Advancing quantitative PCR with color cycle multiplex amplification.

Quantitative PCR (qPCR) is the gold standard for detection and quantitation of known DNA targets, but the scarcity of spectrally distinct fluorophores and filter sets limits the number of detectable targets. Here, we introduce color cycle multiplex amplification (CCMA) to significantly increase the number of detectable DNA targets in a single qPCR reaction using standard instrumentation. In CCMA, presence of one DNA target species results in a pre-programmed pattern of fluorescence increases. This pattern is distinguished by cycle thresholds (Cts) through rationally designed delays in amplification. For example, we design an assay wherein Staphylococcus aureus sequentially induces FAM, then Cy5.5, then ROX fluorescence increases with more than 3 cycles between each signal. CCMA offers notably higher potential for multiplexing because it uses fluorescence permutation rather than combination. With 4 distinct fluorescence colors, CCMA theoretically allows the detection of up to 136 distinct DNA target sequences using fluorescence permutation. Experimentally, we demonstrated a single-tube qPCR assay screening 21 sepsis-related bacterial DNA targets in samples of blood, sputum, pleural effusion and bronchoalveolar lavage fluid, with 89% clinical sensitivity and 100% clinical specificity, showing its potential as a powerful tool for advanced quantitative screening in molecular diagnostics.

Self-Correcting Method for Highly Effective Office-Based Helicobacter pylori Therapy Using Cumulative Test of Cure Data.

BACKGROUND AND AIM: Helicobacter pylori infections have become resistant to many previously highly effective antimicrobial regimens resulting in clarithromycin, metronidazole, or fluoroquinolone-containing therapies becoming unsuccessful. Pretreatment susceptibility testing is only widely available in the United States but is still rarely done. Here, we propose a framework to monitor H. pylori eradication in small clinical settings by routinely assessing the effectiveness of therapy. METHODS: Because of the small sample size in individual practice's, we assume an acceptable cure rate of ≥80% (preferred cure rate ≥85%) in adherent patients, with a dichotomous outcome (cured vs. failed) and consecutive patient enrollment. To obtain results (feedback) in a timely manner, for individual practices, cure rates can be estimated after 10 patients. Large practices which acquire patients more rapidly can delay analysis until a total of 104 H. pylori-infected patients, assuming a baseline cure rate of at least 85% with the preferred regimen. RESULTS: We show how data from individual practices can be utilized to improve the effectiveness of H. pylori treatment decisions. The method consists of recording and accumulating the confirmation of cure data for successive small groups of patients. These data are then analyzed as binary outcomes (pass-fail) and serve as the basis for studying and improving the effectiveness of H. pylori treatment decisions. CONCLUSION: A simple actuarial method can serve outpatient clinics to ensure a reliable test-to-cure method and avoid futile Hp regimens.

Efficacy of Flaxseed Compared to ACE Inhibition in Treating Anthracycline- and Trastuzumab-Induced Cardiotoxicity.

Background: Although the current combination of surgery, radiation, and chemotherapy is used in the breast-cancer setting, the administration of the anticancer drugs doxorubicin and trastuzumab is associated with an increased risk of developing heart failure. The aim of this study is to determine whether dietary flaxseed is comparable and/or synergistic with the angiotensin-converting enzyme inhibitor perindopril in the treatment of doxorubicin- and trastuzumab-mediated cardiotoxicity. Methods: In a chronic in vivo murine model (n = 110), doxorubicin and trastuzumab (8 mg/kg and 3 mg/kg, respectively) were administered weekly for 3 weeks. Following this period, the mice were randomized to daily consumption of a 10% flaxseed supplemented diet, administration of perindopril (3 mg/kg) via oral gavage, or a combination of both flaxseed and perindopril for an additional 3 weeks. Results: In mice treated with doxorubicin and trastuzumab, the left ventricular ejection fraction decreased from 74% ± 4% at baseline to 30% ± 2% at week 6. Treatment with either flaxseed or perindopril, or with flaxseed and perindopril improved left ventricular ejection fraction to 52% ± 4%, 54% ± 4%, and 55% ± 3%, respectively (P < 0.05). Although histologic analyses confirmed significant loss of sarcomere integrity and vacuolization in the doxorubicin- and trastuzumab-treated mice, treatment with flaxseed or perindopril, or with flaxseed and perindopril improved myocyte integrity. Finally, the level of Bcl-2 interacting protein 3, high-mobility group box 1 protein expression, and the levels of select oxylipins, were significantly elevated in mice receiving doxorubicin and trastuzumab; these markers were attenuated by treatment with either flaxseed or perindopril, or with flaxseed and perindopril. Conclusions: Flaxseed was equivalent to perindopril at improving cardiovascular remodelling by reducing biomarkers of inflammation, mitochondrial damage, and cell death.

