Quantitative Real-time Reverse Transcriptase PCR Research Articles

Antibiotic-Induced Changes in Efflux Transporter Expression: A Key Factor in Pseudomonas aeruginosa Biofilm Resistance

Listed by WHO as an antibiotic-resistant priority pathogen, Pseudomonas aeruginosa (P.A.) is a serious threat in nosocomial infections. Its high antibiotic resistance is attributed to major mechanisms that can be categorized into intrinsic, acquired, and adaptive resistance. This study tests the ability of three commonly used antibiotics to inhibit new biofilm formation and eradicate mature biofilm growth, as well as investigate changes in the expression levels of selected genes coding for multidrug efflux pumps in P.A. planktonic cells and biofilms before and after treatment with antibiotics to provide a conceptual estimate of the activity of the efflux transporters that work to extrude antibiotics leading to a reduction in their effectiveness. Antimicrobial susceptibility testing was conducted with Ofloxacin (OFLX), Tobramycin (TOB), and Ceftazidime (CAZ) to determine Mean Inhibitory Concentration (MIC) and Mean Bactericidal Concentration (MBC) using microtiter plate-based biofilm assay and spectrophotometric quantification. Extraction of total RNA was performed from planktonic cultures, inhibition phase, and eradication phase P.A. biofilms. Real-time quantitative reverse transcriptase PCR was utilized to analyze the changes in expression of the mexAB, mexXY, and oprM genes. Three (3) antibiotics that have proven to show less resistance are OFLX, TOB, and CAZ when tested against overnight cultures of P.A. strain PA01. Results showed that OFLX is best for bactericidal properties, which is also supported by the viability assay data obtained from Propidium Iodide staining. Our study showed that the PAO1 strain is susceptible to OFLX for both inhibition and eradication of mature biofilms. TOB was most effective at higher concentrations in the eradication phase.

In vitro cytotoxic activity on KATO-III cancer cell lines of mangiferolic acid purified from Thai Tetragonula laeviceps propolis

Gastric cancer is a global health concern, but current treatment with chemotherapy and surgery is often inadequate, prompting the exploration of alternative treatments. Propolis is a natural substance collected by bees known for its diverse properties linked to floral sources. The Dichloromethane Partitioned Extract (DPE) from Tetragonula laeviceps propolis, in Bankha district, Thailand was previously shown to possess significant cytotoxicity against KATO-III gastric cancer cells, while showing lower cytotoxicity toward WI-38 normal fibroblast cells. Here, the DPE was further fractionated by column chromatography, identified active fractions, and subjected to structural analysis using nuclear magnetic resonance spectroscopy. Cytotoxicity against KATO-III cells was reevaluated, and programmed cell death was analyzed using flow cytometry. Expression levels of cancer-related genes were measured using quantitative real-time reverse transcriptase PCR. Cardol C15:2 (compound 1) and mangiferolic acid (MF; compound 2) were discovered in the most active fractions following structural analysis. MF exhibited strong cytotoxicity against KATO-III cells (IC50 of 4.78–16.02 μg/mL), although this was less effective than doxorubicin (IC50 of 0.56–1.55 μg/mL). Morphological changes, including decreased cell density and increased debris, were observed in KATO-III cells treated with 30 μg/mL of MF. Significant induction of late-stage apoptosis and necrosis, particularly at 48 and 72 h, suggested potential DNA damage and cell cycle arrest, evidenced by an increased proportion of sub-G1 and S-phase cells. Doxorubicin, the positive control, triggered late apoptosis but caused more necrosis after 72 h. Furthermore, MF at 30 μg/mL significantly increased the expression level of COX2 and NFκB genes linked to inflammation and cell death pathways. This upregulation was consistent at later time points (48 and 72 h) and was accompanied by increased expression of CASP3 and CASP7 genes. These findings suggest MF effectively induces cell death in KATO-III cells through late apoptosis and necrosis, potentially mediated by upregulated inflammation-related genes.

FLNA overexpression promotes papillary thyroid cancer aggression via the FAK/AKT signaling pathway.

