Identifying MiR-140-3p as a stable internal reference to normalize MicroRNA qRT-PCR levels of plasma small extracellular vesicles in lung cancer patients.

Exploration of a stably expressed gene as a reference is critical for the accurate evaluation of miRNAs isolated from small extracellular vesicles (sEVs). In this study, we analyzed small RNA sequencing on plasma sEV miRNAs in the training dataset (n = 104) and found that miR-140-3p was the most stably expressed candidate reference for sEV miRNAs. We further demonstrated that miR-140-3p expressed most stably in the validation cohort (n = 46) when compared to two other reference miRNAs, miR-451a and miR-1228-3p, and the commonly-used miRNA reference U6. Finally, we compared the capability of miR-140-3p and U6 as the internal reference for sEV miRNA expression by evaluating key miRNAs expression in lung cancer patients and found that miR-140-3p was more suitable as a sEV miRNA reference gene. Taken together, our data indicated miR-140-3p as a stable internal reference miRNA of plasma sEVs to evaluate miRNA expression profiles in lung cancer patients.

Methylene blue dose-dependently induces cutaneous inflammation and heat hyperalgesia in a novel rat model.

Methylene blue (MB) has been shown to reduce mortality and morbidity in vasoplegic patients after cardiac surgery. Though MB is considered to be safe, extravasation of MB leading to cutaneous toxicity has been reported. In this study, we sought to characterize MB-induced cutaneous toxicity and investigate the underlying mechanisms. To induce MB-induced cutaneous toxicity, we injected 64 adult male Sprague-Dawley rates with 200 µL saline (vehicle) or 1%, 0.1%, or 0.01% MB in the plantar hind paws. Paw swelling, skin histologic changes, and heat and mechanical hyperalgesia were measured. Injection of 1%, but not 0.1% or 0.01% MB, produced significant paw swelling compared to saline. Injection of 1% MB produced heat hyperalgesia but not mechanical hyperalgesia. Pain behaviors were unchanged following injections of 0.1% or 0.01% MB. Global transcriptomic analysis by RNAseq identified 117 differentially expressed genes (111 upregulated, 6 downregulated). Ingenuity Pathway Analysis showed an increased quantity of leukocytes, increased lipids, and decreased apoptosis of myeloid cells and phagocytes with activation of IL-1ß and Fos as the two major regulatory hubs. qPCR showed a 16-fold increase in IL-6 mRNA. Thus, using a novel rat model of MB-induced cutaneous toxicity, we show that infiltration of 1% MB into cutaneous tissue causes a dose-dependent pro-inflammatory response, highlighting potential roles of IL-6, IL-1ß, and Fos. Thus, anesthesiologists should administer dilute MB intravenously through peripheral venous catheters. Higher concentrations of MB (1%) should be administered through a central venous catheter to minimize the risk of cutaneous toxicity.

Advancing COVID-19 Diagnosis: Enhancement in SERS-PCR with 30-nm Au Nanoparticle-Internalized Nanodimpled Substrates.

Real-time polymerase chain reaction (RT-PCR) with fluorescence detection is the gold standard for diagnosing coronavirus disease 2019 (COVID-19) However, the fluorescence detection in RT-PCR requires multiple amplification steps when the initial deoxyribonucleic acid (DNA) concentration is low. Therefore, this study has developed a highly sensitive surface-enhanced Raman scattering-based PCR (SERS-PCR) assay platform using the gold nanoparticle (AuNP)-internalized gold nanodimpled substrate (AuNDS) plasmonic platform. By comparing different sizes of AuNPs, it is observed that using 30 nm AuNPs improves the detection limit by approximately ten times compared to 70 nm AuNPs. Finite-difference time-domain (FDTD) simulations show that multiple hotspots are formed between AuNPs and the cavity surface and between AuNPs when 30 nm AuNPs are internalized in the cavity, generating a strong electric field. With this 30 nm AuNPs-AuNDS SERS platform, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ribonucleic acid (RNA)-dependent RNA polymerase (RdRp) can be detected in only six amplification cycles, significantly improving over the 25 cycles required for RT-PCR. These findings pave the way for an amplification-free molecular diagnostic system based on SERS.