Contexte: Si l'association actuelle de la chirurgie, de la radiothérapie et de la chimiothérapie est utilisée pour le traitement du cancer du sein, on observe néanmoins que l'administration de la doxorubicine et du trastuzumab, deux anticancéreux, augmente les risques d'insuffisance cardiaque. Cette étude vise à déterminer si les graines de lin alimentaires ont un effet comparable et/ou synergique à celui du périndopril, un inhibiteur de l'enzyme de conversion de l'angiotensine, dans le traitement de la cardiotoxicité liée à la doxorubicine et au trastuzumab. Méthodologie: Dans un modèle murin chronique in vivo (n = 110), la doxorubicine et le trastuzumab (8 mg/kg et 3 mg/kg, respectivement) ont été administrés une fois par semaine pendant trois semaines. Après cette période, les souris ont été réparties de façon aléatoire dans trois groupes : l'un recevant tous les jours un régime alimentaire contenant 10 % de graines de lin, un autre recevant du périndopril (3 mg/kg) par gavage oral et un troisième recevant à la fois des graines de lin et du périndopril pendant trois semaines supplémentaires. Résultats: Chez les souris recevant la doxorubicine et le trastuzumab, la fraction d'éjection ventriculaire gauche est passée de 74 % ± 4 % au départ à 30 % ± 2 % à la semaine 6. Avec le traitement par les graines de lin seules, le périndopril seul ou les graines de lin et le périndopril en association, la fraction d'éjection ventriculaire gauche est passée à 52 % ± 4 %, à 54 % ± 4 % et à 55 % ± 3 %, respectivement (p < 0,05). Bien que les analyses histologiques aient permis de confirmer une perte significative de l'intégrité des sarcomères et une vacuolisation chez les souris recevant la doxorubicine et le trastuzumab, le traitement par les graines de lin seules, le périndopril seul ou les graines de lin et le périndopril en association a amélioré l'intégrité des myocytes. Enfin, les taux de protéine 3 interagissant avec BCL-2, l'expression de la protéine HMGB1 (high-mobility group box 1) et les taux de certaines oxylipines étaient significativement élevés chez les souris recevant la doxorubicine et le trastuzumab. Ces marqueurs ont été atténués par les graines de lin, le périndopril ou l'association des deux. Conclusions: En diminuant les biomarqueurs de l'inflammation, les dommages aux mitochondries et la mort cellulaire, les graines de lin ont un effet équivalant à celui du périndopril quant à l'amélioration du remodelage cardiovasculaire.

Recruitment of FBXO22 for targeted degradation of NSD2.

Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain-of-function mutation p.E1099K, resulting in growth suppression, apoptosis and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 to recruit the SCFFBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCFFBXO22. Overall, we present a potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a new FBXO22-recruitment strategy for TPD.

Implementation of Digital Pathology and Artificial Intelligence in Routine Pathology Practice.

