Clinical Samples Research Articles

Development and Clinical Application of a Molecular Assay for Four Common Porcine Enteroviruses

Porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA) are the four main pathogens that cause viral diarrhea in pigs, and they often occur in mixed infections, which are difficult to distinguish only according to clinical symptoms. Here, we developed a multiplex TaqMan-probe-based real-time RT-PCR method for the simultaneous detection of PEDV, TGEV, PDCoV, and PoRVA for the first time. The specific primers and probes were designed for the M protein gene of PEDV, N protein gene of TGEV, N protein gene of PDCoV, and VP7 protein gene of PoRVA, and corresponding recombinant plasmids were constructed. The method showed extreme specificity, high sensitivity, and excellent repeatability; the limit of detection (LOD) can reach as low as 2.18 × 102 copies/μL in multiplex real-time RT-PCR assay. A total of 97 clinical samples were used to compare the results of the conventional reverse transcription PCR (RT-PCR) and this multiplex real-time RT-PCR for PEDV, TGEV, PDCoV, and PoRVA detection, and the results were 100% consistent. Subsequently, five randomly selected clinical samples that tested positive were sent for DNA sequencing verification, and the sequencing results showed consistency with the detection results of the conventional RT-PCR and our developed method in this study. In summary, this study developed a multiplex real-time RT-PCR method for simultaneous detection of PEDV, TGEV, PDCoV, and PoRVA, and the results of this study can provide technical means for the differential diagnosis and epidemiological investigation of these four porcine viral diarrheic diseases.

Prospective modeling and estimating the epidemiologically informative match rate within large foodborne pathogen genomic databases

ObjectivesMuch has been written about the utility of genomic databases to public health. Within food safety these databases contain data from two types of isolates—those from patients (i.e., clinical) and those from non-clinical sources (e.g., a food manufacturing environment). A genetic match between isolates from these sources represents a signal of interest. We investigate the match rate within three large genomic databases (Listeria monocytogenes, Escherichia coli, and Salmonella) and the smaller Cronobacter database; the databases are part of the Pathogen Detection project at NCBI (National Center for Biotechnology Information).ResultsCurrently, the match rate of clinical isolates to non-clinical isolates is 33% for L. monocytogenes, 46% for Salmonella, and 7% for E. coli. These match rates are associated with several database features including the diversity of the organism, the database size, and the proportion of non-clinical BioSamples. Modeling match rate via logistic regression showed relatively good performance. Our prediction model illustrates the importance of populating databases with non-clinical isolates to better identify a match for clinical samples. Such information should help public health officials prioritize surveillance strategies and show the critical need to populate fledgling databases (e.g., Cronobacter sakazakii).

Understanding Internalized Stigma’s Role in Sex-Specific Suicidal Ideation among Individuals with Bipolar Disorder

Background: The experience of stigma can exert a profound impact on the mental health and well-being of individuals with bipolar disorder (BD). Our study explores which factors of internalized stigma are associated with suicidal ideation and how they differ between the two sexes in a clinical sample of BD patients. Methods: The study follows a cross-sectional study design, employing the Clinical Global Impression for Bipolar Patients (CGI-BP) to evaluate the overall severity of illness and the alteration in patients affected by bipolar disorder, the Internalized Stigma of Mental Illness (ISMI) assessing self-stigma among individuals experiencing mental disorders, and the Columbia Suicide Severity Rating Scale (C-SSRS) identifying and assessing individuals vulnerable to suicide. Descriptive analyses, analysis of variance (ANOVA), and logistic regression analysis were conducted, and 344 BD subjects were recruited. Results: Our ANOVA results revealed a significant association between sex and suicide across ISMI sub-items (p = 0.000). Logistic regression analysis comprised three phases: Discrimination was consistently significant across all phases (p < 0.001), while Alienation and Stereotype emerged as additional predictors of suicide in later phases of the analysis (p < 0.001). Conclusions: Our study contributes to the growing body of literature on internalized stigma, sex, and suicidality among individuals with bipolar disorder. Early intervention programs and prevention strategies are needed.

