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The accurate detection and quantification of biological species that are rarely present but potentially devastating is of paramount importance for the life sciences, biosecurity, food safety, and environmental monitoring. Consequently, there has been significant interest in the sensitive and accurate detection of nucleic acids, leveraging both chemical and biological methods. Among these, quantitative polymerase chain reaction (qPCR) is regarded as the gold standard due to its sensitivity and precision in identifying specific nucleic acid targets. Despite the widespread adoption of qPCR for nucleic acid detection, the analysis of qPCR data typically depends on the use of calibrated standard curves and a threshold method to interpret signal measurements. In this study, we use a stochastic simulation to show the limitations of the threshold method due to its assumptions on amplification kinetics. We propose a new approach for the absolute quantification of nucleic acids that overcomes these limitations by reconstructing the efficiency profile across amplification cycles and using cumulative amplification folds to build a standard curve, thus avoiding the constant efficiency assumption. Our method, validated through experiments with nucleic acid amplification in the presence of potent inhibitors, demonstrates improved accuracy in quantifying nucleic acids, avoiding the systematic errors of the threshold method. This innovation enhances the reliability of nucleic acid quantification, especially where traditional methods struggle with kinetic variability. [ABSTRACT FROM AUTHOR]

We report a cluster of clade I monkeypox virus infections linked to sexual contact in the Democratic Republic of the Congo. Case investigations resulted in 5 reverse transcription PCR-confirmed infections; genome sequencing suggest they belonged to the same transmission chain. This finding demonstrates that mpox transmission through sexual contact extends beyond clade IIb.

Purpose: To investigate the possible antimicrobial activity of glycine air polishing by comparing peri-implant microbiota before and after treatment. Materials and Methods: A total of 15 patients who received implant-supported full-arch fixed rehabilitations were included. After prosthesis removal (T0), Plaque Index (PI), probing depth (PD), and bleeding on probing (BOP) were recorded. In each hemiarch, the implant with the highest PD score was selected for microbiologic sample collection from the peri-implant sulcus (T0). All patients received two different hygienic protocols (randomly administered, one per each hemiarch): glycine air-polishing (G) and cleansing with cotton pellets soaked in saline (C). At 7 days (T1) and 3 months (T2) after the intervention, PI and BOP were recorded, and new microbiologic samples were taken. Traditional polymerase chain reaction (PCR) and quantitative PCR real-time were employed for microbiologic analysis to investigate how the presence of different bacterial species varied according to the hygienic treatment performed. Results: Treatment G provided a significantly higher PI score reduction around implants compared to treatment C (P = .015). No statistical difference was found in the microbial population around G and C implant sites, with Tannerella forsythia being the most commonly detected bacterial species in both G and C groups. No statistical differences were found between the antimicrobial activity of treatments C and G. Conclusions: Glycine powder air polishing is a valid method for professional hygienic care of implants and was more effective in PI reduction compared to the control treatment. However, its antimicrobial efficacy cannot be confirmed by the outcomes of the present study. [ABSTRACT FROM AUTHOR]

Microbial infections of the brain can lead to dementia, and for many decades microbial infections have been implicated in Alzheimers disease (AD) pathology. However, a causal role for infection in AD remains contentious, and the lack of standardized detection methodologies has led to inconsistent detection/identification of microbes in AD brains. There is a need for a consensus methodology; the Alzheimers Pathobiome Initiative aims to perform comparative molecular analyses of microbes in post mortem brains versus cerebrospinal fluid, blood, olfactory neuroepithelium, oral/nasopharyngeal tissue, bronchoalveolar, urinary, and gut/stool samples. Diverse extraction methodologies, polymerase chain reaction and sequencing techniques, and bioinformatic tools will be evaluated, in addition to direct microbial culture and metabolomic techniques. The goal is to provide a roadmap for detecting infectious agents in patients with mild cognitive impairment or AD. Positive findings would then prompt tailoring of antimicrobial treatments that might attenuate or remit mounting clinical deficits in a subset of patients.

Limit of detection (LOD), speed, and cost for some of the most important diagnostic tools, i.e., lateral flow assays (LFA), enzyme-linked immunosorbent assays (ELISA), and polymerase chain reaction (PCR), all benefited from both the financial and regulatory support brought about by the pandemic. From those three, PCR has gained the most in overall performance. However, implementing PCR in point of care (POC) settings remains challenging because of its stringent requirements for a low LOD, multiplexing, accuracy, selectivity, robustness, and cost. Moreover, from a clinical point of view, it has become very desirable to attain an overall sample-to-answer time (t) of 10 min or less. Based on those POC requirements, we introduce three parameters to guide the design towards the next generation of PCR reactors: the overall sample-to-answer time (t); lambda (λ), a measure that sets the minimum number of copies required per reactor volume; and gamma (γ), the systems thermal efficiency. These three parameters control the necessary sample volume, the number of reactors that are feasible (for multiplexing), the type of fluidics, the PCR reactor shape, the thermal conductivity, the diffusivity of the materials used, and the type of heating and cooling systems employed. Then, as an illustration, we carry out a numerical simulation of temperature changes in a PCR device, discuss the leading commercial and RT-qPCR contenders under development, and suggest approaches to achieve the PCR reactor for RT-qPCR of the future.

In most people living with HIV (PLWH) on effective antiretroviral therapy (ART), cell-associated viral transcripts are readily detectable in CD4+ T cells despite the absence of viremia. Quantification of HIV RNA species provides insights into the transcriptional activity of proviruses that persist in cells and tissues throughout the body during ART (HIV reservoir). One such technique for HIV RNA quantitation, HIV transcription profiling, developed in the Yukl laboratory, measures a series of HIV RNA species using droplet digital PCR. To take advantage of advances in digital (d)PCR, we adapted the HIV transcription profiling technique to Qiagens dPCR platform (QIAcuity) and compared its performance to droplet digital (dd)PCR (Bio-Rad QX200 system). Using RNA standards, the two technologies were tested in parallel and assessed for multiple parameters including sensitivity, specificity, linearity, and intra- and inter-assay variability. The newly validated dPCR assays were then applied to samples from PLWH to determine HIV transcriptional activity relative to HIV reservoir size. We report that HIV transcriptional profiling was readily adapted to dPCR and assays performed similarly to ddPCR, with no differences in assay characteristics. We applied these assays in a cohort of 23 PLWH and found that HIV reservoir size, based on genetically intact proviral DNA, does not predict HIV transcriptional activity. In contrast, levels of total DNA correlated with levels of most HIV transcripts (initiated, proximally and distally elongated, unspliced, and completed, but not multiply spliced), suggesting that a considerable proportion of HIV transcripts likely originate from defective proviruses. These findings may have implications for measuring and assessing curative strategies and clinical trial outcomes.

A definitive diagnosis of nevus or melanoma is not always possible for histologically ambiguous melanocytic neoplasms. In such cases, ancillary molecular testing can support a diagnosis of melanoma if certain chromosomal aberrations are detected. Current technologies for copy number variation (CNV) detection include chromosomal microarray analysis (CMA) and fluorescence in situ hybridization. Although CMA and fluorescence in situ hybridization are effective, their utilization can be limited by cost, turnaround time, and inaccessibility outside of large reference laboratories. Droplet digital polymerase chain reaction (ddPCR) is a rapid, automated, and relatively inexpensive technology for CNV detection. We investigated the ability of ddPCR to quantify CNV in cyclin-dependent kinase inhibitor 2A ( CDKN2A ), the most commonly deleted tumor suppressor gene in melanoma. CMA data were used as the gold standard. We analyzed 57 skin samples from 52 patients diagnosed with benign nevi, borderline lesions, primary melanomas, and metastatic melanomas. In a training cohort comprising 29 randomly selected samples, receiver operator characteristic curve analysis revealed an optimal ddPCR cutoff value of 1.73 for calling CDKN2A loss. In a validation cohort comprising the remaining 28 samples, ddPCR detected CDKN2A loss with a sensitivity and specificity of 94% and 90%, respectively. Significantly, ddPCR could also identify whether CDKN2A losses were monoallelic or biallelic. These pilot data suggest that ddPCR can detect CDKN2A deletions in melanocytic tumors with accuracy comparable with CMA. With further validation, ddPCR could provide an additional CNV assay to aid in the diagnosis of challenging melanocytic neoplasms.