The advent of affordable technology has significantly influenced the practice of digital pathology, leading to its growing adoption within the pathology community. This review article aims to outline the latest developments in digital pathology, the cutting-edge advancements in artificial intelligence (AI) applications within this field, and the pertinent U.S. regulatory frameworks. The content is based on a thorough analysis of original research articles and official U.S. federal guidelines. Findings from our review indicate that several FDA-approved digital scanners and image management systems are establishing a solid foundation for the seamless integration of advanced technologies into everyday pathology workflows, which may reduce device and operational costs in the future. AI is particularly transforming the way morphological diagnoses are automated, notably in cancers like prostate and colorectal, within screening initiatives, albeit challenges such as data privacy issues and algorithmic biases remain. The regulatory environment, shaped by standards from the FDA, CMS/CLIA, and CAP, is evolving to accommodate these innovations while ensuring safety and reliability. CMS/CLIA have issued policies to allow pathologists to review and render diagnoses using digital pathology remotely. Moreover, the introduction of new digital pathology CPT codes designed to complement existing pathology CPT codes is facilitating reimbursement processes. Overall, these advancements are heralding a new era in pathology that promises enhanced diagnostic precision and efficiency through digital and AI technologies, potentially improving patient care as well as bolstering educational and research activities.

Comparison of perioperative and subacute postoperative complications between LDR and HDR monotherapy brachytherapy for prostate cancer.

PURPOSE: We aim to investigate perioperative and subacute postoperative complications in patients undergoing LDR or HDR monotherapy for prostate cancer. We hypothesize a low rate of complications, and a favorable toxicity profile in patients treated with HDR compared to LDR. MATERIALS AND METHODS: A prospectively collected institutional database was queried for patients treated with HDR or LDR prostate monotherapy between 1998 and 2021. Toxicities were determined per CTCAE. Claims based billing codes were obtained to identify additional events. Events occurring within 4 months of treatment were defined as perioperative or subacute postoperative complications. RESULTS: 759 patients were identified, 446 received LDR with 125I, and 313 received HDR with 192Ir. HDR patients had higher risk features: 75.7% with Gleason score 7+ versus 2.4% of LDR, and 16% with initial PSA 10+ ng/mL versus 2.7% of LDR. Toxicities were mild with the most common being grade 1 GU frequency and nocturia at ∼50%. HDR patients had significantly less grade 2 dysuria (2.6% vs. 9.0%), frequency (4.8% vs. 9.4%), hematuria (1.0% vs. 5.2%), nocturia (3.8% vs. 9.4%), and urinary obstructive symptoms (7.3% vs. 11.2%), all statistically significant. 11 (1.4%) patients had infection requiring antibiotics: 8 (1.8%) from the LDR group and 3 (1%) from the HDR group. Cardiopulmonary events were low at <2% overall, without difference between HDR and LDR. CONCLUSIONS: Overall toxicity rates support the safety of prostate brachytherapy. HDR monotherapy is associated with significantly less perioperative and subacute postoperative GU events when compared to LDR monotherapy. Cardiopulmonary events were equally rare in both groups.

Oxidative stress modulating nanomaterials and their biochemical roles in nanomedicine.

Many pathological conditions are predominantly associated with oxidative stress, arising from reactive oxygen species (ROS); therefore, the modulation of redox activities has been a key strategy to restore normal tissue functions. Current approaches involve establishing a favorable cellular redox environment through the administration of therapeutic drugs and redox-active nanomaterials (RANs). In particular, RANs not only provide a stable and reliable means of therapeutic delivery but also possess the capacity to finely tune various interconnected components, including radicals, enzymes, proteins, transcription factors, and metabolites. Here, we discuss the roles that engineered RANs play in a spectrum of pathological conditions, such as cancer, neurodegenerative diseases, infections, and inflammation. We visualize the dual functions of RANs as both generator and scavenger of ROS, emphasizing their profound impact on diverse cellular functions. The focus of this review is solely on inorganic redox-active nanomaterials (inorganic RANs). Additionally, we deliberate on the challenges associated with current RANs-based approaches and propose potential research directions for their future clinical translation.

A Randomized, Controlled Trial Evaluating Perioperative Risk-Stratification and Risk-Based, Protocol-Driven Management After Elective Major Cancer Surgery.

