Latent Epstein-Barr virus infection collaborates with Myc over-expression in normal human B cells to induce Burkitt-like Lymphomas in mice.

Epstein-Barr virus (EBV) is an important cause of human lymphomas, including Burkitt lymphoma (BL). EBV+ BLs are driven by Myc translocation and have stringent forms of viral latency that do not express either of the two major EBV oncoproteins, EBNA2 (which mimics Notch signaling) and LMP1 (which activates NF-κB signaling). Suppression of Myc-induced apoptosis, often through mutation of the TP53 (p53) gene or inhibition of pro-apoptotic BCL2L11 (BIM) gene expression, is required for development of Myc-driven BLs. EBV+ BLs contain fewer cellular mutations in apoptotic pathways compared to EBV-negative BLs, suggesting that latent EBV infection inhibits Myc-induced apoptosis. Here we use an EBNA2-deleted EBV virus (ΔEBNA2 EBV) to create the first in vivo model for EBV+ BL-like lymphomas derived from primary human B cells. We show that cord blood B cells infected with both ΔEBNA2 EBV and a Myc-expressing vector proliferate indefinitely on a CD40L/IL21 expressing feeder layer in vitro and cause rapid onset EBV+ BL-like tumors in NSG mice. These LMP1/EBNA2-negative Myc-driven lymphomas have wild type p53 and very low BIM, and express numerous germinal center B cell proteins (including TCF3, BACH2, Myb, CD10, CCDN3, and GCSAM) in the absence of BCL6 expression. Myc-induced activation of Myb mediates expression of many of these BL-associated proteins. We demonstrate that Myc blocks LMP1 expression both by inhibiting expression of cellular factors (STAT3 and Src) that activate LMP1 transcription and by increasing expression of proteins (DNMT3B and UHRF1) known to enhance DNA methylation of the LMP1 promoters in human BLs. These results show that latent EBV infection collaborates with Myc over-expression to induce BL-like human B-cell lymphomas in mice. As NF-κB signaling retards the growth of EBV-negative BLs, Myc-mediated repression of LMP1 may be essential for latent EBV infection and Myc translocation to collaboratively induce human BLs.

Epstein-Barr virus LMP1 protein promotes proliferation and inhibits differentiation of epithelial cells via activation of YAP and TAZ.

Latent Epstein-Barr virus (EBV) infection promotes undifferentiated nasopharyngeal carcinomas (NPCs) in humans, but the mechanism(s) for this effect has been difficult to study because EBV cannot transform normal epithelial cells in vitro and the EBV genome is often lost when NPC cells are grown in culture. Here we show that the latent EBV protein, LMP1 (Latent membrane protein 1), induces cellular proliferation and inhibits spontaneous differentiation of telomerase-immortalized normal oral keratinocytes (NOKs) in growth factor-deficient conditions by increasing the activity of the Hippo pathway effectors, YAP (Yes-associated protein) and TAZ (Transcriptional coactivator with PDZ-binding motif). We demonstrate that LMP1 enhances YAP and TAZ activity in NOKs both by decreasing Hippo pathway-mediated serine phosphorylation of YAP and TAZ and increasing Src kinase-mediated Y357 phosphorylation of YAP. Furthermore, knockdown of YAP and TAZ is sufficient to reduce proliferation and promote differentiation in EBV-infected NOKs. We find that YAP and TAZ are also required for LMP1-induced epithelial-to-mesenchymal transition. Importantly, we demonstrate that ibrutinib (an FDA-approved BTK inhibitor that blocks YAP and TAZ activity through an off-target effect) restores spontaneous differentiation and inhibits proliferation of EBV-infected NOKs at clinically relevant doses. These results suggest that LMP1-induced YAP and TAZ activity contributes to the development of NPC.

A Randomized Trial of Two Remote Health Care Delivery Models on the Uptake of Genetic Testing and Impact on Patient-Reported Psychological Outcomes in Families With Pancreatic Cancer: The Genetic Education, Risk Assessment, and Testing (GENERATE) Study.