We report a case of a 53-year-old HIV-negative patient in San Francisco, California, USA, with no classic mpox prodromal symptoms or skin lesions who experienced fulminant, vision-threatening scleritis, keratitis, and uveitis. Deep sequence analysis identified monkeypox virus RNA in the aqueous humor. We confirmed the virus on the cornea and sclera by PCR.

Background and Aim: Leptospirosis is a re-emerging zoonosis that is under-reported in tropical countries, and canines can be a potential reservoir of the disease. The objective of this study was to diagnose Leptospira spp. that is actively infected and re-infected in stray dogs and cats from Bogota, D.C., Colombia. Materials and Methods: A sample of 200 animals, including dogs and cats from the animal protection programs of Bogota, Colombia, were used in this study. Blood was collected from these animals for serum and DNA analysis. Conventional polymerase chain reaction (PCR) was performed using the 16s rRNA primer set, and higher-quality amplification products were sequenced by Sanger. For serodiagnosis, a group of PCR-positive samples was tested using the microagglutination test (MAT). Results: The overall PCR positivity of stray dogs and cats was 56%, 52.9%, and 65.3% in dogs and cats, respectively. The MAT seropositivity was 77.3%, and only dogs showed titers higher than 1:400. Canicola, Icterohaemorrhagiae, Pomona, Hardjo Prajitno, and Canicola and Hardjo prajitno were the serogroups associated with dogs and cats, respectively. Phylogenetic analysis revealed that the strains belonging to Leptospira interrogans serovars related to isolated samples of American, European, and Asian bats (Myotis myotis), dogs, and bovines of American origin. Conclusion: These results showed that stray dogs and cats were previously exposed to different serovars of Leptospira spp. and re-infected with other serovars that actively participated in the transmission cycle. These findings highlight the importance of actively diagnosing infectious animals to design effective intervention strategies.

Abstract Purpose The clinical significance of negative toxin enzyme immunoassays (EIA) for Clostridioides difficile infections (CDIs) is unclear. Our study aimed to investigate the significance of toxin EIA-negative in the diagnosis and prognosis of CDI. Methods All stool specimens submitted for C. difficile toxin EIA testing were cultured to isolate C. difficile. In-house PCR for tcdA, tcdB, cdtA, and cdtB genes were performed using C. difficile isolates. Stool specimens were tested with C. difficile toxins A and B using EIA kit (RIDASCREEN Clostridium difficile toxin A/B, R-Biopharm AG, Darmstadt, Germany). Characteristics and subsequent CDI episodes of toxin EIA-negative and -positive patients were compared. Results Among 190 C. difficile PCR-positive patients, 83 (43.7%) were toxin EIA-negative. Multivariate analysis revealed independent associations toxin EIA-negative results and shorter hospital stays (OR = 0.98, 95% CI 0.96–0.99, p = 0.013) and less high-risk antibiotic exposure in the preceding month (OR = 0.38, 95% CI 0.16–0.94, p = 0.035). Toxin EIA-negative patients displayed a significantly lower white blood cell count rate (11.0 vs. 35.4%, p

Background: Even though a few years have passed since the coronavirus disease 2019 (COVID-19) outbreak, information regarding certain aspects of the disease, such as post-infection immunity, is still quite limited. This study aimed to evaluate post-infection protection and COVID-19 features among healthcare workers (HCWs), during three successive surges, as well as the rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection, reactivation, re-positivity, and severity.Methods: This cross-sectional population-level observational study was conducted from 20 April 2020 to 18 February 2021. The study population included all HCWs in public or private hospitals in Fars Province, Southern Iran. The infection rate was computed as the number of individuals with positive polymerase chain reaction (PCR) tests divided by the total number of person-days at risk. The re-infection was evaluated after 90 days. Results: A total of 30,546 PCR tests were performed among HCWs, of which 13,749 (61.94% of total HCWs) were positive. Considering the applied 90-day threshold, there were 44 (31.2%) cases of reactivation and relapse, and 97 (68.8% of infected and 1.81% of total HCWs) cases of reinfection among 141 (2.64%) diagnosed cases who experienced a second episode of COVID-19. There was no significant difference in symptoms (P=0.65) or the necessity for ICU admission (P=0.25). The estimated protection against repeated infection after a previous SARS-CoV-2 infection was 94.8% (95% CI=93.6-95.7).Conclusion: SARS-CoV-2 re-positivity, relapse, and reinfection were rare in the HCW population. After the first episode of infection, an estimated 94.8% protection against recurring infections was achieved.

Shuai Li, Xue Han, Jing Ma, Guo-Hong Huang, Shu-Ting Yang, Chang-Min Wang Clinical Laboratory Center, People’s Hospital of Xinjiang Uygur Autonomous Region, Urumqi, Xinjiang, 830001, People’s Republic of ChinaCorrespondence: Chang-Min Wang, Department of Clinical Laboratory Center, People’s Hospital of Xinjiang Uygur Autonomous Region, No. 91 Tianchi Street, Tianshan District, Urumqi, Xinjiang, 830001, People’s Republic of China, Tel +86 13609992616, Fax +86− 9918564168, Email wang_c3223min@yeah.netObjective: To investigate the value of metagenomic Next-Generation Sequencing (mNGS) in diagnosing Pneumocystis jirovecii pneumonia (PJP) in non-human immunodeficiency virus (HIV)-infected patients.Methods: In this retrospective study, non-HIV-infected patients with PJP and those diagnosed with non-PJP from August 2022 to December 2024 were selected as subjects. The presence of Pneumocystis jirovecii (PJ) and other co-pathogens in bronchoalveolar lavage fluid (BALF) was analyzed, and the diagnostic efficacy of NGS, polymerase chain reaction (PCR) and serum 1,3-β-D-glucan (BDG) in PJP was compared with the reference standard of clinical compound diagnosis.Results: Eighty-nine non-HIV-infected patients were recruited, with dyspnea as the primary symptom (69.66%) and solid malignant tumor as the most common underlying disease (20.22%). Taking clinical compound diagnosis as the reference standard, the sensitivity, specificity, negative predictive value and positive predictive value of mNGS were higher than those detected by PCR and serum BDG. Among 42 non-HIV-infected patients with PJP who underwent mNGS and conventional pathogen detection of BALF, 6 had simple PJ infection and 36 had combined PJ infection. The detection rate of mNGS in mixed infections was significantly higher than that of conventional pathogen detection (85.71 vs 61.70%, P = 0.012). A total of 127 pathogens were detected in BALF using mNGS, among which fungi had the highest detection rate (46.46%). The fungi, viruses and bacteria detected were mainly Pneumocystis jirovecii, human gammaherpesvirus 4 and Acinetobacter baumannii.Conclusion: mNGS is highly effective in diagnosing non-HIV-infected patients with PJP and exhibits ideal performance in the detection of co-pathogens. In addition, it has certain value for clinical diagnosis and guidance of targeted anti-infective drug treatment.Keywords: metagenomic next-generation sequencing, Pneumocystis jirovecii pneumonia, polymerase chain reaction, non-HIV infection

Introduction: Duchenne Muscular Dystrophy (DMD) is an X-linked recessive neuromuscular disorder characterised by progressive, irreversible muscle weakness. It is caused by a mutation in the dystrophin or DMD gene, leading to the absence of the essential muscle protein Dystrophin in DMD. Muscle strength continually diminishes, and death usually occurs from chronic respiratory insufficiency and/or cardiac failure. Due to the high mortality rate, early diagnosis is crucial to allow appropriate planning for patient care and treatment. In India, the traditional multiplex Polymerase Chain Reaction (PCR) assay is the most common method to detect Dystrophin gene deletion mutations. Aim: To describe the spectrum of deletion patterns in the dystrophin gene in muscular dystrophy patients attending a tertiary care hospital in Northeast India. Materials and Methods: A hospital-based cross-sectional study was conducted on a total of 53 suspected DMD patients attending the Department of Neurology and Department of Paediatrics at Assam Medical College, Dibrugarh, Assam, India from January 2016 to December 2022. Multiplex PCR was performed to study the deletion patterns for the 25 most common exons of the DMD gene for all patients at the Genetic lab, Department of Anatomy. Deletion mutations at different multiple exons were found, and the results were statistically analysed using Statistical Package for Social Sciences (SPSS) to calculate the mean and standard deviation. The results were presented in tabular form as percentages. R esults: Out of the 53 cases suspected to have DMD on the basis of clinical presentation and high serum Creatine Kinase (CK) levels, 34 patients (64.2%) showing deletion mutations in the 25 most common exons of the DMD gene were included in the study. Deletions were most common in 15 (44.1%) patients in the distal hotspot region of exons 44-55. The most common gene deleted was exon 50 in 11 (32.4%) patients. The age at which symptoms were noticed was 4.7±2.01 years. The mean age at diagnosis was 8.4±2.4 years. C onclusion: In the present study, most patients suspected of DMD based on clinical and laboratory findings had deletions in the DMD gene, with the most common region for deletion in the dystrophin gene being the distal hotspot region, and exon 50 being the most commonly deleted.