OBJECTIVE: We evaluated the efficacy of risk-based, protocol-driven management versus (vs) usual management after elective major cancer surgery to reduce 30-day rates of postoperative death or serious complications (DSC) . SUMMARY BACKGROUND DATA: Major cancer surgery is associated with significant perioperative risks which result in worse long-term outcomes. METHODS: Adults scheduled for elective major cancer surgery were stratified/randomized to risk-based escalating levels of care, monitoring, and co-management vs usual management. The primary study outcome was 30-day rate of DSC. Additional outcomes included complications, adverse events, health care utilization, health-related quality of life (HRQOL), and disease-free and overall survival (DFS and OS). RESULTS: Between August 2014 and June 2020, 1529 patients were enrolled and randomly allocated to the study arms; 738 patients in the Intervention Arm and 732 patients in the Control Arm were eligible for analysis. 30-day rate of DSC with the intervention was 15.0% (95% CI, 12.5-17.6%) vs 14.1%, (95% CI, 11.6-16.6%) with usual management (P=0.65). There were no differences in 30-day rates of complications or adverse events (including return to the operating room); postoperative length of stay; rate of discharge to home; or 30, 60, or 90-day HRQOL or rates of hospital readmission or receipt of anti-neoplastic therapy between the study arms. At median follow-up of 48 months, OS (P=0.57) and DFS (P=0.91) were similar. CONCLUSIONS: Risk-based, protocol-driven management did not reduce 30-day rate of DSC after elective major cancer surgery compared to usual management, nor improve postoperative health care utilization, HRQOL, or cancer outcomes. Trials are needed to identify cost-effective, tailored perioperative strategies to optimize outcomes after major cancer surgery.

Clinical effectiveness and safety of time-lapse imaging systems for embryo incubation and selection in in-vitro fertilisation treatment (TILT): a multicentre, three-parallel-group, double-blind, randomised controlled trial.

BACKGROUND: Time-lapse imaging systems for embryo incubation and selection might improve outcomes of in-vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) treatment due to undisturbed embryo culture conditions, improved embryo selection, or both. However, the benefit remains uncertain. We aimed to evaluate the effectiveness of time-lapse imaging systems providing undisturbed culture and embryo selection, and time-lapse imaging systems providing only undisturbed culture, and compared each with standard care without time-lapse imaging. METHODS: We conducted a multicentre, three-parallel-group, double-blind, randomised controlled trial in participants undergoing IVF or ICSI at seven IVF centres in the UK and Hong Kong. Embryologists randomly assigned participants using a web-based system, stratified by clinic in a 1:1:1 ratio to the time-lapse imaging system for undisturbed culture and embryo selection (time-lapse imaging group), time-lapse imaging system for undisturbed culture alone (undisturbed culture group), and standard care without time-lapse imaging (control group). Women were required to be aged 18-42 years and men (ie, their partners) 18 years or older. Couples had to be receiving their first, second, or third IVF or ICSI treatment and could not participate if using donor gametes. Participants and trial staff were masked to group assignment, embryologists were not. The primary outcome was live birth. We performed analyses using the intention-to-treat principle and reported the main analysis in participants with primary outcome data available (full analysis set). The trial is registered on the International Trials Registry (ISRCTN17792989) and is now closed. FINDINGS: 1575 participants were randomly assigned to treatment groups (525 participants per group) between June 21, 2018, and Sept 30, 2022. The live birth rates were 33·7% (175/520) in the time-lapse imaging group, 36·6% (189/516) in the undisturbed culture group, and 33·0% (172/522) in the standard care group. The adjusted odds ratio was 1·04 (97·5% CI 0·73 to 1·47) for time-lapse imaging arm versus control and 1·20 (0·85 to 1·70) for undisturbed culture versus control. The risk reduction for the absolute difference was 0·7 percentage points (97·5% CI -5·85 to 7·25) between the time-lapse imaging and standard care groups and 3·6 percentage points (-3·02 to 10·22) between the undisturbed culture and standard care groups. 79 serious adverse events unrelated to the trial were reported (n=28 in time-lapse imaging, n=27 in undisturbed culture, and n=24 in standard care). INTERPRETATION: In women undergoing IVF or ICSI treatment, the use of time-lapse imaging systems for embryo culture and selection does not significantly increase the odds of live birth compared with standard care without time-lapse imaging. FUNDING: Barts Charity, Pharmasure Pharmaceuticals, Hong Kong OG Trust Fund, Hong Kong Health and Medical Research Fund, Hong Kong Matching Fund.

Hippocampal infarction and generalized seizures predict early mortality after endovascular middle cerebral artery occlusion in mice.

