METAPHOR: Metabolic evaluation through phasor-based hyperspectral imaging and organelle recognition for mouse blastocysts and oocytes.

Only 30% of embryos from in vitro fertilized oocytes successfully implant and develop to term, leading to repeated transfer cycles. To reduce time-to-pregnancy and stress for patients, there is a need for a diagnostic tool to better select embryos and oocytes based on their physiology. The current standard employs brightfield imaging, which provides limited physiological information. Here, we introduce METAPHOR: Metabolic Evaluation through Phasor-based Hyperspectral Imaging and Organelle Recognition. This non-invasive, label-free imaging method combines two-photon illumination and AI to deliver the metabolic profile of embryos and oocytes based on intrinsic autofluorescence signals. We used it to classify i) mouse blastocysts cultured under standard conditions or with depletion of selected metabolites (glucose, pyruvate, lactate); and ii) oocytes from young and old mouse females, or in vitro-aged oocytes. The imaging process was safe for blastocysts and oocytes. The METAPHOR classification of control vs. metabolites-depleted embryos reached an area under the ROC curve (AUC) of 93.7%, compared to 51% achieved for human grading using brightfield imaging. The binary classification of young vs. old/in vitro-aged oocytes and their blastulation prediction using METAPHOR reached an AUC of 96.2% and 82.2%, respectively. Finally, organelle recognition and segmentation based on the flavin adenine dinucleotide signal revealed that quantification of mitochondria size and distribution can be used as a biomarker to classify oocytes and embryos. The performance and safety of the method highlight the accuracy of noninvasive metabolic imaging as a complementary approach to evaluate oocytes and embryos based on their physiology.

Preoperative vs Postoperative Opioid Prescriptions and Prolonged Opioid Refills Among US Youths.

Importance: High-risk practices, including dispensing an opioid prescription before surgery when not recommended, remain poorly characterized among US youths and may contribute to new persistent opioid use. Objective: To characterize changes in preoperative, postoperative, and refill opioid prescriptions up to 180 days after surgery. Design, Setting, and Participants: This retrospective cohort study was performed using national claims data to determine opioid prescribing practices among a cohort of opioid-naive youths aged 11 to 20 years undergoing 22 inpatient and outpatient surgical procedures between 2015 and 2020. Statistical analysis was performed from June 2023 to April 2024. Main Outcomes and Measures: The primary outcome was the percentage of initial opioid prescriptions filled up to 14 days prior to vs 7 days after a procedure. Secondary outcomes included the likelihood of a refill up to 180 days after surgery, including refills at 91 to 180 days, as a proxy for new persistent opioid use, and the opioid quantity dispensed in the initial and refill prescriptions in morphine milligram equivalents (MME). Exposures included patient and prescriber characteristics. Multivariable logistic regression models were used to estimate the association between prescription timing and prolonged refills. Results: Among 100 026 opioid-naive youths (median [IQR] age, 16.0 [14.0-18.0] years) undergoing a surgical procedure, 46 951 (46.9%) filled an initial prescription, of which 7587 (16.2%) were dispensed 1 to 14 days before surgery. The mean quantity dispensed was 227 (95% CI, 225-229) MME; 6467 youths (13.8%) filled a second prescription (mean MME, 239 [95% CI, 231-246]) up to 30 days after surgery, and 1216 (3.0%) refilled a prescription 91 to 180 days after surgery. Preoperative prescriptions, increasing age, and procedures not typically associated with severe pain were most strongly associated with new persistent opioid use. Conclusions and Relevance: In this retrospective study of youths undergoing surgical procedures, of which, many are typically not painful enough to require opioid use, opioid dispensing declined, but approximately 1 in 6 prescriptions were filled before surgery, and 1 in 33 adolescents filled prescriptions 91 to 180 days after surgery, consistent with new persistent opioid use. These findings should be addressed by policymakers and communicated by professional societies to clinicians who prescribe opioids.

Direct-Acting Oral Anticoagulants and Antiseizure Medications for Atrial Fibrillation and Epilepsy and Risk of Thromboembolic Events.

