Thyroid cancer is the most common endocrine malignancy. Although surgery remains the primary treatment, advanced disease frequently recurs or metastasizes, and subsequent therapies yield suboptimal outcomes. Immunotherapy holds promise, yet the underlying immune-evasion mechanisms remain incompletely understood. HBO1, a multifunctional acyltransferase, plays a significant role in tumor immune regulation. Our previous research revealed that HBO1 is highly expressed in thyroid cancer tissues, promotes malignant progression, and inhibits CD8+ T cell function. To further elucidate the role and mechanism of HBO1 in thyroid cancer immune regulation, we conducted the following investigations. Firstly, using an in vitro co-culture model analyzed by flow cytometry and ELISA assays, we found that HBO1 overexpression induces CD8+ T cell apoptosis and significantly suppresses their cytotoxic function and secretion of effector molecules. Secondly, leveraging bioinformatic analysis of CUT&Tag datasets combined with in vitro cellular experiments, we found that HBO1 was associated with increased VTCN1 expression in an H3K14 acetylation-dependent manner, thereby contributing to CD8+ T cell dysfunction. Subsequently, employing RNA-seq analysis, ChIP-qPCR, dual-luciferase reporter assays, co-immunoprecipitation (Co-IP), and in vivo experiments, we further explored the underlying mechanism and found that HBO1 may facilitate BRD4 enrichment at the VTCN1 promoter through H3K14 acetylation, thereby promoting VTCN1 transcription and contributing to anti-tumor immune suppression in thyroid cancer. Finally, through IP-MS and Co-IP experiments, we identified the E3 ligase TRIM21 as a potential upstream regulator of HBO1. TRIM21 modulates HBO1 acetyltransferase activity by promoting its ubiquitination-mediated degradation, consequently influencing HBO1-associated immune escape in thyroid cancer. In summary, this study suggests that HBO1 may regulate VTCN1 expression through an H3K14ac-BRD4-associated mechanism in thyroid cancer, thereby contributing to suppression of CD8+ T cell anti-tumor immunity. In addition, TRIM21 may participate in the regulation of HBO1 protein stability and its associated immune-related signaling. Our findings provide new experimental evidence for understanding immune escape mechanisms and exploring potential immunotherapeutic targets in thyroid cancer.
Owing to the increasing demand for research on large-scale wildfires, it is crucial to accumulate investigation records, with fire marks being a key focus in postfire investigations. In this research, we have measured the spatial distribution of char directions on stembark surfaces and the heights of fire marks at 1019 points across the wildfire burned area in Japan covering 413 hectares. The aims were to (i) characterize the spatial distributions of fire marks within a large wildfire; (ii) identify and distinguish terrain-related determinants of stembark char directions from those influenced by other factors; and (iii) elucidate the fire spread process, highlighting the advantages of ground-based surveys. The findings revealed that (A) stembark char directions exhibited contrasting trends, aligning with the upslope direction on slopes and the eastward direction on ridges, typically within 60 degrees. The eastward alignment on ridges corresponded to the leeward direction of strong regional background winds during the first day, when most of the area burned; and (B) the surface fire moved upslope from the estimated ignition point, intensified on the windy ridge, and transitioned into a crown fire. While strong westerly winds lasted only about six hours after ignition, the fire likely reached the eastern coastline.