Endovascular middle cerebral artery occlusion (MCAO) is a widely used experimental ischemic stroke model. However, the model carries high early mortality. Our aim was to investigate the factors that influence early mortality within 48 h of reperfusion after transient MCAO. Using C57BL/6 mice, we induced 1-hour endovascular filament MCAO. To introduce heterogeneity of infarct volumes, a subset of animals had additional tandem common carotid artery occlusion (MCAO+CCAO). Continuous video monitoring was used to gain insight into the cause of death. Mortality within 48 h was 25% in the pooled cohort. All animals with early mortality suffered from infarcts in the hippocampus, sometimes accompanied by infarcts in the thalamus and midbrain, which occurred exclusively in the MCAO+CCAO group. All animals with early mortality developed convulsive seizures captured on video monitoring. None of the animals that did not develop convulsive seizures died. Among the three regions, hippocampal infarction appeared necessary for convulsive seizures and early mortality. Our data highlight seizures as the primary cause of mortality within the first 48 h after endovascular filament MCAO, linked to hippocampal infarction. Since hippocampal blood supply is mainly from the posterior cerebral artery (PCA), avoiding concurrent PCA ischemia can decrease mortality in proximal MCAO models.

The biological and therapeutic assessment of a P(3HB-co-4HB)-bioactive glass-graphene composite biomaterial for tissue regeneration.

An ideal wound dressing should create a healing environment that relieves pain, protects against infections, maintains moisture, removes debris, and speeds up wound closure and repair. However, conventional options like gauze often fall short in fulfilling these requirements, especially for chronic or nonhealing wounds. Hence there is a critical need for inventive formulations that offer efficient, cost-effective, and eco-friendly alternatives. This study focuses on assessing the innovative formulation based on a microbial-derived copolymer known as poly(3-hydroxybutyrate-co-4-hydroxybutyrate), P(3HB-co-4HB) bioactive glass and graphene particles, and exploring their biological response in vitro and in vivo-to find the best combination that promotes cell adhesion and enhances wound healing. The formulation optimized at concentration of bioactive glass (1 w/w%) and graphene (0.01 w/w%) showed accelerated degradation and enhanced blood vessel formation. Meanwhile biocompatibility was evaluated using murine osteoblasts, human dermal fibroblasts, and standard cell culture assays, demonstrating no adverse effects after 7 days of culture and well-regulated inflammatory kinetics. Whole thickness skin defect using mice indicated the feasibility of the biocomposites for a faster wound closure and reduced inflammation. Overall, this biocomposite appears promising as an ideal wound dressing material and positively influencing wound healing rates.

Vertical transmission of microbiomes into embryo culture media and its association with assisted reproductive outcomes.

RESEARCH QUESTION: Can microbes vertically transmit from semen and follicular fluid to embryo culture media during assisted reproductive technology (ART) treatment? DESIGN: Spent embryo culture media (SECM), seminal fluid and follicular fluid samples were collected from 61 couples with infertility undergoing ART treatment at the Prince of Wales Hospital, Hong Kong SAR, China. Metagenomic analysis was conducted using 16s rRNA sequencing to identify the source of microbes in SECM, correlation between the semen microbiome and male infertility, and correlation between the follicular fluid microbiome and female infertility. RESULTS: Microbial vertical transmission into SECM was reported in 82.5% of cases, and semen was the main source of contamination in conventional IVF cases. The increased abundances of Staphylococcus spp. and Streptococcus anginosus in semen had negative impacts on total motility and sperm count, respectively (P < 0.001). Significant increases in abundance of the genera Prophyromonas, Neisseria and Facklamia were observed in follicular fluid in women with anovulation, uterine factor infertility and unexplained infertility, respectively (P < 0.01). No significant correlation was found between the bacteria identified in all sample types and ART outcomes, including fertilization rate, embryo development, number of available embryos, and clinical pregnancy rate. CONCLUSION: Embryo culture media can be contaminated during ART treatment, not only by seminal microbes but also by follicular fluid and other sources of microbes. Strong correlations were found between specific microbial taxa in semen and sperm quality, and between the follicular fluid microbiome and the aetiology of female infertility. However, no significant association was found between the microbiomes of SECM, semen and follicular fluid and ART outcomes.

