Clinical Samples Research Articles

FGF8 protects against polymicrobial sepsis by enhancing the host's anti-infective immunity

Abstract Background Sepsis is characterized by a life-threatening syndrome caused by an unbalanced host response to infection. Fibroblast Growth Factor 8 (FGF8) has been newly identified to play important roles in inflammation and innate immunity, but its role in host response to sepsis is undefined. Methods A cecal ligation and puncture (CLP) -induced mouse sepsis model was established to evaluate the immunomodulatory function of FGF8 during sepsis. The underlying molecular mechanisms were elucidated by cell models using relevant molecular biology experiments. The clinical value of FGF8 in the adjuvant diagnosis of sepsis was evaluated using clinical samples. Results FGF8 protein concentrations were elevated in CLP-induced septic mice compared to controls. In vivo, FGF8 blockade using anti-FGF8 antibody significantly increased mortality and bacterial burden and was paralleled by significantly aggravated tissue injury after CLP. Therapeutic administration of recombinant FGF8 (rFGF8) improved the bacterial clearance and mortality of septic mice in a FGFR1-dependent manner. In vitro, FGF8 directly enhanced bacterial phagocytosis and killing of macrophages by enhancing the phosphorylation of the ERK1/2 signaling pathway, which could be abrogated with the ERK1/2 pathway inhibitor U0126. Clinically, serum FGF8 levels in both adult and pediatric patients with sepsis from ICU were significantly higher than those in healthy controls. Conclusions These results present a previously unrecognized role of FGF8 in improving survival of sepsis by enhancing host immune defense. Therefore, targeting FGF8 may provide new strategies for the diagnosis and immunotherapy of sepsis.

Determinants of long‐term paramagnetic rim lesion evolution in people with multiple sclerosis

AbstractObjectiveBaseline paramagnetic rim lesion (PRL) load predicts disease progression in people with multiple sclerosis (pwMS). Understanding how PRLs relate to other known MS‐related factors, and the practical utility of PRLs in clinical trials, is crucial for informing clinical decision‐making and guiding development of novel disease‐modifying treatments (DMTs).MethodsThis study included 152 pwMS enrolled in a larger prospective, longitudinal cohort study who had 3T MRI scans and clinical assessments at baseline and 5‐ or 10‐year follow‐ups. PRLs were identified on baseline 3T quantitative susceptibility maps and classified as persisting, disappearing, or newly appearing at follow‐up. The relationships between PRL evolution and clinical, radiological, environmental, and genetic characteristics were assessed, and clinical trial sample sizes were estimated using PRL appearance or disappearance as outcome measures.ResultsDMT use was associated with lower odds of new PRL appearance (for high‐efficacy DMTs: odds ratio = 0.088, p = 0.024), but not disappearance. Current smoking status was associated with greater baseline PRL number (B = 0.527 additional PRLs, p = 0.013). A 24‐month clinical trial in people with progressive MS for a DMT that doubles the rate of PRL rim disappearance would require an estimated 118 people with progressive MS per group at 80% statistical power.InterpretationEarly MS diagnosis and subsequent DMT initiation may reduce new chronic active inflammation. However, the utility of PRL disappearance or new PRL appearance as outcome measures in clinical trials is limited by potentially large sample sizes that are needed for moderate efficacy drugs.

Diurnal Profiles of the Endocrine Stress Response in Internet Gaming Disorder

Background: Differences in subjective stress perception and acute response of the hypothalamic-pituitary-adrenal axis have been reported in internet gaming disorder (IGD). The present study aimed to further investigate alterations in diurnal profiles of the endocrine stress response system in IGD compared to healthy controls (HCs). Methods: The diurnal course of endocrine markers (salivary cortisol and α-amylase) was investigated in a clinical sample of n = 29 adolescents with IGD compared to n = 26 HC. Further, the effect of unrestricted gaming versus restricted gaming was examined within the IGD group. Results: No significant differences in salivary cortisol and α-amylase were observed comparing adolescents with IGD and HC. In addition, in the IGD group, there were no significant differences in salivary cortisol and α-amylase between conditions of unrestricted gaming versus restricted gaming. Compared to the HC group, the IGD group showed a significantly higher body mass index. Conclusions: Our results indicate no alteration in diurnal profiles of the endocrine stress response in IGD.