Importance: Direct-acting oral anticoagulants (DOACs) are commonly prescribed with antiseizure medications (ASMs) due to concurrency of and the association between atrial fibrillation (AF) and epilepsy. However, enzyme-inducing (EI) ASMs may reduce absorption and accelerate metabolism of DOACs, potentially lowering DOAC levels and elevating thromboembolism risk. Objective: To assess the rates of thromboembolic and major bleeding events in adults with AF and epilepsy dispensed DOACs and EI ASMs vs DOACs with non-EI ASMs. Design, Setting, and Participants: This active-comparator, new-user cohort study included US health care data from the Clinformatics Data Mart database from October 2010 to September 2021 for a nationally representative population of adults with AF and epilepsy. Exposure: Evaluations included episodes of contiguous coadministration of DOACs for AF with EI ASMs (exposed) or non-EI ASMs (referent) for epilepsy. Main Outcomes and Measures: Thromboembolic events (primary outcome) and major bleeding events (secondary outcome) were identified based on a series of validated, diagnosis-based coding algorithms. Data-adaptive, high-dimensional propensity score matching was used to control for observed confounders and proxies for unobserved confounders. Adjusted hazard ratios (AHRs) were estimated using Cox proportional hazards regression models with robust variance estimators to account for clustering within matched pairs. Results: This study included 14 078 episodes (median age, 74 [IQR, 67-81]; 52.4% female) and 14 158 episodes (median age, 74 [IQR, 67-81]; 52.4% female) of incident DOAC and ASM use that met eligibility criteria for assessment of thromboembolic and major bleeding outcomes, respectively. Incidence was 88.5 per 1000 person-years for thromboembolic events and 68.3 per 1000 person-years for bleeding events. Compared with use of non-EI ASMs, use of EI ASMs with DOACs was not associated with a difference in risk of thromboembolic events (AHR, 1.10; 95% CI, 0.82-1.46) but was associated with a reduction in risk of major bleeding events (AHR, 0.63; 95% CI, 0.44-0.89). Conclusions and Relevance: In this cohort study, EI ASMs were not associated with alteration in DOAC efficacy. Further research is needed on the reduction in bleeding risk associated with EI ASMs, as this may suggest that pharmacokinetic interactions are associated with lowering DOAC levels without negating therapeutic effects.

Incidence of Sexually Transmitted Infections in Youth with HIV During Pre-COVID and COVID Era.

Adolescents and young adults (AYAs) living with HIV have high rates of co-sexually transmitted infections (STIs). During the coronavirus disease (COVID) pandemic, STI prevention strategies, including access to testing/treatment facilities, availability of health care workers, and condom availability, may have decreased. This study aimed to determine if differences in STI incidence for first infection and reinfection existed between the pre-COVID and COVID eras in a cohort of AYAs living with HIV in Atlanta, GA. Retrospective chart review was conducted for all patients between ages 13 and 24 at the Grady Ponce Clinic. Two eras were identified: a pre-COVID era (January 1, 2009-December31, 2019) and a COVID era (January 1, 2020-June 30, 2021). STIs recorded included gonorrhea, chlamydia, human papillomavirus, syphilis, trichomonas, herpes simplex virus, lymphogranuloma venereum, hepatitis C, bacterial vaginosis, and chancroid. First and recurrent incidence rates for any STIs were reported. Our sample included 766 sexually active AYAs with HIV. A total of 721 patients were included in the pre-COVID era and 583 (80.9%) had at least one STI. A total of 337 patients were included in the COVID era, and 158 had at least one STI (46.9%). The overall first STI incidence rate increased from 42.47 to 58.67 per 100 person-years (PY) and the recurrent STI incidence rate increased from 121.50 to 169.85 per 100 PY from the pre-COVID to the COVID era (p < 0.001). Our study demonstrated significantly higher incidence rates of first and recurrent STIs in AYAs living with HIV in the COVID era. We urge continuation of existing STI prevention programs to avoid secondary clinical and economic adverse effects of increased infections.

Comparison of outcomes of abdominoperineal resection vs low anterior resection in very-low rectal cancer.