Correlation of Ablation Volume with Renal Function Loss after Cryoablation in Solitary Functioning Kidneys.

PURPOSE: To determine the degree of renal function deterioration after renal cryoablation in patients with a solitary functioning kidney on the basis of ablation volume. MATERIALS AND METHODS: Over a 15-year period, 81 percutaneous cryoablations were performed in solitary functioning kidneys. After exclusion of patients with baseline end-stage renal disease (ESRD) and insufficient follow-up, analysis was performed on 65 procedures in 52 patients (40 men; mean age, 63.5 years). The postcryoablation renal function was based on the lowest serum creatinine within 6 months after procedure. Renal function change was defined as percentage glomerular filtration rate (GFR) change. Volumetric analysis was performed on the target lesion, renal parenchyma, and ablation zone. RESULTS: The median tumor diameter was 2.0 cm (range, 0.8-4.7 cm). The median baseline GFR decreased from 56.4 mL/min/1.73 m2 (range, 17.5-89.7 mL/min/1.73 m2) to 46.9 mL/min/1.73 m2 (range, 16.5-89.7 mL/min/1.73 m2) at median of 95 days (P < .001), equating to a -7.9% median renal function change (range, -45.0% to +30.7%). All patients had Stage 2 or worse chronic kidney disease, and baseline function did not correlate with renal function change. The median volume of ablated parenchyma was 19.7 mL (range, 2.4-87.3 mL), equating to 8.1% (range, 0.7%-37.2%) of total parenchyma. The volume of parenchymal volume ablated correlated significantly with renal function loss, whereas age, hypertension, and diabetes mellitus did not. No patient developed ESRD within 1 year after cryoablation. CONCLUSIONS: Cryoablation in solitary functioning kidneys resulted in a modest reduction in renal function, even in patients with chronic kidney disease and ablations up to 20% of renal parenchymal volume.

A Description and Safety Overview of Irreversible Electroporation for Prostate Tissue Ablation in Intermediate-Risk Prostate Cancer Patients: Preliminary Results from the PRESERVE Trial.

The PRESERVE study (NCT04972097) aims to evaluate the safety and effectiveness of the NanoKnife System to ablate prostate tissue in patients with intermediate-risk prostate cancer (PCa). The NanoKnife uses irreversible electroporation (IRE) to deliver high-voltage electrical pulses to change the permeability of cell membranes, leading to cell death. A total of 121 subjects with organ-confined PCa ≤ T2c, prostate-specific antigens (PSAs) ≤ 15 ng/mL, and a Gleason score of 3 + 4 or 4 + 3 underwent focal ablation of the index lesion. The primary endpoints included negative in-field biopsy and adverse event incidence, type, and severity through 12 months. At the time of analysis, the trial had completed accrual with preliminary follow-up available. Demographics, disease characteristics, procedural details, PSA responses, and adverse events (AEs) are presented. The median (IQR) age at screening was 67.0 (61.0-72.0) years and Gleason distribution 3 + 4 (80.2%) and 4 + 3 (19.8%). At 6 months, all patients with available data (n = 74) experienced a median (IQR) percent reduction in PSA of 67.6% (52.3-82.2%). Only ten subjects (8.3%) experienced a Grade 3 adverse event; five were procedure-related. No Grade ≥ 4 AEs were reported. This study supports prior findings that IRE prostate ablation with the NanoKnife System can be performed safely. Final results are required to fully assess oncological, functional, and safety outcomes.

Proteolytic degradation of atrial sarcomere proteins underlies contractile defects in atrial fibrillation.