Abstract Thermostable DNA polymerases, such as Taq isolated from the thermophilic bacterium Thermus aquaticus, enable one-pot exponential DNA amplification known as polymerase chain reaction (PCR). However, properties other than thermostability - such as fidelity, processivity, and compatibility with modified nucleotides - are important in contemporary molecular biology applications. Here, we describe the engineering and characterization of a fusion between a DNA polymerase identified in the marine archaea Nanoarchaeum equitans and a DNA binding domain from the thermophile Sulfolobus solfataricus. The fusion creates a highly active enzyme, Neq2X7, capable of amplifying long and GC-rich DNA, unaffected by replacing dTTP with dUTP in PCR, and tolerant to various known PCR inhibitors. This makes it an attractive DNA polymerase for use, e.g., with uracil excision (USER) DNA assembly and for contamination-free diagnostics. Using a magnification via nucleotide imbalance fidelity assay, Neq2X7 was estimated to have an error rate lower than 2 ∙ 10−5 bp−1 and an approximately 100x lower fidelity than the parental variant Neq2X, indicating a trade-off between fidelity and processivity – an observation that may be of importance for similarly engineered DNA polymerases. Neq2X7 is easy to produce for routine application in any molecular biology laboratory, and the expression plasmid is made freely available.

Trypanosoma evansi is a widespread hemoprotozoa that causes trypanosomiasis in both livestock and wildlife. One of the susceptible animals is the Sumatran elephant, which is included in the endangered species category. The infection of this parasite often develops into chronic and sub-clinical forms in elephants, therefore it may become unnoticed and hard to diagnose. This study aimed to analyze and evaluate the infection of T. evansi in semi-captive Sumatran elephants in Way Kambas National Park, Indonesia, using various diagnostic tests. The prevalence of T. evansi in a total of 53 Sumatran elephants was estimated using a card agglutination test for trypanosomiasis (CATT) in 2016. A longitudinal study was later conducted in 2019 using Giemsa stained blood smear (GSBS) and polymerase chain reaction (PCR). The results showed that 26.4% of samples (14/53) were positively detected by both CATT in 2016 and PCR in 2019, while GSBS was unable to detect the parasites in all samples. Furthermore, four individuals were confirmed to have persistent infections. This study concluded that the ability of CATT and PCR were more convincing over GSBS for the diagnosis of sub-clinical trypanosomiasis in Sumatran elephants. However, it is recommended to use a combination of CATT as a screening tool and PCR as a confirmatory test for reliable results.

OBJECTIVE: To determine the Frequency of Germinal Center B (GCB) type Diffuse Large B Cell Lymphoma (DLBCL) in Karachi, Pakistan, and to screen the DLBCL patients for EZH2 mutation. METHODOLOGY: A cross-sectional study was conducted on DLBCL patients, and IHC analysis, PCR, and sequencing of the EZH2 gene were used. This study was conducted from August 2016 to July 2020. Surgical specimens received in the histopathology section of DDRRL centrally and from different collection units throughout Sindh having been diagnosed with DLBCL were collected. Three markers (CD10, BCL6, and MUM1) were used for subtyping. DNA was manually extracted from the FFPE Blocks of DLBCL cases using phenol: chloroform: isopropanol alcohol. Polymerase chain reaction (PCR) was performed, and samples were sent from Dow University to Korea (Macrogen) for sequencing. The amplified product was 189bp. Primers were used for EZH2 to flank the region containing the tyrosine domain on exon 16 and exon 18. RESULTS: Out of 116 cases, 60.3% and 39.7% were male and female, respectively. The mean age was 49.73±16.38 years. The mean survival duration of cases diagnosed with GCB was 8.21±7.39 months. Polymorphism of EZH2 mutation (Tyr 701 Asn) was found to be positive in GCB-DLBCL for three samples (16.6%). CONCLUSION: In the current study, Sanger sequencing was used to detect any mutation in the coding region of EZH2, and a unique mutation was reported (Tyr701Asn).

Background: High-risk Human Papillomavirus (HPV) genotypes are found in malignant oral epithelial lesions, and HPV infection is proposed as a risk factor for initiating Squamous cell carcinoma (SCC) in the head and neck region. This study suggests a practical approach to detect HPV in HPV-associated oral epithelial dysplasia (HAOED).Methods: Fifty-four oral epithelial dysplasia specimens were examined, comprising twenty-seven cases diagnosed with high-grade dysplasia and twenty-seven cases diagnosed with low-grade dysplasia using a binary grading system. To assess the cases for HPV, the specimens were examined for p16 protein using an immunohistochemical (IHC) study, and then, the Chromatin In Situ Hybridization (CISH) test was performed for all positive cases. Chromatin Immunoprecipitation-Polymerase Chain Reaction (ChIP-PCR) was performed on CISH-positive specimens to assess the outcome. This cross-sectional study was conducted in 2020 at Tehran University of Medical Science. SPSS software version 22.0 was used to perform the Chi square or Fisher’s exact test to examine the relationship between variables (statistically significant level P0.99), and in the nine cases, undergone the ChIP-PCR study, two cases (22.2%) showed positivity for HPV-16, while one case (11.1%) demonstrated positivity for HPV-51.Conclusion: Regarding HAOED, here, we proposed a step-by-step combination approach using different diagnostic methods, including IHC for p16 protein, CISH, and ChIP-PCR based on a complementary algorithm.

Detecting Ganoderma boninense in Indonesia is crucial for effectively controlling and mitigating the spread of basal stem disease in oil palm fields. While there is ongoing development of tolerant plants, no such plant has been successfully created yet. Consequently, researchers are actively studying detection methods for Ganoderma boninense. One established and highly accurate approach is the use of polymerase chain reaction (PCR) techniques for molecular detection. However, this method requires time‐consuming sample preparation, which can pose challenges in plantation settings. To address this problem, a portable lab‐on‐chip device has been introduced. This technology enables easy and automatic DNA retrieval from liquid samples by absorbing lysed DNA using magnetic beads. An efficient mechanism for manipulating the magnetic bead within the semiconductor has been successfully implemented. The extraction process typically takes around 15 minutes using a modified methodology on the chip device approach. The chip facilitates the retrieval of two samples with a capacity of 120 µL for each sample. The PCR method was utilized to validate the equivalence of the lab‐on‐chip device extraction to the standard extraction method. This represents a promising alternative for expedited and simplified detection of Ganoderma boninense in field conditions.

Aims: Continuous local antibiotic perfusion (CLAP) has recently attracted attention as a new drug delivery system for orthopaedic infections. CLAP is a direct continuous infusion of high-concentration gentamicin (1,200 μg/ml) into the bone marrow. As it is a new system, its influence on the bone marrow is unknown. This study aimed to examine the effects of high-concentration antibiotics on human bone tissue-derived cells. Methods: Cells were isolated from the bone tissue grafts collected from six patients using the Reamer-Irrigator-Aspirator system, and exposed to different gentamicin concentrations. Live cells rate, apoptosis rate, alkaline phosphatase (ALP) activity, expression of osteoblast-related genes, mineralization potential, and restoration of cell viability and ALP activity were examined by in vitro studies. Results: The live cells rate (the ratio of total number of cells in the well plate to the absorbance-measured number of live cells) was significantly decreased at ≥ 500 μg/ml of gentamicin on day 14; apoptosis rate was significantly increased at ≥ 750 μg/ml, and ALP activity was significantly decreased at ≥ 750 μg/ml. Real-time reverse transcription-polymerase chain reaction results showed no significant decrease in the ALP and activating transcription factor 4 transcript levels at ≥ 1,000 μg/ml on day 7. Mineralization potential was significantly decreased at all concentrations. Restoration of cell viability was significantly decreased at 750 and 1,000 μg/ml on day 21 and at 500 μg/ml on day 28, and ALP activity was significantly decreased at 500 μg/ml on day 28. Conclusion: Our findings suggest that the exposure concentration and duration of antibiotic administration during CLAP could affect cell functions. However, further in vivo studies are needed to determine the optimal dose in a clinical setting. Cite this article: Bone Joint Res 2024;13(3):92–101.

