Adenoid hypertrophy (AH) is associated with excessive proliferation of germinal center B (GC-B) cells, yet the mechanisms underlying GC regression and termination remain poorly understood. This study used single-cell RNA sequencing (scRNA-seq) to profile the cellular composition of GC-B cells in AH. A unique subset of GC-B cells expressing CD9 was identified through comprehensive scRNA-seq, flow cytometry, and Mass cytometry (CyToF) analyses, with subsequent studies assessing the roles of CD9 and FOXP1 in GC-B cell differentiation and apoptosis pathways. CD9+ B cells were detected throughout GC-B cell development and were regulated by FOXP1. These cells also exhibited activation of cell death-related pathways, particularly during adenoid development. AH samples showed an increase in T follicular helper (Tfh) cells, accompanied by a reduction in CD9+ B cells. In Cd9 knockout mice, CD9 deficiency led to a significant decrease in serum IgG levels. Further analysis revealed that CD9+ B cells promoted Tfh cell apoptosis via pathways such as ALCAM-CD6. CD9+ B cells may regulate GC-B cell regression by inducing Tfh cell apoptosis, and FOXP1 may play a role in their differentiation. These findings clarify mechanisms of GC degeneration and termination, offering potential targets for AH treatment.

Clinical quality registries (CQRs) are central to improving surgical safety and quality by enabling systematic monitoring of outcomes and proactive identification of outliers. Published guidelines provide important advice on methods and activities for CQRs. Guideline flexibility allows for the varied and unique requirements of each CQR, but some aspects around guideline implementation are less certain, particularly for outlier management. In order to describe activities in Australia and the UK, searches were undertaken for published examples of CQR analysis and reporting of outliers and the subsequent management of outlier surgeons. There are many examples of high quality CQRs which actively report relevant performance indicators, with dashboards that show inter-hospital variation including unwarranted (outlier) variance. A small number of reports from the UK provide insight to local responses to suspected outlier status, commonly identifying data errors or systemic failures of local care processes. In Australia, challenges include voluntary participation, incomplete datasets, varied benchmarks, complex governance across public and private systems, and in some the restrictions imposed by qualified privilege. Worryingly, no single body is responsible for the management of the outlier surgeon. Outlier management can build on existing governance practices such as morbidity and mortality meetings and professional development activities. The publication of outlier processes and outcomes provides transparency to participants and closes the feedback loop. Active participation in CQRs and effective local outlier management actions should be encouraged as part of quality improvement, to learn from mistakes, celebrate excellence, and improve patient outcomes.

Polycaprolactone (PCL) is a biodegradable polyester widely used in industry, but it degrades slowly in the environment. Enzymatic hydrolysis catalyzed by cutinases represents a promising approach for PCL waste utilization. Through screening, we selected the Aureobasidium pullulans VKM 1116 strain capable of degrading PCL. We amplified the ApCUT1 cutinase gene from the A. pullulans VKM 1116 genomic DNA. Heterologous expression in Komagataella phaffii yielded ~60 mg/L of the target protein. The recombinant enzyme exhibited maximum activity at pH 6.0-6.5 and 30-40°C. Using site-directed mutagenesis, we generated single mutants (Y58W and L186F) and a double mutant (Y58W/L186F). The double mutant demonstrated the highest catalytic efficiency toward the model substrate 4-nitrophenyl 16-methylsulfonyl hexadecanoate (4-NP-(16-MS-C16)), exhibiting a two-fold increase in the hydrolysis rate compared to the wild-type enzyme. During PCL hydrolysis, the Y58W mutant showed the highest activity, providing a 1.8-fold increase in the suspension degradation rate and complete polymer granule degradation at a rate of 1 mg/h per 1 mg of protein. Our molecular modeling suggests that the hydrophobic bridge at the entrance to the active site functions as a "molecular trap." For the low-molecular-weight substrate (4-NP-(16-MS-C16)), enzymatic activity is limited by the kinetics of the substrate retention step, which is consistent with the maximum catalytic efficiency of the Y58W/L186F mutant with a closing trap. Conversely, activity toward PCL is limited by the initial adsorption stage, for which an open conformation of the free enzyme - as observed in the Y58W variant - is critically important. These results demonstrate the potential of the ApCUT1_Y58W cutinase for developing advanced PCL recycling technologies.