BACKGROUND & AIMS: Genetic testing uptake for cancer susceptibility in family members of patients with cancer is suboptimal. Among relatives of patients with pancreatic ductal adenocarcinoma (PDAC), The GENetic Education, Risk Assessment, and TEsting (GENERATE) study evaluated 2 online genetic education/testing delivery models and their impact on patient-reported psychological outcomes. METHODS: Eligible participants had ≥1 first-degree relative with PDAC, or ≥1 first-/second-degree relative with PDAC with a known pathogenic germline variant in 1 of 13 PDAC predisposition genes. Participants were randomized by family, between May 8, 2019, and June 1, 2021. Arm 1 participants underwent a remote interactive telemedicine session and online genetic education. Arm 2 participants were offered online genetic education only. All participants were offered germline testing. The primary outcome was genetic testing uptake, compared by permutation tests and mixed-effects logistic regression models. We hypothesized that Arm 1 participants would have a higher genetic testing uptake than Arm 2. Validated surveys were administered to assess patient-reported anxiety, depression, and cancer worry at baseline and 3 months postintervention. RESULTS: A total of 424 families were randomized, including 601 participants (n = 296 Arm 1; n = 305 Arm 2), 90% of whom completed genetic testing (Arm 1 [87%]; Arm 2 [93%], P = .014). Arm 1 participants were significantly less likely to complete genetic testing compared with Arm 2 participants (adjusted ratio [Arm1/Arm2] 0.90, 95% confidence interval 0.78-0.98). Among participants who completed patient-reported psychological outcomes questionnaires (Arm 1 [n = 194]; Arm 2 [n = 206]), the intervention did not affect mean anxiety, depression, or cancer worry scores. CONCLUSIONS: Remote genetic education and testing can be a successful and complementary option for delivering genetics care. (Clinicaltrials.gov, number NCT03762590).

Reduced IRF4 expression promotes lytic phenotype in Type 2 EBV-infected B cells.

Humans are infected with two types of EBV (Type 1 (T1) and Type 2 (T2)) that differ substantially in their EBNA2 and EBNA 3A/B/C latency proteins and have different phenotypes in B cells. T1 EBV transforms B cells more efficiently than T2 EBV in vitro, and T2 EBV-infected B cells are more lytic. We previously showed that both increased NFATc1/c2 activity, and an NFAT-binding motif within the BZLF1 immediate-early promoter variant (Zp-V3) contained in all T2 strains, contribute to lytic infection in T2 EBV-infected B cells. Here we compare cellular and viral gene expression in early-passage lymphoblastoid cell lines (LCLs) infected with either T1 or T2 EBV strains. Using bulk RNA-seq, we show that T2 LCLs are readily distinguishable from T1 LCLs, with approximately 600 differentially expressed cellular genes. Gene Set Enrichment Analysis (GSEA) suggests that T2 LCLs have increased B-cell receptor (BCR) signaling, NFAT activation, and enhanced expression of epithelial-mesenchymal-transition-associated genes. T2 LCLs also have decreased RNA and protein expression of a cellular gene required for survival of T1 LCLs, IRF4. In addition to its essential role in plasma cell differentiation, IRF4 decreases BCR signaling. Knock-down of IRF4 in a T1 LCL (infected with the Zp-V3-containing Akata strain) induced lytic reactivation whereas over-expression of IRF4 in Burkitt lymphoma cells inhibited both NFATc1 and NFATc2 expression and lytic EBV reactivation. Single-cell RNA-seq confirmed that T2 LCLs have many more lytic cells compared to T1 LCLs and showed that lytically infected cells have both increased NFATc1, and decreased IRF4, compared to latently infected cells. These studies reveal numerous differences in cellular gene expression in B cells infected with T1 versus T2 EBV and suggest that decreased IRF4 contributes to both the latent and lytic phenotypes in cells with T2 EBV.

