Detection of Mycobacterium tuberculosis transrenal DNA in urine samples among adults in Peru.

Diagnosis of pulmonary tuberculosis (TB) relies on a sputum sample, which cannot be obtained from all symptomatic individuals. Mycobacterium tuberculosis (Mtb) transrenal DNA (trDNA) has been detected in urine, an easily obtainable, noninvasive, alternative sample type. However, reported sensitivities have been variable and likely depend on collection and assay procedures and aspects of trDNA biology. We analyzed three serial urine samples from each of 75 adults with culture-confirmed pulmonary TB disease in Lima, Peru for detection of trDNA using short-fragment real-time PCR. Additionally, we examined host, urine, and sampling factors associated with detection. Overall per-sample sensitivity was 38 % (95 % Confidence Interval [CI] 30-45 %). On an individual level (i.e., any of the three samples positive), sensitivity was 73 % (95 % CI: 62-83 %). Sensitivity was highest among samples from patients with smear-positive TB, 92 % (95 % CI: 62-100 %). Specificity from a single sample from each of 10 healthy controls was 100 % (95 % CI: 69-100 %). Adjusting our assay positivity threshold increased individual-level sensitivity to 88 % (95 % CI: 78-94 %) overall without affecting the specificity. We did not find associations between Mtb trDNA detection and individual characteristics or urine sample characteristics. Overall, our results support the potential of trDNA detection for TB diagnosis.

Hemoplasmas in wild rodents and marsupials from the Caatinga Biome, Brazil.

A total of 231 blood samples from wild mammals belonging to the orders Rodentia (n = 142) and Didelphimorphia (n = 89) were screened by real-time PCR assay (qPCR), being six Rhipidomys sp., 118 Thrichomys laurentius, nine Rattus rattus, four Kerodon rupestris, five Necromys lasiurus, 42 Didelphis albiventris and 47 Monodelphis domestica. Results using qPCR showed that 32 of the total 231 (13.85 %) samples were positive for hemoplasma sequences of the 16S rRNA gene. Sequences from two D. albiventris showed 99.77-99.89 % identity with 'Candidatus Mycoplasma haemoalbiventris' and 99.09 % with 'Candidatus Mycoplasma haemodidelphidis', respectively. Furthermore, one M. domestica and five T. laurentius showed 99.72-99.77 % identity with Mycoplasma sp., and one K. rupestris showed 98.13 % identity with 'Candidatus Mycoplasma haematohydrochaerus'; and from two Rattus rattus showed 99.65-99.89 % identity with Mycoplasma sp. and 'Candidatus Mycoplasma haemomuris'. The 23S rRNA gene sequences obtained from the two D. albiventris showed 100 % identity with 'Ca. M. haemoalbiventris' whereas the sequences from the R. rattus showed only 85.31 % identity with 'Candidatus Mycoplasma haematohydrochaerus'. Two T. laurentius and one K. rupestris showed 84.66-92.97 % identity with 'Candidatus Mycoplasma haemosphiggurus'. Based on phylogenetic and Neighbor-Net network analyses of the 16S and 23S rRNA genes, potential novel species are described. In addition, 'Ca. M. haemoalbiventris' was detected in Didelphis albiventris, and Mycoplasma sp. was detected in Rattus sp. rodents from the Caatinga biome, Brazil.

Development and comparative assessment of RT-qPCR and duplex RT-LAMP assays for the monitoring of Aichi virus A (AiV-A) in untreated wastewater samples.