Analytical evaluation of the automated genotyping system (GenPlex) compared to a traditional real-time PCR assay for the detection of high-risk human papillomaviruses.

The detection of high-risk human papillomaviruses (HPVs) is crucial for early screening and preventing cervical cancer. However, the substantial workload in high-level hospitals or the limited resources in primary-level hospitals hinder widespread testing. To address this issue, we explored a sample-to-answer genotyping system and assessed its performance by comparing it with the traditional real-time polymerase chain reaction (PCR) method conducted manually. Samples randomly selected from those undergoing routine real-time PCR detection were re-analyzed using the fully automatic GenPlex® system. This system identifies 24 types of HPV through a combination of ordinary PCR and microarray-based reverse hybridization. Inconsistent results were confirmed by repeated testing with both methods, and the κ concordance test was employed to evaluate differences between the two methods. A total of 365 samples were randomly selected from 7259 women. According to real-time PCR results, 76 were high-risk HPV negative, and 289 were positive. The GenPlex® system achieved a κ value greater than 0.9 (ranging from 0.920 to 1.000, p < 0.0001) for 14 types of high-risk HPV, except HPV 51 (κ = 0.697, p < 0.0001). However, the inconsistent results in high-risk HPV 51 were revealed to be false positive in real-time PCR by other method. When counting by samples without discriminating the high-risk HPV type, the results of both methods were entirely consistent (κ = 1.000, p < 0.0001). Notably, the GenPlex® system identified more positive cases, with 73 having an HPV type not covered by real-time PCR, and 20 potentially due to low DNA concentration undetectable by the latter. Compared with the routinely used real-time PCR assay, the GenPlex® system demonstrated high consistency. Importantly, the system's advantages in automatic operation and a sealed lab-on-chip format respectively reduce manual work and prevent aerosol pollution. For widespread use of GenPlex® system, formal clinical validation following international criteria should be warranted.

Identification and Validation of an Invasion-Related Disease-Free Survival Prognostic Model for Tongue Squamous Cell Carcinoma.

INTRODUCTION: Tongue squamous cell carcinoma (TSCC) is a common malignant tumour type with aggressive invasion and a poor prognosis. To date, invasion-related gene expression signatures for the prognostic stratification of TSCC patients are unavailable in clinical practice. This study aimed to assess the impact of invasion-related genes on the prognosis of TSCC patients. METHODS: We obtained mRNA profiles and clinical data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases (TCGA-TSCC and GSE41116, respectively). The TSCC samples from the TCGA-TSCC cohort were randomly divided into TCGA training and TCGA test datasets at a 7:3 ratio. Next, a disease-free survival (DFS) prognostic risk model was established on the basis of univariate and stepwise multivariate Cox regression analyses of the TCGA training cohort. Moreover, prognostic genes were screened. The model was subsequently evaluated and validated using the TCGA test and GSE41116 datasets. In addition, the prognostic genes were validated in the human TSCC cell line UM1 and the human oral keratinocyte (HOK) cell line using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. RESULTS: A total of 70 candidate genes related to invasion were identified in the TCGA-TSCC cohort. DFS data were subsequently constructed, and 6 prognostic genes, HMGN2, MYL12B, ACTB, PPP1CA, PSMB9, and IFITM3, were identified. The TSCC samples were divided into high- and low-risk groups in the TCGA training, TCGA test, and GSE41116 cohorts, respectively. In particular, patients with TSCC in the low-risk group had longer DFS than those in the high-risk group. Furthermore, qRT-PCR analysis confirmed that the expression levels of the 6 prognostic genes were significantly greater in the TSCC cell line UM1 than in the HOK cell line. CONCLUSION: This study identified new invasion-related target genes related to poor prognosis in TSCC patients, providing new insights into the underlying mechanisms of TSCC invasion.

Cytomegalovirus infection and disease in systemic lupus erythematosus patients at a high-complexity hospital in southwestern Colombia.

