large doses of SQHT intervention dramatically increased AMPK protein expression, AMPK-α, FOXO3a, MnSOD, and PPARγ, indicating that SQHT may reduce oxidative stress by activating the PPARγ-mediated AMPK/FOXO3a signaling pathway. Similar results were obtained using RT-qPCR. SQHT is effective for COPD treatment. The mechanism of action may be related to the activation of the PPARγ-mediated AMPK/FOXO3a signaling pathway to improve oxidative stress in lung tissue.

Association of Demographic, Clinical, and Vaccination Characteristics with COVID-19 Viral Load Assessed by qRT-PCR.

The effect of vaccination on the SARS-CoV-2 baseline viral load and clearance during COVID-19 infection is debatable. This study aimed to assess the effects of demographic and vaccination characteristics on the viral load of SARS-CoV-2. We included the patients referred for outpatient SARS-CoV-2 qRT-PCR (reverse transcriptase quantitative polymerase chain reaction) test between July and September 2022. Cycle threshold (Ct) data were compared based on the demographic and vaccination characteristics. A generalized linear model was used to determine the factors associated with the SARS-CoV-2 PCR Ct value. Of 657 participants, 390 (59.4%) were symptomatic and 308 (47.1%) were COVID-19 positive. Among 590 individuals with known vaccination status, 358 (60.6%) were booster vaccinated, 193 (32.6%) were fully vaccinated, 13 (2.2%) were partially vaccinated, and 26 (4.4%) were unvaccinated. Most vaccinated patients received inactivated vaccines (70.5%). The median Ct value was 20 [IQR: 18-23.75] with no significant difference between individuals with different vaccination statuses (P value = 0.182). There were significant differences in Ct value in terms of both symptom presence and onset (both P values < 0.001). Our regression model showed that inactivated vaccines (P value = 0.027), mRNA vaccines (P value = 0.037), and the presence and onset of symptoms (both P values < 0.001) were independent factors significantly associated with the viral load. The SARS-CoV-2 baseline viral load is unaffected by vaccination status, yet vaccination might accelerate viral clearance. Furthermore, we demonstrated that the presence and onset of symptoms are independent variables substantially associated with the patient's viral load.