Filamin A (FLNA) is a member of the filamin family and has been found to be critical for the progression of several cancers. However, its biological function in papillary thyroid cancer (PTC) remains largely unexplored. Data from The Cancer Genome Atlas (TCGA) databases were utilized to analyze the FLNA expression level and its influence on the clinical implications of patients with PTC. Gene Expression Omnibus (GEO) and qRT-PCR was used to verify the expression levels of FLNA in PTC. Kaplan-Meier survival analysis was conducted to evaluate the prognostic value of FLNA in PTC. Transwell assays and wound healing were performed to examine the biological function of FLNA knockdown in PTC cells. Gene set enrichment analysis (GSEA) and Western blotting were conducted to investigate the potential mechanisms underlying the role of FLNA in PTC progression. In addition, the relationship between FLNA expression and the tumor immune microenvironment (TME) in PTC was explored. FLNA was significantly upregulated in PTC tissues. High expression levels of FLNA was correlated with advanced TNM stage, T stage, and N stage, as well as poor disease-free interval (DFI) and progression-free interval (PFI) time in PTC patients. Moreover, we found that FLNA knockdown inhibited the migration and invasion of PTC cells. Mechanistically, FLNA knockdown inhibited epithelial-mesenchymal transition (EMT) in PTC and affected the activation of the FAK/AKT signaling pathway. In addition, FLNA expression was associated with TME in PTC. FLNA may be regarded as a new therapeutic target for PTC patients.

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.

Comparative analysis of genes expression involved in type II toxin-antitoxin system in Staphylococcus aureus following persister cell formation.

The formation of persister cells is the main reason for persistent infections. They are associated with antibiotic treatment failure and subsequently chronic infection. The study aimed to assess the expression of type II toxin/antitoxin (TA) system genes in persister cells of Staphylococcus aureus in the presence of the following antibiotics vancomycin, ciprofloxacin, and gentamicin in exponential and stationary phases. The colony count was used to evaluate the effect of different types of antibiotics on S. aureus persister cell formation during exponential and stationary phases. Moreover, the expression level of TA systems and clpP genes in the persister population in exponential and stationary phases were measured by quantitative reverse transcriptase real-time PCR (qRT-PCR). The results of the study showed the presence of persister phenotype of S. aureus strains in the attendance of bactericidal antibiotics in comparison to the control group during the exponential and stationary phases. Moreover, qRT-PCR resulted in the fact that the role of TA systems involved in the persister cell formation depends on the bacterial growth phase and the type of strain and antibiotic. In total, the present study provides some data on the persister cell formation and the possible role of TA system genes in this process.

TAZ acting as a potential pathogenic biomarker to promote the development of lichen planus.

Lichen planus is a chronic inflammatory disorder. Transcriptional coactivator with PDZ-binding motif (TAZ/WWTR1) is an important downstream effector of the Hippo pathway which regulates organ size and tissue homeostasis. But little is known about the role of TAZ in lichen planus so far. To explore the expression of TAZ in lichen planus and normal skin, and to discover the relationship between TAZ expression and the clinical characteristics of lichen planus patients. The method of immunohistochemistry was performed to quantify the expression of TAZ in 262 patients with lichen planus and 90 control tissues. Western blot and quantitative real-time reverse transcriptase-PCR (qRT-PCR) analysis were performed to examine and compare TAZ expression in 4 cases of fresh lichen planus lesions and normal skin tissues. TAZ was weakly expressed in the basal layers of the epidermis in normal skin tissues with a positive rate of 52.22% (47/90). But in lichen planus, TAZ was strongly expressed in almost the entire epidermis with a positive rate of 81.30% (213/262), and the difference between the two groups was statistically significant (p<0.05). Additionally, TAZ expression was significantly related to the location of the lichen planus, clinical phenotype, smoking, and alcohol preference (p<0.05). Western blot and qRT-PCR showed that the expression of TAZ in protein and mRNA levels in four cases of lichen planus lesions was significantly higher than that in normal skin tissues. TAZ may play a regulatory role in the occurrence and development of lichen planus, which might provide a new perspective for studying pathogenesis and theoretical treatment targets.

Quantitative analysis of infectious precocity virus load and stunted growth syndrome via an immunohistochemical assay and reverse transcriptase quantitative real-time PCR in Macrobrachium rosenbergii