Abstract A017: HER2 overexpression initiates breast tumorigenesis non-cell-autonomously by altering energy metabolism in the tissue microenvironment

Abstract Breast cancer is the most common cancer globally, with approximately 70% of pre-malignant breast tumors, known as ductal carcinoma in situ (DCIS), and 20% of invasive breast tumors overexpressing human epidermal growth factor receptor 2 (HER2). While the biological role of HER2 in invasive breast cancer has been extensively studied, its impact on breast cancer stem cells (BCSCs) and early-stage tumorigenesis, particularly within the tumor microenvironment, remains unclear. Here, we explore the role of HER2 overexpression in driving cellular events within the tumor microenvironment during the premalignant stage of breast tumorigenesis, using in vitro, in vivo, and ex vivo models. We conducted multi-omics analyses on mammary ducts from a HER2+ breast cancer mouse model at the pre-cancerous stage. Our results showed that HER2/Neu overexpression alters glucose and lipid metabolism and pentose phosphate pathway in mammary epithelial cells. Ex vivo organ culture of mammary ducts confirmed that these metabolic changes lead to increased reactive oxygen species (ROS) levels within the tumor microenvironment suggesting that oxidative stress and subsequent DNA damage are early events in HER2-driven tumorigenesis. Immunostaining of histological sections from mouse mammary glands further revealed that elevated oxidative stress and DNA damage not only in HER2+ cells but also in neighboring HER2- cells. Moreover, whole genome sequencing of tumor cells confirmed that both HER2+ and HER2- tumor cells have similar mutational burden. This suggests a non-cell-autonomous effect, where HER2 overexpression in one cell population affects the tumor microenvironment and adjacent cells, contributing to early-stage cancer development. To investigate the involvement of BCSCs during tumor initiation, we performed mammosphere assays on primary cells from HER2+ mouse models and clinical DCIS samples. Our findings showed that neither BCSCs nor their cells of origin express HER2 in both DCIS samples and murine mammary glands and tumors. This suggests that HER2 overexpression may not directly drive BCSC formation but rather creates a genotoxic microenvironment that facilitates the transformation of HER2- cell populations through a non-cell-autonomous mechanism. In summary, our findings indicate that HER2 overexpression contributes to the creation of a genotoxic tumor microenvironment by altering cellular energy metabolism and increasing ROS levels. This, in turn, may lead to the transformation of BCSCs in HER2- cell populations during early breast tumorigenesis. Understanding the role of HER2 overexpression in shaping the tumor microenvironment could provide new insights into therapeutic and preventive strategies against breast cancer. Citation Format: Sevim B. Gurler, Oliver Wagstaff, Lili Dimitrova, Fuhui Chen, Robert Pedley, William Weston, Ian J. Donaldson, Brian A. Telfer, David Novo, Kyriaki Pavlou, George Taylor, Yaqing Ou, Kaye Williams, Andrew Gilmore, Keith Brennan, Ahmet Ucar. HER2 overexpression initiates breast tumorigenesis non-cell-autonomously by altering energy metabolism in the tissue microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A017.

Abstract PR001: An immunomodulatory crosstalk between cancer cells and the hepatic microenvironment underlies mutant estrogen receptor-driven breast cancer-to-liver metastasis