Diverse enteric pathogens, transmitted through human and animal feces, can cause gastroenteritis. Enteric viruses, such as human Aichi virus, specifically genotype A (AiV-A), are emerging pathogens that cause illnesses even at low doses and are spreading globally. This research developed a reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay targeting the 3CD junction and a reverse transcription colorimetric loop-mediated isothermal amplification (RT-cLAMP) duplex assay targeting junctions 2BC and 3CD of the AiV-A genome for rapid and sensitive detection of this virus in metropolitan and regional wastewater samples in Queensland, Australia. The performance of these assays was evaluated using control materials and by analyzing wastewater samples. In serially diluted control materials, RT-qPCR provided quantifiable data (mean 1.51 log10 GC/2 µL of nucleic acid) down to a dilution of 1 × 10-5 pg/µL. In comparison, the duplex RT-cLAMP assay detected down to 1 × 10-4 pg/µL, indicating that its sensitivity was one order of magnitude less than that of RT-qPCR. Of the 38 wastewater samples from 38 metropolitan and regional wastewater treatment plants (WWTPs) in Queensland, Australia, 21 (55.3 %) tested positive by RT-qPCR with concentrations ranging from 3.60 to 6.23 log10 GC/L. In contrast, only 15 (39.5 %) of 38 wastewater samples were positive using the duplex RT-cLAMP assay. The methods demonstrated substantial qualitative agreement (κ = 0.730), with a concordance of 86.5 %, demonstrating the reliability of RT-cLAMP for detecting AiV-A in wastewater samples. The duplex RT-cLAMP assay, despite demonstrating reduced detection sensitivity, has proven effective and holds promise as a supplementary approach, especially in settings with limited resources where rapid and affordable testing is crucial.

Do SOD2 and SOD3 gene polymorphisms impact the oral health-related quality of life in Para athletes?

The aim of this study was to evaluate whether polymorphisms in SOD2 and SOD3 genes modulate the oral health-related quality of life (OHRQoL) of Para athletes with dental caries experience. The cross-sectional study included 264 Para athletes (143 in athletics, 61 in weightlifting and 60 in swimming). A trained and calibrated team recorded the decayed, missing and filled teeth index (DMFT). The Brazilian version of the Oral Health Impact Profile (OHIP-14) was used to measure OHRQoL. Genomic DNA was extracted from the athletes' saliva, and genetic polymorphisms in the SOD2 (rs5746136 and rs10370) and SOD3 (rs2855262 and rs13306703) genes were analyzed by real-time polymerase chain reaction. Univariate and multivariate analyses were performed. A multivariate General Linear Model analysis, adjusted for sex, revealed that the SOD3 gene polymorphism (rs2855262) had a significant effect on the psychological disability domain [codominant (p = 0.045) and recessive (p=0.038) models]. The SOD2 gene polymorphism (rs5746136) had a significant effect on the total OHIP-14 score [dominant model (p = 0.038)] and the psychological discomfort [dominant model (p = 0.034)] and physical disability [codominant model (p=0.037)] domains. Presence of the SOD2 rs10370 polymorphism led to statistical differences in the total score [codominant (p = 0.026) and dominant (p = 0.023) models] and the handicap domain scores [codominant (p = 0.027) and dominant (p = 0.032) models]. Polymorphisms of the SOD2 and SOD3 genes may be important biomarkers of OHRQoL in Para athletes with dental caries experience.

Selection of references for quantitative real-time PCR analysis of microRNAs in Nile tilapia (Oreochromis niloticus) under osmotic stress.

MicroRNAs play crucial regulatory roles in various aspects of development and physiology, including environmental adaptation and stress responses in teleosts. RT-qPCR is the most commonly used method for studying microRNA expression, with the accuracy and reliability of results depending on the use of an appropriate reference gene for normalization. This study aimed to evaluate seven miRNAs (U6, Let-7a, miR-23a, miR-25-3, miR-103, miR-99-5, and miR-455) expression stability in different tissues of Nile tilapia subjected to osmotic stress. Fish were divided into two groups: a control and an experimental group, raised in 0 and 12 ppt salinity water respectively. After 21 days, brain, gills, liver, and posterior intestine were collected for analysis. Different mathematical algorithms (geNorm, NormFinder, BestKeeper, and the comparative ΔCt method) were employed to identify the most suitable reference miRNAs. The results indicate that the miR-455/miR-23a combination is a robust reference for normalizing miRNA expression levels in studies of osmotic stress responses in Nile tilapia. The stability of miRNA expression can vary depending on specific stress conditions and biological processes, underscoring the necessity of selecting appropriate normalizing miRNAs for each experimental context. This study identifies reliable reference genes for future RT-qPCR analyses of miRNA expression, thereby enhancing our understanding of molecular responses in fish to environmental challenges. These insights are fundamental to the development of new technologies for the improved management and sustainability of aquaculture practices.