A Programmable Microfluidic Paper-Based Analytical Device for Simultaneous Colorimetric and Photothermal Visual Sensing of Multiple Enzyme Activities.

New strategies for the simultaneous and portable detection of multiple enzyme activities are highly desirable for clinical diagnosis and home care. However, the methods developed thus far generally suffer from high costs, cumbersome procedures, and heavy reliance on large-scale instruments. To satisfy the actual requirements of rapid, accurate, and on-site detection of multiple enzyme activities, we report herein a smartphone-assisted programmable microfluidic paper-based analytical device (μPAD) that utilizes colorimetric and photothermal signals for simultaneous, accurate, and visual quantitative detection of alkaline phosphatase (ALP) and butyrylcholinesterase (BChE). Specifically, the operation of this μPAD sensing platform is based on two sequential steps. Cobalt-doped mesoporous cerium oxide (Co-m-CeO2) with remarkable peroxidase-like activities under neutral conditions first catalytically decomposes H2O2 for effectively converting colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxidized TMB (oxTMB). The subsequent addition of ALP or BChE to their respective substrates produces a reducing substance that can somewhat inhibit the oxTMB transformation for compromised colorimetric and photothermal signals of oxTMB. Notably, these two-step bioenzyme-nanozyme cascade reactions strongly support the straightforward and excellent processability of this platform, which exhibit lower detection limits for ALP and BChE with a detection limit for BChE an order of magnitude lower than those of the other reported paper-based detection methods. The practicability and efficiency of this platform are further demonstrated through the analysis of clinical serum samples. This innovative platform exhibits great potential as a facile yet robust approach for simultaneous, accurate, and on-site visual detection of multiple enzyme activities in authentic samples.

Characterizing clinical heterogeneity in an inpatient service treating mental, substance use and concurrent disorders

Patients diagnosed with concurrent disorders (CD)—comorbid substance use disorder with other psychiatric conditions—experience poorer clinical outcomes, and significant gaps remain in defining the optimal care path for treating CD. Toward this goal, the primary aim of this study was to characterize individual differences in substance use and psychiatric symptomology in an inpatient clinical sample using a person-centred approach. Admission assessment data from a private inpatient service treating mental disorders, substance use, and concurrent disorders was used (n = 177). Latent profile analysis (LPA) was performed to classify individuals into statistically distinct latent profiles based on their psychiatric symptoms and polysubstance use as covariates. LPA revealed four profiles. Profile 1 (20%) was identified as having low SUD and low psychiatric disorders, profile 2 (65%) was identified as having low SUD and high psychiatric disorders, profile 3 (8%) was characterized as high substance use and moderate psychiatric disorders and profile 4 (7%) was identified as the high SUD and high psychiatric disorders. The participants in the two profiles endorsing high SUDs, Profiles 3 and 4, showed significantly higher impulsivity in terms of higher positive urgency sensation-seeking scores compared to the other profiles and the highest use of cocaine/stimulants than the other two. Identifying clinical heterogeneity by classifying individuals into distinct profiles is a first step toward designing more targeted and personalized interventions in clinically complex inpatient populations.

Infection prevention, control and management: assessing the knowledge among nurses and student nurses in the Eastern Province of Sri Lanka