Cytomegalovirus (CMV) infection and disease is a condition usually described in immunocompromised patients, but among them, those with connective tissue diseases are poorly represented. Here we present the clinical, laboratory characteristics, management and outcomes of systemic lupus erythematosus (SLE) patients who presented with a CMV infection/disease to a high complexity hospital in southwestern Colombia between 2011 and 2020. 16 SLE patients were found to have a CMV infection. SLE was predominantly characterized by renal involvement (10 patients; 62.50%), and 14 patients (87.5%) were receiving steroids previous to the CMV infection. The entire sample required hospital admission, mainly related to acute kidney injury, and nine patients were admitted to the intensive care unit (ICU). Gastrointestinal organ damage was the most common CMV disease manifestation. All patients received ganciclovir, five of them (31.25%) suffered from septic shock, and seven (43.75%) died. Age ≥38 years and the presence of septic shock at admission were correlated to the mortality outcome. To our knowledge, this is the first publication evaluating SLE patients with CMV infection/disease in a Colombian population.

Comparison of telomere length in patients with bone marrow failure syndromes and healthy controls.

INTRODUCTION: During normal aging, telomeric DNA is gradually lost in dividing somatic cells, and critically short telomeres lead to replicative senescence, apoptosis, or chromosomal instability. We studied telomere length in bone marrow failure syndromes (BMFS) compared to normal healthy population. METHODS: Peripheral blood was collected from the participants, and genomic DNA was extracted. Relative telomere length was measured using a quantitative polymerase chain reaction. Statistical analysis was performed using SPSS and GraphPad Prism 8.2 software. RESULTS: The median age of normal Indian population was 31 (0-60) years. As expected, telomere length (TL) showed a decline with age and no difference in TL between males and females. The median age of 650 patients with aplastic anemia (AA) was 30 (1-60) years. TL was significantly shorter in patients with AA compared to healthy controls (p < .001). In FA and MDS patients, TL was significantly shorter than age-matched healthy controls (p = .028; p < .001), respectively. There was no difference between the median TL in age-matched AA and FA patients (p = .727). However, patients with MDS had shorter TL than age-matched AA (p = .031). CONCLUSION: TL in BMF syndrome patients was significantly shorter than age-matched healthy controls.

Immunohistochemistry and real-time Polymerase Chain Reaction: importance in the diagnosis of intestinal tuberculosis in a Peruvian population.

INTRODUCTION: The diagnosis of intestinal tuberculosis is challenging even nowadays. This study aims to report the positivity rates of new diagnostic methods such as immunohistochemistry and Real-Time Polymerase Chain Reaction in patients with intestinal tuberculosis, as well as describe the pathological and endoscopic features of intestinal tuberculosis in our population. METHODS: This was a retrospective observational study conducted in patients diagnosed with intestinal tuberculosis, between 2010 to 2023 from the Hospital Nacional Daniel Alcides Carrion and a Private Pathology Center, both located in Peru. Clinical data was obtained, histologic features were independently re-evaluated by three pathologists; and immunohistochemistry and real-time Polymerase Chain Reaction evaluation were performed. The 33 patients with intestinal tuberculosis who fulfilled the inclusion criteria were recruited. RESULTS: Immunohistochemistry was positive in 90.9% of cases, while real-time Polymerase Chain Reaction was positive in 38.7%. The ileocecal region was the most affected area (33.3%), and the most frequent endoscopic appearance was an ulcer (63.6%). Most of the granulomas were composed solely of epithelioid histiocytes (75.8%). Crypt architectural disarray was the second most frequent histologic finding (78.8%) after granulomas, but most of them were mild. CONCLUSION: Since immunohistochemistry does not require an intact cell wall, it demonstrates higher sensitivity compared to Ziehl-Neelsen staining. Therefore, it could be helpful for the diagnosis of paucibacillary tuberculosis.

Abundance of Colistin-Resistance Genes in Retail Meats in Vietnam.