Recruiting and retaining mother-infant dyads in clinical research is often complex, particularly in socioeconomically disadvantaged and ethnically diverse communities. This paper reports on the recruitment and retention strategies employed within a prospective longitudinal cohort study (First Tooth Study) that investigated the oral colonization of Candida albicans and Streptococcus mutans as risk factors for Severe Early Childhood Caries (S-ECC) and the vertical transmission of these oral microorganisms. The study population was underserved mother-infant dyads, a group historically challenging to engage and retain due to factors such as socioeconomic barriers, busy schedules, and logistical challenges, which were further complicated by the COVID-19 pandemic. Few studies have systematically described recruitment and retention strategies for this population in the context of oral health research. The purpose of this paper is to describe and evaluate the recruitment and retention strategies used to enroll and maintain 186 third-trimester pregnant women and their infants within a primary care setting, and to assess the relative efficiency of each approach based on enrollment conversion rates, contribution to overall recruitment, and time allocation. Participants were recruited from two facilities of the University of Rochester: Highland Family Medicine (HFM), and the Perinatal dental clinic at Eastman Institute for Oral Health in Rochester, NY using a combination of direct recruitment, community networking, and outreach within prenatal groups. Retention efforts included scheduling study visits alongside routine pediatric appointments to reduce participant burden, offering financial incentives, and implementing a Reminder, Recall, and Outreach (RRO) system managed by a dedicated outreach worker. During the COVID-19 pandemic, additional adaptations were made, including offering home visits and curbside data collection, to maintain participant safety and adherence. Over 27 months, the study successfully enrolled 186 mother-infant dyads, meeting the recruitment target despite initial challenges and COVID-19 disruptions. Retention rates remained high, with an approximate 15% annual attrition during the two-year follow-up period, attributed to strategic visit alignment, personalized outreach, and practical supports like parking vouchers and home visits. The study underscores the importance of customized recruitment and retention strategies in overcoming socioeconomic and logistical barriers in underserved populations. Findings contribute valuable insights into best practices for engaging mother-infant dyads in longitudinal oral health research.
The development of various autologous platelet concentrates (APCs) has garnered recent attention for various applications in tissue regeneration. The aim of this study was to investigate 3 protocols to produce APCs on the biological capacity of human dental pulp cells (hDPCs) cultured under both normal and inflammatory conditions (induced by LPS from E. coli) in vitro. HDPCs were cultured in culture media from either 1) PRP (first centrifugation at 900 RCF for 5 minutes and second centrifugation at 2000 RCF for 15 minutes), 2) i-PRF (700 RPM for 3 minutes) or 3) C-PRF (2000 RCF for 8 minutes) when cultured under normal and inflammatory conditions. Cell migration was assessed using a scratch and transwell assay. Cell proliferation was tested using EdU assay and Ki67 immunofluorescence. HDPCs differentiation was assessed via Alizarin Red Staining, DSPP staining and genes encoding DMP1, DSPP, and COL1A1. Additionally, hDPCs conditioned under an inflammatory condition were further monitored for genes encoding P65 and IL-1B. All APC groups demonstrated the ability to promote migration and proliferation with C-PRF demonstrating significantly highest values. PRP and i-PRF moderately increased mineralized nodule formation and odontogenic marker expression, whereas C-PRF showed the strongest enhancement of Alizarin Red staining and the expression of DMP1, DSPP, and COL1A1. While culture conditions under inflammatory conditions induced by LPS induced higher IL-1B, P65, and lower hDPCs differentiation, the use of APCs improved all outcomes and the C-PRF group showed the greatest reduction in negative effects caused by LPS/inflammation. Based on these findings, the use of APCs was able to improve hDPCs activity in vitro with the C-PRF group showing the best results. Noteworthy, while LPS conditioned media typically led to higher hDPCs inflammation, the use of each APC group, especially C-PRF, was able to attenuate some of this impact. Future large animal and clinical studies are needed to further validate these findings.
Rapid and ultrasensitive detection of botulinum neurotoxin serotype A (BoNT/A), the most lethal known toxin, is critical for food safety and clinical diagnosis. Herein, we present a novel visual detection platform integrating a programmable DNA-guided nuclease, mutant Pyrococcus furiosus Argonaute (mPfAgo), with a pregnancy test strip (PTS), collectively termed mPfAgo-PTS. In this design, BoNT/A is first captured by an immunocomplex functionalized with a DNA tag. After amplification, the resulting amplicon directs mPfAgo to cleave a customized probe conjugated to human chorionic gonadotropin (hCG), thereby releasing free hCG for visual readout on the PTS. This cascaded signal transduction and amplification strategy enables the mPfAgo-PTS platform to detect BoNT/A down to 10 fg/mL within 60 min, representing a 1000-fold improvement in sensitivity over the gold-standard mouse bioassay. High precision and sensitivity validate its robustness and versatility for practical applications in food safety and clinical screening.