Background and aims: The knowledge of infection control among health-care workers is a crucial factor in the success of infection control pro-grams. This study compares the understanding of infection control and prevention practices among nurses and student nurses in eastern province of Sri Lanka. Methods: We utilized a pre-tested self-administered questionnaire with 25 questions. Descriptive statistics and the independent t-test were used to analyze survey responses and compare scores between groups. Results: Out of 178 responses analyzed 112 were nurses and 66 were student-nurses. The average scores were 44% for nurses and 51% for student-nurses. Both groups had less than 10% knowledge of notifiable diseases in Sri Lanka and medical abbreviations. Knowledge about immunization, vaccines, clinical sample transportation, and MRSA decolonization was under 40% for both groups. Overall knowledge of infection control and prevention practices among nurses was significantly poorer than that of student-nurses (p<0.001). Conclusion: These findings underscore the generally inadequate knowledge of infection control and prevention practices among both nurses and student-nurses. It is imperative to implement policies and measures to as-sess and enhance the training curricula for student-nurses and to ensure ongoing training programs for nurses.

Direct retrospective measurement of therapeutic changes: an example using the Czech version of the Questionnaire of Personal Changes (Q-PC)

Objective: The study aimed to test the psychometric properties of the Czech translation of the Questionnaire of Personal Changes (Q-PC), a measure designed for retrospective (direct) measurement of change in psychotherapy. Methods: A sample of group psychotherapy clients (N = 222) and a nonclinical sample (N = 167) sample were used. Clients in the clinical sample were administered the Q-PC in addition to several pre–post outcome measures. Confirmatory factor analysis, correlational analysis, and structural equation modeling were used to test the Q-PC’s factor structure, longitudinal measurement invariance, reliability, convergent validity, sensitivity to change, and other psychometric properties. Results: The Q-PC demonstrated a unidimensional structure that was strictly invariant between two follow-up measurement waves. The measure also demonstrated excellent reliability and sensitivity to change and good convergent validity. Furthermore, it demonstrated a similar relationship to baseline severity as the pre–post outcome measures. Conclusions: The retrospective measurement of change is a promising approach that has the potential to complement the traditional pre–post measurement of change.

The Brief Symptom Inventory in the Swiss general population: Presentation of norm scores and predictors of psychological distress.

Psychological distress is an important and frequent health problem. The Brief Symptom Inventory (BSI) allows screening for psychological distress in clinical, general and research populations. We aimed to provide normative data for the BSI and the BSI-18 for the Swiss general population: We 1) present psychometric properties, 2) develop a Swiss T-standardization and validate it using a clinical sample, 3) describe psychological distress in the Swiss general population and the clinical sample, and 4) compare the means and T-standardized scores of the Swiss general population to different German norm populations. Using a cross-sectional study design, we invited a representative sample of the Swiss general population aged 18-75 years to the study. A sample of psychotherapy outpatients had competed the BSI before start of their therapy. We calculated scores for the nine scales of the BSI (three of them constitute the BSI-18), the T-standardization and the following BSI indices: Global Severity Index (GSI), Positive Symptom Total (PST), Positive Symptom Distress Index (PSDI), and Caseness (reaching T≥63 on the GSI or T≥63 on at least two of the scales). A total of 1238 general population participants completed the BSI (41.8% male; mean age 48.9 years). The BSI had good psychometric properties. The Swiss T-standardization showed good validity when applied in the clinical sample. Females reached a significantly higher GSI score than males (p<0.001). Older participants (p = 0.026), those with higher education (p <0.001), and those employed or retired (p<0.001) reached lower scores than participants aged 18-25 years, those with compulsory schooling, and unemployed participants, respectively. A total of 18.1% (CI: 16.0-20.5) participants of the general population and 75.2% (CI: 73.7-76.7) of the psychotherapy patients were considered cases with psychological distress. Our study presents detailed normative data for the BSI and the BSI-18 based on a representative sample of the Swiss general population. This information will be helpful for clinical applications and research in the Swiss and international context.