BACKGROUND: The management of very-low rectal cancer is one of the most challenging issues faced by general and colorectal surgeons. Many feel compelled to pursue abdominoperineal resection (APR) over low anterior resection (LAR) to optimize oncologic outcomes. This study aimed to determine differences in long-term oncologic outcomes between patients undergoing APR or LAR for very-low rectal cancer. METHODS: The United States Rectal Cancer Consortium (2010-2016) was queried for adults who underwent either APR or LAR for stage I-III rectal cancers < 5 cm from anorectal junction and met inclusion criteria. The primary outcome was disease-free survival. Secondary outcomes included overall survival, length of stay, complications, recurrence location, and perioperative factors. RESULTS: A total of 431 patients with very-low rectal cancer who underwent APR or LAR were identified; 154 (35.7%) underwent APR. The overall recurrence rate was 19.6%. The median follow-up was 42.5 months. An analysis adjusted for demographics and pathologic stage observed no difference in disease-free survival between operative types (APR-hazard ratio [HR] = 0.90, 95% CI: 0.53-1.52, P = .70). Secondary outcomes demonstrated no significant difference between operation types, including overall survival (HR = 1.29, 95% CI: 0.71-2.32, P = .39), complications (OR = 1.53, 95% CI: 0.94-2.50, P = .12), or length of stay (estimate: 0.04, SE = 0.25, P = .54). CONCLUSION: We observed no significant difference in disease-free survival or overall survival between patients undergoing APR or LAR for very-low rectal cancer. This analysis supports the treatment of very-low rectal cancer, without sphincter involvement, by either APR or LAR.

Deep mutational scanning of H5 hemagglutinin to inform influenza virus surveillance.

H5 influenza is considered a potential pandemic threat. Recently, H5 viruses belonging to clade 2.3.4.4b have caused large outbreaks in avian and multiple non-human mammalian species1,2. Previous studies have identified molecular phenotypes of the viral hemagglutinin (HA) protein that contribute to pandemic potential in humans, including cell entry, receptor preference, HA stability, and reduced neutralization by polyclonal sera3-6. However, prior experimental work has only measured how these phenotypes are affected by a handful of the >10,000 different possible amino-acid mutations to HA. Here we use pseudovirus deep mutational scanning7 to measure how all mutations to a 2.3.4.4b H5 HA affect each phenotype. We identify mutations that allow HA to better bind α2-6-linked sialic acids, and show that some viruses already carry mutations that stabilize HA. We also measure how all HA mutations affect neutralization by sera from mice and ferrets vaccinated against or infected with 2.3.4.4b H5 viruses. These antigenic maps enable rapid assessment of when new viral strains have acquired mutations that may create mismatches with candidate vaccine strains. Overall, the systematic nature of deep mutational scanning combined with the safety of pseudoviruses enables comprehensive measurements of the phenotypic effects of mutations that can inform real-time interpretation of viral variation observed during surveillance of H5 influenza.

Responses of wheat (Triticum aestivum L.) constitutively expressing four different monolignol biosynthetic genes to Fusarium head blight caused by Fusarium graminearum.

The Fusarium head blight (FHB) pathogen Fusarium graminearum produces the trichothecene mycotoxin deoxynivalenol (DON) and reduces wheat yield and grain quality. Spring wheat (Triticum aestivum L.) genotype CB037 was transformed with constitutive expression (CE) constructs containing sorghum (Sorghum bicolor L. (Moench)) genes encoding monolignol biosynthetic enzymes, caffeoyl-Coenzyme A (CoA) 3-O-methyltransferase (SbCCoAOMT), 4-coumarate-CoA ligase (Sb4CL), or coumaroyl shikimate 3-hydroxylase (SbC3'H), or monolignol pathway transcriptional activator, SbMyb60. Spring wheats were screened for Type I (resistance to initial infection, using spray inoculations) and Type II (resistance to spread within the spike, using single floret inoculations) resistances in the field (spray) and greenhouse (spray and single floret). Following field inoculations, disease index, percent Fusarium damaged kernels (FDK), and DON measurements of CE plants were similar to or greater than CB037. For greenhouse inoculations, the area under the disease progress curve (AUDPC) and FDK were determined. Following screens, focus was placed on two each, SbC3'H and SbCCoAOMT CE lines because of trends towards decreased AUDPC and FDK observed following single floret inoculations. These four lines were as susceptible as CB037 following spray inoculations. However, single floret inoculations showed that these CE lines had significantly reduced AUDPC (P<0.01) and FDK (P≤0.02) compared with CB037, indicating improved Type II resistance. None of these CE lines had increased acid detergent lignin, as compared with CB037, indicating that lignin concentration may not be a major factor in FHB resistance. The SbC3'H and SbCCoAOMT CE lines are valuable for investigating phenylpropanoid-based resistance to FHB.