The degree of contamination of retail meat with colistin-resistant bacteria and its potential contribution to dissemination within communities remains to be determined. Thus, we aimed to elucidate the contamination status of colistin-resistance genes, indicative of colistin-resistant bacteria, in retail meats in Vietnam. In total, 46 chicken and 49 pork meats from stores in Vietnam and Japan were examined. Multiplex real-time polymerase chain reaction with TaqMan probes was performed for detecting mcr-1, mcr-3, and Escherichia coli 16S rRNA. Colistin-resistant bacteria in meats were isolated using selective media. The minimum inhibitory concentrations of colistin were determined using the broth microdilution method. The results showed that 70.7% of chicken meats in Vietnam were contaminated with both mcr-1 and mcr-3. Meanwhile, mcr-1 and mcr-3 were detected in 15.9% and 40.9% of pork meat, respectively. Only mcr-3 was detected in 40% of chicken in Japan. In addition, mcr-1-harboring E. coli and mcr-3-harboring Aeromonas were isolated from chicken meats in Vietnam. Some of these isolates showed colistin resistance. These results showed that most retail meats were highly contaminated with colistin-resistance genes. Notably, our results suggest that mcr-3 is more prevalent in the contaminated samples compared with mcr-1.

Effect of two irrigating solutions on antimicrobial activity and clinical and radiographic success after endodontic treatment in primary teeth: a randomized clinical trial.

OBJECTIVES: This study evaluated the antimicrobial activity, clinical and radiographic outcome of pulpectomy in primary teeth using either 1% sodium hypochlorite (NaOCl) or 2% chlorhexidine (CHX) as irrigants. MATERIALS AND METHODS: A randomized double-blind controlled clinical study in which primary teeth were allocated to 1% NaOCl (n = 20) and 2% CHX (n = 20) groups. Microbiological collections were performed before and after irrigation for agar culture and real-time polymerase chain reaction (qPCR). Clinical and radiographic success was assessed at different times. Data were submitted to descriptive analysis, chi-square, Mann-Whitney, and Wilcoxon tests (p  < .05). RESULTS: For 1% NaOCl, the following clinical and radiographic success rates were observed: 7 days (93%/80%); 30 days, 3 and 6 months (100%). For 2% CHX: 7 days (73%/53%); 30 days (93%); 3 months (100%/93%); 6 months (100%) (p > .05). One percent NaOCl and 2% CHX effectively reduced total microorganisms (p < .05) but not mutans streptococci (p > .05). In qPCR analysis, the solutions promoted a reduction of total bacteria and Streptococcus mutans, and no difference was observed between times and groups (p > .05). CONCLUSIONS: One percent NaOCl and 2% CHX were effective for clinical and radiographic success and antimicrobial activity in primary teeth submitted to pulpectomy. CLINICAL RELEVANCE: Studying the antimicrobial activity and clinical and radiographic outcomes of pulpectomy in primary teeth using NaOCl and CHX as irrigants is clinically relevant because it provides information for optimizing treatment protocols and improving the quality of care for pediatric patients. It contributes to evidence-based practice and can potentially lead to better outcomes, reduced complications, and enhanced patient experiences.

Clinical and microbiological efficacy of intra-pocket application of diode laser in grade C periodontitis: a randomized controlled clinical trial.