Improvement of osteoblast adhesion, viability, and mineralization by restoring the cell cytoskeleton after bisphosphonate discontinuation in vitro.

OBJECTIVE: Bisphosphonates are prescribed to treat excessive bone resorption in patients with osteoporosis. However, its use is associated with potential adverse effects such as medication-related osteonecrosis of the jaw, prompting the introduction of the drug holiday concept in patients prior to dentoalveolar surgery. Furthermore, bisphosphonate discontinuation has been studied in vivo, in humans, and in animal models. However, it is not known whether this approach could affect bone cells in vitro. Therefore, the objective of this study was to investigate the potential effects of bisphosphonate discontinuation on pre-osteoblast and osteoblast activities in vitro. METHODOLOGY: Pre-osteoblasts (MC3T3) and osteoblasts were treated with bisphosphonate (alendronate) at concentrations of 1, 5, and 10 µM. Alendronate was then withdrawn at different time points. The negative control consisted of untreated cells (0 µM), while the positive control consisted of cells incubated with alendronate throughout the experiment. Cell viability, cell adhesion, cell cytoskeleton, mineralization, and gene expressions were investigated. RESULTS: Pre-osteoblasts and osteoblasts showed a decrease in cell viability after treatment with 5-10 µM alendronate for 4 days or longer. Two days of alendronate discontinuation significantly increased cell viability compared with the positive control. However, these levels did not reach those of the negative control. Bone nodule formation was reduced by alendronate. Discontinuation of alendronate regained bone nodule formation. Longer periods of discontinuation were more effective in restoring nodule formation than shorter periods. Addition of alendronate resulted in an increase in the percentage of dead cells, which, in turn, decreased when alendronate was discontinued. Alendronate affected the cell cytoskeleton by disassembling actin stress fibers. Cell adhesion and cell morphological parameters were also affected by alendronate. Discontinuation of alendronate restored cell adhesion and these parameters. Overall, the highest improvement after alendronate discontinuation was seen at 10 µM. However, alendronate treatment and discontinuation did not affect osteoblast gene expression. CONCLUSION: Discontinuation of alendronate helps to reverse the negative effects of the drug on cell viability, cell adhesion, and mineralization by restoring the cell cytoskeleton. Our data suggest the benefits of drug holiday and/or intermittent strategies for alendronate administration at the cellular level.

Hyaluronic acid-mediated epithelial-mesenchymal transition of human lens epithelial cells via CD44 and TGF-ß2.

PURPOSE: The epithelial-mesenchymal transition of human lens epithelial cells plays a role in posterior capsule opacification, a fibrotic process that leads to a common type of cataract. Hyaluronic acid has been implicated in this fibrosis. Studies have investigated the role of transforming growth factor (TGF)-ß2 in epithelial-mesenchymal transition. However, the role of TGF-ß2 in hyaluronic acid-mediated fibrosis of lens epithelial cell remains unknown. We here examined the role of TGF-ß2 in the hyaluronic acid-mediated epithelial-mesenchymal transition of lens epithelial cells. METHODS: Cultured human lens epithelial cells (HLEB3) were infected with CD44-siRNA by using the Lipofectamine 3000 transfection reagent. The CCK-8 kit was used to measure cell viability, and the scratch assay was used to determine cell migration. Cell oxidative stress was analyzed in a dichloro-dihydro-fluorescein diacetate assay and by using a flow cytometer. The TGF-ß2 level in HLEB3 cells was examined through immunohistochemical staining. The TGF-ß2 protein level was determined through western blotting. mRNA expression levels were determined through quantitative real-time polymerase chain reaction. RESULTS: Treatment with hyaluronic acid (1.0 µM, 24 h) increased the epithelial-mesenchymal transition of HLEB3 cells. The increase in TGF-ß2 levels corresponded to an increase in CD44 levels in the culture medium. However, blocking the CD44 function significantly reduced the TGF-ß2-mediated epithelial-mesenchymal transition response of HLEB3 cells. CONCLUSIONS: Our study showed that both CD44 and TGF-ß2 are critical contributors to the hyaluronic acid-mediated epithelial-mesenchymal transition of lens epithelial cells, and that TGF-ß2 in epithelial-mesenchymal transition is regulated by CD44. These results suggest that CD44 could be used as a target for preventing hyaluronic acid-induced posterior capsule opacification. Our findings suggest that CD44/TGF-ß2 is crucial for the hyaluronic acid-induced epithelial-mesenchymal transition of lens epithelial cells.