Evidence supports developmental surveillance and integrated care hubs for early identification and intervention in child developmental vulnerabilities. However, data on their use in community settings including schools and multicultural populations is limited. This study examined the implementation of the Watch Me Grow-Electronic (WMG-E) developmental surveillance tool and staff perspectives at the Mirrung wellbeing hub integrated with a preschool in South-Western Sydney. A mixed-methods study recruited parents/carers of children aged 3-6 years enrolled at Ashcroft Public School to complete WMG-E developmental screening assessments. Additionally, qualitative interviews with Mirrung staff assessed implementation metrics including acceptability, adoption, appropriateness, coverage and sustainability. Multilevel binary logistic regression models were conducted to determine whether sociodemographic and clinical characteristics were associated with the occurrence of developmental concern. Qualitative data was analysed thematically. Lower parent/carer education was associated with three-fold higher risk of child developmental concern (AOR 3.36, 95% CI 1.06-12.44). Thematic analysis revealed three themes: Barriers to Service Access, Enablers to Service Access and Uptake of WMG-E at Mirrung. WMG-E is a feasible, appropriate and acceptable developmental screening tool for multicultural school-based settings. Findings emphasise the need for culturally relevant health literacy resources to improve parents/carers' engagement to support early child development.
This randomized controlled trial investigated the effects of combining Global Postural Therapy (GPT) with a customized intraoral device (Byte) on stiffness-related performance and perceived stiffness in elite rhythmic gymnasts with low back pain. Seventeen athletes were randomly assigned to a Byte group (n = 8) or a control No Byte group receiving GPT alone (n = 9). Both groups completed a 12-week supervised postural re-education program focused on global muscle chain stretching, spinal alignment, and neuromuscular control. Functional stiffness was assessed across four post-intervention sessions using a Baiobit sensor to measure jump performance variables (maximum speed, height, force, concentric and eccentric phases, flight, and contact time), while perceived stiffness was evaluated using a visual analogue scale (VAS). Both groups improved in maximum speed, height, and force (η² = 0.25-0.31; p < 0.05), with no significant group × time interaction (p > 0.05). A significant reduction in perceived stiffness was observed in the Byte group compared with controls (p = 0.015; η² = 0.35). These findings suggest that adding a customized intraoral device to GPT did not produce additional improvements in objective stiffness-related performance over 12 weeks. However, the Byte group showed descriptively lower subjective stiffness values, which may indicate a potential perceptual effect associated with the device. Further research is required to clarify the clinical relevance and underlying mechanisms of these findings.
Helicobacter pylori (H. pylori) has evolved multiple immune evasion mechanisms that enable its persistent colonization in the gastric mucosa of approximately half of the global population. The chronic inflammation resulting from this persistent infection is widely recognized as a major contributor to gastric carcinogenesis. The specific goal of this study is to elucidate how H. pylori infection regulates dendritic cell (DC) maturation via the Serpinb8/ITGAX axis. First, a bioinformatics analysis was performed on the mouse gastric mucosal injury dataset GSE13873 from the GEO database. Using single-sample gene set enrichment analysis (ssGSEA), we evaluated immune cell infiltration profiles. Next, the phenotypic characteristics of DCs following H. pylori infection were comprehensively analyzed using flow cytometry and multiplex detection techniques. To further assess functional outcomes, the secretion level of IFN-γ in a co-culture system with CD4⁺ T cells was measured by ELISPOT. For histopathological evaluation, hematoxylin and eosin (H&E) staining was used to assess gastric tissue alterations in a mouse model, while Giemsa staining was performed to determine H. pylori colonization within the gastric mucosa. In this study, we found that DCs accumulated and displayed an activated phenotype in the H. pylori-induced gastric mucosal injury model. Serpinb8 protein expression was upregulated upon infection and positively correlated with ITGAX, a key DCs maturation marker. RNA interference-mediated knockdown of Serpinb8 significantly reduced ITGAX expression and the levels of CD80, CD103 and MHC II, indicating a crucial role for Serpinb8 in DCs maturation. Functionally, Serpinb8 knockdown impaired DCs-mediated T cell activation, attenuating the inflammatory response and gastric tissue damage. ELISA confirmed elevated pro-inflammatory cytokines in the infection group, which decreased following Serpinb8 knockdown. Together, these results suggest that the Serpinb8/ITGAX axis promotes DCs maturation and inflammation in H. pylori-induced gastric injury and may represent a potential immunotherapeutic target. This study demonstrates that H. pylori upregulates Serpinb8 expression, which in turn promotes ITGAX expression and modulates dendritic cell function. These findings provide new insights into the immunoregulatory mechanisms underlying H. pylori infection and suggest a potential precision medicine approach for treating gastric inflammatory diseases.