Background and Aim: Bovine tuberculosis (TB) is a zoonotic disease of great public health importance, particularly in Indonesia, where control measures are limited or are not implemented. This study aimed to detect the presence of Mycobacterium pathogens in milk samples from dairy cattle in Pasuruan regency and Surabaya City, East Java, using Ziehl–Neelsen acid-fast staining and polymerase chain reaction (PCR). Materials and Methods: Milk samples were aseptically collected from 50 cattle in the Lekok Subdistrict, Pasuruan Regency, and 44 from dairy farms in the Lakarsantri Subdistrict, Wonocolo Subdistrict, Mulyorejo Subdistrict, and Kenjeran Subdistrict, Surabaya, East Java. To detect Mycobacteria at the species level, each sample was assessed by Ziehl–Neelsen staining and PCR using the RD1 and RD4 genes. Results: The results of PCR assay from 50 samples in Lekok Subdistrict, Pasuruan Regency showed that 30 samples (60%) were positive for Mycobacterium tuberculosis and two samples (4%) were positive for Mycobacterium bovis, although Ziehl–Neelsen staining did not show the presence of Mycobacterium spp. In the Surabaya region, 31 samples (70.45%) were positive for M. tuberculosis and three samples (6.8%) were positive for M. bovis. Six samples (13.63%) from all PCR-positive samples could be detected microscopically with Ziehl–Neelsen. Conclusion: The presence of bovine TB in this study supports the importance of using a molecular tool alongside routine surveillance for a better understanding of the epidemiology of bovine TB in East Java.

Morinda officinalis is a traditional Chinese tonic herb, and have been used in the treatment of multiple diseases. Here, three iridoid glycosides isolated from M. officinalis were evaluated for their roles in the autophagy-lysosomal pathway. All three iridoid glycosides could induce TFEB/TFE3-mediated lysosomal biogenesis and trigger autophagy. Interestingly, they promoted the nuclear import of TFEB/TFE3 without affecting their nuclear export, suggesting their role in the maintenance of lysosomal homeostasis. The results from this study shed light on the identification of autophagy activators from M. officinalis and provide a basis for developing them in the treatment of oxidative stress-involved diseases. [ABSTRACT FROM AUTHOR]

Infectious pediatric uveitis is a rare disease that can cause severe ocular damage if not detected rapidly and treated properly. Additionally, early identification of an infection can protect the child from life-threatening systemic infection. Infectious uveitis can be congenital or acquired and may manifest as a primary ocular infection or as a reactivation. Nevertheless, publications on infectious paediatric uveitis are usually limited to a small number of patients or a case report. So far, most studies on uveitis in children have focused primarily on noninfectious uveitis, and a systematic study on infectious uveitis is lacking. In this review, we summarize the literature on infectious uveitis in pediatric populations and report on the epidemiology, pathophysiology, clinical signs, diagnostic tests, and treatment. We will describe the different possible pathogens causing uveitis in childhood by microbiological group (i.e. parasites, viruses, bacteria, and fungi). We aim to contribute to early diagnosis and management of infectious pediatric uveitis, which in turn might improve not only visual outcome, but also the general health outcome. [ABSTRACT FROM AUTHOR]

Primary vitreoretinal lymphoma is a potentially aggressive intraocular malignancy with poor systemic prognosis and sometimes significant diagnostic delays as it may masquerade as chronic uveitis. Despite the variety of diagnostic techniques, it is unclear which modality is most accurate in the diagnosis of PVRL. A systematic literature search was conducted on Ovid MEDLINE, EMBASE and the Cochrane Controlled Register of Trials for studies published between January, 2000, and June, 2023. Randomized controlled trials (RCTs) reporting on the following diagnostic tools used to diagnose patients with PVRL were included: cytology, flow cytometry, MYD88 L265P mutation, CD79B mutation, interleukin 10/interleukin-6 (IL-10/IL-6) ratio, polymerase chain reaction (PCR) for monoclonal immunoglobulin heavy chain (IgH) and immunoglobulin kappa light chain (IgK) rearrangements, and imaging findings. The aggregated sensitivity of each diagnostic modality was reported and compared using the chi-squared (χ2) test. A total of 662 eyes from 29 retrospective studies reporting on patients diagnosed with PVRL were included. An IL-10/IL-6 ratio greater than 1 had the highest sensitivity (89.39%, n = 278/311 eyes, n = 16 studies) for PVRL, where the sensitivity was not significantly different when only vitreous samples were drawn (88.89%, n = 232/261 eyes, n = 13 studies) compared to aqueous samples (83.33%, n = 20/24, n = 2) (p = 0.42). Flow cytometry of vitreous samples gave a positive result in 66/75 eyes (88.00%, n = 6 studies) with PVRL, and monoclonal IgH rearrangements on PCR gave a positive result in 354/416 eyes (85.10%, n = 20 studies) with PVRL. MYD88 L265P and CD79B mutation analysis performed poorly, yielding a positive result in 63/90 eyes (70.00%, n = 8 studies) with PVRL, and 20/57 eyes (35.09%, n = 4 studies) with PVRL, respectively. Overall, our systematic review found that an IL-10/IL-6 ratio greater or equal to one may provide the highest sensitivity in identifying patients with PVRL. Future studies are needed to employ multiple diagnostic tools to aid in the detection of PVRL and to further establish nuanced guidelines when determining the optimal diagnostic tool to use in diverse patient populations. [ABSTRACT FROM AUTHOR]

Particulate matter with an aerodynamic diameter ≤2.5 μm (PM2.5) is a public health risk. We investigate PM2.5 on metabolites in cardiomyocytes and the influence of vitamin C on PM2.5 toxicity. For 24 hours, H9C2 were exposed to various concentrations of PM2.5 (0, 100, 200, 400, 800 μg/ml), after which the levels of reactive oxygen species (ROS) and cell viability were measured using the cell counting kit-8 (CCK-8) and 2′,7′-dichlorofluoresceindiacetate (DCFH2-DA), respectively. H9C2 were treated with PM2.5 (200 μg/ml) in the presence or absence of vitamin C (40 μmol/L). mRNA levels of interleukin 6(IL-6), caspase-3, fatty acid-binding protein 3 (FABP3), and hemeoxygenase-1 (HO-1) were investigated by quantitative reverse-transcription polymerase chain reaction. Non-targeted metabolomics by LC-MS/MS was applied to evaluate the metabolic profile in the cell. Results revealed a concentration-dependent reduction in cell viability, death, ROS, and increased expression of caspase-3, FABP3, and IL-6. In total, 15 metabolites exhibited significant differential expression (FC > 2, p < 0.05) between the control and PM2.5 group. In the PM2.5 group, lysophosphatidylcholines (LysoPC,3/3) were upregulated, whereas amino acids (5/5), amino acid analogues (3/3), and other acids and derivatives (4/4) were downregulated. PM2.5 toxicity was lessened by vitamin C. It reduced PM2.5-induced elevation of LysoPC (16:0), LysoPC (16:1), and LysoPC (18:1). PM2.5 induces metabolic disorders in H9C2 cardiomyocytes that can be ameliorated by treatment with vitamin C. [ABSTRACT FROM AUTHOR]