Immunostaining and gene expression of epidermal growth factor receptors (HER1/HER2) in canine cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is a neoplasm type often diagnosed in dogs. However, studies focused on further investigating its molecular biology, mainly biomarkers to help implementing new therapies, remain scare in the literature. Thus, immunostaining and the gene expression of epidermal growth factor receptors (HER1 and HER2) in canine cSCC presenting different cell differentiation degrees were herein assessed. Thirty-two (32) canine cSCC were selected, classified based on to their cell differentiation degree and subjected to immunohistochemical study to assess HER1 and HER2 immunostaining intensity and distribution. In addition, HER1 and HER2 gene expression was investigated through real-time PCR. Membranous and cytoplasmic immunostaining were observed in both markers. HER2 prevailed in poorly differentiated cSCC; there was positive protein expression correlation between both markers. Mean HER1 gene expression was higher in moderately differentiated, whereas mean HER2 gene expression was higher in poorly differentiated cSCC. Moreover, there was gene expression correlation between markers, regardless of cell differentiation degree. Thus, HER2 protein immunostaining and gene expression were higher in poorly differentiated canine cSCC and it enabled understanding that increase observed in this epidermal growth factor receptor is proportional to this neoplasm's cell differentiation degree in canine species. Results in the current study helped better understanding canine cSCC's molecular biology; however, it is relevant studying other markers aiming to investigate signaling pathways.

RNA-seq validation: software for selection of reference and variable candidate genes for RT-qPCR.

BACKGROUND: Real-time quantitative PCR (RT-qPCR) is one of the most widely used gene expression analyses for validating RNA-seq data. This technique requires reference genes that are stable and highly expressed, at least across the different biological conditions present in the transcriptome. Reference and variable candidate gene selection is often neglected, leading to misinterpretation of the results. RESULTS: We developed a software named "Gene Selector for Validation" (GSV), which identifies the best reference and variable candidate genes for validation within a quantitative transcriptome. This tool also filters the candidate genes concerning the RT-qPCR assay detection limit. GSV was compared with other software using synthetic datasets and performed better, removing stable low-expression genes from the reference candidate list and creating the variable-expression validation list. GSV software was used on a real case, an Aedes aegypti transcriptome. The top GSV reference candidate genes were selected for RT-qPCR analysis, confirming that eiF1A and eiF3j were the most stable genes tested. The tool also confirmed that traditional mosquito reference genes were less stable in the analyzed samples, highlighting the possibility of inappropriate choices. A meta-transcriptome dataset with more than ninety thousand genes was also processed successfully. CONCLUSION: The GSV tool is a time and cost-effective tool that can be used to select reference and validation candidate genes from the biological conditions present in transcriptomic data.

The association of CYP11A1 gene polymorphisms with the polycystic ovary syndrome patients.

OBJECTIVE: The objective of this study was to investigate the allele frequencies of polymorphisms in genes CYP11A1 rs4886595 and CYP11A1 rs4887139 that are responsible for the steroidogenesis mechanism in polycystic ovary syndrome patients and control females. METHODS: Samples were obtained from the Department of Obstetrics and Gynecology in the Near East University Hospital from September 2019 to December 2019. Only the nonobese patients between the ages of 18-40 years were included in this study following informed consent. Obese patients and patients more than 40 years of age were excluded from the study. Nonobese women and normal ovulation were included in the control group. DNA was isolated from blood samples. Real-time polymerase chain reaction (PCR) was used to analyze single nucleotide polymorphisms (SNPs) in various genes linked to polycystic ovary syndrome. The studies were carried out using the samples obtained from 120 women, of whom 55 were nonobese and had normal ovulation, and 65 were polycystic ovary syndrome patients. The allelic frequencies of SNPs in genes linked to polycystic ovary syndrome were calculated using real-time PCR outcomes. RESULTS: The variation of the CYP11A1 rs4887139 G>A did not show any significance, while the variation of CYP11A1 rs4886595 C>A showed significant differences between the patient and the control groups (p=0.01), respectively. CONCLUSION: Future research ought to focus on elucidating the susceptible causes of polycystic ovary syndrome with a wide range of SNPs and more sample size. The genome-wide association studies in polycystic ovary syndrome patients of different origin will be important to identify candidate genes as well as proteins that are implied in polycystic ovary syndrome risk.