The vertebrate visual system contains a sophisticated physiological assembly of highly-specialized proteins that mediate light stimuli to be processed by the brain. Two types of cells, the rod and cone cells, are involved in detecting dim and bright light, respectively. The rod cell contains rhodopsin whose structure and function has been extensively studied and the molecular interactions involving rhodopsin and the other proteins of the phototransduction cascade have been dissected in great detail, although several mechanistic features remain to be determined. The cone phototransduction process has been less studied, particularly at the cone opsin structural level, and some conformational and mechanistic information has been inferred from analogy to the rod system. In spite of this, current efforts of investigation are focused on the study of the structure and function of cone opsins and other proteins of the cone phototransduction system. In this review we cover the current knowledge on the genetic and molecular aspects of cone opsins and the consequences of cone opsin mutations on the structure and function of these photoreceptor proteins causing vision disorders ranging from mild color vision abnormalities to severe cone-mediated retinal degeneration. A second part of the review is dedicated to the analysis of the molecular interactions involving calcium-binding proteins (such as recoverin and guanylate cyclase-activating proteins) in zebrafish which has emerged as a very useful model organism for the study of cone opsin phototransduction particularly in the deactivation steps of the visual phototransduction cascade.

Retinoic acid (RA), a biologically active metabolite of vitamin A, regulates gene expression through retinoic acid receptor/retinoid X receptor (RAR/RXR)-dependent transcription and is important for various biological phenomena. To understand the role of forebrain RA signaling in synaptic plasticity and memory, we generated transgenic mice expressing a dominant-negative form of retinoic acid receptor α (dnRARα) in adult forebrain. We previously showed that dnRARα expression in the adult forebrain impairs α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated synaptic transmission and long-term potentiation (LTP) in hippocampal CA1 neurons and hippocampus-dependent memory. To investigate the molecular basis of these impairments, we here examined expressions of synaptic plasticity-related molecules in the hippocampus of dnRARα mice. We found that protein levels of GluA1 and postsynaptic density protein 95 (PSD-95) were significantly reduced in the hippocampus of dnRARα mice in a dnRARα expression-dependent manner. To further examine protein expression changes in dnRARα mice, we performed proteome analysis and found that expression of actin-related protein 3 (ARP3), a molecule implicated in activity-dependent spine enlargement and maturation, was also reduced. Combined with our previous findings, these results suggest that forebrain RAR/RXR signaling is crucial for maintaining synaptic transmission, plasticity, and memory formation by upregulating GluA1, PSD-95, and ARP3.

Equol (Eq), produced by intestinal bacteria, is a more active metabolite than its precursor, daidzein; however, its production varies greatly among individuals. Bile acids (BAs) influence the balance of intestinal microbiota and may affect Eq production. Therefore, this study aimed to investigate the effects of BAs on Eq production in humans. Twenty-four-hour urine and feces were collected simultaneously from 23 participants after a 3-d soy challenge. Fecal samples from three participants with confirmed Eq-production ability, as determined by urinary analysis and in vitro fecal incubation, were used for incubation with BAs. Furthermore, the effects of co-addition of substances with BA-binding ability were examined. Fecal incubation from the three Eq producers showed distinctive daidzin (D) metabolic patterns, and the inhibitory concentrations of BAs on Eq production differed among individual samples. These differences were thought to be due to variations in individual microbiota. However, in all samples, cholic, chenodeoxycholic, and deoxycholic acids (CA, CDCA, and DCA, respectively), the major human BAs, inhibited D metabolism to Eq in a concentration-dependent manner at physiological concentrations. CDCA and DCA had approximately 10 times stronger inhibitory effects than CA. Co-addition of cholestyramine attenuated the inhibitory effects of BAs; therefore, ingestion of substances with BA-binding ability was expected to improve Eq production. However, the addition of sodium alginate, which has BA-binding ability and may affect the microbiota, showed variable response among individual samples. These results strongly suggest that BAs secreted into the intestinal tract are significant factors affecting Eq production in humans.