BACKGROUND: Periodontitis is a microbially induced disease destroying structures anchoring teeth to jaw bones. Although metronidazole in combination with spiramycin is the effective conventional treatment of stage III grade C periodontitis, it has several systemic side effects. Laser therapy is widely used nowadays as an adjunct to scaling and root planing (SRP) to modulate inflammatory host response and eradicate microbes, due to bactericidal and detoxifying effects. Since microbiological analysis is one of the diagnostic methods identifying periodontal risk; our research aimed to investigate the efficacy of intra-pocket application of diode laser (980 nm) versus antibiotic therapy in enhancing clinical and microbiological parameters in stage III grade C periodontitis. METHODS: A randomized controlled clinical trial was conducted on fifty patients with stage III grade C periodontitis, divided equally into two groups. We managed test group by SRP with intra-pocket application of diode laser (980 nm) and the control group by SRP with systemic antibiotic administration (spiramycin and metronidazole). Then, we measured periodontal pocket depth (PPD) and clinical attachment loss (CAL) for both groups, before treatment (baseline), four and twelve weeks after. Moreover, we collected gingival crevicular fluid from both groups at baseline, four and twelve weeks after treatment and analyzed by real-time polymerase chain reaction to detect the relative count of Aggregatibacter actinomycetemcomitans and Porhyromonas gingivalis. RESULTS: Compared to baseline, all assessed clinical and microbiological parameters attested improvement at the end of the study period in each group individually with no significant difference between the two studied groups. Although, at twelve weeks, flare up of bacterial levels was detected with systemic antibiotic administration. CONCLUSION: Laser therapy can be considered as an effective treatment modality in stage III grade C periodontitis, avoiding the systemic antibiotic side effects and solving the recurrence problems due to bacterial resistance by long term usage. TRIAL REGISTRATION: NCT05222737 retrospectively on 03/02/2022, Clinicaltrial.gov.

Next-generation molecular diagnostics: Leveraging digital technologies to enhance multiplexing in real-time PCR.

Real-time polymerase chain reaction (qPCR) enables accurate detection and quantification of nucleic acids and has become a fundamental tool in biological sciences, bioengineering and medicine. By combining multiple primer sets in one reaction, it is possible to detect several DNA or RNA targets simultaneously, a process called multiplex PCR (mPCR) which is key to attaining optimal throughput, cost-effectiveness and efficiency in molecular diagnostics, particularly in infectious diseases. Multiple solutions have been devised to increase multiplexing in qPCR, including single-well techniques, using target-specific fluorescent oligonucleotide probes, and spatial multiplexing, where segregation of the sample enables parallel amplification of multiple targets. However, these solutions are mostly limited to three or four targets, or highly sophisticated and expensive instrumentation. There is a need for innovations that will push forward the multiplexing field in qPCR, enabling for a next generation of diagnostic tools which could accommodate high throughput in an affordable manner. To this end, the use of machine learning (ML) algorithms (data-driven solutions) has recently emerged to leverage information contained in amplification and melting curves (AC and MC, respectively) - two of the most standard bio-signals emitted during qPCR - for accurate classification of multiple nucleic acid targets in a single reaction. Therefore, this review aims to demonstrate and illustrate that data-driven solutions can be successfully coupled with state-of-the-art and common qPCR platforms using a variety of amplification chemistries to enhance multiplexing in qPCR. Further, because both ACs and MCs can be predicted from sequence data using thermodynamic databases, it has also become possible to use computer simulation to rationalize and optimize the design of mPCR assays where target detection is supported by data-driven technologies. Thus, this review also discusses recent work converging towards the development of an end-to-end framework where knowledge-based and data-driven software solutions are integrated to streamline assay design, and increase the accuracy of target detection and quantification in the multiplex setting. We envision that concerted efforts by academic and industry scientists will help advance these technologies, to a point where they become mature and robust enough to bring about major improvements in the detection of nucleic acids across many fields.

Application of droplet digital PCR combined with TaqMan real-time PCR in Dombrock blood group genotyping in Northwest China.

BACKGROUND AND OBJECTIVES: The Dombrock blood group system is based on the DO gene. DO*A and DO*B antigens are the result of a single-nucleotide polymorphism (SNP) on this gene. The introduction of Do antigens through blood transfusion or other invasive factors like infection may result in the production of Do antibodies, which may cause serious haemolytic transfusion reactions. In this study, TaqMan real-time PCR and droplet digital PCR were used to detect rare DO*A allele, guide the search for rare DO*A allele donors, and calculate DO alleles frequencies in mixed populations in Northwest China. MATERIALS AND METHODS: In this study, the highly sensitive and accurate TaqMan real-time polymerase chain reaction (PCR) method was used to detect and screen DO genotype SNPs in combination with droplet digital PCR. We also searched for rare DO*A allele donors and calculated the frequencies of DO alleles in mixed populations. RESULTS: A total of 1202 donor DNA samples were collected from Northwest China, of which 202 were used to detect DO allele SNPs using TaqMan real-time PCR. The rare DO*A allele was detected in the other 1000 blood donors by droplet digital PCR, and gene frequencies were inferred from dual channel droplet digital PCR data. Among 1202 donors from Northwest China, the allele frequencies of DO*A and DO*B were 0.1128 and 0.8872, respectively. CONCLUSION: The sequencing results confirmed that this new way of detecting DO alleles by droplet digital PCR with specific probes can detect rare DO*A allele to predict the presence of the rare antigen Doa and infer DO allele frequencies. This method is highly sensitive and specific.