Detection through the use of RT-MqPCR of asymptomatic reservoirs of malaria in samples of patients from the indigenous Comarca of Guna Yala, Panama: Essential method to achieve the elimination of malaria.

BACKGROUND: Plasmodium vivax is the main causative agent of malaria in Panama. However, the prevalence of asymptomatic infections in the different endemic regions remains unknown. Understanding the epidemiological behavior of asymptomatic infections is essential for the elimination of malaria. This study aimed to determine the prevalence of asymptomatic malarial infections in one of the main endemic regions of Panama using multiplex real-time reverse transcription RT-MqPCR. METHODS: A cross-sectional study was conducted in three communities in the Guna Yala Comarca. A total of 551 thick blood smears and their respective samples on filter paper were collected from volunteers of different ages and sexes from June 20 to 25, 2016. Infections by the Plasmodium spp. were diagnosed using microscopy and RT-MqPCR. All statistical analyses were performed using the R software. RESULTS: The average prevalence of asymptomatic infections by P. vivax in the three communities detected by RT-MqPCR was 9.3%, with Ukupa having the highest prevalence (13.4%), followed by Aidirgandi (11.1%) and Irgandi (3.3%). A total of 74 samples were diagnosed as asymptomatic infections using RT-MqPCR. Light microscopy (LM) detected that 17.6% (13/74) of the asymptomatic samples and 82.4% (61/74) were diagnosed as false negatives. A 100% correlation was observed between samples diagnosed using LM and RT-MqPCR. A total of 52.7% (39/74) of the asymptomatic patients were female and 85.1% (63/74) were registered between the ages of 1 and 21 years. Factors associated with asymptomatic infection were community (aOR = 0.38 (95% CI 0.17-0.83), p < 0.001) and age aOR = 0.98 (95% CI 0.97-1.00), p < 0.05); F = 5.38; p < 0.05). CONCLUSIONS: This study provides novel evidence of the considerable prevalence of asymptomatic P. vivax infections in the endemic region of Kuna Yala, representing a new challenge that requires immediate attention from the National Malaria Program. The results of this study provide essential information for the health authorities responsible for developing new policies. Furthermore, it will allow program administrators to reorient and design effective malaria control strategies that consider asymptomatic infections as a fundamental part of malaria control and move towards fulfilling their commitment to eliminate it.

Development of novel species-specific and genus-specific primers for the detection of Babaco Mosaic Virus (BabMV).

Babaco is a hybrid cultivar native to the Andean region of Ecuador and Colombia, commercially attractive for its fruit. Babaco production in Ecuador faces losses from plant pathogens like babaco mosaic virus (BabMV), an RNA virus that causes chlorosis, leaf mottling, and deformation. Phylogenetic studies link BabMV to papaya mosaic virus (PapMV), alternanthera mosaic virus, and senna mosaic virus. To address this threat, we developed novel species-specific primers to detect BabMV targeting a 165 bp region of the coat protein (CP). Genus-specific primers were designed to validate the species-specific primers and attest their ability to discriminate between BabMV and its closest relatives. These primers targeted a 175 bp fragment of the CP region. The most effective sets of primers were chosen for reverse transcription polymerase chain reaction (RT-PCR) and SYBR® Green-based quantitative reverse transcription polymerase chain reaction (RT-qPCR) in symptomatic and asymptomatic babaco plants. Among 28 plants tested, 25 were positive and 3 were negative for BabMV using species-specific and genus-specific primers in RT-PCR and RT-qPCR, while the PapMV positive control was detected with the genus-specific primers and was negative for the species-specific primers. These primers represent a valuable molecular tool for detecting BabMV, potentially enhancing crop management.

Assessment of reference genes for qRT-PCR normalization to elucidate host response to African swine fever infection.