Abstract While major advancements have been made in treatment of primary tumors in estrogen receptor-positive (ER+) breast cancer, reducing mortality from metastatic breast cancer (mBC) in treated patients who develop endocrine resistance to first line therapies remains an unmet clinical need. Wild-type ER activity or availability of its cognate ligand 17-beta estradiol (E2) are major factors in primary tumor progression, which are lost/reduced due to endocrine treatments, such as use of selective estrogen degraders (SERDs) and aromatase inhibitors (AIs), respectively. Over time, this leads to selection of rare cancer cells bearing point mutations in ESR1, the gene encoding ER-alpha, which become the dominant population at metastatic sites, and such mutations are now recognized as a frequent driver of endocrine resistance and metastasis, especially in patients with advanced ER+ breast cancer who have been heavily pre-treated with AIs. Analyses of clinical samples have shown a statistically significant association enrichment of ESR1 mutations in liver metastases, compared to the primary tumor. Other distant tissues commonly colonized by breast cancer cells, such as bones, lungs, or brain, although prevalent, do not display this strong association. While this might be due to ease of biopsy sampling in the liver compared to other sites, an alternate and intriguing hypothesis is that the liver offers a fertile soil for metastatic colonization by mutant ER-expressing cells, compared to other distant sites, such as lungs. Using a combination of pre-clinical models, we assessed if mutant ER could drive metastatic colonization at distant sites, such as the liver. We have uncovered a novel link between cancer cell-intrinsic immunomodulatory signaling pathways that are specifically upregulated in mutant ER-expressing breast cancer cells, which represents at least one major molecular mechanism underlying the observed liver tropism of mutant ER-associated, endocrine resistant ER+ breast cancer. We show that disseminated, mutant ER-expressing BC cells engage in crosstalk with the liver stromal and immune populations post-extravasation to further fuel metastatic outgrowth. Citation Format: Sunny Das, Joana Liu Donaher, Lisa Verhoeven, Ranjan Mishra, Ferenc Reinhardt, Sumeet Gupta, Zheqi Li, Kornelia Polyak, Robert A. Weinberg. An immunomodulatory crosstalk between cancer cells and the hepatic microenvironment underlies mutant estrogen receptor-driven breast cancer-to-liver metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR001.

Psychometric properties of the Maladjustment Inventory in a Spanish clinical and student sample.

The various systems of diagnosis and classification of mental disorders underline the need to evaluate the interference caused by the different disorders in a person's daily life. The Maladjustment Inventory (MI) evaluates the impairment in the individual's functioning in a brief and self-applied way, through six items. The objective of this research was to explore the psychometric properties of the MI scores through two studies, one with a Spanish clinical sample (Study 1) and another with a Spanish university students' sample (Study 2). The total sample was made up of 928 participants (81.1% women, n = 495 clinical sample). Descriptive analyses, exploration of internal structure and reliability, exploratory and confirmatory factor analyses, relationship with other variables (quality of life, anxiety, depression, neuroticism and extraversion), and percentiles and T-scores were performed. The results showed good psychometric properties of the MI, with a good fit model for one factor solution in both samples, Cronbach's alpha coefficient of 0.84-88, and evidence of validity based on the relationship with other variables. The good psychometric properties of the MI, together with its brevity, make it a recommended instrument for the evaluation of interference in both clinical and research contexts.

Comprehensive analysis and validation of TP73 as a biomarker for calcium oxalate nephrolithiasis using machine learning and in vivo and in vitro experiments.

Calcium oxalate (CaOx) nephrolithiasis constitutes approximately 75% of nephrolithiasis cases, resulting from the supersaturation and deposition of CaOx crystals in renal tissues. Despite their prevalence, precise biomarkers for CaOx nephrolithiasis are lacking. With advances in high-throughput sequencing, we aimed to identify biomarkers of CaOx nephrolithiasis by combining twoCaOx nephrolithiasis datasets (GSE73680 and GSE117518). Utilizing weighted gene co-expression network analysis (WGCNA) and four machine learning, we identified six hub genes (DLK2, BHLHA15, C12orf5, ICMT, LOXHD1, and TP73) as potential biomarkers. Additionally, CIBERSORT immune infiltration analysis suggested that these core genes may influence immune cell recruitment and infiltration in CaOx nephrolithiasis. Then, TP73 emerged as a significant hub gene in CaOx nephrolithiasis via receiver operating characteristic (ROC) analysis (AUC = 0.885). Furthermore, the role of TP73 was validated in CaOx nephrolithiasis rat models induced by 1% ethylene glycol, as well as clinical samples and renal tubular epithelial cell models treated with 1mM oxalate. Immunohistochemistry, RNA-Sequencing, and RT-qPCR experiments demonstrated an increased expression of TP73 in CaOx nephrolithiasis rat models and clinical samples. After transfection with TP73 lentivirus, CCK-8 assays suggested that TP73 could inhibit the proliferation of HK-2 and NRK-52E cells. In oxalate-induced cell models, dihydroethidium staining and flow cytometry apoptosis assays indicated that TP73 could enhance ROS levels and cell apoptosis. In summary, our study preliminarily identified TP73 as a diagnostic biomarker and elucidated the promoting role of TP73 in CaOx nephrolithiasis, providing a deeper understanding of the clinical diagnosis and pathogenesis.