Detection of influenza A(H3N2) viruses with polymerase acidic subunit substitutions after and prior to baloxavir marboxil treatment during the 2022-2023 influenza season in Japan

Baloxavir marboxil (baloxavir), approved as an anti-influenza drug in Japan in March 2018, can induce reduced therapeutic effectiveness due to PA protein substitutions. We assessed PA substitutions in clinical samples from influenza-infected children and adults pre- and post-baloxavir treatment, examining their impact on fever and symptom duration. During the 2022-2023 influenza season, the predominant circulating influenza subtype detected by cycling-probe RT-PCR was A(H3N2) (n=234), with a minor circulation of A(H1N1)pdm09 (n=10). Of the 234 influenza A(H3N2) viruses collected prior to baloxavir treatment, 2 (0.8%) viruses carry PA/I38T substitution. One virus was collected from a toddler and one from an adult, indicating the presence of viruses with reduced susceptibility to baloxavir, without prior exposure to the drug. Of the 54 paired influenza A(H3N2) viruses collected following baloxavir treatment, 8 (14.8%) viruses carried E23K/G, or I38M/T substitutions in PA. Variant calling through next-generation sequencing (NGS) showed varying proportions (6 to 100 %), a polymorphism and a mixture of PA/E23K/G, and I38M/T substitutions in the clinical samples. These eight viruses were obtained from children aged 7-14 years, with a median fever duration of 16.7 hours and a median symptom duration of 93.7 hours, which were similar to those of the wild type. However, the delayed viral clearance associated with the emergence of PA substitutions was observed. No substitutions conferring resistance to neuraminidase inhibitors were detected in 37 paired samples collected before and following oseltamivir treatment. These findings underscore the need for ongoing antiviral surveillance, informing public health strategies and clinical antiviral recommendations for seasonal influenza.

When Being Bad Feels Good: A Systematic Review of the Relationship Between Positive Emotion and Antisocial Behavior in Children and Adolescents.

Antisocial behavior in childhood and adolescence is associated with poor family and peer relationships, and a higher risk of mental and physical health problems in adulthood, as well as criminality. Emotions play a central role in children's moral development, but most research has focused on negative emotions (e.g., shame and guilt), in relation to childhood antisocial behavior. Research in adult populations indicates that positive emotions experienced in anticipation of, during, and after antisocial acts may play an important role in the development and maintenance of antisocial behavior. Consequently, this systematic review aimed to investigate the relationship between positive emotion and antisocial behavior in children and adolescents. A systematic search in five databases was conducted, yielding 52 studies that used different methodological approaches, samples, designs and methods to examine this association. Results provide support for a positive relationship between positive emotion and antisocial behavior across community, forensic and clinical samples. This link appeared to be stronger for younger children, boys, and for children high in social dominance, callous-unemotional or sensation-seeking traits. Results suggested that positive affect may act in concert with negative emotion, cognitive, personality and motivational processes, as well as peer influences to determine the initiation and maintenance of antisocial behavior. This review presents directions for future research and discusses the implications of findings for prevention and intervention programs for youth with antisocial behavior.

Single Nucleus Total RNA Sequencing of Formalin-Fixed Paraffin-Embedded Gliomas.

Gliomas, the predominant form of brain cancer, comprise diverse malignant subtypes with limited curative therapies available. The insufficient understanding of their molecular diversity and evolutionary processes hinders the advancement of new treatments. Technical complexities associated with formalin-fixed paraffin-embedded (FFPE) clinical samples hinder molecular-level analyses of gliomas. Current single-cell RNA sequencing (scRNA-seq) platforms are inadequate for large-scale clinical applications. In this study, automated snRandom-seq is developed, a high-throughput single-nucleus total RNA sequencing platform optimized for archival FFPE samples. This platform integrates automated single-nucleus isolation and droplet barcoding systems with the random primer-based scRNA-seq chemistry, accommodating a broad spectrum of sample types. The automated snRandom-seq is applied to analyze 116492 single nuclei from 17 FFPE samples of various glioma subtypes, including rare clinical samples and matched primary-recurrent glioblastomas (GBMs). The study provides comprehensive insights into the molecular characteristics of gliomas at the single-cell level. Abundant non-coding RNAs (ncRNAs) with distinct expression profiles across different glioma clusters and uncovered promising recurrence-related targets and pathways in primary-recurrent GBMs are identified. These findings establish automated snRandom-seq as a robust tool for scRNA-seq of FFPE samples, enabling exploration of molecular diversities and tumor evolution. This platform holds significant implications for large-scale integrative and retrospective clinical research.