Atrial fibrillation (AFib) is the most common cardiac rhythm disturbance, often treated via electrical cardioversion. Following rhythm restoration, a period of depressed mechanical function known as atrial stunning occurs, suggesting that defects in contractility occur in AFib and are revealed upon restoration of rhythm. This project aims to define the contractile remodeling that occurs in AFib. To assess contractile function, we used a canine atrial tachypacing model of induced AFib. Mass spectrometry analysis showed dysregulation of contractile proteins in samples from AFib compared with sinus rhythm atria. Atrial cardiomyocytes show reduced force of contraction, decreased resting tension, and increased calcium sensitivity in skinned single cardiomyocyte studies. These alterations correlated with degradation of myofilament proteins including myosin heavy chain altering force of contraction, titin altering resting tension, and troponin I altering calcium sensitivity. We measured degradation of other myofilament proteins, including cardiac myosin binding protein C and actinin, that show degradation products in the AFib samples that are absent in the sinus rhythm atria. Many of the degradation products appeared as discrete cleavage products that are generated by calpain proteolysis. We assessed calpain activity and found it to be significantly increased. These results provide an understanding of the contractile remodeling that occurs in AFib and provide insight into the molecular explanation for atrial stunning and the increased risk of atrial thrombus and stroke in AFib.NEW & NOTEWORTHY Atrial fibrillation is the most common cardiac rhythm disorder, and remodeling during atrial fibrillation is highly variable between patients. This study has defined the biophysical changes in contractility that occur in atrial fibrillation along with identifying potential molecular mechanisms that may drive this remodeling. This includes proteolysis of several myofilament proteins including titin, troponin I, myosin heavy chain, myosin binding protein C, and actinin, which is consistent with the observed contractile deficits.

We've all been wrong about provisional tic disorder.

BACKGROUND: Provisional Tic Disorder (PTD) is common in childhood. The received wisdom among clinicians is that PTD is short-lived and mild, with at most a few tics, and rarely includes complex tics, premonitory phenomena or comorbid illnesses. However, such conclusions come from clinical experience, with biased ascertainment and limited follow-up. METHODS: Prospective study of 89 children with tics starting 0-9 months ago (median 4 months), fewer than half from clinical sources. Follow-up at 12 (± 24, 36, 48) months after the first tic. RESULTS: At study entry, many children had ADHD (39), an anxiety disorder (27), OCD (9) or enuresis (17). All had at least two current tics, with a mean total since onset of 6.9 motor and 2.0 phonic tics. Forty-one had experienced a complex tic, and 69 could suppress some tics. Tics were clinically meaningful: 64 had tics severe enough for a clinical trial, and 76 families sought medical attention for the tics. At 12 months, 79 returned, and 78 still had tics. Of these, 29 manifested no tics during history and extended examination, but only via audio-visual monitoring when the child was seated alone. Only 12/70 now had plans to see a doctor for tics. Most who returned at 2-4 years still had tics known to the child and family, but medical impact was low. CONCLUSIONS: Our results do not contradict previous data, but overturn clinical lore. The data strongly argue against the longstanding but arbitrary tradition of separating tic disorders into recent-onset versus chronic.

Current global status of male reproductive health.