Tropodithietic acid (TDA) is a secondary metabolite with antimicrobial and iron-binding properties, produced by several marine Roseobacter group bacteria. Although classified as a siderophore, TDA is also produced under iron-replete conditions. To address this paradox, we fused the tdaCDE promoter region of Phaeobacter piscinae S26 with a promoter-less gfp reporter gene to monitor the transcription of TDA biosynthetic genes under different conditions and validated its accuracy using RT-qPCR. In both complex (½YTIO) and low-complexity (IOCGH) marine media, iron supplementation repressed tdaCDE transcription and reduced iron-chelation activity. However, TDA production responded differently depending on medium composition: iron suppressed TDA production in IOCGH but increased it in ½YTIO. Antimicrobial activity of culture supernatants declined in late-stage iron-limited ½YTIO-based cultures, even though tdaCDE transcription and iron-chelating activity increased. Acidification restored antimicrobial activity and increased detectable TDA levels, while several non-antimicrobial structural analogues - detected by LC-MS in non-acidified samples - diminished upon acidification, suggesting pH-driven interconversion. These findings reveal that TDA biosynthesis is regulated by iron availability but that the antimicrobial output of the producer depends on medium composition and pH-driven chemical transformations between TDA and its related structural analogues.
Antibiotic resistance gene (ARG) dissemination has long been attributed primarily to antibiotic contamination. However, this paradigm is increasingly challenged by emerging evidence that non antibiotic drugs (NADs) constitute a pervasive and previously overlooked driver of ARG spread. Here, we summarize that widely-used NADs (such as antidepressants and non-steroidal anti-inflammatory drugs) can actively promote horizontal gene transfer (HGT) and accelerate the emergence of multidrug resistance within microbial communities. Notably, significant enhancement of ARG dissemination has been reported even at clinically or environmentally relevant concentrations (e.g., 0.005-0.05 mg/L) commonly detected in wastewater and aquatic systems, by inducing stress responses in bacteria. The stress consequently elevated reactive oxygen species production, increased membrane permeability, and activation of efflux systems, collectively facilitating ARG mobility and persistence. These findings suggest that the ecological risks associated with NADs extend beyond conventional toxicity concerns and into the realm of antimicrobial resistance evolution. To address this emerging challenge, we propose several paradigm-shifting strategies to reduce relevant environmental risk of NADs, including molecular-level risk prediction based on chemical properties, artificial intelligence-assisted drug design, engineered microbial degradation systems, and advanced wastewater treatment technologies. Recognizing and mitigating NAD-driven ARG dissemination is essential for safeguarding environmental and public health in the post-antibiotic era.