YAP1 suppression by ZDHHC7 is associated with ferroptosis resistance and poor prognosis in ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC), which has unique clinical characteristics, arises from benign endometriotic cysts, forming an oxidative stress environment due to excess iron accumulation, and exhibits poor prognosis, particularly in advanced stages owing to resistance to conventional therapeutics. Ferroptosis is an iron-dependent form of programmed cell death induced by lipid peroxidation and controlled by Hippo signaling. We hypothesized that overcoming ferroptosis resistance is an attractive strategy because OCCC acquires oxidative stress resistance during its development and exhibits chemoresistant features indicative of ferroptosis resistance. This study aimed to determine whether OCCC is resistant to ferroptosis and clarify the mechanism underlying resistance. Unlike ovarian high-grade serous carcinoma cells, OCCC cells were exposed to oxidative stress. However, OCCC cells remained unaffected by lipid peroxidation. Cell viability assays revealed that OCCC cells exhibited resistance to the ferroptosis inducer erastin. Moreover, Samroc analysis showed that the Hippo signaling pathway was enriched in OCCC cell lines and clinical samples. Furthermore, patients with low expression of nuclear Yes-associated protein 1(YAP1) exhibited a significantly poor prognosis of OCCC. Moreover, YAP1 activation enhanced ferroptosis in OCCC cell lines. Furthermore, suppression of zinc finger DHHC-type palmitoyltransferase 7 (ZDHHC7) enhanced ferroptosis by activating YAP1 in OCCC cell lines. Mouse xenograft models demonstrated that ZDHHC7 inhibition suppressed tumor growth via YAP1 activation by erastin treatment. In conclusion, YAP1 activation regulated by ZDHHC7 enhanced ferroptosis in OCCC. Thus, overcoming ferroptosis resistance is a potential therapeutic strategy for OCCC.

Prognostic value of oxidative phosphorylation-related genes in hepatocellular carcinoma

PurposeHepatocellular carcinoma (HCC) is the most prevalent malignancies worldwide. Recently, oxidative phosphorylation (OXPHOS) has received extensive concern as an emerging target in antitumor therapy. However, the OXPHOS-involved underlying genes and clinical utilization in HCC remain worth exploring. The present research aimed to create an OXPHOS-relevant signature in HCC.Patients and methodsIn this study, the prognostic signature genes linked with OXPHOS were identified, and prognostic models were built using least absolute shrinkage and selection operator (LASSO) cox regression analysis. Furthermore, the combination study of immune microenvironment and signature genes looked into the involvement of immune cells in signature-based genes in HCC. Following that, chemotherapeutic drug sensitivity and immunotherapy analysis was implemented to predict clinical efficacy in HCC patients. Finally, clinical samples were collected to measure the expression of OXPHOS-related signature genes.ResultsFollowing a series of screens, six prognostic signature genes related with OXPHOS were identified: MRPS23, MPV17, MAPK3, IGF2BP2, CDK5, and IDH2, on which a risk model was built. The findings revealed a significant drop in the survival rate of HCC patients as their risk score increased. Meanwhile, independent prognostic study demonstrated that the risk score could accurately identify HCC patients. Immuno-microenvironmental correlation research suggested that the prognostic characteristics could serve as a reference index for both immunotherapy and chemotherapy. Finally, RT-qPCR exhibited a trend in signature gene expression that was consistent with the results.ConclusionIn this study, a total of six prognostic genes associated with OXPHOS were selected and a prognostic model was constructed, providing an essential reference for the study of OXPHOS in HCC.