Association of Surgical Approaches and Outcomes in Total Mesorectal Excision and Margin Status for Rectal Cancer.

INTRODUCTION: Despite being a key metric with a significant correlation with the outcomes of patients with rectal cancer, the optimal surgical approach for total mesorectal excision (TME) has not yet been identified. The aim of this study was to assess the association of the surgical approach on the quality of TME and surgical margins and to characterize the surgical and long-term oncologic outcomes in patients undergoing robotic, laparoscopic, and open TME for rectal cancer. METHODS: Patients with primary, nonmetastatic rectal adenocarcinoma who underwent either lower anterior resection or abdominoperineal resection via robotic (Rob), laparoscopic (Lap), or open approaches were selected from the US Rectal Cancer Consortium database (2007-2017). Quasi-Poisson regression analysis with backward selection was used to investigate the relationship between the surgical approach and outcomes of interest. RESULTS: Among the 664 patients included in the study, the distribution of surgical approaches was as follows: 351 (52.9%) underwent TME via the open approach, 159 (23.9%) via the robotic approach, and 154 (23.2%) via the laparoscopic approach. There were no significant differences in baseline demographics among the three cohorts. The laparoscopic cohort had fewer patients with low rectal cancer (<6 cm from the anal verge) than the robotic and open cohorts (Lap 28.6% versus Rob 59.1% versus Open 45.6%, P = 0.015). Patients who underwent Rob and Lap TME had lower intraoperative blood loss compared with the Open approach (Rob 200 mL [Q1, Q3: 100.0, 300.0] versus Lap 150 mL [Q1, Q3: 75.0, 250.0] versus Open 300 mL [Q1, Q3: 150.0, 600.0], P < 0.001). There was no difference in the operative time (Rob 243 min [Q1, Q3: 203.8, 300.2] versus Lap 241 min [Q1, Q3: 186, 336] versus Open 226 min [Q1, Q3: 178, 315.8], P = 0.309) between the three approaches. Postoperative length of stay was shorter with robotic and laparoscopic approach compared to open approach (Rob 5.0 d [Q1, Q3: 4, 8.2] versus Lap 5 d [Q1, Q3: 4, 8] versus Open 7.0 d [Q1, Q3: 5, 9], P < 0.001). There was no statistically significant difference in the quality of TME between the robotic, laparoscopic, and open approaches (79.2%, 64.9%, and 64.7%, respectively; P = 0.46). The margin positivity rate, a composite of circumferential margin and distal margin, was higher with the robotic and open approaches than with the laparoscopic approach (Rob 8.2% versus Open 6.6% versus Lap 1.9%, P = 0.17), Rob versus Lap (odds ratio 0.21; 95% confidence interval 0.05, 0.83) and Rob versus Open (odds ratio 0.5; 95% confidence interval 0.22, 1.12). There was no difference in long-term survival, including overall survival and recurrence-free survival, between patients who underwent robotic, laparoscopic, or open TME (Figure 1). CONCLUSIONS: In patients undergoing surgery with curative intent for rectal cancer, we did not observe a difference in the quality of TME between the robotic, laparoscopic, or open approaches. Robotic and open TME compared to laparoscopic TME were associated with higher margin positivity rates in our study. This was likely due to the higher percentage of low rectal cancers in the robotic and open cohorts. We also reported no significant differences in overall survival and recurrence-free survival between the aforementioned surgical techniques.