Type 1 and Type 2 Epstein-Barr viruses induce proliferation, and inhibit differentiation, in infected telomerase-immortalized normal oral keratinocytes.

Differentiated epithelial cells are an important source of infectious EBV virions in human saliva, and latent Epstein-Barr virus (EBV) infection is strongly associated with the epithelial cell tumor, nasopharyngeal carcinoma (NPC). However, it has been difficult to model how EBV contributes to NPC, since EBV has not been shown to enhance proliferation of epithelial cells in monolayer culture in vitro and is not stably maintained in epithelial cells without antibiotic selection. In addition, although there are two major types of EBV (type 1 (T1) and type 2 (T2)), it is currently unknown whether T1 and T2 EBV behave differently in epithelial cells. Here we inserted a G418 resistance gene into the T2 EBV strain, AG876, allowing us to compare the phenotypes of T1 Akata virus versus T2 AG876 virus in a telomerase-immortalized normal oral keratinocyte cell line (NOKs) using a variety of different methods, including RNA-seq analysis, proliferation assays, immunoblot analyses, and air-liquid interface culture. We show that both T1 Akata virus infection and T2 AG876 virus infection of NOKs induce cellular proliferation, and inhibit spontaneous differentiation, in comparison to the uninfected cells when cells are grown without supplemental growth factors in monolayer culture. T1 EBV and T2 EBV also have a similar ability to induce epithelial-to-mesenchymal (EMT) transition and activate canonical and non-canonical NF-κB signaling in infected NOKs. In contrast to our recent results in EBV-infected lymphoblastoid cells (in which T2 EBV infection is much more lytic than T1 EBV infection), we find that NOKs infected with T1 and T2 EBV respond similarly to lytic inducing agents such as TPA treatment or differentiation. These results suggest that T1 and T2 EBV have similar phenotypes in infected epithelial cells, with both EBV types enhancing cellular proliferation and inhibiting differentiation when growth factors are limiting.

Sensitivity of North American grassland birds to weather and climate variability.

Grassland birds in North America have declined sharply over the last 60 years, driven by the widespread loss and degradation of grassland habitats. Climate change is occurring more rapidly in grasslands relative to some other ecosystems, and exposure to extreme and novel climate conditions may affect grassland bird ecology and demographics. To determine the potential effects of weather and climate variability on grassland birds, we conducted a systematic review of relationships between temperature and precipitation and demographic responses in grassland bird species of North America. Based on 124 independent studies, we used a vote-counting approach to quantify the frequency and direction of significant effects of weather and climate variability on grassland birds. Grassland birds tended to experience positive and negative effects of higher temperatures and altered precipitation. Moderate, sustained increases in mean temperature and precipitation benefitted some species, but extreme heat, drought, and heavy rainfall often reduced abundance and nest success. These patterns varied among climate regions, temporal scales of temperature and precipitation (<1 or ≥1 month), and taxa. The sensitivity of grassland bird populations to extreme weather and altered climate variability will likely be mediated by regional climates, interaction with other stressors, life-history strategies of various species, and species' tolerances for novel climate conditions.

Sensibilidad de las aves norteamericanas de pastizales ante la variabilidad climática y el clima Resumen Las aves de los pastizales norteamericanos han declinado gravemente durante los últimos 60 años, principalmente debido a la pérdida generalizada y la degradación del hábitat. El cambio climático ocurre cada vez más rápido en los pastizales en relación con otros ecosistemas, y la exposición a las condiciones climáticas nuevas y extremas puede afectar la demografía y la ecología aviar en los pastizales. Realizamos un análisis sistemático de las relaciones entre la temperatura y la precipitación y las respuestas demográficas de las especies de aves de pastizales en Norteamérica para determinar los efectos potenciales del clima y la variabilidad climática sobre estas aves. Usamos un método de conteo de votos basado en 124 estudios independientes para cuantificar la frecuencia y dirección de los efectos significativos del clima y la variabilidad climática sobre las aves de pastizal. Las aves de pastizal tendieron a experimentar los efectos positivos y negativos de las altas temperaturas y la precipitación alterada. El incremento moderado y sostenido en las medias de temperatura y precipitación beneficiaron a algunas especies, pero el calor extremo, la sequía y las lluvias torrenciales redujeron con frecuencia la abundancia y el éxito de anidación. Estos patrones variaron entre las regiones climáticas, las escalas temporales de temperatura y precipitación (< 1 mes o ≥ 1 mes) y los taxones. La sensibilidad de las poblaciones de aves de pastizal ante el clima extremo y la variabilidad climática alterada probablemente será mediada por los climas regionales, la interacción con otros estresantes, las estrategias de vida de varias especies y la tolerancia de las especies a las condiciones climáticas nuevas.