Viral infection disrupts the normal regulation of the host gene's expression. In order to normalise the expression of dysregulated host genes upon virus infection, analysis of stable reference housekeeping genes using quantitative real-time-PCR (qRT-PCR) is necessary. In the present study, healthy and African swine fever virus (ASFV) infected porcine tissues were assessed for the expression stability of five widely used housekeeping genes (HPRT1, B2M, 18 S rRNA, PGK1 and H3F3A) as reference genes using standard algorithm. Total RNA from each tissue sample (lymph node, spleen, kidney, heart and liver) from healthy and ASFV-infected pigs was extracted and subsequently cDNA was synthesized, and subjected to qRT-PCR. Stability analysis of reference genes expression was performed using the Comparative delta CT, geNorm, BestKeeper and NormFinder algorithm available at RefFinder for the different groups. Direct Cycle threshold (CT) values of samples were used as an input for the web-based tool RefFinder. HPRT1 in spleen, 18 S rRNA in liver and kidney and H3F3A in heart and lymph nodes were found to be stable in the individual healthy tissue group (group A). The majority of the ASFV-infected organs (liver, kidney, heart, lymph node) exhibited H3F3A as stable reference gene with the exception of the ASFV-infected spleen, where HPRT1 was found to be the stable gene (group B). HPRT1 was found to be stable in all combinations of all CT values of both healthy and ASFV-infected porcine tissues (group C). Of five different reference genes investigated for their stability in qPCR analysis, the present study revealed that the 18 S rRNA, H3F3A and HPRT1 genes were optimal reference genes in healthy and ASFV-infected different porcine tissue samples. The study revealed the stable reference genes found in healthy as well as ASF-infected pigs and these reference genes identified through this study will form the baseline data which will be very useful in future investigations on gene expression in ASFV-infected pigs.

Quadruplex qPCR for detection and discrimination of C. Coli,C. fetus, and C. Jejuni from other Campylobacter species in chicken and sheep meat.

Campylobacter is gram-negative bacteria considered the predominant genera isolated from poultry samples and associated with gastroenteritis. Due to the problems in conventional cultural methods of time-consuming and technically demanding requirements, a rapid and feasible method for their identification and discrimination of the closely related spp. Including Campylobacter coli, Campylobacter fetus, and Campylobacter jejuni is needed. This study analyzes the chicken and sheep meats samples (n = 125) using culture and pre-enrichment-based Quadraplex real-time PCR by targeting OrfA, CstA, HipO, and 16 S rRNA genes of C. coli, C. fetus, C. jejuni and Campylobacter spp. Respectively. The analysis of 125 chicken and sheep meat samples by culture and real-time PCR showed high concordance between the results of the two methods. The present study show high prevalence of Campylobacter species (35% and 32% from chicken and meat respectively) of which C. jejuni were the most abundant. Reaction efficiencies were between 90 and 110%, and detect as low as 8.9 fg in C. jejuni. The need for quick detection and discrimination methods in sheep and chicken meat can be met using the described Quadraplex real-time PCR methodology.

Prevalence of Mycobacterium leprae and Mycobacterium lepromatosis in roadkill armadillos in Brazil.

To evaluate the prevalence of Mycobacterium leprae and Mycobacterium lepromatosis in road killed armadillos identified along Brazilian regions, samples of liver, spleen, muscle, ear, nose and tail were collected on highways from 78 animals. The armadillos were of four different species, Cabassous tatouay, Dasypus novemcinctus, Dasypus septemcinctus and Euphractus sexcinctus. After DNA extraction from two tissues, specific primers were used for the detection of each pathogen using SYBR green qualitative Real-Time PCR, and amplicons were sequenced. The species with the highest prevalence was D. novemcinctus, mainly in the Central-West, South, and Southeast regions of Brazil. We detected M. leprae DNA in 32 (41 %) of the 78 individuals and M. lepromatosis DNA was not identified in any of the examined samples. The zoonotic component of leprosy may play a role in the transmission of the disease in endemic areas in which environmental conditions and contact with reservoirs must be investigated.

Modulation of osteoclastogenesis by macrogeometrically designed hydrophilic dual acid-etched titanium surfaces.