2,3,5,4’-tetrahydroxy-stilbene-2-O-β-D-glucoside promotes liver regeneration after partial hepatectomy in mice: the potential involvement of PPARα-mediated fatty acid metabolism

Ethnopharmacological relevance2,3,5,4’-tetrahydroxy-stilbene-2-O-β-D-glucoside (TSG) is the principal bioactive compound contained in Polygonum multiflorum Thunb. (PMT), which is traditionally recorded to possess tonic and anti-aging efficacy. Aim of the studyTo identify the TSG-provided promotion on liver regeneration (LR) following partial hepatectomy (PHx) in mice and to explicate its involved mechanism. Materials and methodsThe promotion of TSG on LR was evaluated by hematoxylin and eosin (H&E), 5-bromodeoxyuridinc (BrdU) and Ki-67 staining, and measuring the level of proliferating cell nuclear antigen (PCNA) and Cyclin D1 in mice with PHx at different time points. Gene Expression Omnibus (GEO, GSE15239) database and the label-free quantitative proteomics from liver of mice at 24 h after PHx were integrated to identify potential involved critical proteins, which were verified by Western-blot, Real-time polymerase chain reaction (RT-PCR), molecular docking and luciferase activity assay. Primary hepatocytes isolated from mice were used to investigate the TSG-provided promotion on proliferation in vitro. ResultsTSG (20 mg/kg) promoted LR in mice after PHx. Results from RNA expression data from clinical samples and proteomic analysis from liver tissues indicated that peroxisome proliferator-activated receptor α (PPARα)-mediated fatty acid metabolism pathway were crucially associated with the TSG-provided promotion on LR. TSG enhanced the nuclear translocation of PPARα and the mRNA expression of a series of PPARα-regulated downstream genes. In addition, TSG lowered hepatic triglyceride (TG) and non-esterified fatty acid (NEFA) amounts and increased hepatic adenosine triphosphate (ATP) level in mice after PHx. TSG up-regulated the transcriptional activity of PPARα in vitro. Next results displayed that TSG promoted cell proliferation as well as ATP level in mice primary hepatocytes, which were abolished when PPARα was suppressed. Meanwhile, the cell viability was also elevated in mice primary hepatocytes treated with ATP. ConclusionActivating PPARα-mediated fatty acid β-oxidation (FAO) pathway led to the production of ATP, which contributed to the TSG-provided promotion on LR after PHx in mice.

Solving selectivity issues in LBAs: case study using Gyrolab to quantify CB307, a bispecific Humabody in human serum.

Aim: Endogenous interferents can cause nonselectivity in ligand binding pharmacokinetic assays, leading to inaccurate quantification of drug concentrations. We describe the development of a Gyrolab immunoassay to quantify a new modality, CB307 and discuss strategies implemented to overcome matrix effects and achieve selectivity at the desired sensitivity. Results: Matrix effects were mitigated using strategies including increasing minimum required dilution (MRD) and lower limit of quantification, optimization of antibody orientation, assay buffer and solid phase. Conclusion: The strategies described resulted in a selective method for CB307 in disease state matrix that met bioanalytical method validation (BMV) guidance and is currently used to support clinical pharmacokinetic sample analysis in the first-in-human POTENTIA clinical study (NCT04839991) as a secondary clinical end point.

Boronic Acid-Rich Lanthanide Metal-Organic Frameworks Enable Deep Proteomics with Ultratrace Biological Samples.