Highly Specific and Rapid Multiplex Identification of Candida Species Using Digital Microfluidics Integrated with a Semi-Nested Genoarray.

Candida species are the most common cause of fungal infections around the world, associated with superficial and even deep-seated infections. In clinical practice, there is great significance in identifying different Candida species because of their respective characteristics. However, current technologies have difficulty in onsite species identification due to long turnover time, high cost of reagents and instruments, or limited detection performance. We developed a semi-nested recombinase polymerase amplification (RPA) genoarray as well as an integrated system for highly specific identification of four Candida species with a simple design of primers, high detection sensitivity, fast turnover time, and good cost-effectiveness. The system constructed to perform the assay consists of a rapid sample processing module for nucleic acid release from fungal samples in 15 min and a digital microfluidic platform for precise and efficient detection reactions in 35 min. Therefore, our system could automatically identify specific Candida species, with a reagent consumption of only 2.5 μL of the RPA reaction mixture per target and no cross-reaction. Its detection sensitivity for four Candida species achieved 101-102 CFU/mL, which was 10-fold better than conventional RPA and even comparable to a common polymerase chain reaction. Evaluated by using cultured samples and 24 clinical samples, our system shows great applicability to onsite multiplex nucleic acid analysis.

Clinical measures in chronic neuropathic pain are related to the Kennedy and endocannabinoid pathways.

Chronic neuropathic pain (CNP) is a debilitating condition, often refractory to currently available drugs. Understanding biochemical alterations in peripheral tissues such as blood will be useful for understanding underlying pathophysiological processes relating to CNP. We collected blood from two independent cohorts of CNP and pain-free controls (CNP n = 129/Controls n = 127) in the UK and Ireland to investigate the relationship between CNP-associated molecular/biochemical alterations and a range of clinical and pain metric parameters. Multiple statistical comparisons were conducted on the data, with selected variables included in one or more of the intended inferential analyses (six models). Gene expression analysis showed that choline phosphotransferase (CHPT1) was increased (p < .001) in the CNP group compared to controls. The levels of phosphatidylcholine, a metabolite of CHPT1 in the Kennedy Pathway, were significantly (p = .008) decreased in the plasma of patients with CNP. Given the relationship between the Kennedy pathway and endocannabinoids, plasma endocannabinoids and related N-acylethanolamines were quantified in clinical samples by HPLC-Tandem Mass Spectrometry. Plasma levels of the endocannabinoid 2-arachidonoylglycerol were higher in CNP samples compared to controls, and in the statistical models applied, 2-arachidonoylglycerol significantly increased the odds of CNP (p < .001). The expression of genes related to the synthesis and catabolism of endocannabinoids also corroborated the increased plasma 2-arachidonoylglycerol levels in patients with CNP. Endocannabinoid levels, expression of genes related to endocannabinoid metabolism, age, sex, depression and anxiety state together were strong predictors of CNP. The observed molecular changes indicate that lipid metabolism is altered in CNP and thus may represent a viable target for novel analgesics or biomarker development.

Human Tendon-on-a-Chip for Modeling the Myofibroblast Microenvironment in Peritendinous Fibrosis.