The long maturation period and strong environmental reliance of Tuber borchii's fruiting bodies limit its commercial and medicinal utilization, leading to unstable yield and limited supply of bioactive compounds. Developing a controlled cultivation strategy for consistent production of truffle-derived metabolites, therefore, remains a critical challenge. In this study, grain-based solid-state fermentation was investigated as an alternative platform for mycelial biomass generation and triterpenoid production under controlled conditions. During the main solid-state cultivation phase, adlay was identified as the most effective substrate, yielding the highest biomass (45.32±1.58mg/g substrate) and triterpenoid content (1.00±0.13mg/g substrate) on day 28. Optimization of fermentation parameters was conducted by evaluating initial pH, moisture content, and inoculum age. The optimal conditions were found to be pH 7, 50% moisture content, and an inoculum age of 21 days, balancing both mycelial growth and triterpenoid accumulation. Regarding nutrient supplementation, 3% (w/v) glucose was identified as the most effective carbon source, resulting in the highest biomass and triterpenoid yield per gram of substrate (1.37±0.20mg/g substrate), while 1% (w/v) yeast extract proved to be the most effective nitrogen source, yielding 88.42±1.77mg/g substrate mycelial biomass and 1.91±0.12mg/g substrate triterpenoid content. Furthermore, co-culturing with 1g Quercus glauca somatic embryos increased the mycelial biomass and triterpenoid content to 100.72±1.98mg/g substrate and 2.39±0.14mg/g substrate, respectively. Overall, the study demonstrated the potential of solid-state fermentation as a sustainable method for the production of T. borchii mycelia and triterpenoids under controlled conditions.
Pneumonia causes significant mortality in intensive care unit (ICU) patients, yet traditional culture-based pathogen detection lacks sufficient sensitivity. While bronchoalveolar lavage fluid (BAL) provides optimal diagnostic yield, bronchoscopy is often contraindicated in critically ill patients. This study compares the respiratory microbiome profiles of paired tracheal aspirate (ETA) and BAL samples from pneumonia patients in a tertiary hospital ICU (n=23, November 2019-September 2022). Using 16S rRNA next-generation sequencing, we analyzed microbial diversity (Shannon Index), taxonomic composition, and differential abundance (edgeR). Results showed comparable diversity indices and microbial communities between ETA and BAL samples, with ETA successfully capturing key pneumonia-related microbial signatures. These findings validate ETA as a reliable, less invasive alternative to BAL for respiratory microbiome analysis in critically ill patients, establishing the groundwork for future clinical applications.
Environmental health monitoring (EHM) has gained attention as a sensitive, animal-free alternative to soiled bedding sentinel (SBS) programs for microbiologic surveillance in laboratory animal facilities. Although several EHM approaches have been validated in facilities using IVC systems, empirical evidence supporting room and equipment monitoring (REM) remains limited, particularly in open-top housing environments. This study evaluated the feasibility and practical applicability of environment-derived dust-based REM in an open-top housing facility using open-top cages and open racks. Microbial capture at room exhaust outlets was first assessed by culture-based analysis of exhaust prefilters in mouse, rat, and rabbit rooms. In parallel, exhaust louver-derived dust (ELD) was collected from downstream exhaust duct louvers and analyzed by PCR. To enable more frequent and standardized sampling, floor-derived dust (FDD) was collected using robotic cleaning devices and subjected to PCR analysis. The impact of robotic cleaning on environmental hygiene and welfare was evaluated using ATP-based cleanliness testing and noise measurements. Environmental microorganisms were consistently captured on exhaust prefilters, including in rooms without animals. PCR-based testing successfully detected microbial nucleic acids in both ELD and FDD samples across multiple facility zones, demonstrating the feasibility of REM in an open-top housing system. Robotic-based dust collection provided sufficient material for repeated testing under routine facility operation, without compromising environmental cleanliness or exceeding acceptable noise levels. These findings suggest that environment-derived dust-based REM can be implemented as a facility-level surveillance approach in open-top housing facilities, offering an animal-free, time-integrated monitoring strategy aligned with the principles of the 3Rs (Replacement, Reduction, and Refinement).