The giant freshwater prawn Macrobrachium rosenbergii (M. rosenbergii) is susceptible to a flavivirus known as the infectious precocity virus (IPV). The symptoms of infection, such as sexual precocity and stunted growth, collectively constitute what is referred to as iron prawn syndrome (IPS). IPV manifests as a persistent, long-term infection marked by subtle morphological changes that pose challenges for detection. Prawns suspected of early-stage infection do not necessarily present clear clinical signs. This study investigated IPV diagnostics based on clinical signs, immunohistochemistry (IHC), and reverse transcriptase quantitative real-time PCR (RT-qPCR) in prawns suspected of IPV-infection. We also explored the correlations among the three diagnostic approaches. Some clinical signs are easily observable, such as shorter body length, mature gonads, elongated blue claws, blue tailfins, and darker body coloration; however, they are not absolute signs of infection. Additionally, many infected prawns do not exhibit clear clinical signs during the early stages. We produced anti-IPV serum and then applied it via IHC staining to highlight the location of the virus. Positive signals were observed in the perinuclear cytoplasm of haemocytes in the hepatopancreas, gills, and pereiopods. IPV-positive signals were also detected in support cells within the lamina ganglionaris as well as in neurosecretory cells within the bellonci organ, sinus glands, and the X organ. The detection of IPV-positive signals in prawns without clinical signs demonstrates the value of this diagnostic method. IHC analysis was used to distinguish between IPV-positive and IPV-negative prawns, and also used in conjunction with RT-qPCR to quantify viral loads in these two groups. The IPV viral loads were as follows: IHC-positive prawns (> 103.5 copies/ μg) and IHC-negative prawns (< 103.5 copies/ μg). Note that the highest numbers of viral copies were observed in pleopods, which are easily obtained and do not necessitate sacrificing the animal. In conclusion, the correlation between viral load and IHC pathological diagnosis could be used to identify cases of IPV infection, while providing valuable insights into disease prevention and prognosis.

Potential roles of Culicoides spp. (Culicoides imicola, Culicoides oxystoma) as biological vectors of bluetongue virus in Yuanyang of Yunnan, P. R. China.

Culicoides plays a crucial role as an insect vector in the field of veterinary medicine. The transmission of significant viruses such as bluetongue virus (BTV) and African horse sickness virus (AHSV) by this insect poses a substantial threat, leading to the development of severe diseases in domestic animals. This study aimed to explore the Culicoides species, identify their blood-meal sources, and assess the presence of BTV and AHSV carried by Culicoides in Yuanyang County, Yunnan Province. The aim was to gain insights into the potential vectors of these two viruses and elucidate their potential roles in the transmission of pathogens. The midges were collected from cattle (Bos indicus), pig (Sus scrofa), and goat (Capra hircus) pens in Yuanyang County, Yunnan Province in June 2020. Initial identification of midges was conducted through morphological characteristics, followed by molecular identification using the cytochrome C oxidase subunit I (COI) gene. The determination of Culicoides blood-meal sources was accomplished using specific primers targeting the cytochrome b (Cyt b) gene from potential hosts. BTV and AHSV RNA were identified in Culicoides pools through the application of reverse transcriptase PCR and quantitative real-time PCR. Nucleotide homology and phylogenetic analysis were performed using MegAlign (DNAStar) and Mega 6.0 software. A total of 6,300 Culicoides, consisting of C. oxystoma, C. arakawai, C. imicola, and C. innoxius, were collected from cattle, pigs, and goat pens. The engorgement rates for these species were 30.2%, 54.6%, 75%, and 66.7%, respectively. In the cattle pen, the prevailing species is C. oxystoma (100%). In the pig pen, C. arakawai dominates (70%), with C. oxystoma following at 30%. In the goat pen, C. imicola holds the majority (45.45%), trailed by C. oxystoma (25%), C. innoxius (20.45%), and C. arakawai (9.09%). These Culicoides species were identified as feeding on cattle, pigs, goats, chickens (Gallus gallus), and humans (Homo sapiens). The positivity rates for BTV were 20.00% and 11.54% in blood-fed specimens of C. imicola and C. oxystoma, respectively. Conversely, the positivity rates for BTV in non-blood-fed specimens were 0.00% and 6.67% for C. imicola and C. oxystoma, respectively. BTV was not detected in C. arakawai and C. innoxius. The specimens (YY86) from C. imicola that tested positive for BTV had the closest genetic relationship to YTS-4 isolated from Mangshi, Yunnan Province in 1996. All test results for the nucleic acid of AHSV were negative. The study reveals variations in the species distribution, community composition, blood sucking rate, and blood-feeding sources of Culicoides across different habitats. Notably, C. imicola and C. oxystoma emerge as potential vectors for the transmission of BTV in local animals. Accordingly, this investigation provides crucial insights that can serve as a valuable reference for the prevention and control of BTV in local animals, particularly from the perspective of vector management.

Analysis of Sporulation in Bacillus cereus Biovar anthracis Which Contains an Insertion in the Gene for the Sporulation Factor σK.