* Context.--Droplet digital polymerase chain reaction (ddPCR) is a sensitive method to detect common pathogenic EGFR mutations in non-small cell lung cancer. Although targeted assays have not been specifically designed to detect them, uncommon EGFR mutations have been linked to response to targeted therapy. Objective.--To describe atypical ddPCR patterns that correspond to uncommon but clinically actionable EGFR mutations. Design.--A cohort of 1134 consecutive non-small cell lung cancers that underwent targeted next-generation sequencing was reviewed. Uncommon EGFR mutations involving probe binding sites were evaluated by ddPCR. Results.--Two hundred fifty-five of 1134 cancers (22.5%) harbored pathogenic EGFR mutations. One hundred eighty-six of 255 (72.9%) had canonical EGFR exon 19 deletion or exon 21 p.L858R variants designed for detection by ddPCR. An additional 25 of 255 cases (9.8%) had uncommon EGFR mutations within the probe-binding site, including 1 case with concurrent uncommon mutations in both exon 19 and exon 21. These mutations included uncommon EGFR exon 19 deletions (n = 6), EGFR exon 19 substitutions p.L747P (n = 3) and p.L747A (n = 1), dinucleotide substitutions leading to EGFR p.L858R (n = 5), EGFR exon 21 substitutions p.K860I (n = 1) and p.L861Q (n = 9), and EGFR p.[L858R;K860I] (n = 1). Droplet digital polymerase chain reaction generated atypical but reproducible signal for each of these uncommon variants. Conclusions.--Droplet digital polymerase chain reaction analysis of uncommon pathogenic EGFR variants can yield unique and reproducible results. Recognition of atypical patterns in EGFR ddPCR testing can prompt confirmatory molecular testing and aid appropriate targeted therapy selection for patients with non--small cell lung cancer. [ABSTRACT FROM AUTHOR]

Background: DNA damage response (DDR) mutation-related genes and composition of immune cells are core factors affecting the effectiveness of immune checkpoint inhibitor therapy. The aim of this study is to combine DDR with immune-related genes to screen the prognostic signature for prostate cancer (PCa). Methods: Gene expression profile and somatic mutation were downloaded from The Cancer Genome Atlas (TCGA). DDR-related genes were obtained from published study. After identification of prognostic-related DDR genes, samples were divided into mutation and nonmutation groups. Differentially expressed genes between these two groups were screened, followed by selection of immune-related DDR genes. Univariate and multivariate Cox analyses were performed to screen genes for constructing prognostic model. Nomogram model was also developed. The expression level of signature was detected by quantitative real-time PCR (qPCR). Results: Two genes (MYBBP1A and PCDHA9) were screened to construct the prognostic model, and it showed good risk prediction of PCa prognosis. Survival analysis showed that patients in high-risk group had worse overall survival than those in low-risk group. Cox analyses indicated that risk score could be used as an independent prognostic factor for PCa. qPCR results indicated that MYBBP1A was upregulated, whereas PCDHA9 was downregulated in PCa cell lines. Conclusions: A prognostic model based on DDR mutation-related genes for PCa was established, which serves as an effective tool for prognostic differentiation in patients with PCa. [ABSTRACT FROM AUTHOR]

Background: Long noncoding RNA (lncRNA) small nucleolar RNA host gene 6 (SNHG6) has been reported to be an oncogene in a variety of cancers. However, the role of SNHG6 and its associated mechanisms in Wilms' tumor progression remain largely unknown. Methods: The expression of SNHG6, microRNA-429 (miR-429), and FGF receptor substrates 2 (FRS2) messenger RNA (mRNA) was detected by quantitative real-time polymerase chain reaction. Cell proliferation was analyzed through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and plate colony assay. The apoptosis was assessed by flow cytometry. Cell glycolytic metabolism was analyzed through detecting the lactate dehydrogenase activity, glucose uptake, lactate production, and ATP level. The target relationship between miR-429 and SNHG6 or FRS2 was predicted by miRcode or Starbase and then validated by dual-luciferase reporter assay and RNA pull-down assay. Murine xenograft model was established to validate the function of SNHG6 in vivo. Results: The level of SNHG6 was elevated in Wilms' tumor tissues and cells, and SNHG6 played an oncogenic role to promote the proliferation and glycolysis and restrain the apoptosis of Wilms' tumor cells. MiR-429 was identified as a target of SNHG6, and miR-429 interference partly reversed the inhibitory effects induced by SNHG6 silencing on the malignant behaviors of Wilms' tumor cells. FRS2 mRNA bound to miR-429 in Wilms' tumor cells. SNHG6 upregulated the expression of FRS2 through acting as a sponge of miR-429. MiR-429-induced influences in Wilms' tumor cells were largely counteracted by the overexpression of FRS2. SNHG6 silencing suppressed the Wilms' tumor growth through miR-429/FRS2 axis in vivo. Conclusion: SNHG6 accelerated Wilms' tumor progression through regulating miR-429/FRS2 signaling in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Background: Circular RNAs (circRNAs) play critical roles in the tumorigenesis and radiosensitivity of multiple cancers. Nevertheless, the biological functions of circRNA periostin (circ‐POSTN) in esophageal cancer (EC) progression and radiosensitivity have not been well elucidated. Methods: The expression of circ‐POSTN, microRNA‐876‐5p (miR‐876‐5p), and proto‐oncogene tyrosine‐protein kinase (FYN) was analyzed by quantitative reverse transcription PCR (RT‐qPCR). Cell proliferation was assessed by MTT, colony formation, and 5‐ethynyl‐2′‐deoxyuridine (EDU) assays. All protein levels were detected by western blot assay. Cell apoptosis and invasion were assessed by flow cytometry analysis and transwell assay, respectively. Dual‐luciferase reporter and RNA immunoprecipitation (RIP) assays were used to validate the interaction between miR‐876‐5p and circ‐POSTN or FYN. The role of circ‐POSTN in vivo was explored by establishing mice xenograft model. Results: Circ‐POSTN was overexpressed in EC tissues and cells. Knockdown of circ‐POSTN inhibited cell proliferation and invasion and elevated apoptosis and radiosensitivity in EC cells. MiR‐876‐5p was a direct target of circ‐POSTN, and its knockdown reversed the role of sh‐circ‐POSTN in EC cells. FYN was a direct target of miR‐876‐5p, and FYN elevation weakened the effects of miR‐876‐5p overexpression on the progression and radiosensitivity of EC cells. Moreover, circ‐POSTN acted as a miR‐876‐5p sponge to regulate FYN expression. Circ‐POSTN interference also suppressed tumor growth and enhanced radiosensitivity in vivo. Conclusion: Circ‐POSTN knockdown inhibited proliferation and invasion, but increased apoptosis and enhanced radiosensitivity in EC cells via modulating miR‐876‐5p/FYN axis, which might be a potential diagnostic and therapeutic target for EC. [ABSTRACT FROM AUTHOR]

Aims/Introduction: Mitochondrial damage caused by oxidative stress is a main driver of pancreatic β‐cell dysfunction in the pathogenesis of type 2 diabetes mellitus. Prohibitin2 (PHB2) is a vital inner mitochondrial membrane protein that participates in mitophagy to remove the damaged mitochondria. This study aimed to investigate the role and mechanisms of PHB2‐mediated mitophagy in oxidative stress‐induced pancreatic β‐cell dysfunction. Materials and Methods: PHB2 and mitophagy‐related protein expression were analyzed by real‐time polymerase chain reaction and western blotting in RINm5F cells treated with H2O2 and islets of diabetic rats. Mitophagy was observed by mitochondrial and lysosome colocalization. RINm5F cells were transfected by phb2 siRNA or overexpression plasmid to explore the role of PHB2 in mitophagy of RINm5F cells. The mechanism of Nrf2 regulating PHB2 was explored by Nrf2 inhibitor and agonist. Results: The expression of PHB2, mitophagy related protein PINK1, and Parkin were decreased in RINm5F cells incubated with H2O2 and in islets of diabetic rats. Overexpression of PHB2 protected β‐cells from oxidative stress by promoting mitophagy and inhibiting cell apoptosis, whereas transfection with PHB2 siRNA suppressed mitophagy. Furthermore, PHB2‐mediated mitophagy induced by oxidative stress was through the Nrf2/PHB2 pathway in β‐cells. Antioxidant NAC alleviated oxidative stress injury by promoting PHB2‐mediated mitophagy. Conclusion: Our study suggested that PHB2‐mediated mitophagy can protect β‐cells from apoptosis via the Nrf2/PHB2 pathway under oxidative stress. Antioxidants may protect β‐cell from oxidative stress by prompting PHB2‐mediated mitophagy. PHB2‐mediated mitophagy as a potential mechanism takes part in the oxidative stress induced β‐cell injury. [ABSTRACT FROM AUTHOR]