To retrospectively assess the comprehensive impact of transitioning from routine to refined quality management on medical quality, patient safety, and healthcare workers' perceived competence and satisfaction within the same medical team. A retrospective self‑controlled before‑and‑after study was conducted in a single psychiatric hospital. A stable cohort of 90 healthcare workers was followed through two periods: the control period (January-December 2024) with routine quality management, and the intervention period (January-December 2025) with refined quality management. Data were extracted from hospital information systems, quality management archives, and specialized databases. Outcome measures included medical quality scores as measured by internally developed scales, patient safety events measured as annual incidence rates using denominator data from hospital systems, and healthcare workers' self‑reported competence and satisfaction. Paired t‑tests were used for continuous scores, McNemar tests were applied for paired staff-level binary and ordinal data, while Pearson chi-square tests were used for patient safety incidence rates. Compared with the control period, the intervention period showed significant improvements across all domains. Scores for safety culture, management evaluation, and self‑assessed competence increased significantly (all P<0.001). All patient safety event rates decreased significantly (P<0.05), with adverse drug reactions and medical complaints showing the largest reductions (P<0.001). Overall satisfaction with the management model increased from 75.56% to 97.78% (P<0.001). Refined quality management was associated with substantial improvements in healthcare workers' perceptions, self‑assessed competence, patient safety, and overall satisfaction in this psychiatric hospital setting. Although limited by its retrospective, single‑center design, these findings suggest that refined quality management may serve as a promising strategy for improving hospital outcomes. Further validation through multicenter prospective studies is warranted to confirm the generalizability and sustainability of these effects.
This dataset includes in vivo electrical bioimpedance data collected from different biological tissues of two male Yorkshire pigs. The tissues probed include muscle, kidney, liver, bladder, intestine, skin, subcutaneous tissue, stomach, tendon, spleen, peritoneum, and blood from different vessels. Measurements were performed using a custom-made electrical impedance analyzer with two different types of sensing electrodes (monopolar and bipolar) and two sensing modalities (single fixed frequency and frequency scan). This data can be used to support a wide range of research and development efforts towards medical technologies that use electrical bioimpedance sensing as a key source of information to support clinical decisions, for example during diagnosis, treatment administration, or surgery. Specifically, this dataset may be used to support the development of technologies for autonomous identification of different tissues on contact, as demonstrated by the illustrative results presented in this paper. This capability may support future research towards assistive systems for enhancing procedural precision, safety and effectiveness, and may contribute to the development of autonomous robotic functionalities.
To evaluate outcomes of the Dutch policy permitting expectant management up to 72 h after term prelabour rupture of membranes (PROM) and to assess whether maternal or neonatal infection risk increase approaching this threshold. Retrospective cohort study. Two-site regional teaching hospital providing secondary obstetric care, Northwest Clinics, the Netherlands (2019-2024). Women with singleton term PROM (≥37 weeks) who have given birth >24 h after rupturing of membranes. Women were stratified by latency: 24-48, 48-72 h and >72 h. Primary outcomes were composite neonatal infection (suspected or culture-confirmed early-onset sepsis (EOS) requiring antibiotics treatment or high-care admission for suspected infection) and composite maternal infection (intrapartum or postpartum infection requiring antibiotic treatment). Primary outcomes were analysed using logistic regressions adjusted for Group B streptococcus (GBS) status. Maternal and neonatal infectious morbidity. A total of 914 women were included. Composite neonatal infection occurred in 13.9% in the 24-48 h group, 13.0% in the 48-72 h group and 17.7% in the >72 h group (p = 0.47). Composite maternal infection occurred in 9.7%, 8.9% and 12.4% (p = 0.58). No culture-confirmed EOS occurred. Suspected EOS increased with latency 5.2%, 9.7%, 10.6% (p = 0.02). Neonatal acidosis was significantly higher after >72 (17.6%) as compared to the 48-72 h group (8.9%) and the 24-48 h group (9.8%) (p = 0.03). Caesarean birth rates were 13.6%, 10.7% and 19.6% across the three latency groups; although rates appeared higher beyond 72 h, these differences did not reach statistical significance. No neonatal deaths occurred. Women undergoing expectant management for term PROM should be counselled regarding the potential increase in adverse outcomes with prolonged latency, while recognizing that this observational study does not allow conclusions regarding the effectiveness of induction at specific time points. Further studies are needed to determine the optimal timing of intervention.