Bacillus cereus biovar anthracis (Bcbva) is an untypical pathogen causing a fatal anthrax-like disease in a variety of wildlife species in African rainforest areas. In contrast to Bacillus anthracis and most species of the B. cereus group, all strains of the Bcbva cluster contain a 22 kb insertion in the sigK gene which encodes the essential late sporulation sigma factor σK. This insertion is excised during sporulation in a site-specific recombination process resulting in an intact sigK gene and a circular molecule. The sporulation kinetics of two strains each of Bcbva and B. anthracis were compared by the expression analysis of eight sporulation-associated genes, including sigK, using reverse transcriptase quantitative real-time PCR. In addition, morphological sporulation stages were analyzed and quantified by electron microscopy. Our results indicated that the necessary excision of the insertion in Bcbva neither delayed nor inhibited its sporulation. In two spontaneous mutants of Bcbva, the excision of the sigK insertion and sporulation were impeded due to mutations in the spo0A and spoVG regulator genes, respectively. The spo0A frameshift mutation was overcome by intragenic suppression in a revertant which was able to sporulate normally, despite an M171S amino acid exchange in the global regulator Spo0A. A screening of the NCBI database identified further strains of the B. cereus group which possess unrelated insertions in the sigK gene, and two strains containing almost identical insertions at the same gene position. Some of the sigK insertions encode putative prophages, whereas the Bcbva insertion encoded a type I restriction-modification system. The function of these insertions and if they are possibly essential for sporulation remains to be assessed.

MiR-184-5p represses neuropathic pain by regulating CCL1/CCR8 signaling interplay in the spinal cord in diabetic mice

ABSTRACTBackgroundDiabetic neuropathic pain (DNP) is a serious complication for diabetic patients involving nervous system. MicroRNAs (miRNAs) are small-noncoding RNAs which are dysregulated in neuropathic pain, and might be critical molecules for pain treatment. Our previous study has shown miR-184-5p was significantly downregulated in DNP. Therefore, the mechanism of miR-184-5p in DNP was investigated in this study.MethodsA DNP model was established through streptozotocin (STZ). The pharmacological tools were injected intrathecally, and pain behavior was evaluated by paw withdrawal mechanical thresholds (PWMTs). Bioinformatics analysis, Dual-luciferase reporter assay and fluorescence-in-situ-hybridization (FISH) were used to seek and confirm the potential target genes of miR-184-5p. The expression of relative genes and proteins was analyzed by quantitative reverse transcriptase real-time PCR (qPCR) and western blotting.ResultsMiR-184-5p expression was down-regulated in spinal dorsal on days 7 and 14 after STZ, while intrathecal administration of miR-184-5p agomir attenuates neuropathic pain induced by DNP and intrathecal miR-184-5p antagomir induces pain behaviors in naïve mice. Chemokine CC motif ligand 1 (CCL1) was found to be a potential target of miR-184-5p and the protein expression of CCL1 and the mRNA expression of CCR8 were up-regulated in spinal dorsal on days 7 and 14 after STZ. The luciferase reporter assay and FISH demonstrated that CCL1 is a direct target of miR-184-5p. MiR-184-5p overexpression attenuated the expression of CCL1/CCR8 in DNP; intrathecal miR-184-5p antagomir increased the expression of CCL1/CCR8 in spinal dorsal of naïve mice.ConclusionThis research illustrates that miR-184-5p alleviates DNP through the inhibition of CCL1/CCR8 signaling expression.

Inhibitory checkpoint molecule mRNA expression in canine soft tissue sarcoma.

Canine soft tissue sarcomas (STS) are common neoplasms and considered immune deserts. Tumour infiltrating lymphocytes are sparse in STS and, when present, tend to organize around blood vessels or at the periphery of the neoplasm. This pattern is associated with an immunosuppressive tumour microenvironment linked to overexpression of molecules of the PD-axis. PD-1, PD-L1 and PD-L2 expression correlates with malignancy and poor prognosis in other neoplasms in humans and dogs, but little is known about their role in canine STS, their relationship to tumour grade, and how different therapies affect expression. The objective of this study was to evaluate the expression of checkpoint molecules across STS tumour grades and after tumour ablation treatment. Gene expression analysis was performed by reverse-transcriptase real-time quantitative PCR in soft tissue sarcomas that underwent histotripsy and from histologic specimens of STS from the Virginia Tech Animal Laboratory Services archives. The expression of PD-1, PD-L1 and PD-L2 was detected in untreated STS tissue representing grades 1, 2, and 3. Numerically decreased expression of all markers was observed in tissue sampled from the treatment interface relative to untreated areas of the tumour. The relatively lower expression of these checkpoint molecules at the periphery of the treated area may be related to liquefactive necrosis induced by the histotripsy treatment, and would potentially allow TILs to infiltrate the tumour. Relative increases of these checkpoint molecules in tumours of a higher grade and alongside immune cell infiltration are consistent with previous reports that associate their expression with malignancy.