Epidural analgesia in labour: separating fact from fiction for autism and neurodevelopment.

Having epidural analgesia in labour has been associated with a later diagnosis of autism spectrum disorder in the offspring, resulting in concerns about childhood wellbeing. Neurodevelopmental changes are inconsistently reported in the literature, creating challenges in the interpretation of these findings. Here we explore the limitations of the current evidence base, and why findings differ between studies, concluding that the current body of evidence does not support a causal association between use of epidural analgesia in labour and autism spectrum disorder.

Association of Opioid Disposal Practices with Parental Education and a Home Opioid Disposal Kit Following Pediatric Ambulatory Surgery.

BACKGROUND: The majority of opioid analgesics prescribed for pain after ambulatory pediatric surgery remain unused. Most parents do not dispose of these leftover opioids or dispose of them in an unsafe manner. We aimed to evaluate the association of optimal opioid disposal with a multidisciplinary quality improvement (QI) initiative that proactively educated parents about the importance of optimal opioid disposal practices and provided a home opioid disposal kit before discharge after pediatric ambulatory surgery. METHODS: Opioid disposal behaviors were assessed during a brief telephone interview pre- (Phase I) and post-implementation (Phase II) after surgery. For each phase, we aimed to contact the parents of 300 pediatric patients ages 0 to 17 years who were prescribed an opioid after an ambulatory surgery. The QI initiative included enhanced education and a home opioid disposal kit including DisposeRX®, a medication disposal packet that renders medications inert within a polymeric gel when mixed with water. Weighted segmented regression models evaluated the association between the QI initiative and outcomes. We considered the association between the QI initiative and outcome significant if the beta coefficient for the change in intercept between the end of Phase I and the beginning of Phase II was significant. Safe opioid disposal and any opioid disposal were evaluated as secondary outcomes. RESULTS: The analyzed sample contained 161 pediatric patients in Phase I and 190 pediatric patients in Phase II. Phase II (post-QI initiative) cohort compared to Phase I cohort reported higher rates of optimal (58%, n = 111/190 vs 11%, n = 18/161) and safe (66%, n = 125/190 vs 34%, n = 55/161) opioid disposal. Weighted segmented regression analyses demonstrated significant increases in the odds of optimal (odds ratio [OR], 26.5, 95% confidence interval [CI], 4.0-177.0) and safe (OR, 4.4, 95% CI, 1.1-18.4) opioid disposal at the beginning of Phase II compared to the end of Phase I. The trends over time (slopes) within phases were nonsignificant and close to 0. The numbers needed to be exposed to achieve one new disposal event were 2.2 (95% CI, 1.4-3.7]), 3.1 (95% CI, 1.6-7.4), and 4.3 (95% CI, 1.7-13.6) for optimal, safe, and any disposal, respectively. CONCLUSIONS: A multidisciplinary approach to educating parents on the importance of safe disposal of leftover opioids paired with dispensing a convenient opioid disposal kit was associated with increased odds of optimal opioid disposal.

Long-term cardiometabolic health in people born after assisted reproductive technology: a multi-cohort analysis.