Objective: It has been estimated that about 5 million people of those affected with otitis media have cholesteatoma, however, its pathophysiology is unclear. In this study we aimed to detect Helicobacter pylori via polymerase chain reaction and real-time polymerase chain reaction in young patients with chronic otitis media. Methods: Patients included in our prospective cross-sectional study had middle-ear/mastoid inflammation and underwent surgical procedures. Middle-ear mucosa samples were collected, and genomic DNA was extracted for H pylori detection by polymerase chain reaction and real-time polymerase chain reaction analyses. Sociodemographic data and gastroesophageal reflux symptoms were analysed. Results: We included 49 patients with mean age of 12.7 ± 3.8 years. Twenty per cent of the patients were diagnosed with cholesteatoma. No increase in H pylori -amplified fluorescence was observed, indicating absence of H pylori. Conclusion: Due to the absence of amplification for H pylori and the fact that albumin was amplified in all samples, we conclude that H pylori does not appear to be a causal factor. [ABSTRACT FROM AUTHOR]

BACKGROUND AND OBJECTIVES: Test-to-stay concepts apply serial testing of children in daycare after exposure to SARS-CoV-2 without use of quarantine. This study aims to assess the safety of a test-to-stay screening in daycare facilities. METHODS: 714 daycare facilities and approximately 50 000 children ≤6 years in Cologne, Germany participated in a SARS-CoV-2 Pool-polymerase chain reaction (PCR) screening from March 2021 to April 2022. The screening initially comprised post-exposure quarantine and was adapted to a test-to-stay approach during its course. To assess safety of the test-to-stay approach, we explored potential changes in frequencies of infections among children after the adaptation to the test-to-stay approach by applying regression discontinuity in time (RDiT) analyses. To this end, PCR-test data were linked with routinely collected data on reported infections in children and analyzed using ordinary least squares regressions. RESULTS: 219 885 Pool-PCRs and 352 305 Single-PCRs were performed. 6440 (2.93%) Pool-PCRs tested positive, and 17 208 infections in children were reported. We estimated that during a period of 30 weeks, the test-to-stay concept avoided between 7 and 20 days of quarantine per eligible daycare child. RDiT revealed a 26% reduction (Exp. Coef: 0.74, confidence interval 0.52-1.06) in infection frequency among children and indicated no significant increase attributable to the test-to-stay approach. This result was not sensitive to adjustments for 7-day incidence, season, SARS-CoV-2 variant, and socioeconomic status. CONCLUSIONS: Our analyses provide evidence that suggest safety of the test-to-stay approach compared with quarantine measures. This approach offers a promising option to avoid use of quarantine after exposure to respiratory pathogens in daycare settings. [ABSTRACT FROM AUTHOR]

BACKGROUND AND OBJECTIVES: Respiratory viral infections increase risk of asthma in infants and children. Infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus can cause severe lung inflammation and prolonged respiratory symptoms. We sought to determine whether SARS-CoV-2 infection modified pediatric incident asthma risk. METHODS: This retrospective cohort study examined children ages 1 to 16 within the Children's Hospital of Philadelphia Care Network who received polymerase chain reaction (PCR) testing for SARS-CoV-2 between March 1, 2020 and February 28, 2021. Multivariable Cox regression models assessed the hazard ratio of new asthma diagnosis between SARS-CoV-2 PCR positive and SARS-CoV-2 PCR negative groups within an 18-month observation window. Models were adjusted for demographic characteristics, socioeconomic variables, and atopic comorbidities. RESULTS: There were 27 423 subjects included in the study. In adjusted analyses, SARS-CoV-2 PCR positivity had no significant effect on the hazard of new asthma diagnosis (hazard ratio [HR]: 0.96; P = .79). Black race (HR: 1.49; P = .004), food allergies (HR: 1.26; P = .025), and allergic rhinitis (HR: 2.30; P < .001) significantly increased the hazard of new asthma diagnosis. Preterm birth (HR: 1.48; P = .005) and BMI (HR: 1.13; P < .001) significantly increased the hazard of new asthma diagnosis for children <5 years old. CONCLUSIONS: SARS-CoV-2 PCR positivity was not associated with new asthma diagnosis in children within the observation period, although known risk factors for pediatric asthma were confirmed. This study informs the prognosis and care of children with a history of SARS-CoV-2 infection. [ABSTRACT FROM AUTHOR]

Objective: The major histocompatibility complex in chicken demonstrates a great range of variations within varities, breeds, populations and that can eventually influence their immuneresponses. The preset study was conducted to understand the major histocompatibility complex-B (MHC-B) variability in five major populations of Bangladesh native chicken: Aseel, Hilly, Junglefowl, Non-descript Deshi, and Naked Neck. Methods: These five major populations of Bangladesh native chicken were analyzed with a subset of 89 single nucleotide polymorphisms (SNPs) in the high-density MHC-B SNP panel and Kompetitive Allele-Specific polymerase chain reaction genotyping was applied. To explore haplotype diversity within these populations, the results were analyzed both manually and computationally using PHASE 2.1 program. The phylogenetic investigations were also performed using MrBayes program. Results: A total of 136 unique haplotypes were identified within these five Bangladesh chicken populations, and only one was shared (between Hilly and Naked Neck). Phylogenetic analysis showed no distinct haplotype clustering among the five populations, although they were shared in distinct clades; notably, the first clade lacked Naked Neck haplotypes. Conclusion: The present study discovered a set of unique MHC-B haplotypes in Bangladesh chickens that could possibly cause varied immune reponses. However, further investigations are required to evaluate their relationships with global chicken populations. [ABSTRACT FROM AUTHOR]

Cancer is understood as a multifactorial disease that involve multiple cell types and phenotypes in the tumor microenvironment (TME). The components of the TME can interact directly or via soluble factors (cytokines, chemokines, growth factors, extracellular vesicles, etc.). Among the cells composing the TME, mesenchymal stem cells (MSCs) appear as a population with debated properties since it has been seen that they can both promote or attenuate tumor progression. For various authors, the main mechanism of interaction of MSCs is through their secretome, the set of molecules secreted into the extracellular milieu, recruiting, and influencing the behavior of other cells in inflammatory environments where they normally reside, such as wounds and tumors. Natural products have been studied as possible cancer treatments, appealing to synergisms between the molecules in their composition; thus, extracts obtained from Petiveria alliacea (Anamu-SC) and Caesalpinia spinosa (P2Et) have been produced and studied previously on different models, showing promising results. The effect of plant extracts on the MSC secretome has been poorly studied, especially in the context of the TME. Here, we studied the effect of Anamu-SC and P2Et extracts in the human adipose-derived MSC (hAMSC)–tumor cell interaction as a TME model. We also investigated the influence of the hAMSC secretome, in combination with these natural products, on tumor cell hallmarks such as viability, clonogenicity, and migration. In addition, hAMSC gene expression and protein synthesis were evaluated for some key factors in tumor progression in the presence of the extracts by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Multiplex, respectively. It was found that the presence of the hAMSC secretome did not affect the cytotoxic or clonogenicity-reducing activities of the natural extracts on cancer cells, and even this secretome can inhibit the migration of these tumor cells, in addition to the fact that the profile of molecules can be modified by natural products. Overall, our findings demonstrate that hAMSC secretome participation in TME interactions can favor the antitumor activities of natural products. [ABSTRACT FROM AUTHOR]

A total of 17 Legionnaires' disease cases were identified in an outbreak in a western town in New York. The cases had symptom onset dates ranging from August 1, 2019, to July 1, 2021. Staff from the New York State Department of Health and Chemung County Health Department employed numerous environmental investigation techniques. Environmental health program staff collected 74 environmental samples, including samples from cooling towers, residences, and a wastewater treatment plant. Wind direction was analyzed to determine the potential for the dispersion of aerosolized Legionella. In total, five cases were found to be genetically related by whole genome sequencing. Further testing revealed that isolates recovered from a cooling tower at an industrial facility were genetically related to four of the 2019 cases. Excessive iron was found in the tower and might have suppressed the oxidizing biocide residual and interfered with the evaluation of routine culture and other testing methods. In 2021, clinical and environmental samples from a private hot tub were found to be genetically related to the four matching cases and cooling tower samples recovered in 2019. [ABSTRACT FROM AUTHOR]