Coinfection of Canine Distemper Virus and Rabies Virus in Wildlife Samples Submitted for Routine Rabies Testing.

Canine distemper virus (also known as Canine morbillivirus), the etiologic agent of canine distemper, is a highly contagious pathogen causing a multisystemic infection in carnivores globally. Canine distemper may be clinically indistinguishable from rabies, and outbreaks of either disease are major concerns. In the US, both diseases are endemic and managed by parenteral vaccination in domestic animals. In wildlife, oral vaccination and trap-vaccinate-release programs are available for rabies prevention, but no such strategies exist for canine distemper. We evaluated the prevalence at which canine distemper virus occurred concurrently in animals infected with rabies virus. Real-time quantitative reverse transcriptase PCR (qRT-PCR) was performed on specimens previously diagnosed with rabies during 2017-19 by the New York State Rabies Laboratory. Real-time qRT-PCR detected concurrent canine distemper virus infection in 73 of 1,302 animals with rabies virus. Coinfection rates were approximately 9% in Procyon lotor, 2% in Vulpes vulpes, and 0.4% in Mephitis mephitis, with an overall prevalence of 5.6%. As comorbidities in wildlife occur, laboratory-based surveillance and confirmatory testing are critical to rapid decision making for disease prevention. Rabies virus incursions are expensive and difficult to manage, and spillover events create health risks to humans and domestic animals as well as to free-roaming wildlife.

The anti-psoriatic effect of Gallic acid is associated with the suppression of keratin 6 and Nrf2

Background: Psoriasis is recognized as an autoimmune dermatosis, and keratin 6 (KRT 6) is a hallmark of psoriasis. Gallic acid (GA) is a natural small molecule with a series of biological activities. However, the effect of GA on psoriasis has not been clarified. background: Psoriasis is recognized as an autoimmune dermatosis and keratin 6 (KRT 6) is a hallmark of psoriasis. Gallic acid (GA) is a natural small molecule with a series of biological activities. However, the effect of GA on psoriasis has not been clarified. Aims: This study aims to investigate the anti-psoriatic activity of GA in psoriasis-like mice and in vitro. objective: This study aimed to investigate the antipsoriatic activity of GA both in psoriasis-like mice and in vitro. Methods: The transcriptions of the Homo sapiens KRT6 gene, and Mus musculus KRT6 gene, were identified using a quantitative real-time reverse transcriptase PCR (qRT-PCR) assay. Expressions of KRT 6, STAT3, pSTAT3, Nrf2, and pNrf2 in HaCaT cells and skin biopsies were determined with a western blotting assay. The immunofluorescence (IF) assay was used to examine the expression of KRT6, pSTAT3, and pNrf2 in HaCaT cells. The expression of KRT 6, PCNA, Ki67, and CD3 was evaluated on the skin of psoriasis-like mice and quantified with histochemical scores (H scores). method: The transcriptions of the Homo sapiens KRT6 gene and Mus musculus KRT6 gene were identified using a quantitative real-time reverse transcriptase PCR (qRT-PCR) assay. Expressions of KRT 6, STAT3, pSTAT3, Nrf2, and pNrf2 in HaCaT cells and skin biopsies were determined with a Western blotting assay. The immunofluorescence (IF) assay was used to examine the expression of KRT6, pSTAT3 and pNrf2 in HaCaT cells. Expression of KRT 6, PCNA, Ki67, and CD3 was evaluated on skin from psoriasis-like mice and quantified with histochemical scores (H scores). Results: GA significantly inhibited KRT 6 gene transcription and expression in psoriasis-like disease both in vitro and in vivo. It significantly inhibited the expression of keratinocyte proliferation markers (PCNA and Ki67), suppressed the expression of CD3 (a marker of T cells), and decreased the thickness of the folded skin, as well as improved the splenomegaly in imiquimod-induced mice similar to psoriasis. Furthermore, the suppressing effect of GA on KRT 6 was abolished by the continuous activation of Nrf2 rather than STAT3, although GA significantly inhibited Nrf2 and STAT3 activation in IL-17A-induced HaCaT cells. result: GA significantly inhibited KRT 6 gene transcription and expression in psoriasis-like disease both in vitro and in vivo. GA significantly inhibited the expression of keratinocyte proliferation markers (PCNA and Ki67), suppressed the expression of CD3 (a marker of T cells), and decreased the thickness of the folded skin, as well as improved the splenomegaly in imiquimod-induced mice similar to psoriasis. Furthermore, the suppressing effect of GA on KRT 6 was abolished by continuously activated Nrf2 rather than STAT3, although GA significantly inhibited Nrf2 and STAT3 activation in IL-17A-induced HaCaT cells. Conclusions: KRT 6 acts as a potential target for GA against psoriasis, and the anti-psoriatic effect of GA could be related to Nrf2 signaling. other: By pharmacological development, this bioactivity of GA could be applied in the treatment of psoriasis in the future.