Understanding the myofibroblast microenvironment is critical to developing therapies for fibrotic diseases. Here the development of a novel human tendon-on-a-chip (hToC) is reported to model this crosstalk in peritendinous adhesions, which currently lacks biological therapies. The hToC facilitates cellular and paracrine interactions between a vascular component, which contains endothelial cells and monocytes, and a tissue hydrogel component that houses tendon cells and macrophages. It is found that the hToC replicates some aspects of in vivo inflammatory and fibrotic phenotypes in preclinical and clinical samples, including activated mTOR signaling, vascular inflammation, tissue contraction induced by myofibroblast activation, inflammatory cytokines secretion, and transendothelial migration of monocytes to the tissue hydrogel. Transcriptional analysis demonstrates significant overlap in enriched pathways in the hToC with human tenolysis samples, including the activation of mTOR signaling. Rapamycin suppresses the vascular inflammation and fibrotic phenotype in the hToC, which provides proof-of-concept of its utility as an in vitro tool for investigating multicellular crosstalk in fibrosis and testing therapeutics to mitigate it.

KGF secreted from HSCs activates PAK4/BMI1, promotes HCC stemness through PI3K/AKT pathway.

In our present study, we investigated the interaction between HSCs and HCC, also explored the molecular mechanism. Clinical samples were collected from HCC and adjacent tissue with different degree of liver fibrosis. HCC cells were co-cultured with LX-2 cell by Transwell system or cultured with conditioned medium (CM), which was collected from LX-2. The tumor spheroid growth and colony formation analyses were performed to evaluate the cell stemness. Flow cytometry analysis was conducted on cell apoptosis after 5-Fu treatment. Co-immunoprecipitation assay confirmed the interaction between BMI1 and PAK4. Our results showed that BMI1 was highly expressed in HCC and was correlated with HCC liver fibrosis. Both co-cultured with LX-2 and cultured with CM promoted HCC stemness, also increased KGF level and BMI1 expression. KGF treatment had a similar effect with co-culture with LX-2 on HCC. BMI1 overexpression promoted HCC stemness and activated PI3K/AKT pathway, which was reversed by PI3K inhibition. PAK4 was activated by KGF, then phosphorylated S315 site and promoted protein stability of BMI1, therefore enhanced HCC stemness. BMI1 also had a promote effect on liver fibrosis. In summary, we found that KGF secreted by HSCs activated PAK4, which phosphorylated S315 and promoted protein stability of BMI1, and further promoted liver fibrosis and HCC stemness through the PI3K/AKT signaling pathway. Our present study deeply studied the interaction and mechanism between HSCs and HCC, which might provide a new insight for HCC therapy.

Application of metagenomic next-generation sequencing in the diagnosis of infectious diseases

Metagenomic next-generation sequencing (mNGS) is a transformative approach in the diagnosis of infectious diseases, utilizing unbiased high-throughput sequencing to directly detect and characterize microbial genomes from clinical samples. This review comprehensively outlines the fundamental principles, sequencing workflow, and platforms utilized in mNGS technology. The methodological backbone involves shotgun sequencing of total nucleic acids extracted from diverse sample types, enabling simultaneous detection of bacteria, viruses, fungi, and parasites without prior knowledge of the infectious agent. Key advantages of mNGS include its capability to identify rare, novel, or unculturable pathogens, providing a more comprehensive view of microbial communities compared to traditional culture-based methods. Despite these strengths, challenges such as data analysis complexity, high cost, and the need for optimized sample preparation protocols remain significant hurdles. The application of mNGS across various systemic infections highlights its clinical utility. Case studies discussed in this review illustrate its efficacy in diagnosing respiratory tract infections, bloodstream infections, central nervous system infections, gastrointestinal infections, and others. By rapidly identifying pathogens and their genomic characteristics, mNGS facilitates timely and targeted therapeutic interventions, thereby improving patient outcomes and infection control measures. Looking ahead, the future of mNGS in infectious disease diagnostics appears promising. Advances in bioinformatics tools and sequencing technologies are anticipated to streamline data analysis, enhance sensitivity and specificity, and reduce turnaround times. Integration with clinical decision support systems promises to further optimize mNGS utilization in routine clinical practice. In conclusion, mNGS represents a paradigm shift in the field of infectious disease diagnostics, offering unparalleled insights into microbial diversity and pathogenesis. While challenges persist, ongoing technological advancements hold immense potential to consolidate mNGS as a pivotal tool in the armamentarium of modern medicine, empowering clinicians with precise, rapid, and comprehensive pathogen detection capabilities.