AIMS: To examine associations of assisted reproductive technology (ART) conception (vs. natural conception: NC) with offspring cardiometabolic health outcomes and whether these differ with age. METHODS AND RESULTS: Differences in systolic (SBP) and diastolic blood pressure (DBP), heart rate (HR), lipids, and hyperglycaemic/insulin resistance markers were examined using multiple linear regression models in 14 population-based birth cohorts in Europe, Australia, and Singapore, and results were combined using meta-analysis. Change in cardiometabolic outcomes from 2 to 26 years was examined using trajectory modelling of four cohorts with repeated measures. 35 938 (654 ART) offspring were included in the meta-analysis. Mean age ranged from 13 months to 27.4 years but was <10 years in 11/14 cohorts. Meta-analysis found no statistical difference (ART minus NC) in SBP (-0.53 mmHg; 95% CI:-1.59 to 0.53), DBP (-0.24 mmHg; -0.83 to 0.35), or HR (0.02 beat/min; -0.91 to 0.94). Total cholesterol (2.59%; 0.10-5.07), HDL cholesterol (4.16%; 2.52-5.81), LDL cholesterol (4.95%; 0.47-9.43) were statistically significantly higher in ART-conceived vs. NC offspring. No statistical difference was seen for triglycerides (TG), glucose, insulin, and glycated haemoglobin. Long-term follow-up of 17 244 (244 ART) births identified statistically significant associations between ART and lower predicted SBP/DBP in childhood, and subtle trajectories to higher SBP and TG in young adulthood; however, most differences were not statistically significant. CONCLUSION: These findings of small and statistically non-significant differences in offspring cardiometabolic outcomes should reassure people receiving ART. Longer-term follow-up is warranted to investigate changes over adulthood in the risks of hypertension, dyslipidaemia, and preclinical and clinical cardiovascular disease.

Clinical compatibility of magnetic resonance imaging with magnetic intramedullary nails: a feasibility study.

INTRODUCTION: The use of magnetic resonance imaging (MRI) with a magnetic intramedullary lengthening nail in place is contraindicated per the manufacturer due to the concern of implant activation and migration. A prior in vitro study did not confirm these complications only noting that a 3.0 T MRI weakened the internal magnet. Therefore, a retrospective analysis of patients who underwent an MRI with a magnetic nail in place was performed to determine if any adverse effects occurred in the clinical setting. MATERIALS AND METHODS: A retrospective review of all patients who underwent an MRI with a magnetic lengthening nail in place was performed. The time spent being imaged in the MRI, number of times the patient entered the MRI suite, and the images obtained were recorded. Radiographs were performed before and after the MRI to determine if any hardware complications occurred. The patients were monitored for any adverse symptoms while they were in the suite. RESULTS: A total of 12 patients with 13 nails were identified. Two patients underwent imaging with a 3.0 T MRI while the remaining 10 underwent imaging with a 1.5 T MRI. Each patient entered the MRI suite 2.1 times and spent an average of 84.7 min being imaged in the MRI (range 21-494). No patients noted any adverse symptoms related to the nail while in the suite and no hardware complications were identified. CONCLUSION: MRI appears to be safe with a magnetic nail in place and did not result in any complications. Given the manufacturer's recommendations, informed consent should be obtained prior to an MRI being performed and a 3.0 T MRI should be avoided when possible if further activation of the nail is required.

Plasma membrane proteoglycans syndecan-2 and syndecan-4 engage with EGFR and RON kinase to sustain carcinoma cell cycle progression.