The aim of this study was to evaluate the influence of implant macrodesign and surface hydrophilicity on osteoclast (OC) differentiation, activation, and survival in vitro. Titanium disks were produced with a sandblasted, dual acid-etched surface, with or without additional chemical modification for increasing hydrophilicity (SAE-HD and SAE, respectively) and different macrodesign comprising trapezoidal (HLX) or triangular threads (TMX). This study evaluated 7 groups in total, 4 of which were experimental: HLX/SAE-HD, HLX-SAE, TMX/SAE-HD, and TMX/SAE; and 3 control groups comprising OC differentiated on polystyrene plates (CCPC): a positive CCPC (+), a negative CCPC (-), and a lipopolysaccharide-stimulated assay positive control group, CCPC-LPS. Murine macrophage RAW264.7 cells were seeded on the disks, differentiated to OC (RAW-OC) by receptor activator of nuclear factor-κB ligand (RANKL) treatment and cultured for 5 days. Osteoclast differentiation and cell viability were respectively assessed by specific enzymatic Tartrate-Resistant Acid Phosphatase (TRAP) activity and MTT assays. Expression levels of various OC-related genes were measured at the mRNA level by quantitative polymerase chain reaction (qPCR). HLX/SAE-HD, TMX/SAE-HD, and HLX/SAE significantly suppressed OC differentiation when compared to CCPC (+). Cell viability was significantly increased in TMX/SAE and reduced in HLX/SAE-HD. In addition, the expression of Interleukin (IL)-6 and Tumour Necrosis Factor (TNF)-α was upregulated in TMX/SAE-HD compared to CCPC (+). Hydrophilic surfaces negatively modulate macrophage/osteoclast viability. Specifically, SAE-HD with double triangular threads increases the cellular pro-inflammatory status, while surface hydrophilicity and macrodesign do not seem to have a distinct impact on osteoclast differentiation, activation, or survival.

16S rRNA amplicon sequencing and antimicrobial resistance profile of intensive care units environment in 41 Brazilian hospitals.

Introduction: Infections acquired during healthcare setting stay pose significant public health threats. These infections are known as Healthcare-Associated Infections (HAI), mostly caused by pathogenic bacteria, which exhibit a wide range of antimicrobial resistance. Currently, there is no knowledge about the global cleaning process of hospitals and the bacterial diversity found in ICUs of Brazilian hospitals contributing to HAI. Objective: Characterize the microbiome and common antimicrobial resistance genes present in high-touch Intensive Care Unit (ICU) surfaces, and to identify the potential contamination of the sanitizers/processes used to clean hospital surfaces. Methods: In this national, multicenter, observational, and prospective cohort, bacterial profiles and several antimicrobial resistance genes from 41 hospitals across 16 Brazilian states were evaluated. Using high-throughput 16S rRNA amplicon sequencing and real-time PCR, the bacterial abundance and resistance genes presence were analyzed in both ICU environments and cleaning products. Results: We identified a wide diversity of microbial populations with a recurring presence of HAI-related bacteria among most of the hospitals. The median bacterial positivity rate in surface samples was high (88.24%), varying from 21.62 to 100% in different hospitals. Hospitals with the highest bacterial load in samples were also the ones with highest HAI-related abundances. Streptococcus spp., Corynebacterium spp., Staphylococcus spp., Bacillus spp., Acinetobacter spp., and bacteria from the Flavobacteriaceae family were the microorganisms most found across all hospitals. Despite each hospital particularities in bacterial composition, clustering profiles were found for surfaces and locations in the ICU. Antimicrobial resistance genes mecA, bla KPC-like, bla NDM-like, and bla OXA-23-like were the most frequently detected in surface samples. A wide variety of sanitizers were collected, with 19 different active principles in-use, and 21% of the solutions collected showed viable bacterial growth with antimicrobial resistance genes detected. Conclusion: This study demonstrated a diverse and spread pattern of bacteria and antimicrobial resistance genes covering a large part of the national territory in ICU surface samples and in sanitizers solutions. This data should contribute to the adoption of surveillance programs to improve HAI control strategies and demonstrate that large-scale epidemiology studies must be performed to further understand the implications of bacterial contamination in hospital surfaces and sanitizer solutions.