Systematic comparison of fluorescence imaging in the near-infrared and shortwave-infrared spectral range using clinical tumor samples containing cetuximab-IRDye800CW

Systematic comparison of fluorescence imaging in the near-infrared and shortwave-infrared spectral range using clinical tumor samples containing cetuximab-IRDye800CW

Targeting PRMT7-mediated monomethylation of MAVS enhances antiviral innate immune responses and inhibits RNA virus replication

RIG-I-like receptors (RLRs)-mitochondrial antiviral signaling protein (MAVS) are crucial for type I interferon (IFN) signaling pathway and innate immune responses triggered by RNA viruses. However, the regulatory molecular mechanisms underlying RNA virus-activated type I IFN signaling pathway remain incompletely understood. Here, we found that protein arginine methyltransferase 7 (PRMT7) serves as a negative regulator of the type I IFN signaling pathway by interacting with MAVS and catalyzing monomethylation of arginine 232 (R232me1) in MAVS. RNA virus infection leads to the downregulation and dissociation of PRMT7 from MAVS as well as the decrease of R232me1 methylation, enhancing MAVS/RIG-I interaction, MAVS aggregation, type I IFN signaling activation, and antiviral immune responses. Knock-in mice with MAVS R232 substituted with lysine (MavsR232K-KI) are more resistant to Vesicular Stomatitis Virus infection due to enhanced antiviral immune responses. PiPRMT7-MAVS, a short peptide inhibitor designed to interrupt the interaction between PRMT7 and MAVS, inhibits R232me1 methylation, thereby enhancing MAVS/RIG-I interaction, promoting MAVS aggregation, activating type I IFN signaling, and bolstering antiviral immune responses to suppress RNA virus replication. Moreover, the clinical relevance of PRMT7 is highlighted that it is significantly downregulated in RNA virus-infected clinical samples, such as blood samples from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Ebola virus, as well as H1N1-infected bronchial epithelial cells. Our findings uncovered that PRMT7-mediated arginine methylation plays critical roles in regulating MAVS-mediated antiviral innate immune responses, and targeting arginine methylation might represent a therapeutic avenue for treating RNA viral infection.

Precise Genotyping Via Surface‐Enhanced Raman Spectroscopy‐Based Optical Sensing Chip for Guiding Targeted Therapy in Lung Cancer

AbstractThe emergence of “precision medicine” marks a notable shift in cancer treatment, moving from a tumor type–oriented approach to a more targeted, gene‐oriented approach. Detecting low‐abundance mutant genes in blood is challenging but crucial for personalized treatment plans. Herein, a novel platform combining catalytic hairpin self‐assembly (CHA)‐mediated self‐calibrating surface‐enhanced Raman spectroscopy (SERS) with a high‐throughput Raman system (CCSPS) was designed. This platform enables ultrasensitive and rapid genotype analysis of gene mutations. The development of CCSPS specifically targets EGFR mutations, which serve as crucial therapeutic targets for precision therapy in lung cancer. This system shows excellent sensitivity and selectivity, capable of detecting multiple EGFR mutations (Del‐19, L858R, and T790M) with a detection limit as low as attomolar levels. Additionally, precise genotyping analysis was successfully conducted on 42 clinical samples using the CCSPS, yielding results consistent with those obtained through next‐generation sequencing. These results underscore the efficacy of the CCSPS in noninvasively identifying circulating tumor DNA (ctDNA) mutations, facilitating immediate therapeutic decision making at the bedside. In summary, the CCSPS is a fast, accurate, versatile, and compact testing system capable of precisely screening individuals who stand to benefit from targeted therapy, thus promoting personalized and precise healthcare.