Within-host influenza viral diversity in the pediatric population as a function of age, vaccine, and health status.

Seasonal influenza virus predominantly evolves through antigenic drift, marked by the accumulation of mutations at antigenic sites. Because of antigenic drift, influenza vaccines are frequently updated, though their efficacy may still be limited due to strain mismatches. Despite the high levels of viral diversity observed across populations, most human studies reveal limited intrahost diversity, leaving the origin of population-level viral diversity unclear. Previous studies show host characteristics, such as immunity, might affect within-host viral evolution. Here we investigate influenza A viral diversity in children aged between 6 months and 18 years. Influenza virus evolution in children is less well characterized than in adults, yet may be associated with higher levels of viral diversity given the lower level of pre-existing immunity and longer durations of infection in children. We obtained influenza isolates from banked influenza A-positive nasopharyngeal swabs collected at the Children's Hospital of Philadelphia during the 2017-18 influenza season. Using next-generation sequencing, we evaluated the population of influenza viruses present in each sample. We characterized within-host viral diversity using the number and frequency of intrahost single-nucleotide variants (iSNVs) detected in each sample. We related viral diversity to clinical metadata, including subjects' age, vaccination status, and comorbid conditions, as well as sample metadata such as virus strain and cycle threshold. Consistent with previous studies, most samples contained low levels of diversity with no clear association between the subjects' age, vaccine status, or health status. Further, there was no enrichment of iSNVs near known antigenic sites. Taken together, these findings are consistent with previous observations that the majority of intrahost influenza virus infection is characterized by low viral diversity without evidence of diversifying selection.

RSV Vaccination in the adult pulmonary patient.

TOPIC IMPORTANCE: Since its discovery in 1957, respiratory syncytial virus (RSV) has been widely recognized as a common and deadly pathogen. While early studies focused on the impact of RSV on the health of children, more recent data show that RSV imposes a significant burden on individuals aged 70 years or greater. RSV also substantially harms the health of individuals with cardiopulmonary diseases. REVIEW FINDINGS: Early efforts to develop an RSV vaccine were hampered by toxicity due to antibody-enhanced viral pneumonia and a lack of efficacy in vaccines that targeted the post-fusion configuration of the F fusion protein, which is crucial to the pathogenesis of RSV-mediated injury. A newer wave of vaccines has targeted a stabilized prefusion F protein, generating effective neutralizing antibodies and reducing the burden of mild and severe RSV lower respiratory tract injury. This review focuses on the burden of RSV in patients with pulmonary diseases, highlights the tumultuous path from the early days of RSV vaccine development to the modern era, and offers insights into key gaps in knowledge that must be addressed in order to adequately protect the vulnerable population of patients with severe pulmonary diseases.

Structural and Interactional Analysis of the Flavonoid Pathway Proteins: Chalcone Synthase, Chalcone Isomerase and Chalcone Isomerase-like Protein.

Chalcone synthase (CHS) and chalcone isomerase (CHI) catalyze the first two committed steps of the flavonoid pathway that plays a pivotal role in the growth and reproduction of land plants, including UV protection, pigmentation, symbiotic nitrogen fixation, and pathogen resistance. Based on the obtained X-ray crystal structures of CHS, CHI, and chalcone isomerase-like protein (CHIL) from the same monocotyledon, Panicum virgatum, along with the results of the steady-state kinetics, spectroscopic/thermodynamic analyses, intermolecular interactions, and their effect on each catalytic step are proposed. In addition, PvCHI's unique activity for both naringenin chalcone and isoliquiritigenin was analyzed, and the observed hierarchical activity for those type-I and -II substrates was explained with the intrinsic characteristics of the enzyme and two substrates. The structure of PvCHS complexed with naringenin supports uncompetitive inhibition. PvCHS displays intrinsic catalytic promiscuity, evident from the formation of p-coumaroyltriacetic acid lactone (CTAL) in addition to naringenin chalcone. In the presence of PvCHIL, conversion of p-coumaroyl-CoA to naringenin through PvCHS and PvCHI displayed ~400-fold increased Vmax with reduced formation of CTAL by 70%. Supporting this model, molecular docking, ITC (Isothermal Titration Calorimetry), and FRET (Fluorescence Resonance Energy Transfer) indicated that both PvCHI and PvCHIL interact with PvCHS in a non-competitive manner, indicating the plausible allosteric effect of naringenin on CHS. Significantly, the presence of naringenin increased the affinity between PvCHS and PvCHIL, whereas naringenin chalcone decreased the affinity, indicating a plausible feedback mechanism to minimize spontaneous incorrect stereoisomers. These are the first findings from a three-body system from the same species, indicating the importance of the macromolecular assembly of CHS-CHI-CHIL in determining the amount and type of flavonoids produced in plant cells.