Evaluation of nasal swab and nasal wipe for detection of Influenza A in swine using Bayesian latent class analysis.

Influenza A virus (IAV) is an important pathogen in Brazilian swine herds, and monitoring the viral circulation is essential to control and reduce the transmission. Surveillance programs for IAV are often based on individual piglets level sampling, making the evaluation of the available diagnostic tools crucial to assessing IAV circulation in herds. Thus, two sample collection methodologies were compared in pig herds in southern Brazil to detect IAV by RT-qPCR: nasal swab (NS) and nasal wipe (NW). A Bayesian latent class model (BLCM) was set for two tests and two populations. The NW and NS used are more specific (higher than 95 % for both) than sensitive. The sensitivity for NW was lower than the NS, 84.14 % (70 % - 95 %; posterior probability interval (PPI): 95 %) and 87.15 % (73 % - 97 %; PPI: 95 %), respectively, and the specificity was 95 % (90 % - 99 %; PPI: 95 %) and 99 % (96 % - 100 %; PPI: 95 %), respectively. Although the wipe sample collection loses both sensitivity and specificity compared with nasal swab, differences in test performance were very limited and PPIs largely overlapped. Therefore NW can also be considered a valuable tool. The decision about the use of both techniques should be based on the trade-off between their performance limitations and feasibility in routine monitoring.

Selection of reference miRNAs for RT-qPCR assays in endometriosis menstrual blood-derived mesenchymal stem cells.

Choosing appropriate reference genes or internal controls to normalize RT-qPCR data is mandatory for the interexperimental reproducibility of gene expression data obtained by RT-qPCR in most studies, including those on endometriosis. Particularly for miRNAs, the choice for reference genes is challenging because of their physicochemical and biological characteristics. Moreover, the retrograde menstruation theory, mesenchymal stem cells in menstrual blood (MenSCs), and changes in post-transcriptional regulatory processes through miRNAs have gained prominence in the scientific community as important players in endometriosis. Therefore, we originally explored the stability of 10 miRNAs expressions as internal control candidates in conditions involving the two-dimensional culture of MenSCs from healthy women and patients with endometriosis. Here, we applied multiple algorithms (geNorm, NormFinder, Bestkeeper, and delta Ct) to screen reference genes and assessed the comprehensive stability classification of miRNAs using RefFinder. Pairwise variation calculated using geNorm identified three miRNAs as a sufficient number of reference genes for accurate normalization. MiR-191-5p, miR-24-3p, and miR-103a-3p were the best combination for suitable gene expression normalization. This study will benefit similar research, but is also attractive for regenerative medicine and clinics that use MenSCs, miRNA expression, and RT-qPCR.

Characterization of the MicroRNA profile in rheumatoid arthritis plasma exosomes and their roles in B-cell responses.

OBJECTIVE: This study aimed to identify differentially expressed microRNAs (miRNAs) in exosomes derived from the blood plasma of Rheumatoid Arthritis (RA) patients and explore their clinical significance and biological roles. METHODS: Illumina high-throughput sequencing was employed to measure miRNA expression levels in plasma exosomes, followed by validation using qRT-PCR. The correlation between exosomal miRNAs and disease activity was systematically analyzed. Additionally, the pathogenic effects of RA exosomes were investigated through bioinformatics analysis and in vitro experiments. RESULTS: Significantly reduced levels of exosomal miR-144-3p and miR-30b-5p were observed in RA patients, which were negatively correlated with DAS28 scores and anti-CCP antibody levels. ROC curve analysis showed that miR-144-3p and miR-30b-5p in plasma exosomes could effectively distinguish RA patients from healthy controls, with AUC values of 0.725 and 0.773, respectively. Combining bioinformatics analysis and in vitro experiments, it was demonstrated that plasma exosomes contribute to ongoing autoantibody production in RA by promoting B-cell differentiation and antibody production. CONCLUSION: The present study indicates that plasma exosomes from RA patients may be potentially pathogenic. Exosomal miR-144-3p and miR-30b-5p exhibit significant decreases in RA patients and are associated with disease activity, suggesting their potential as valuable biomarkers for RA.