Epidermal growth factor receptor (EGFR) is a causal factor in carcinoma, yet many carcinoma patients are resistant to EGFR inhibitors. Potential insight into this resistance stems from prior work that showed EGFR in normal epithelial cells docks to the extracellular domain of the plasma membrane proteoglycan syndecan-4 (Sdc4) engaged with α3ß1 and α6ß4 integrins. We now report that this receptor complex is modified by the recruitment of syndecan-2 (Sdc2), the Recepteur d'Origine Nantais (RON) tyrosine kinase, and the cellular signaling mediator Abelson murine leukemia viral oncogene homolog 1 (ABL1) in triple-negative breast carcinoma and head and neck squamous cell carcinoma, where it contributes to EGFR kinase-independent proliferation. Treatment with a peptide mimetic of the EGFR docking site in the extracellular domain of Sdc4 (called SSTNEGFR) disrupts the entire complex and causes a rapid, global arrest of the cell cycle. Normal epithelial cells do not recruit these additional receptors to the adhesion mechanism and are not arrested by SSTNEGFR. Although EGFR docking with Sdc4 in the tumor cells is required, cell cycle progression does not depend on EGFR kinase. Instead, progression depends on RON kinase, activated by its incorporation into the complex. RON activates ABL1, which suppresses p38 mitogen-activated protein kinase and prevents a p38-mediated signal that would otherwise arrest the cell cycle. These findings add to the growing list of receptor tyrosine kinases that support tumorigenesis when activated by their association with syndecans at sites of matrix adhesion and identify new potential targets for cancer therapy.

Hippo signaling effectors YAP and TAZ induce Epstein-Barr Virus (EBV) lytic reactivation through TEADs in epithelial cells.

The Epstein-Barr virus (EBV) human herpesvirus is associated with B-cell and epithelial-cell malignancies, and both the latent and lytic forms of viral infection contribute to the development of EBV-associated tumors. Here we show that the Hippo signaling effectors, YAP and TAZ, promote lytic EBV reactivation in epithelial cells. The transcriptional co-activators YAP/TAZ (which are inhibited by Hippo signaling) interact with DNA-binding proteins, particularly TEADs, to induce transcription. We demonstrate that depletion of either YAP or TAZ inhibits the ability of phorbol ester (TPA) treatment, cellular differentiation or the EBV BRLF1 immediate-early (IE) protein to induce lytic EBV reactivation in oral keratinocytes, and show that over-expression of constitutively active forms of YAP and TAZ reactivate lytic EBV infection in conjunction with TEAD family members. Mechanistically, we find that YAP and TAZ interact with, and activate, the EBV BZLF1 immediate-early promoter. Furthermore, we demonstrate that YAP, TAZ, and TEAD family members are expressed at much higher levels in epithelial cell lines in comparison to B-cell lines, and find that EBV infection of oral keratinocytes increases the level of activated (dephosphorylated) YAP and TAZ. Finally, we have discovered that lysophosphatidic acid (LPA), a known YAP/TAZ activator that plays an important role in inflammation, induces EBV lytic reactivation in epithelial cells through a YAP/TAZ dependent mechanism. Together these results establish that YAP/TAZ are powerful inducers of the lytic form of EBV infection and suggest that the ability of EBV to enter latency in B cells at least partially reflects the extremely low levels of YAP/TAZ and TEADs in this cell type.

Lifelong Imaging Surveillance is Indicated for Patients with Primary Retroperitoneal Liposarcoma.

BACKGROUND: Surveillance imaging of patients with retroperitoneal liposarcoma (RP-LPS) after surgical resection is based on a projected risk of locoregional and distant recurrence. The duration of surveillance is not well defined because the natural history of RP-LPS after treatment is poorly understood. This study evaluated the long-term risk of recurrence and disease-specific survival (DSS) for a cohort of patients with at least 10 years of progression-free survival (10yr-PFS) from their primary resection. METHODS: The prospective University of California, Los Angeles (UCLA) Sarcoma Database identified RP-LPS patients with 10yr-PFS after initial resection. The patients in the 10yr-PFS cohort were subsequently evaluated for recurrence and DSS. The time intervals start at date of initial surgical resection. Cox proportional hazards models were used to determine factors associated with recurrence and DSS. RESULTS: From 1972 to 2010, 76 patients with RP-LPS had at least 10 years of follow-up evaluation. Of these 76 patients, 39 (51%) demonstrated 10yr-PFS. The median follow-up period was 15 years (range 10-33 years). Among the 10yr-PFS patients, 49% (19/39) experienced a recurrence at least 10 years after surgery. Of those who experienced recurrence, 42% (8/19) died of disease. Neither long-term recurrence nor DSS were significantly associated with age, sex, tumor size, LPS subtype, surgical margin, or perioperative treatment with radiation or chemotherapy. CONCLUSION: Patients who have primary RP-LPS treated with surgical resection ± multimodality therapy face a long-term risk of recurrence and disease-specific death unacknowledged by current surveillance imaging guidelines. Among the patients with 10yr-PFS, 49% experienced a recurrence, and 42% of those died of disease. These findings suggest a need for lifelong surveillance imaging for patients with RP-LPS.