Aims: To find clinical and immunological signatures of the SARS‐CoV‐2 and the COVID‐19 pandemic on children newly diagnosed with type 1 diabetes (T1D). Methods: A single‐centre, retrospective, observational study comparing the clinical and immunological characteristics of children diagnosed with T1D the year before and during the first 2 years of the COVID‐19 pandemic. Data extracted from the medical records included clinical and demographic parameters, COVID‐19 PCR results and the presence of anti‐islet, thyroid and celiac‐related antibodies. Also obtained from the medical records was a family history of T1D, celiac disease and autoimmune thyroid disease in a first‐degree family member. Results: A total of 376 children were diagnosed with T1D during the study period. A total of 132 in the pre‐COVID era and 246 in the first 2 years of the pandemic. At diagnosis, the pH in children with DKA was lower, and HbA1c tended to be higher in the COVID‐19 group compared to the pre‐COVID‐19 group (7.30 [7.18, 7.35] vs 7.33 [7.19, 7.36], p = 0.046) and (110.9 [86.9, 129.5] vs 100 [80.3, 129.5], p = 0.067]) respectively. Multiple islet antibodies (IA) were significantly more common among patients in the pre‐COVID‐19 group compared to the COVID‐19 group (72% vs 61%, p = 0.032). Tissue transglutaminase antibodies were more common among children diagnosed in the COVID‐19 compared to the pre‐COVID group (16.6% vs 7.9%, p = 0.022). Conclusions: Our findings suggest that SARS‐CoV‐2 and the environmental alterations caused by the pandemic affected the clinical characteristics and the immunological profile of children diagnosed with T1D. It is, therefore, plausible that the virus plays a role in the autoimmune process causing T1D. [ABSTRACT FROM AUTHOR]

The regeneration of osteochondral lesions by tissue engineering techniques is challenging due to the lack of physicochemical characteristics and dual‐lineage (osteogenesis and chondrogenesis). A scaffold with better mechanical properties and dual lineage capability is required for the regeneration of osteochondral defects. In this study, a hydrogel prepared from decellularized human umbilical cord tissue was developed and evaluated for osteochondral regeneration. Mesenchymal stem cells (MSCs) isolated from the umbilical cord were seeded with hydrogel for 28 days, and cell‐hydrogel composites were cultured in basal and osteogenic media. Alizarin red staining, quantitative polymerase chain reaction, and immunofluorescent staining were used to confirm that the hydrogel was biocompatible and capable of inducing osteogenic differentiation in umbilical cord‐derived MSCs. The findings demonstrate that human MSCs differentiated into an osteogenic lineage following 28 days of cultivation in basal and osteoinductive media. The expression was higher in the cell‐hydrogel composites cultured in osteoinductive media, as evidenced by increased levels of messenger RNA and protein expression of osteogenic markers as compared to basal media cultured cell‐hydrogel composites. Additionally, calcium deposits were also observed, which provide additional evidence of osteogenic differentiation. The findings demonstrate that the hydrogel is biocompatible with MSCs and possesses osteoinductive capability in vitro. It may be potentially useful for osteochondral regeneration. [ABSTRACT FROM AUTHOR]

Introduction: In 1969, Li-Fraumeni syndrome (LFS), which is a rare cancer predisposition syndrome, was reported for the first time. The main problem in LFS is the mutation in the TP53 gene, which is a crucial tumor suppressor gene in the cell cycle. A hereditary syndrome is inherited in an autosomal dominant pattern. There is a significant correlation between this syndrome and various cancers such as sarcoma, breast cancer, brain tumors, and different other types of malignancies. This study aimed to identify the possibility of LFS in cancer patients in the East Azarbaijan, Iran. Methods: In this experimental study, 45 children with cancer in the Northwest of Iran were investigated for LFS. DNA was extracted from the whole blood cells using the salting-out method. The region within the exons 5-8 of the TP53 gene has been replicated via Polymerase Chain Reaction (PCR) method. The PCR products were sent for Sanger sequencing, and finally, the data were analyzed by Chromas software. Results: In the studied probands, in 12 (26.67%) cases, polymorphisms in Exon 6 and Introns 6 and Intron 7 were identified, and no mutation was observed in exons 5-8 of the TP53 gene. Conclusion: Our results show that there were no mutations in exons 5-8 of the TP53 gene as an indication of LFS possibility in these families. Further studies are needed to be done in a bigger population, and Next-Generation Sequencing (NGS) needs to be done to evaluate the whole genome of these patients to complete our data. [ABSTRACT FROM AUTHOR]

This study aimed to assess the global status and genetic diversity of Microsporidia infection in different birds. An online search was conducted in international databases from 1 January 1990 to 30 June 2022. A total of 34 articles (including 37 datasets) were included for the final meta-analysis. The pooled global prevalence of Microsporidia infection in birds was 14.6% (95% CI: 11.6–18.1). The highest prevalence of Microsporidia was found in wild waterfowl which was 54.5% (28.1–78.6). In terms of detection methods, the pooled prevalence was estimated to be 21.2% (95% CI: 12.1–34.4) and 13.4% (95% CI: 10.3–17.3) for using microscopic and molecular detection methods, respectively. Enterocytozoon bieneusi was the most common pathogen (24/31; 77.42% of the studies) according to PCR-based methods, and genotype D was the highest reported genotype (nine studies). In conclusion, designing strategies for the control and prevention of Microsporidia infection in birds should be recommended. [ABSTRACT FROM AUTHOR]

Long COVID can include impaired cognition ('brain fog'; a term encompassing multiple symptoms) and mental health conditions. We performed a systematic review and meta-analysis to estimate their prevalence and to explore relevant factors associated with the incidence of impaired cognition and mental health conditions. Searches were conducted in Medline and PsycINFO to cover the start of the pandemic until August 2023. Included studies reported prevalence of mental health conditions and brain fog in adults with long COVID after clinically-diagnosed or PCR-confirmed SARS-CoV-2 infection. 17 studies were included, reporting 41,249 long COVID patients. Across all timepoints (3–24 months), the combined prevalence of mental health conditions and brain fog was 20·4% (95% CI 11·1%-34·4%), being lower among those previously hospitalised than in community-managed patients(19·5 vs 29·7% respectively; p = 0·047). The odds of mental health conditions and brain fog increased over time and when validated instruments were used. Odds of brain fog significantly decreased with increasing vaccination rates (p = ·000). Given the increasing prevalence of mental health conditions and brain fog over time, preventive interventions and treatments are needed. Research is needed to explore underlying mechanisms that could inform further research in development of effective treatments. The reduced risk of brain fog associated with vaccination emphasizes the need for ongoing vaccination programs. • The all-time prevalence of mental health conditions and brain fog is 20.4% in long COVID. • The odds of mental health conditions and brain fog significantly increased over time. • The odds for brain fog being diagnosed significantly increased when validated instruments and cognitive tests were used. • The odds of brain fog significantly decreased with increasing vaccination rates. [ABSTRACT FROM AUTHOR]