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Chronic overconsumption of high-glycemic index (GI) starchy foods is a well-established risk factor for metabolic disorders. Unlike previous reviews focused on fragmented topics, a complete logical chain centered on starch digestion and resulting GI fluctuations is constructed. Various models and emerging methods (bionics, AI, and omics) are employed for evaluation. Native starch digestibility is jointly governed by amylose/amylopectin ratio, chain-length distribution, crystalline organization, and granular architecture. Furthermore, proteins, lipids, fibers, and polyphenols affect starch hydrolysis via spatial resistance, viscosity alteration, or enzyme inhibition. Processing is equally critical, typically covering thermal and non-thermal ways. Afterwards, we proposed an integrative framework linking structural determinants, enzyme accessibility, and final outcomes followed by discussing effective low-GI strategies accordingly. However, there is still a gap in accomplishing industrialization and standardization. For combining scientific understanding with industrial applications, cross-scale prediction model, precise customization, nutrition-sensory trade-off, and standard ecosystem for inclusive scale-up may represent future trends.
to assess whether implant mucosal tunnel depth is associated with differences in cultivable microbial diversity in clinically healthy peri-implant tissues. fifty-two patients with single molar implants were included after a standardized 3-month healing period and stratified into three groups according to implant mucosal tunnel depth: <3 mm, 3-5 mm, and > 5 mm. Microbial samples from the inner lining of the implant mucosal tunnel were cultured under aerobic and anaerobic conditions and identified using MALDI-TOF mass spectrometry. As a secondary exploratory outcome, TNF-α expression in peri-implant mucosa was quantified using real-time PCR. a total of 262 cultivable isolates representing 46 species were identified, with facultative anaerobic species predominating (81.68%). The highest Shannon diversity index was observed in the 3-5 mm group, whereas tunnels > 5 mm demonstrated reduced cultivable diversity, with clinically significant growth predominantly represented by streptococci. TNF-α expression showed a progressive increase with increasing implant mucosal tunnel depth, reaching approximately 38-fold higher levels in the > 5 mm group compared with < 3 mm (p < 0.017). within the limitations of this exploratory cross-sectional study, implant mucosal tunnel depth was associated with differences in cultivable microbial composition and local TNF-α expression in clinically stable peri-implant tissues. Deeper mucosal tunnels (> 5 mm) demonstrated reduced cultivable species diversity without a consistent predominance of cultivable periodontal pathogens under the applied culture conditions. The observed increase in TNF-α expression should be interpreted as a biologic association rather than evidence of active inflammation or disease. Due to the culture-based methodology and the absence of longitudinal clinical data, no conclusions regarding disease risk or progression can be drawn. ClinicalTrials.gov, NCT05870774. Registered on 20,230,425.
As a globally cultivated edible fungus, Pleurotus ostreatus is highly nutritious and rich in proteins, vitamins, minerals, and amino acids. However, viral infections cause significant losses in quality and yield during cultivation. In our survey, weakened growth and malformed fruiting bodies were observed in Sanbaoxiang Agricultural Technology Park, which is located in Tongzhou District of Beijing, but the associated viruses were not clear. The mycelia were isolated, purified and used for ribosomal RNA-depleted total RNA deep sequencing. A total of 118,973,697 clean reads were obtained and subsequently assembled into 53,864 contigs. BLAST analysis indicated that most contigs originated from the host P. ostreatus; however several contigs showed high amino acid sequence identities to known mycoviruses. Further complete genome sequencing with RT‒PCR and rapid amplification of cDNA ends for the terminal sequences revealed that mixed infection with seven RNA viruses was associated with the observed symptoms: four reported viruses, including oyster mushroom spherical virus, pleurotus ostreatus deltaflexivirus 2, pleurotus ostreatus alpha-like virus, and pleurotus ostreatus ambivirus 2, and three new viruses tentatively named pleurotus ostreatus deltaflexivirus 3, pleurotus ostreatus ambivirus 3 and pleurotus ostreatus lentinuvirus 1. The sequence characterization, genomic structure and phylogenetic relationships of these new viruses with other known viruses were also characterized. Further virus distribution patterns were explored with RT-PCR analysis of different strains isolated from different locations. In this study, a mixed infection with seven RNA viruses from a P. ostreatus strain was identified via deep sequencing, and their genomes were characterized. These findings advance our understanding of the diversity of viruses infecting P. ostreatus and provide critical insights for further research on virus‒host interactions and the development of viral elimination strategies.