One Hundred Years of Colposcopy: Reconciling Its Auschwitz Past.

The centennial anniversary of Hans Hinselmann's initial publication describing colposcopy is approaching. In the 100 years since the inventor's seminal paper, colposcopy has become indispensable in the diagnosis and management of cervical cancer. It remains central in diagnosing precancerous and cancerous cervical lesions and has dramatically reduced cervical cancer incidence and mortality since the mid-20th century.Previous descriptions of colposcopy's development in medical literature obscure the dark history of its earliest days, arising within the center of German Nazism. The pioneers of colposcopy benefited from the Nazi government's public health focus and exploited the environment fostered by the Nazi medical establishment. They made use of the apparatus of the Auschwitz concentration camp to position colposcopy for expanded postwar adoption, ultimately accomplishing Hinselmann's stated goal that colposcopy become a routine part of gynecologic examination and care. This historical exposition clarifies the Nazi past of colposcopy, highlights the important role that unethical treatment of victims of Auschwitz played in cementing this procedure within standard cervical cancer screening programs globally, and offers steps to reckon with this tragic legacy.

Reducing firearm access for youth at risk for suicide in a pediatric emergency department.

Background: Firearm-related suicide is the second leading cause of pediatric firearm death. Lethal means counseling (LMC) can improve firearm safe-storage practices for families with youth at risk of suicide. Objectives: This study aims to evaluate the feasibility of pediatric emergency department (ED) behavioral mental health (BMH) specialists providing LMC to caregivers of youth presenting with BMH complaints and to test for changes in firearm safety practices, pre-post ED LMC intervention, as measures of preliminary efficacy. Methods: Prospective pilot feasibility study of caregivers of youth presenting to a pediatric ED with BMH complaints. Caregivers completed an electronic survey regarding demographics and firearm safe-storage knowledge/practices followed by BMH specialist LMC. Firearm owners were offered a free lockbox and/or trigger lock. One-week follow-up surveys gathered self-reported data on firearm safety practices and intervention acceptability. One-month interviews with randomly sampled firearm owners collected additional firearm safety data. Primary outcomes were feasibility measures, including participant accrual/attrition and LMC intervention acceptability. Secondary outcomes included self-reported firearm safety practice changes. Feasibility benchmarks were manually tabulated, and Likert-scale acceptability responses were dichotomized to strongly agree/agree vs. neutral/disagree/strongly disagree. Descriptive statistics were used for univariate and paired data responses. Results: In total, 81 caregivers were approached; of which, 50 (81%) caregivers enrolled. A total of 44% reported having a firearm at home, 80% completed follow-up at one week. More than 80% affirmed that ED firearm safety education was useful and that the ED is an appropriate place for firearm safety discussions. In total, 58% of participants reported not having prior firearm safety education/counseling. Among firearm owners (n = 22), 18% reported rarely/never previously using a safe-storage device, and 59% of firearm owners requested safe storage devices.At 1-week follow-up (n = 40), a greater proportion of caregivers self-reported asking about firearms before their child visited other homes (+28%). Among firearm owners that completed follow-up (n = 19), 100% reported storing all firearms locked at one week (+23% post-intervention). In total, 10 caregivers reported temporarily/permanently removing firearms from the home. Conclusion: It is feasible to provide LMC in the pediatric ED via BMH specialists to families of high-risk youth. Caregivers were receptive to LMC and reported finding this intervention useful, acceptable, and appropriate. Additionally, LMC and device distribution led to reported changes in safe storage practices.

