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.

Role of histopathological, serological and molecular findings for the early diagnosis of treatment failure in leprosy.

BACKGROUND: Treatment failure (TF) in leprosy following multidrug therapy (MDT) presents a significant challenge. The current World Health Organization (WHO) fixed-duration MDT regimen, based on lesion count, might not be adequate. Leprosy lacks clear-cut objective cure criteria, and the predictive value of post-MDT histopathological findings remains uncertain. This study aims to identify predictive factors for TF among leprosy patients who have completed the WHO-recommended MDT. METHODS: An analysis was conducted on 80 individuals from a national leprosy reference center, comprising 40 TF cases (with a mean relapse at 13.0 months) and 40 controls (with a mean of 113.1 months without disease signs). Various epidemiological and clinical-laboratory parameters were assessed post-MDT. RESULTS: In skin samples, the presence of foamy granuloma (OR = 7.36; 95%CI2.20-24.60; p = 0.0012) and histological bacillary index (hBI) ≥ 1+ (OR = 1.55; 95%CI1. 22-1.99; p = 0.0004) were significantly associated with TF, with odds ratios of 7.36 and 1.55, respectively. Individuals who experienced TF had a mean hBI of 3.02+ (SD ± 2.02), while the control group exhibited a mean hBI of 1.8+ (SD ± 1.88). An hBI ≥ 3 + showed a sensitivity of 73% and a specificity of 78% for TF detection (AUC: 0.75; p = 0.0001). Other histopathological features like epithelioid granulomas, and skin changes did not show significant associations (p > 0.05). Additionally, higher anti-phenolic glycolipid-I (anti-PGL-I) ELISA index (EI) levels were linked to a 1.4-fold increased likelihood for TF (OR = 1.4; 95%CI1.13-1.74; p = 0.0019). A mean EI of 4.48 (SD ± 2.80) was observed, with an EI ≥ 3.95 showing a sensitivity of 79% and a specificity of 59% for TF detection (AUC: 0.74; p = 0.0001). Moreover, the presence of Mycobacterium leprae (M. leprae) DNA in real-time polymerase chain reaction (qPCR) was associated with a 3.43-fold higher likelihood of TF. Multivariate regression analysis indicated that concurrent presentation of neural/perineural lymphocytic infiltrate, foamy granuloma, hBI ≥ 1+, and EI ≥ 1 markedly increased the likelihood of TF by up to 95.41%. CONCLUSION: Persistence of nerve-selective lymphocytic infiltrate, foamy granulomas, and bacilli in skin biopsies, and elevated EI post-MDT, may serve as predictive factors for identifying individuals at higher probability of TF.

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.