Creatine supplementation can improve muscle outcomes in older adults, particularly when combined with resistance exercise; however, its efficacy without exercise is less consistent. Guanidinoacetic acid (GAA), the immediate precursor of creatine, may bypass age-related limitations in creatine synthesis and transport and exert distinct effects on muscle energetics and protein metabolism. The main aim of the study was compare the effects of 8 wk of GAA versus creatine supplementation on muscle strength, body composition, and selected protein metabolism biomarkers in older adults. In this randomized, double-blind, exploratory pilot trial, 25 apparently healthy adults aged ≥65 y were assigned to receive GAA (2.0 g/d) or an equimolar dose of creatine monohydrate for 8 wk. Twenty-one participants completed the study (GAA, n=11; creatine, n=10). Handgrip strength, body composition and fluid compartments (via multifrequency bioelectrical impedance analysis), and circulating myostatin, insulin-like growth factor-1, mammalian target of rapamycin, total homocysteine, albumin, and total protein were assessed at baseline and follow-up. Intervention effects were evaluated using repeated-measures ANOVA, and between-group effect sizes for change scores were estimated using Hedges' g with 95% confidence intervals. Handgrip strength increased significantly with GAA (p=0.01) but not with creatine, with a significant time-by-treatment interaction (p=0.04) and a large between-group effect favoring GAA (g=0.91, 95% CI: 0.00 to 1.81). GAA supplementation was associated with significant within-group increases in fat-free mass, body cell mass, muscle mass, and total body water, whereas creatine supplementation did not produce comparable changes. Effect size analyses generally favored GAA for strength, muscle-related indices, and hydration-related compartments, although confidence intervals were wide. Both supplements reduced circulating myostatin, with greater suppression observed with GAA. Creatine supplementation produced a modest reduction in serum albumin that remained within physiological reference ranges. Serum IGF-1 concentrations were below the assay detection limit in most participants, precluding evaluation of the GH-IGF-1 axis. No adverse events were reported, and compliance exceeded 97% in both groups. In this exploratory pilot trial conducted without structured exercise, GAA supplementation was associated with numerically greater improvements in muscle strength and several muscle-related indices compared with creatine; however, these findings are preliminary and hypothesis-generating and require confirmation in adequately powered trials targeting sarcopenia.

The complement system, which plays a key role in innate immunity, facilitates the elimination of foreign and endogenous substances in the body. Consequently, while a balance between complement activation and regulation is typically maintained, a disruption of this balance can lead to various diseases. Paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome (aHUS) are diseases in which abnormal complement regulatory factors cause excessive complement activation. To accurately diagnose diseases such as aHUS, international standard complement tests including activation markers such as Ba and sC5b-9 are used. The Japanese Society for Complement Research has contributed to the standardization of the International Complement Society and has established this testing system in Japan. In the present study, we revealed that the Ba level serves as a good predictive marker for the onset of thrombotic microangiopathy, a complication that typically occurs following hematopoietic stem cell transplantation. We also showed that complement activation levels rise more frequently postpartum than during pregnancy. Considering the growing number of drugs targeting the complement pathway, it is speculated that these complement markers will become available for testing in clinical settings in Japan.

We report the case of an 80-year-old woman in which marked prolongation of prothrombin time (PT) and activated partial thromboplastin time (APTT) noted upon diagnosis of autoimmune hemolytic anemia revealed severe factor V (FV) deficiency. A mixing study showed a downward-convex pattern and the Bethesda assay for inhibitors was negative, which initially suggested congenital FV deficiency. Although the patient had concomitant early-stage esophageal cancer, infusions of fresh frozen plasma (FFP) failed to correct the coagulopathy, necessitating postponement of endoscopic therapy. Subsequently, a comprehensive evaluation by the Standardization Committee of the Japanese Ministry of Health, Labour and Welfare detected anti- FV IgG, establishing the diagnosis of hyperclearance-type autoimmune FV deficiency (AiFVD). Despite corticosteroid therapy, conventional coagulation screening test results remained markedly abnormal, complicating periprocedural management. In contrast, viscoelastic testing (VET) and thrombin generation testing (TGT), including ex vivo supplementation assays, indicated that adequate hemostasis could be achieved, allowing the endoscopic treatment to proceed safely. This case underscores that, even in FV deficiency with a negative inhibitor screen, testing for anti-FV IgG is essential for identifying hyperclearance-type AiFVD. Furthermore, ex vivo VET and TGT provided actionable assessments of hemostatic capacity and proved useful for guiding management in AiFVD.