Selection and Validation of Reference Genes for Gene Expression Studies in an Equine Adipose-Derived Mesenchymal Stem Cell Differentiation Model by Proteome Analysis and Reverse-Transcriptase Quantitative Real-Time PCR.

Adipose-derived stem cells (ADSCs) are used in tissue regeneration therapies. The objective of this study is to identify stable reference genes (RGs) for use in gene expression studies in a characterized equine adipose-derived mesenchymal stem cell (EADMSC) differentiation model. ADSCs were differentiated into adipocytes (ADs) or osteoblasts (OBs), and the proteomes from these cells were analyzed by liquid chromatography tandem mass spectrometry. Proteins that were stably expressed in all three cells types were identified, and the mRNA expression stabilities for their corresponding genes were validated by RT-qPCR. PPP6R1, CCDC97, and then either ACTB or EPHA2 demonstrated the most stable mRNA levels. Normalizing target gene Cq data with at least three of these RGs simultaneously, as per MIQE guidelines (PPP6R1 and CCDC97 with either ACTB or EPHA2), resulted in congruent conclusions. FABP5 expression was increased in ADs (5.99 and 8.00 fold, p = 0.00002 and p = 0.0003) and in OBs (5.18 and 5.91 fold, p = 0.0011 and p = 0.0023) relative to ADSCs. RUNX2 expression was slightly higher in ADs relative to ADSCs (1.97 and 2.65 fold, p = 0.04 and p = 0.01), but not in OBs (0.9 and 1.03 fold, p = 0.58 and p = 0.91).

Expression of cannabinoid (CB1 and CB2) and cannabinoid-related receptors (TRPV1, GPR55, and PPARα) in the synovial membrane of the horse metacarpophalangeal joint.

The metacarpophalangeal joint undergoes enormous loading during locomotion and can therefore often become inflamed, potentially resulting in osteoarthritis (OA). There are studies indicating that the endocannabinoid system (ECS) modulates synovium homeostasis, and could be a promising target for OA therapy. Some cannabinoid receptors, which modulate proliferative and secretory responses in joint inflammation, have been functionally identified in human and animal synovial cells. To characterize the cellular distribution of the cannabinoid receptors 1 (CB1R) and 2 (CB2R), and the cannabinoid-related receptors transient receptor potential vanilloid type 1 (TRPV1), G protein-related receptor 55 (GPR55) and peroxisome proliferator-activated receptor alpha (PPARα) in the synovial membrane of the metacarpophalangeal joint of the horse. The dorsal synovial membranes of 14 equine metacarpophalangeal joints were collected post-mortem from an abattoir. The dorsal synovial membranes of 14 equine metacarpophalangeal joints were collected post-mortem from an abattoir. The expression of the CB1R, CB2R, TRPV1, GPR55, and PPARα in synovial tissues was studied using qualitative and quantitative immunofluorescence, and quantitative real-time reverse transcriptase PCR (qRT-PCR). Macrophage-like (MLS) and fibroblast-like (FLS) synoviocytes were identified by means of antibodies directed against IBA1 and vimentin, respectively. Both the mRNA and protein expression of the CB2R, TRPV1, GPR55, and PPARα were found in the synoviocytes and blood vessels of the metacarpophalangeal joints. The synoviocytes expressed the mRNA and protein of the CB1R in some of the horses investigated, but not in all. Given the expression of the CB1R, CB2R, TRPV1, GPR55, and PPARα in the synovial elements of the metacarpophalangeal joint, these findings encouraged the development of new studies supporting the use of molecules acting on these receptors to reduce the inflammation during joint inflammation in the horse.