BACKGROUND: The widespread interest in male reproductive health (MRH), fueled by emerging evidence, such as the global decline in sperm counts, has intensified concerns about the status of MRH. Consequently, there is a pressing requirement for a strategic, systematic approach to identifying critical questions, collecting pertinent information, and utilizing these data to develop evidence-based strategies. The methods for addressing these questions and the pathways toward their answers will inevitably vary based on the variations in cultural, geopolitical, and health-related contexts. To address these issues, a conjoint ESHRE and Male Reproductive Health Initiative (MRHI) Campus workshop was convened. OBJECTIVE AND RATIONALE: The three objectives were: first, to assess the current state of MRH around the world; second, to identify some of the key gaps in knowledge; and, third, to examine how MRH stakeholders can collaboratively generate intelligent and effective paths forward. SEARCH METHODS: Each expert reviewed and summarized the current literature that was subsequently used to provide a comprehensive overview of challenges related to MRH. OUTCOMES: This narrative report is an overview of the data, opinions, and arguments presented during the workshop. A number of outcomes are presented and can be summarized by the following overarching themes: MRH is a serious global issue and there is a plethora of gaps in our understanding; there is a need for widespread international collaborative networks to undertake multidisciplinary research into fundamental issues, such as lifestyle/environmental exposure studies, and high-quality clinical trials; and there is an urgent requirement for effective strategies to educate young people and the general public to safeguard and improve MRH across diverse population demographics and resources. LIMITATIONS REASONS FOR CAUTION: This was a workshop where worldwide leading experts from a wide range of disciplines presented and discussed the evidence regarding challenges related to MRH. While each expert summarized the current literature and placed it in context, the data in a number of areas are limited and/or sparse. Equally, important areas for consideration may have been missed. Moreover, there are clear gaps in our knowledge base, which makes some conclusions necessarily speculative and warranting of further study. WIDER IMPLICATIONS: Poor MRH is a global issue that suffers from low awareness among the public, patients, and heathcare professionals. Addressing this will require a coordinated multidisciplinary approach. Addressing the significant number of knowledge gaps will require policy makers prioritizing MRH and its funding. STUDY FUNDING/COMPETING INTERESTS: The authors would like to extend their gratitude to ESHRE for providing financial support for the Budapest Campus Workshop, as well as to Microptic S.L. (Barcelona) for kindly sponsoring the workshop. P.B. is the Director of the not-for-profit organization Global Action on Men's Health and receives fees and expenses for his work, (which includes the preparation of this manuscript). Conflicts of interest: C.J.D.J., C.L.R.B., R.A.A., P.B., M.P.C., M.L.E., N.G., N.J., C.K., AAP, M.K.O., S.R.-H., M.H.V.-L.: ESHRE Campus Workshop 2022 (Travel support-personal). C.J.D.J.: Cambridge University Press (book royalties-personal). ESHRE Annual Meeting 2022 and Yale University Panel Meeting 2023 (Travel support-personal). C.L.R.B.: Ferring and IBSA (Lecture), RBMO editor (Honorarium to support travel, etc.), ExSeed and ExScentia (University of Dundee), Bill & Melinda Gates Foundation (for research on contraception). M.P.C.: Previously received funding from pharmaceutical companies for health economic research. The funding was not in relation to this work and had no bearing on the contents of this work. No funding from other sources has been provided in relation to this work (funding was provided to his company Global Market Access Solutions). M.L.E.: Advisor to Ro, Doveras, Next, Hannah, Sandstone. C.K.: European Academy of Andrology (Past president UNPAID), S.K.: CEO of His Turn, a male fertility Diagnostic and Therapeutic company (No payments or profits to date). R.I.M.: www.healthymale.org.au (Australian Government funded not for profit in men's health sector (Employed as Medical Director 0.2 FET), Monash IVF Pty Ltd (Equity holder)). N.J.: Merck (consulting fees), Gedeon Richter (honoraria). S.R.-H.: ESHRE (Travel reimbursements). C.N.: LLC (Nursing educator); COMMIT (Core Outcomes Measures for Infertility Trials) Advisor, meeting attendee, and co-author; COMMA (Core Outcomes in Menopause) Meeting attendee, and co-author; International Federation of Gynecology and Obstetrics (FIGO) Delegate Letters and Sciences; ReproNovo, Advisory board; American Board of Urology Examiner; American Urological Association Journal subsection editor, committee member, guidelines co-author Ferring Scientific trial NexHand Chief Technology Officer, stock ownership Posterity Health Board member, stock ownership. A.P.: Economic and Social Research Council (A collaborator on research grant number ES/W001381/1). Member of an advisory committee for Merck Serono (November 2022), Member of an advisory board for Exceed Health, Speaker fees for educational events organized by Mealis Group; Chairman of the Cryos External Scientific Advisory Committee: All fees associated with this are paid to his former employer The University of Sheffield. Trustee of the Progress Educational Trust (Unpaid). M.K.O.: National Health and Medical Research Council and Australian Research Council (Funding for research of the topic of male fertility), Bill and Melinda Gates Foundation (Funding aimed at the development of male gamete-based contraception), Medical Research Future Fund (Funding aimed at defining the long-term consequences of male infertility). M.H.V.-L.: Department of Sexual and Reproductive Health and Research (SRH)/Human Reproduction Programme (HRP) Research Project Panel RP2/WHO Review Member; MRHI (Core Group Member), COMMIT (member), EGOI (Member); Human Reproduction (Associate Editor), Fertility and Sterility (Editor), AndroLATAM (Founder and Coordinator).