Seasonal reproductive dynamics were investigated in two adult male Pacific white-sided dolphins (Aethalodelphis obliquidens, formerly Lagenorhynchus obliquidens) maintained at the Enoshima Aquarium over a two-year period (December 2018-November 2020). Serum testosterone (T), testicular cross-sectional area (TS), and pixel intensity (PI), an index of testicular echogenicity, were measured along with rectal body temperature at two depths (shallow, 15 cm; deep, 30 cm). Untargeted serum metabolomic profiling was performed using gas chromatography-mass spectrometry (GC-MS). In both individuals, T showed a clear seasonal pattern, with elevations lasting approximately one month during spring and summer, followed by prolonged increases in TS (approximately three months) and PI (three-four months). This temporal sequence was consistent across both years of observation. The T, TS, PI, and photoperiod were strongly and positively correlated with one another (Spearman's rs > 0.74, P < 0.01). Shallow-body temperature varied seasonally with water temperature, whereas deep-body temperature remained relatively stable. GC-MS detected 131 metabolites, and partial least squares discriminant analysis revealed a separation between high-T (> 10 ng/ml) and low-T (< 1 ng/ml) states. The four metabolites with the highest variable importance in projection scores were myoinositol, palmitoleic acid, L-tyrosine, and L-phenylalanine. Myoinositol and palmitoleic acid were positively correlated with the reproductive indices, whereas L-tyrosine and L-phenylalanine were negatively correlated, suggesting coordinated shifts in inositol, lipid, and amino acid metabolism during reproductive activation. To the best of our knowledge, this is the first study to integrate endocrine, ultrasonographic, thermal, and metabolomic approaches to characterize male reproductive seasonality in this species.

Sperm freezability varies considerably among pigs owing to genetic, husbandry, and management differences. Boar sperm cryopreservation remains underutilized owing to its inconsistent freezability. Semen with good freezability (GF) increases sow litter size and reduces breeding costs. Previous studies identified freezability biomarkers in cattle, sheep, and pigs. To comprehensively investigate these biomarkers, we conducted proteomic profiling of sperm with poor freezability (PF) and GF in Wannan Black pigs. We detected 2,485 proteins, identifying 337 significantly differentially expressed proteins (DEPs). Gene Ontology annotation revealed the involvement of DEPs in crucial processes, such as protein homeostasis maintenance, zona pellucida binding, polyspermy blockade, and ATPase activation regulation. Its molecular functions include immune signaling and oxidative stress defense. KEGG pathway analysis highlighted DEPs enrichment in seminal adhesion complexes, nucleic acid helicase families, and mitochondrial membrane protein clusters. Subcellular localization showed predominant DEPs distribution in the cytoplasm, extracellular matrix, nucleus, mitochondria, and plasma membranes. Western blotting validation of differentially expressed proteins (HSP90AA1, ACE, ANXA2, DEFB1, and S100A8) demonstrated results consistent with the proteomic sequencing data. This study delineates the key proteomic differences underlying boar sperm freezability, providing insights into refining cryopreservation protocols and augmenting swine reproductive efficiency.

We describe a male in his fifties with a refractory azole-resistant Candida albicans renal and perirenal walled-off abscess. C. albicans developed acquired azole-resistance during fluconazole (FLCZ) therapy. Despite FLCZ penetrating the walled-off abscess (11.1 μg/g) with levels exceeding those in normal renal tissue, the infection persisted, necessitating nephrectomy. Despite no amino acid substitutions in the ERG11 gene as revealed by genetic analysis, a Rhodamine 6G assay demonstrated a 4.5- to 6.6-fold increase in efflux activity in the resistant isolates in comparison with the susceptible controls, suggesting efflux pump-mediated resistance. Following surgery, the patient developed emphysematous pyelonephritis in the contralateral kidney due to azole-resistant C. albicans. This case demonstrates that even substantial tissue penetration cannot overcome high-level, effluxmediated resistance within walled-off lesions, underscoring the critical importance of surgical source control.