MiR-214 Expression Is Elevated in Chronic Rhinosinusitis Mucosa and Regulates Lipopolysaccharide-Mediated Responses in Undifferentiated Human Nasal Epithelial Cell Culture.

Chronic rhinosinusitis (CRS) is an inflammatory disorder of the upper airways. MicroRNAs (miRs) are reported to regulate several diverse physiological and pathological processes. This study aimed to evaluate the impact of miR-214 on lipopolysaccharide (LPS)-mediated inflammation, and mucin 5AC (MUC5AC) expression in human nasal epithelial cells. The expression of miR-214 was detected in CRS with polyps (CRSwNP) and CRS without polyps (CRSsNP) tissues. Cells were treated with LPS and a miR-214 inhibitor. The level of miR-214 was detected by quantitative real-time reverse transcriptase-PCR (qRT-PCR). The inflammatory cytokines (IL-6, IL-8, TNF, and IL-1β) and MUC5AC production were determined by qRT-PCR and ELISA. MUC5AC protein level was detected using western blot. Similarly, we determined the relationship between miR-214 and Sirtuin 1 (SIRT1) using the Dual luciferase activity assay. miR-214 was increased in CRSwNP and CRSsNP tissues. LPS triggered the expression of miR-214, while miR-214 inhibition diminished the level of miR-214. MiR-214 inhibition prevented LPS-mediated the production of inflammatory cytokines. LPS treatment augmented MUC5AC mRNA, protein levels, and secretion, whereas miR-214 loss inhibited MUC5AC production in the presence of LPS. SIRT1 is a direct target of miR-214. Impairing SIRT1 by siRNA (siSIRT1) or EX527 (a selective SIRT1 inhibitor) reversed the effects of miR-214 inhibitor on inflammation and MUC5AC expression. Furthermore, miR-214 depression inhibited the STAT3/GDF15 pathway via targeting SIRT1. Upregulation of STAT3 or GDF15 partly abolished the anti-inflammatory roles of miR-214 inhibitor. Taken together, miR-214 regulates LPS-mediated inflammation and MUC5AC expression via targeting SIRT1, and STAT3/GDF15 may involve in the regulation of miR-214 inhibitor on inflammation and MUC5AC expression.

P101 Circulating MicroRNA-16 in Inflammatory Bowel Disease Patients - Potential Biomarker to Assess Inflammation

Abstract Background miR-16 is important for the inflammatory processes and the signal pathways that participate in the immune system regulation. As there is an immune mucosal inflammation in IBD, miR-16 could also serve as a potential biomarker to assess inflammation. Methods The study aims to assess the expression of serum miR-16 in IBD patients and to correlate its expression with clinical parameters such as disease extent, activity and severity. 35 patients with UC and 35 patients with CD were included in the study. Serum miR-16 expression was assessed with reverse transcriptase quantitative real time PCR (RT-qPCR), and was then correlated with the miR-16 levels in a group of 30 healthy subjects. Results miR-16 expression is increased in patients with CD, localized in the small intestine (3.54±2.33) and in pancolitis Е3 of UC (2.01±1.54). There is a significant difference in miR-16 expression in inflammatory (B1), stenotic (B2) and penetrating (B3) form of CD (р=0.010) with higher expression levels in the more severe B2 and B3 forms. There is no significant difference in miR-16 expression according to the forms of evolution in patients with UC. In patients with CD, there is a moderate direct correlation between the expression of miR-16 and the Crohn’s disease activity index CDAI (r=0.424; p=0.015). miR-16 expression levels increase progressively in correlation with the activity of CD. Patients with moderate activity of their CD have significantly higher miR-16 expression (4.27±3.00) as compared to patients with mild activity (2.38±1.06) or in remission (2.41±1.23) of the disease (р=0.048). The increased expression levels of miR-16 are a risk factor for CDAI &amp;gt; 150 levels (OR=4.80 (0.506-45.495); p=0.015). In patients with UC, there is no correlation between the miR-16 expression levels and the severity (S) index, measured as per the Montreal classification or the endoscopic activity, measured as per the Partial Mayo score. In patients with severe disease, measured by both S index (1.38±0.89) and Partial Mayo score (1.80±1.12), there is a decrease in miR-16, close to normal controls. MiR-16 expression levels do not correlate with any treatment that was given in patients with either CD or UC. Conclusion The increased miR-16 serum expression corelates with the localization of CD in the small intestine and the stenotic and penetrating form of the disease. Increased miR-16 levels are also found in extensive UC. In patients with CD there is a positive correlation between the expression of miR-16 and the activity of the disease as measured by the CDAI.