Compact highly sensitive photothermal RT-LAMP chip for simultaneous multidisease detection.

Developing instant detection systems with disease diagnostic capabilities holds immense importance for remote or resource-limited areas. However, the task of creating these systems-which are simultaneously easy to operate, rapid in detection, and cost-effective-remains a challenge. In this study, we present a compact highly sensitive photothermal reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) chip (SPRC) designed for the detection of multiple diseases. The nucleic acid (NA) amplification on the chip is achieved through LAMP driven by either LED illumination or simple sunlight focusing. SPRC performs sample addition and amplification within a limited volume and autonomous enrichment of NA during the sample addition process, achieving a limit of detection (LOD) as low as 0.2 copies per microliter. Through 120 clinical samples, we achieved an accuracy of 95%, with a specificity exceeding 97.5%. Overall, SPRC has achieved promising progress in the application of point-of-care testing (POCT) by using light energy to simultaneously detect multiple diseases.

Application of Pan-Viral Metagenomic Sequencing on United States Air Force Academy Wastewater to Uncover Potential Causes of Acute Gastroenteritis.

Wastewater surveillance is an important technique to monitor public health and is being studied extensively for pandemic prevention, force health protection and readiness, and as a potential early warning system for chem-bio defense. Wastewater surveillance has traditionally relied on techniques such as quantitative PCR or targeted sequencing, both of which are microbe- or disease-specific tools. Newer pan-viral metagenomics strategies may provide higher specificity for pathogens of interest, resulting in a lower false negative rate and reduced sequencing of undesired background nucleic acids. One such system, VirCapSeq-VERT, has been developed to targetall vertebrate virus pathogens; until recently, its application has been limited to clinical samples. The objective of this study was to use VirCapSeq-VERT to interrogate wastewater samples from the U.S. Air Force Academy (USAFA), Colorado Springs, Colorado, to determine its utility in assessing complex samples and its potential application in public health surveillance. Biweekly samples were analyzed from February 2022 through May 2023. Samples were collected from the wastewater treatment facility at USAFA before treatment and stored at -20 °C until total nucleic acid (tNA: DNA and RNA) extraction. tNA was then subject to the probe-based capture system, VirCapSeq, and run through a collection of public bioinformatics pipelines to identify captured viral pathogens and perform phylogenetic analysis. It was determined by the USAFA IRB that the study was non-human subject research and was deemed exempt. In total, 68 families of viruses were identified, comprising thousands of individual strains. This study focused on viruses responsible for gastrointestinal dysfunction as a test of the use of the VirCapSeq-VERT to identify human pathogenic viruses within a complex and highly enriched biological sample. Four enteric viruses dominated the wastewater samples, with Adenoviridae most prevalent before the cadet winter recess (December 17, 2022-January 4, 2023) and Astroviridae most abundant thereafter. Although gastroenteritis outbreaks at USAFA are commonly attributed to norovirus because of clinical presentation and the acute nature of the illness-often diagnosed and treated without confirmatory stool testing-this virus was not found in high prevalence in these wastewater samples. Among adenoviruses, F serotype 41 predominated, suggesting a role in gastrointestinal infections among the cadet population. Phylogenetic investigation of adenovirus and norovirus exposed common variants with seasonal distributions. These findings may prompt correlational studies to assess the clinical predictive capability of VirCapSeq-VERT and to determine the utility of wastewater monitoring as an outbreak early warning system.