Assessing severe acute respiratory syndrome coronavirus 2 infectivity by reverse-transcription polymerase chain reaction: A systematic review and meta-analysis.

The cornerstone of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection is reverse-transcription polymerase chain reaction (RT-PCR) of viral RNA. As a surrogate assay SARS-CoV-2 RNA detection does not necessarily imply infectivity. Only virus isolation in permissive cell culture systems can indicate infectivity. Here, we review the evidence on RT-PCR performance in detecting infectious SARS-CoV-2. We searched for any studies that used RT-PCR and cell culture to determine infectious SARS-CoV-2 in respiratory samples. We assessed (i) diagnostic accuracy of RT-PCR compared to cell culture as reference test, (ii) performed meta-analysis of positive predictive values (PPV) and (iii) determined the virus isolation probabilities depending on cycle threshold (Ct) or log10 genome copies/ml using logistic regression. We included 55 studies. There is substantial statistical and clinical heterogeneity. Seven studies were included for diagnostic accuracy. Sensitivity ranged from 90% to 99% and specificity from 29% to 92%. In meta-analysis, the PPVs varied across subgroups with different sampling times after symptom onset, with 1% (95% confidence interval [CI], 0%-7%) in sampling beyond 10 days and 27% (CI, 19%-36%) to 46% (CI, 33%-60%) in subgroups that also included earlier samples. Estimates of virus isolation probability varied between 6% (CI, 0%-100%) and 50% (CI, 0%-100%) at a Ct value of 30 and between 0% (CI, 0%-22%) and 63% (CI, 0%-100%) at 5 log10 genome copies/ml. Evidence on RT-PCR performance in detecting infectious SARS-CoV-2 in respiratory samples was limited. Major limitations were heterogeneity and poor reporting. RT-PCR and cell culture protocols need further standardisation.

A cocktail probe approach to evaluate the effect of hormones on the expression and activity of CYP enzymes in human hepatocytes with conditions simulating late stage of pregnancy.

PURPOSE: Pregnancy-mediated physiological and biochemical changes contribute to alterations in the pharmacokinetics of certain drugs. There is a paucity of data on the systematic evaluation of the underlying mechanisms. The objective of the current study was to examine the impact of changes in circulating and tissue hormonal concentration during the late stage of pregnancy on the activity and expression of hepatic cytochrome P450 (CYP) enzymes using a cocktail probe approach. METHODS: Freshly isolated primary human hepatocytes were incubated with third trimester physiologic (plasma) and projected liver (ten-fold higher) concentrations of female hormones: progesterone (2 µM), estradiol (0.3 µM), estriol (0.8 µM), estrone (0.2 µM), 17α-hydroxyprogesterone (0.1 µM), and human growth hormone (0.005 µM). The metabolic activity of the hepatocytes was assessed using a cocktail of isozyme-specific P450 probe substrates (CYP1A2 (phenacetin), CYP2C9 (diclofenac), CYP2C19 (S-mephenytoin), CYP2D6 (dextromethorphan), and CYP3A4 (testosterone)). A validated LC-MS/MS assay was used to measure the corresponding metabolite concentrations. CYP450 protein and mRNA levels were measured using western blot and qRT-PCR, respectively. RESULTS: Female hormones at projected third-semester hepatic concentrations significantly enhanced mRNA and protein expression and increased the metabolic activity of CYP3A4. The expression and activity of other CYP450 enzymes studied were not affected by mixtures of female hormones at concentrations used. CONCLUSION: The increased activity of CYP3A4 is consistent with the clinically observed increase in clearance of CYP3A4 substrates during pregnancy. Overall expression and activity of CYP450 isozymes are differentially regulated during pregnancy.