The adjunct product with enzymatic activity from Aspergillus oryzae is beneficial for flavor enrichment in the ripened cheese. However, an excessive lipolytic reaction leads to the release of volatile free fatty acids. Accordingly, a strong off-flavor (i.e., rancidity) has been detected when A. oryzae AHU 7139 is used. To identify the rancidity-related lipase from this strain, we evaluated the substrate specificity and lipase distribution using five mutants cultured on a whey-based solid medium under different initial pH conditions. The results showed a higher diacylglycerol lipase activity than triacylglycerol lipase activity. Moreover, an initial pH of 6.5 for the culture resulted in higher lipolytic activity than a pH of 4.0, and most of the activity was found in the extracellular fraction. Based on the gene expression analysis by real-time polymerase chain reaction and location and substrate specificity, five genes (No. 1, No. 19, mdlB , tglA , and cutL) were selected among 25 annotated lipase genes to identify the respective knockout strains. Because Δ tglA and Δ mdlB showed an outstanding involvement in the release of free fatty acids, these strains were applied to in vitro cheese curd experiments. In conclusion, we posit that triacylglycerol lipase (TglA) plays a key role as the trigger of rancidity and the resulting diglycerides have to be exposed to diacylglycerol lipase (MdlB) to stimulate rancidity in cheese made with A. oryzae AHU 7139. This finding could help screen suitable A. oryzae strains as cheese adjuncts to prevent the generation of the rancid-off flavor. • High diacylglycerol lipase activity was found in Aspergillus oryzae AHU 7139 adjuncts. • Extracellular lipase, a major fraction, is highly produced when cultured at pH 6.5. • Initial culture pH influences TG lipase production profile but not DG lipase. • Δ tglA and Δ mdlB could reduce the released free fatty acids in the in vitro curds. • TglA is proposed to be the most rancid-related lipase in Aspergillus oryzae. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This study evaluated whether IgG avidity measured by chemiluminescent microparticle immunoassay (CMIA) compared with enzyme-linked immunosorbent assay (ELISA) was useful to detect primary T. gondii infection during pregnancy and to estimate the risk for congenital T. gondii infection. One hundred six women with positive tests for T. gondii IgG and T. gondii IgM, comprising 21 women (19.8%) with low (<30%), 6 (5.7%) with borderline (30%–35%), and 79 (74.5%) with high (>35%) IgG avidity measured by ELISA were selected. Their stored sera were used for T. gondii IgG avidity measurements by CMIA. In CMIA, 72 (67.9%) women had low (<50%), 12 (11.3%) had borderline (50%–59.9%), and 22 (20.8%) had high (≥60%) IgG avidity. The ratio of low T. gondii IgG avidity index in CMIA was more than three-fold than that in ELISA. Eighteen (85.7%) of 21 women with ELISA low avidity also had CMIA low avidity, and 26 (96.3%) of 27 women with ELISA low or borderline avidity corresponded to CMIA low or borderline avidity, whereas 21 (26.6%) of 79 women with ELISA high avidity were diagnosed with CMIA low avidity. All three cases with congenital T. gondii infection showed coincidentally low IgG avidity in both methods. A positive correlation in IgG avidity indices was found between of ELISA and CMIA. CMIA for T. gondii avidity measurements compared with ELISA was clinically useful to detect pregnant women at a high risk of developing congenital T. gondii infection. [ABSTRACT FROM AUTHOR]

Background: Persistent airway inflammation is a central feature of bronchiectasis. Arachidonate 15-lipoxygenase (ALOX-15) controls production of endogenous lipid mediators, including lipoxins that regulate airway inflammation. Mutations at various positions in ALOX-15 gene can influence airway disease development. We investigated association between ALOX-15,c.-292 C > T gene polymorphism and bronchiectasis unrelated to cystic fibrosis in Egyptian children. Also, lipoxin A4 (LXA4) level in bronchoalveolar lavage (BAL) was studied in relation to polymorphism genotypes and disease phenotypes determined by clinical, pulmonary functions, and radiological severity parameters. Methods: This was an exploratory study that included 60 participants. Thirty children with non-cystic fibrosis bronchiectasis (NCFB) were compared with 30 age and sex-matched controls. ALOX-15,c.-292 C > T polymorphism was genotyped using TaqMan-based Real-time PCR. LXA4 was measured in BAL using ELISA method. Results: There was no significant difference between patients and controls regarding ALOX-15,c.-292 C > T polymorphism genotypes and alleles (OR = 1.75; 95% CI (0.53–5.7), P = 0.35) (OR = 1; 95% CI (0.48-2), p = 1). BAL LXA4 level was significantly lower in patients, median (IQR) of 576.9 (147.6–1510) ng/ml compared to controls, median (IQR) of 1675 (536.8–2542) (p = 0.002). Patients with severe bronchiectasis had a significantly lower LXA4 level (p < 0.001). There were significant correlations with exacerbations frequency (r=-0.54, p = 0.002) and FEV1% predicted (r = 0.64, p = 0.001). Heterozygous CT genotype carriers showed higher LXA4 levels compared to other genotypes(p = 0.005). Conclusions: Low airway LXA4 in children with NCFB is associated with severe disease phenotype and lung function deterioration. CT genotype of ALOX-15,c.-292 C > T polymorphism might be a protective genetic factor against bronchiectasis development and/or progression due to enhanced LXA4 production. [ABSTRACT FROM AUTHOR]

Background. There is no conclusive evidence on the association between interleukin- (IL-) 6 gene polymorphism and type 2 diabetes mellitus (type 2 DM). Thus, this study is aimed at evaluating the role of rs1800795 and rs1800796 polymorphisms in the pathogenesis of type 2 DM among Ghanaians in the Ho Municipality. Materials and Methods. We recruited into this hospital-based case-control study 174 patients with type 2 DM (75 DM alone and 99 with DM+HTN) and 149 healthy individuals between 2018 and 2020. Demographic, lifestyle, clinical, anthropometric, and haemodynamic variables were obtained. Fasting blood samples were collected for haematological, biochemical, and molecular analyses. Genomic DNA was extracted, amplified using Tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) technique, and genotyped for IL-6 gene polymorphism. Logistic regression analyses were performed to assess the association between IL-6 gene polymorphism and type 2 DM. Results. The minor allele frequency (MAF) of the rs1800795 and rs1800796 polymorphisms was higher in DM alone (57.5%, 62.0%) and DM with HTN groups (58.3%, 65.3%) than controls (33.1%, 20.0%). Carriers of the rs1800795GC genotype (aOR = 2.35 , 95% CI: 1.13-4.90, p = 0.022) and mutant C allele (aOR = 2.41 , 95% CI: 1.16-5.00, p = 0.019) as well as those who carried the rs1800796GC (aOR = 8.67 , 95% CI: 4.00-18.90, p < 0.001) and mutant C allele (aOR = 8.84 , 95% CI: 4.06-19.26, p = 0.001) had increased odds of type 2 DM. For both polymorphisms, carriers of the GC genotype had comparable levels of insulin, HOMA-IR, and fasting blood glucose (FBG) with those who carried the GG genotype. IL-6 levels were higher among carriers of the rs1800796GC variant compared to carriers of the rs1800796GG variant (p = 0.023). The rs1800796 polymorphism, dietary sugar intake, and exercise status, respectively, explained approximately 3% (p = 0.046), 3.2% (p = 0.038 , coefficient = 1.456), and 6.2% (p = 0.004 , coefficient = − 2.754) of the variability in IL-6 levels, suggesting weak effect sizes. Conclusion. The GC genotype and mutant C allele are risk genetic variants associated with type 2 DM in the Ghanaian population. The rs1800796 GC variant, dietary sugar intake, and exercise status appear to contribute significantly to the variations in circulating IL-6 levels but with weak effect sizes. [ABSTRACT FROM AUTHOR]

The detection of polymorphisms in genes that control livestock reproduction could be highly beneficial for identifying and enhancing economic traits. One of these genes is pentraxin 3 (PTX3), which affects the reproduction of sheep. Therefore, this study investigated whether the variability of the PTX3 gene was related to the litter size of Awassi and Hamdani ewes. A total of 200 ewes (130 Awassi and 70 Hamdani) were used for genomic DNA extraction. Polymerase chain reaction was used to amplify the sequence fragments of exons 1, 2, 3, and 4 from the PTX3 gene (Oar_v4.0; Chr 1, NC_056054.1), resulting in products of 254, 312, 302, and 253, respectively. Two genotypes, GG and GT, were identified for 302 bp amplicon. A novel mutation was discovered through sequence analysis in the GT genotype at position g.22645332G>T. The statistical analysis revealed a significant association between single nucleotide polymorphism (SNP g.22645332G>T; Oar_v4.0; Chr 1, NC_056054.1) and litter size. The presence of the SNP g.22645332G>T (Oar_v4.0; Chr 1, NC_056054.1) genotype in ewes resulted in a significant difference compared to ewes with GG genotypes. The discrepancy became apparent in several aspects, including litter sizes, twinning rates, lambing rates, litter weight at birth, and days to lambing. There were fewer lambs born to ewes with the GG genotype than to ewes with the GT genotype. The variant SNP g.22645332G>T (Oar_v4.0; Chr 1, NC_056054.1) has positive effects on the litter size of Awassi and Hamdani sheep. The SNP g.22645332G>T (Oar_v4.0; Chr 1, NC_056054.1 has been associated with an increase in litter size and higher prolificacy in ewes. [ABSTRACT FROM AUTHOR]