Label-free proteomics is widely used to identify disease mechanism and potential therapeutic targets. However, deep proteomics with ultratrace clinical specimen remains a major technical challenge due to extensive contact loss during complex sample pretreatment. Here, a hybrid of four boronic acid-rich lanthanide metal-organic frameworks (MOFs) with high protein affinity is introduced to capture proteins in ultratrace samples jointly by nitrogen-boronate complexation, cation-π and ionic interactions. A MOFs Aided Sample Preparation (MASP) workflow that shrinks sample volume and integrates lysis, protein capture, protein digestion and peptide collection steps into a single PCR tube to minimize sample loss caused by non-specific absorption, is proposed further. MASP is validated to quantify ≈1800 proteins in 10 HEK-293T cells. MASP is applied to profile cerebrospinal fluid (CSF) proteome from cerebral stroke and brain damaged patients, and identified ≈3700 proteins in 1µL CSF. MASP is further demonstrated to detect ≈9600 proteins in as few as 50µg mouse brain tissues. MASP thus enables deep, scalable, and reproducible proteome on precious clinical samples with low abundant proteins.

Mucor germinans, a novel dimorphic species resembling Paracoccidioides in a clinical sample: questions on ecological strategy.

Dimorphism is known among the etiologic agents of endemic mycoses as well as in filamentous Mucorales. Under appropriate thermal conditions, mononuclear yeast forms alternate with multi-nucleate hyphae. Here, we describe a dimorphic mucoralean fungus obtained from the sputum of a patient with Burkitt lymphoma and ongoing graft-versus-host reactions. The fungus is described as Mucor germinans sp. nov. Laboratory studies were performed to simulate temperature-dependent dimorphism, with two environmental strains Mucor circinelloides and Mucor kunryangriensis as controls. Both strains could be induced to form multinucleate arthrospores and subsequent yeast-like cells in vitro. Multilateral yeast cells emerge in all three Mucor species at elevated temperatures. This morphological transformation appears to occur at body temperature since the yeast-like cells were observed in the lungs of our immunocompromised patient. The microscopic appearance of the yeast-like cells in the clinical samples is easily confused with that of Paracoccidioides. The ecological role of yeast forms in Mucorales is discussed.IMPORTANCEMucormycosis is a devastating disease with high morbidity and mortality in susceptible patients. Accurate diagnosis is required for timely clinical management since antifungal susceptibility differs between species. Irregular hyphal elements are usually taken as the hallmark of mucormycosis, but here, we show that some species may also produce yeast-like cells, potentially being mistaken for Candida or Paracoccidioides. We demonstrate that the dimorphic transition is common in Mucor species and can be driven by many factors. The multi-nucleate yeast-like cells provide an effective parameter to distinguish mucoralean infections from similar yeast-like species in clinical samples.

Simultaneous differential detection of H5, H7 and H9 subtypes of avian influenza viruses by a triplex fluorescence loop-mediated isothermal amplification assay

H5, H7, and H9 are pivotal avian influenza virus (AIV) subtypes that cause substantial economic losses and pose potential threats to public health worldwide. In this study, a novel triplex fluorescence reverse transcription-loop-mediated isothermal amplification (TLAMP) assay was developed in which traditional LAMP techniques were combined with probes for detection. Through this innovative approach, H5, H7, and H9 subtypes of AIV can be simultaneously identified and differentiated, thereby offering crucial technical support for prevention and control efforts. Three primer sets and composite probes were designed based on conserved regions of the haemagglutinin gene for each subtype. The probes were labelled with distinct fluorophores at their 3′ ends, which were detached to release the fluorescence signal during the amplification process. The detection results were interpreted based on the colour of the TLAMP products. Then, the reaction conditions were optimized, and three primer sets and probes were combined in the same reaction system, resulting in a TLAMP detection assay for the differential diagnosis of AIV subtypes. Sensitivity testing with in vitro-transcribed RNA revealed that the detection limit of the TLAMP assay was 205 copies per reaction for H5, 360 copies for H7, and 545 copies for H9. The TLAMP assay demonstrated excellent specificity, no cross-reactivity with related avian viruses, and 100% consistency with a previously published quantitative polymerase chain reaction (qPCR) assay. Therefore, due to its simplicity, rapidity, sensitivity, and specificity, this TLAMP assay is suitable for epidemiological investigations and is a valuable tool for detecting and distinguishing H5, H7, and H9 subtypes of AIV in clinical samples.