Reducing Bias and Quantifying Uncertainty in Fluorescence Produced by PCR.

We present a new approach for relating nucleic-acid content to fluorescence in a real-time Polymerase Chain Reaction (PCR) assay. By coupling a two-type branching process for PCR with a fluorescence analog of Beer's Law, the approach reduces bias and quantifies uncertainty in fluorescence. As the two-type branching process distinguishes between complementary strands of DNA, it allows for a stoichiometric description of reactions between fluorescent probes and DNA and can capture the initial conditions encountered in assays targeting RNA. Analysis of the expected copy-number identifies additional dynamics that occur at short times (or, equivalently, low cycle numbers), while investigation of the variance reveals the contributions from liquid volume transfer, imperfect amplification, and strand-specific amplification (i.e., if one strand is synthesized more efficiently than its complement). Linking the branching process to fluorescence by the Beer's Law analog allows for an a priori description of background fluorescence. It also enables uncertainty quantification (UQ) in fluorescence which, in turn, leads to analytical relationships between amplification efficiency (probability) and limit of detection. This work sets the stage for UQ-PCR, where both the input copy-number and its uncertainty are quantified from fluorescence kinetics.

Chemical Analysis and in Vitro Antiviral Effects of Northeast Türkiye Propolis Samples against HSV-1.

Propolis is one of the mixtures with the widest biological activity among natural products used in complementary medicine. HSV-1 is a highly contagious and endemic virus. Available drugs are insufficient for recurrent HSV-1 infections. Therefore, new approaches to treat HSV-1 infections are still being developed. In this study, it was aimed to investigate the inhibition effect of ethanolic Anatolian propolis extracts obtained from the Eastern Black Sea Region (Pazar, Ardahan, and Uzungöl) on HSV-1. In addition to the total phenolic (TPC) and the total flavonoid content (TFC), the phenolic profiles of the extracts were analyzed by HPLC-UV. The antiviral activity of the extracts were tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), quantitative Real Time Polymerase Chain Reaction (qRT-PCR), and plaque reduction tests, and the results were evaluated statistically. It was determined that the total amount of phenolic substances varied between 44.12 and 166.91 mg GAE/g, and the total flavonoid content of the samples varied between 12.50 and 41.58 (mg QUE/g). It was shown that all propolis samples used in the current study were effective against HSV-1, but the higher phenolic compounds contained in the samples showed the higher activity. The results show that ethanolic propolis extracts are promising candidates for HSV-1 treatment.

Assessment of interleukin 1 receptor antagonist (IL-1RA) levels in children with and without community acquired pneumonia: a hospital based case-control study.

The primary objective was to compare serum interleukin-1 receptor antagonist (IL-1RA) levels in cases of community acquired pneumonia (CAP) and healthy age-gender-matched controls. The secondary objective was to compare serum IL-1RA levels in cases which were positive or negative for Streptococcus pneumoniae in the blood by real-time-polymerase chain reaction (RT-PCR). Hospitalized children with World Health Organization defined CAP, aged 2-59 months, were included as cases. Healthy controls were recruited from the immunization clinic of the hospital. Enzyme-linked immunosorbent assay (ELISA) test was used to detect serum IL-1RA levels. Identification of S.pneumoniae in blood was done by RT-PCR. From October 2019 to October 2021, 330 cases (123, 37.27% female) and 330 controls (151, 45.75% females) were recruited. Mean serum IL-1RA levels (ng/ml) were 1.36 ± 0.95 in cases and 0.25 ± 0.25 in controls (p < 0.001). Within cases, serum IL-1RA levels were significantly higher in those whose RT-PCR was positive for S.pneumoniae. Thus serum IL-1RA levels may be evaluated as a surrogate marker of S.pneumoniae in future studies.