Establishment of the uterine microbiome following artificial insemination in virgin heifers.

Introduction: The concept of a sterile uterus was challenged by recent studies that have described the microbiome of the virgin and pregnant uterus for species including humans and cattle. We designed two studies that tested whether the microbiome is introduced into the uterus when the virgin heifer is first inseminated and whether the origin of the microbiome is the vagina/cervix. Methods: The uterine microbiome was measured immediately before and after an artificial insemination (AI; Study 1; n = 7 AI and n = 6 control) and 14 d after insemination (Study 2; n = 12 AI and n = 12 control) in AI and non-AI (control) Holstein heifers. A third study (Study 3; n = 5 Holstein heifers) that included additional negative controls was subsequently conducted to support the presence of a unique microbiome within the uterus despite the low microbial biomass and regardless of insemination. Traditional bacteriological culture was performed in addition to 16S rRNA gene sequencing on the same samples to determine whether there were viable organisms in addition to those detected based on DNA sequencing (16S rRNA gene sequence). Results and discussion: Inseminating a heifer did not lead to a large change in the microbiome when assessed by traditional methods of bacterial culture or metataxonomic (16S rRNA gene) sequencing (results of Studies 1 and 2). Very few bacteria were cultured from the body or horn of the uterus regardless of whether an AI was or was not (negative control) performed. The cultured bacterial genera (e.g., Bacillus, Corynebacterium, Cutibacterium, Micrococcus, Staphylococcus, and Streptococcus) were typical of those found in the soil, environment, skin, mucous membranes, and urogenital tract of animals. Metataxonomic sequencing of 16S rRNA gene generated a large number of amplicon sequence variants (ASV), but these larger datasets that were based on DNA sequencing did not consistently demonstrate an effect of AI on the abundance of ASVs across all uterine locations compared with the external surface of the tract (e.g., perimetrium; positive control samples for environment contamination during slaughter and collection). Major genera identified by 16S rRNA gene sequencing overlapped with those identified with bacterial culture and included Cutibacterium, Staphylococcus, and Streptococcus.

Hospital strategies in commercial episode-based reimbursement.

OBJECTIVES: To understand hospitals' approaches to spending reduction in commercial episode-based payment programs and inform incentive design. STUDY DESIGN: Qualitative arm of an explanatory sequential mixed-methods study involving semistructured interviews with hospital leaders participating in a statewide quality improvement collaborative with novel episode-based incentive payments introduced by the state's largest commercial payer. METHODS: We recruited 21 leaders from 8 purposively selected, diverse hospitals with both high and low performance. Video teleconference-based interviews followed a standardized protocol and addressed 4 domains: choice of clinical condition for evaluation, strategies for episode spending reduction, best practices for success in earning incentives, and barriers to achievement. Rapid qualitative analysis with purposeful data reduction was employed to generate a matrix of key themes within the study domains. RESULTS: Strategies were similar between high- and low-performing hospitals. When selecting conditions, some hospitals focused on areas of underperformance, aiming for improvement opportunities, whereas others chose conditions already achieving highest efficiency. Many tried to synergize with other ongoing improvement initiatives and clinical areas with established leaders and champions. Key strategies included data-driven improvement, care standardization, and protocol dissemination. Best practices for success included readmission prevention and postacute care spending containment. CONCLUSIONS: The findings highlighted hospitals' most common strategies and approaches, providing several insights into optimal design of commercial episode-based incentives: They must be lucrative enough to earn attention or consistent with larger federal programs; hospitals need opportunities to succeed through both improved performance and sustained excellence; and programs may incur malalignment between hospitals and credentialed physicians.

Single-molecule imaging reveals the mechanism of bidirectional replication initiation in metazoa.