Glycosylphosphatidylinositol anchor biosynthesis pathway-based biomarker identification with machine learning for prognosis and T cell exhaustion status prediction in breast cancer

As the primary component of anti-tumor immunity, T cells are prone to exhaustion and dysfunction in the tumor microenvironment (TME). A thorough understanding of T cell exhaustion (TEX) in the TME is crucial for effectively addressing TEX in clinical settings and promoting the efficacy of immune checkpoint blockade therapies. In eukaryotes, numerous cell surface proteins are tethered to the plasma membrane via Glycosylphosphatidylinositol (GPI) anchors, which play a crucial role in facilitating the proper translocation of membrane proteins. However, the available evidence is insufficient to support any additional functional involvement of GPI anchors. Here, we investigate the signature of GPI-anchor biosynthesis in the TME of breast cancer (BC)patients, particularly its correlation with TEX. GPI-anchor biosynthesis should be considered as a prognostic risk factor for BC. Patients with high GPI-anchor biosynthesis showed more severe TEX. And the levels of GPI-anchor biosynthesis in exhausted CD8 T cells was higher than normal CD8 T cells, which was not observed between malignant epithelial cells and normal mammary epithelial cells. In addition, we also found that GPI -anchor biosynthesis related genes can be used to diagnose TEX status and predict prognosis in BC patients, both the TEX diagnostic model and the prognostic model showed good AUC values. Finally, we confirmed our findings in cells and clinical samples. Knockdown of PIGU gene expression significantly reduced the proliferation rate of MDA-MB-231 and MCF-7 cell lines. Immunofluorescence results from clinical samples showed reduced aggregation of CD8 T cells in tissues with high expression of GPAA1 and PIGU.

Determine Biofilm Genes in Pseudomonas Aeruginosa Isolated from Clinical and Environmental Samples

Objective: Isolates of Pseudomonas aeruginosa are an extremely adaptable bacterium that causes opportunistic diseases because of its varied metabolic pathways, genes, virulence factors, and considerable antibiotic resistance. Methods: A total of 293 samples were collected from different places: 193 samples (% 65.87) of human samples and 100 samples (% 34.13) of wastewater samples in the period between 3rd September to 15th November 2023). Bacterial isolates were identified according to microscopic, cultural, and genetic characteristics. Antibiotic susceptibility of bacterial isolates was determined against twelve of the selected antibiotics. The biofilm production was done by using phenotypic ways (Congo red agar and Microtiter plate methods) as well as genotypic ways by detection of biofilm genes (algD, pelf, and pslD genes). Results: Hundred-forty eight bacterial isolates were obtained, and sixty of these isolates were identified as Pseudomonas spp. (40.9%), twenty-six isolates of E. coli (17.9%), seventeen isolates of K. pneumoniae (11.3%), and forty-five isolates were beyond to other types of bacteria (30.1%), and out of sixty isolates of Pseudomonas spp., forty-two were identified as P. aeruginosa isolates. P. aeruginosa isolates revealed various resistance levels to antimicrobial agents gradually, ranging from 83.87% to Trimethoprim-sulfamethoxazole (SXT) to 3.22% to Aztreonam (AZT). Biofilm production by using the Congo red method showed that 27 isolates (64.28%) were positive results, while in the microtiter method, all forty-two isolates were positive (100%), the genetic detection showed that the AlgD gene was recognized in thirty-one isolates (73.8%), followed by Pelf and PslD genes in four isolates each (9.5%). Conclusion: The isolation percentage showed a high occurrence of multi-drug resistance biofilm forming Pseudomonas spp. isolates which could be a critical indicator. Methods of biofilm detection showed that the microtiter plate method has accuracy more than the Congo red method; as well AlgD gene was prevalent compared with both other genes Pelf, and PslD.