Strain and Depot-specific Differences in Adipose Tissues of Obese BFMI Mice.

Obesity is associated with the structural and functional disorders related to the molecules of the tissues, cells, and membranes. This study aimed to examine the alterations in the secretion of inflammatory cytokines and metabolic factors and structural changes in inguinal (IF) and gonadal (GF) adipose tissues at the molecular level. The IF and GF tissues of Berlin Fat Mouse Inbred (BFMI) lines namely BFMI852, BFMI856, BFMI860, BFMI861 obese and DBAJ control mouse lines were used for mRNA expression and Attenuated Total Reflection - Fourier Transform Infrared Spectroscopy (ATR-FTIR) studies. The mRNA levels of inflammatory cytokines including leptin, interleukin 6 (IL-6), tumor necrosis factor-alpha (Tnf-α), and insulin-like growth factor-1 (Igf-1), and peroxisome proliferator-activated receptor gamma 2 (Pparγ-2), were investigated using quantitative reverse transcriptase real-time PCR (qRT-PCR). Infrared spectroscopy does not provide information about specific proteins, instead, it gives information about overall (total) proteins, which is called global information. Therefore, in the current study, adequate information about secondary structures of adipose tissues proteins was obtained using artificial neural network (ANN) and secondary derivative-vector normalization methods based on the spectral profiles. According to the mRNA expression studies, high leptin resistance was found in all BFMI lines. Differences were observed in the levels of measured factors except for Igf-1 among BFMI lines. Protein secondary structure studies showed an increase in random coil contents, especially for BFMI860, which indicates denaturation of the proteins. Among the spontaneous obese BFMI mouse lines, the BFMI860 line is the most suitable for obesity studies. Obesity-induced effect on the adipose tissues varies considerably with location, type of adipose tissue, and animal line.

Quantitative Real-Time Gene Profiling of Human Alveolar Osteoblasts Using a One-Step System.

The use of quantitative real-time reverse transcriptase PCR (qRT2-PCR) for the identification of differentially regulated genes is a powerful technology. The protocol presented here uses qRT2-PCR gene arrays to investigate the regulation of 84 angiogenic related genes in human primary alveolar osteoblasts following treatment with the bisphosphonate, zoledronic acid, and geranylgeraniol (GGOH). GGOH has potential as a therapeutic agent for medication-related osteonecrosis of the jaw, a serious side effect resulting from treatment for metastatic cancer (Zafar S, Coates DE, Cullinan MP, Drummond BK, Milne T, Seymour GJ. J Oral Pathol Med 43:711-721, 2014). The isolation of the primary osteoblast cells follows the methods described by Dillon et al. (Method Mol Biol 816:3-18, 2012) with a new RNA extraction technique described fully. The method highlights the importance of obtaining high-quality RNA which is DNA-free. Relative levels of gene expression are normalized against selected reference genes (HKG) and a number of examples of how fold regulation (2-ΔΔCq) and gene expression level (2-ΔCq) data can be presented are given.

Evaluation of compressed sodium chloride on the inactivation of SARS-CoV-2 and surrogates.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes the global COVID-19 pandemic. Limited studies have been performed on various types of disinfectants utilized to control the spread of this highly contagious virus. This study aimed to investigate the inactivation of SARS-CoV-2 using compressed sodium chloride (CSC) surface. A real-time reverse transcriptase quantitative PCR (RT-qPCR) assay was used to evaluate the effectiveness of CSC on the disintegration of viral RNA in a time dependent manner. The effects of CSC on viral infectivity were determined using a TCID50 assay of a surrogate virus, hCoV-229E, in MRC-5 cell culture. The results demonstrated that CSC achieved a 2 to 3- log10 reduction of viral genomic RNA for a laboratory strain of hCoV-229E, and clinical samples of hCoV-229E and hCoV-OC43. A 3 to 4-log10 reduction was observed for SARS-CoV-2 (RdRp and E gene) suggesting that a CSC surface could effectively disintegrate the SARS-CoV-2 RNA genome. CSC was observed to have a 6 log10 inactivation of infectious hCoV-229E using cell culture after 5 minutes of exposure compared to the control, indicating good disinfection efficacy of a CSC surface against virus.