Metazoan genomes are copied bidirectionally from thousands of replication origins. Replication initiation entails the assembly and activation of two CMG helicases (Cdc45⋅Mcm2-7⋅GINS) at each origin. This requires several replication firing factors (including TopBP1, RecQL4, and DONSON) whose exact roles are still under debate. How two helicases are correctly assembled and activated at each origin is a long-standing question. By visualizing the recruitment of GINS, Cdc45, TopBP1, RecQL4, and DONSON in real time, we uncovered that replication initiation is surprisingly dynamic. First, TopBP1 transiently binds to the origin and dissociates before the start of DNA synthesis. Second, two Cdc45 are recruited together, even though Cdc45 alone cannot dimerize. Next, two copies of DONSON and two GINS simultaneously arrive at the origin, completing the assembly of two CMG helicases. Finally, RecQL4 is recruited to the CMG⋅DONSON⋅DONSON⋅CMG complex and promotes DONSON dissociation and CMG activation via its ATPase activity.

Multicenter appraisal of comorbid TANGO2 deficiency disorder in patients with 22q11.2 deletion syndrome.

TANGO2 deficiency disorder (TDD) is a rare, autosomal recessive condition caused by pathogenic variants in TANGO2, a gene residing within the region commonly deleted in 22q11.2 deletion syndrome (22q11.2DS). Although patients with 22q11.2DS are at substantially higher risk for comorbid TDD, it remains underdiagnosed within 22q11.2DS, likely due to overlapping symptomatology and a lack of knowledge about TDD. Initiation of B-vitamin supplementation may provide therapeutic benefit in TDD, highlighting the need for effective screening methods to improve diagnosis rates in this at-risk group. In this retrospective, multicenter study, we evaluated two cohorts of patients with 22q11.2DS (total N = 435) for possible comorbid TDD using two different symptom-based screening methods (free text-mining and manual chart review versus manual chart review alone). The methodology of the cohort 1 screening method successfully identified a known 22q11.2DS patient with TDD. Combined, these two cohorts identified 21 living patients meeting the consensus recommendation for TANGO2 testing for suspected comorbid TDD. Of the nine patients undergoing TANGO2 sequencing with del/dup analysis, none were ultimately diagnosed with TDD. Of the 12 deaths in the suspected comorbid TDD cohort, some of these patients exhibited symptoms (rhabdomyolysis, cardiac arrhythmia, or metabolic crisis) suspicious of comorbid TDD contributing to their death. Collectively, these findings highlight the need for robust prospective screening tools for diagnosing comorbid TDD in patients with 22q11.2DS.

Thinking Three-Dimensionally: A Self- and Externally-Controlled Approach to Screening for Drug-Drug-Drug Interactions Among High-Risk Populations.

The global rise in polypharmacy has increased both the necessity and complexity of drug-drug interaction (DDI) assessments, given the growing potential for interactions involving more than two drugs. Leveraging large-scale healthcare claims data, we piloted a semi-automated, high-throughput case-crossover-based approach for drug-drug-drug interaction (3DI) screening. Cases were direct-acting oral anticoagulant (DOAC) users with either a major bleeding event during ongoing dispensings for potentially interacting, enzyme-inhibiting antihypertensive drugs (AHDs) (Study 1), or a thromboembolic event during ongoing dispensings for potentially interacting, enzyme-inducing antiseizure medications (ASMs) (Study 2). 3DI detection was based on screening for additional drug exposures that served as acute outcome triggers. To mitigate direct effects and confounding by concomitant drugs, self-controlled estimates were adjusted using negative cases (external "control" DOAC users with the same outcomes but co-dispensings for non-interacting AHDs or ASMs). Signal thresholds were set based on P-values and false discovery rate q-values to address multiple comparisons. Study 1: 285 drugs were examined among 3,306 episodes. Self-controlled assessments with q-value thresholds yielded 9 3DI signals (cases) and 40 DDI signals (negative cases). External adjustment generated 10 3DI signals from the P-value threshold and no signals from the q-value threshold. Study 2: 126 drugs were examined among 604 episodes. Assessments with P-value thresholds yielded 3 3DI and 26 DDI signals following self-control, as well as 4 3DI signals following adjustment. No 3DI signals met the q-value threshold. The presented self- and externally-controlled approach aimed to advance paradigms for real-world higher order drug interaction screening among high-susceptibility populations with pre-existent DDI risk.