Virtual Reality (VR) is increasingly used in surgical education. This study examines the impact of VR-integrated training on learning, memory, and psychological responses. A prototype VR system with immersive OR views was tested on 67 clinical clerkship students. OR experiences followed the simulation. Thirty-two questionnaires covered surgical procedures and systems. Mood and personality were assessed using POMS-2, focusing on Total Mood Disturbance (TMD), fatigue-inertia (FI), vigor-activity (VA), and friendliness (F). VR modestly increased interest in surgery (~3.4/5) but training confidence stayed low (2.2/5). Understanding of abdominal surgery improved (2.6/5), with 96% correctly identifying the sterile field. Robotic roles were partially grasped; DCS understanding remained low (2.3/5). VR users better located OR equipment but 22% experienced nausea and 46% disliked the design. Compared to non-VR peers, they had lower procedural knowledge (p<0.01), but reported less stress and improved teamwork awareness (p<0.01). Mood analysis showed increased TMD and FI, decreased VA and F (all p<0.01), and reduced anxiety (TA). Personality traits (low neuroticism, high extraversion) were linked to stronger emotional reactions. VR sickness (51%) correlated with cognitive confusion and higher TMD (p<0.01). This VR approach enhances surgical understanding and team role awareness, offering experiential value in preclinical training.

Temporal lobe epilepsy (TLE) is linked to progressive alterations in brain network dynamics, leading to behavioral comorbidities and emergence of drug resistance; yet the underlying mechanistic and synaptic substrates remain incompletely understood. Here, we performed longitudinal phase-specific characterization of network dysfunction in TLE employing a lithium-pilocarpine-induced model in 7-week male Wistar rats. We integrated behavioral and electroencephalographic (EEG) analyses at five timepoints: baseline, acute, latent, chronic phases, and after assessment of drug-resistant epilepsy (DRE). After the chronic phase, responsiveness to phenobarbital (PB), a standardized approach for DRE characterization, was assessed via video monitoring to identify drug-resistant (DRUG-R) and drug-sensitive (DRUG-S) subpopulations. Our data show that epileptic rats exhibited progressive and phase-dependent behavioral and EEG alterations. Behavioral profiling revealed a hypermotor phenotype, impairment in burrowing natural behavior, altered phase-specific response to anxiety and depressive-associated behavioral paradigms, and progressive memory impairment. Following PB treatment, the identified DRUG-R subpopulation displayed specific hyperactive behavioral traits compared with DRUG-S, especially in anxiety-associated and burrowing behaviors. Spectral EEG analysis revealed modulation of frequency bands across disease stages, particularly in Theta and Delta power, and descriptive analyses attempted to stratify animals based on different responsiveness to PB. Lastly, correlation analyses supported associations between EEG, frequency of seizures and behavioral measures, especially in the anxiety domain and declarative memory. This study, moving beyond a seizure-centric perspective, evidences that TLE induces progressive and phase-specific reorganization of cortical activity that relapse on distinct electrophysiological and behavioral features, offering a novel framework to identify translational stage-related signatures of epileptogenesis and DRE.

Wogonoside, a major flavone 7-O-glucuronide from Scutellaria baicalensis Georgi, has long been used as a quality-control marker for Scutellariae Radix and related traditional herbal formulations. Emerging evidence suggests that wogonoside is not merely an analytical marker or an inactive glucuronide, but a pharmacologically active constituent and potential precursor of bioactive metabolites. In preclinical models, it exhibits anti-inflammatory, antioxidant, anti-angiogenic, anti-fibrotic, antitumor, cardioprotective, neuroprotective, and renoprotective activities. These effects are associated with the regulation of key signaling networks involved in inflammation, oxidative stress, inflammasome activation, autophagy, angiogenesis, fibrosis, and regulated cell death. Representative mechanisms include modulation of TLR4/NF-κB/NLRP3, Nrf2/HO-1, PI3K/AKT/mTOR, AMPK/mTOR, Wnt/β-catenin, Hedgehog/SMO/Gli1, and ferroptosis-related SLC7A11/GPX4 signaling. Recent target-validation studies have identified several candidate molecular targets, including SMO, NF-κB p65, HNF4α, NEK7, GPX4, TLR4, and CD39. However, the strength of evidence varies, and direct target engagement remains to be confirmed for many of these proteins. Pharmacokinetic studies reveal complex disposition characteristics, including intestinal microbial deglycosylation to wogonin, intestinal and hepatic re-glucuronidation, transporter-mediated efflux, enterohepatic or enteric recycling, and frequent multiple-peak plasma profiles. These properties support the value of wogonoside as both a quality-control marker and a dynamic exposure-related marker in Scutellaria-containing preparations. This review summarizes current advances in the chemical analysis, pharmacokinetics, biotransformation, molecular pharmacology, and disease-specific actions of wogonoside, and discusses key challenges for future development, including variable bioavailability, metabolite contribution, target specificity, pharmacokinetic-pharmacodynamic relationships, and clinical translation.