Prevalence of Atypical Bacteria in Patients from Different Paediatric Age Groups Diagnosed with a Respiratory Disease

Atypical bacterial pathogens present the ability to induce pulmonary damage. At present, there are no available phenotypic diagnosis tests that achieve up to 100% reliability. Therefore, clinicians must utilise molecular techniques for the detection and identification of these pathogens. The main objective of this research was to evaluate the prevalence of atypical bacteria in paediatric patients from different age groups. A total of 609 clinical samples were collected from paediatric patients who presented with an adverse respiratory condition during the period from March 2021 to February 2024. DNA was extracted from the samples, and end-point PCR was performed to detect atypical bacteria. Statistical analyses were performed to evaluate the bacterial prevalence and assess clinical data from newborns and mothers that could be related to RDS. A total of 139 patients exhibited at least one atypical organism (22.82%). Ureaplasma parvum was more prevalent in neonates, while M. pneumoniae and C. pneumoniae were more prevalent in older infants. Atypical bacteria can be present in all seasons of the year, but their prevalence increases during hot weather. Mixed infections due to atypical bacteria may occur. The risk factors related to the development of RDS are prematurity, low weight, and orotracheal intubation.

Pattern of Rapidly growing Mycobacteria (RGM) species isolated from clinical samples: A 10-year retrospective study in a tertiary care hospital of Bangladesh.

Infections caused by rapidly growing mycobacteria (RGM) are increasing worldwide. The study was conducted in a microbiological laboratory of Bangladesh to determine the pattern of detection of RGM from clinical samples. All laboratory culture records of RGM from 2012 to 2022 were collected retrospectively and analyzed. A total 62 RGM infected patients with surgical site infection (74.1%), injection site and skin abscess (9.7%), septicemia (4.8%) and UTI (1.6%) were identified. The annual isolation frequency of RGM increased 4.8% to 29.1% in between year 2012 and 2022. RGM infected patients (14.5%) were mistakenly treated with first line anti tubercular drug before correct microbiological diagnosis (median, IQR; 3, 2-5 months). Out of 23 RGM isolates, 86.9% were M. abscessus and rest 13.1% were M. fortuitum. Most of them (≥95%) were sensitive to amikacin, linezolid, clarithromycin where as 27.1% to imipenem and ciprofloxacin, 40% to cefoxitin, 35.3% and 1.7% to doxycycline and co-trimoxazole respectively. Misdiagnosis or delay in diagnosis and erroneous treatment with first line anti tubercular drug may cause prolong morbidity and therapeutic failure to patients with RGM infection. So, early and appropriate diagnosis is crucial for successful outcome.

MALAT1 is important for facilitating HIV-1 latency reversal in latently infected monocytes.

Long non-coding RNAs (lncRNAs) are long RNA transcripts with length > 200 nucleotides that do not encode proteins. They play a crucial role in regulating HIV-1 infection, yet their involvement in myeloid cells remains underexplored. Myeloid cells are susceptible to HIV infection and contribute substantially to the latent HIV reservoir. Therefore, disruption of latency within these reservoirs is crucial for achieving a definite cure. In this study, we aimed to ascertain the role of MALAT1 lncRNA in reversal of HIV-1 latency. Latently HIV-infected cell line, U1 was treated with SAHA, followed by qRT-PCR assays to confirm HIV-1 reactivation, and MALAT1 expression was assessed. The in vitro experiments revealed a significant increase in MALAT1 expression following latency reactivation and HIV-1 infection, while its knockdown using siRNA resulted in suppression of HIV transcription, which implies that MALAT1 play a significant role in facilitating the reversal of HIV-1 latency by promoting HIV transcription. Clinical samples were analysed to measure MALAT1 and pro-inflammatory cytokines expression. The elevated MALAT1 expression in treatment-naïve subjects compared to treated subjects and HIV-negative controls suggests its association with HIV replication. Moreover, correlation analysis revealed a positive association between MALAT1 expression and pro-inflammatory cytokines, TNF-α and IP-10. To conclude, MALAT1 lncRNA emerged as a crucial facilitator of HIV-1 latency reversal in latently infected monocytes by inducing the expression of pro-inflammatory factors. These findings highlight that MALAT1 could potentially be explored as the therapeutic intervention to reactivate latent cells in monocytes.