The main purpose of the study was to compare the levels of a protein in the blood that is part of the immune system, called interleukin-1 receptor antagonist (IL-1RA) which binds to the same site in the body as an antibody does when it is fighting certain diseases, like pneumonia. We then compared the levels of this protein, IL-1RA, in hospitalized cases of community acquired pneumonia (CAP), caused from exposure to germs in the community, rather than obtained or contracted in a hospital, to that found in healthy people or 'controls' recruited from an immunization clinic. Cases and controls were matched for age and gender. The secondary objective of our study was to compare the level of IL-1RA protein in the blood in cases that were positive for the bacteria Streptococcus pneumoniae measured in the blood by a molecular test called real-time-polymerase chain reaction which can detect a very small amounts of a protein that is uniquely found in the S.pneumoniae bacteria that causes CAP. This case­control study was conducted in a large teaching institution that receives referrals from the other hospitals in northern India. It was found that serum IL-1RA levels were raised in cases of CAP, especially those which were possibly due to S.pneumoniae.

Clinical significance of Bacteroides fragilis as a potential prognostic factor in colorectal cancer.

INTRODUCTION: Bacteroides fragilis (B. fragilis) is considered to act in an anti-inflammatory manner on the intestinal tract. On the contrary, enterotoxigenic B. fragilis (ETBF), a subtype of B. fragilis, produces an enterotoxin (BFT; B. fragilis toxin), leading to asymptomatic chronic infections and colonic tumor formation. However, the impact of B. fragilis and ETBF on the clinical outcome of colorectal cancer (CRC) remains unclear. We aim to assess whether their presence affects the outcome in patients with CRC after curative resection. METHODS: We obtained 197 pairs of matched formalin-fixed paraffin-embedded samples from cancerous and adjacent non-cancerous tissues of patients with pathological stage (pstage) II and III CRC after curative resection. The presence of B. fragilis and ETBF were estimated using real-time polymerase chain reaction, and recurrence-free survival (RFS) and overall survival (OS) of the patients were analyzed. RESULTS: 16S rRNA for B. fragilis and bft DNA were detected in 120 (60.9%) and 12 (6.1%) of the 197 patients, respectively. B. fragilis-positive patients had better RFS than B. fragilis-negative patients, although that was not statistically significant. In subgroup analysis, better outcomes on RFS were observed in the presence of B. fragilis in pstage II and left-sided CRC. The association of B. fragilis positivity on OS was accentuated in the depth of T4 subgroup. No significant differences were observed in RFS and OS between ETBF and non-toxigenic B. fragilis. CONCLUSIONS: Our findings suggest that the presence of B. fragilis is associated with better outcomes in patients with pstage II and III CRC after curative resection.

An Aluminum-Based Microfluidic Chip for Polymerase Chain Reaction Diagnosis.

Real-time polymerase chain reaction (real-time PCR) tests were successfully conducted in an aluminum-based microfluidic chip developed in this work. The reaction chamber was coated with silicone-modified epoxy resin to isolate the reaction system from metal surfaces, preventing the metal ions from interfering with the reaction process. The patterned aluminum substrate was bonded with a hydroxylated glass mask using silicone sealant at room temperature. The effect of thermal expansion was counteracted by the elasticity of cured silicone. With the heating process closely monitored, real-time PCR testing in reaction chambers proceeded smoothly, and the results show similar quantification cycle values to those of traditional test sets. Scanning electron microscope (SEM) and atomic force microscopy (AFM) images showed that the surface of the reaction chamber was smoothly coated, illustrating the promising coating and isolating properties. Energy-dispersive X-ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), and inductively coupled plasma-optical emission spectrometer (ICP-OES) showed that no metal ions escaped from the metal to the chip surface. Fourier-transform infrared spectroscopy (FTIR) was used to check the surface chemical state before and after tests, and the unchanged infrared absorption peaks indicated the unreacted, antifouling surface. The limit of detection (LOD) of at least two copies can be obtained in this chip.