Interventions to prevent or reduce the incidence and severity of ovarian hyperstimulation syndrome: a systematic umbrella review of the best clinical evidence.

Ovarian hyperstimulation syndrome (OHSS) is a potentially life-threating iatrogenic complication of the early luteal phase and/or early pregnancy after in vitro fertilization (IVF) treatment. The aim of the current study was to identify the most effective methods for preventing of and reducing the incidence and severity of OHSS in IVF patients. A systematic review of systematic reviews of randomized controlled trials (RCTs) with meta-analysis was used to assess each potential intervention (PROSPERO website, CRD 268626) and only studies with the highest quality were included in the qualitative analysis. Primary outcomes included prevention and reduction of OHSS incidence and severity. Secondary outcomes were maternal death, incidence of hospital admission, days of hospitalization, and reproductive outcomes, such as incidence of live-births, clinical pregnancies, pregnancy rate, ongoing pregnancy, miscarriages, and oocytes retrieved. A total of specific interventions related to OHSS were analyzed in 28 systematic reviews of RCTs with meta-analyses. The quality assessment of the included studies was high, moderate, and low for 23, 2, and 3 studies, respectively. The certainty of evidence (CoE) for interventions was reported for 37 specific situations/populations and resulted high, moderate, and low-to-very low for one, 5, and 26 cases, respectively, while it was not reported in 5 cases. Considering the effective interventions without deleterious reproductive effects, GnRH-ant co-treatment (36 RCTs; OR 0.61, 95% C 0.51 to 0.72, n = 7,944; I2 = 31%) and GnRH agonist triggering (8 RCTs; OR 0.15, 95% CI 0.05 to 0.47, n = 989; I2 = 42%) emerged as the most effective interventions for preventing OHSS with a moderate CoE, even though elective embryo cryopreservation exhibited a low CoE. Furthermore, the use of mild ovarian stimulation (9 RCTs; RR 0.26, CI 0.14 to 0.49, n = 1,925; I2 = 0%), and dopaminergic agonists (10 RCTs; OR 0.32, 95% CI 0.23 to 0.44, n = 1,202; I2 = 13%) coadministration proved effective and safe with a moderate CoE. In conclusion, the current study demonstrates that only a few interventions currently can be considered effective to reduce the incidence of OHSS and its severity with high/moderate CoE despite the numerous published studies on the topic. Further well-designed RCTs are needed, particularly for GnRH-a down-regulated IVF cycles.

Prediction of ovarian response using the automated Elecsys anti-Müllerian hormone assay in gonadotrophin-releasing hormone antagonist cycles.

RESEARCH QUESTION: What is the capability of serum anti-Müllerian hormone (AMH) measured using the automated Elecsys® AMH immunoassay to (Roche Diagnostics International Ltd) determine ovarian response after fertility treatment? DESIGN: Single-centre, retrospective, observational, cohort study including women undergoing ovarian stimulation. Serum AMH concentrations were determined using the Elecsys AMH immunoassay based on one blood sample drawn 6 months or less before treatment. Stimulation was conducted in accordance with a gonadotrophin-releasing hormone (GnRH) antagonist protocol. Patients were divided into four ovarian response categories based on their oocyte yield: low (0-3), suboptimal (4-9), optimal (10-15) and high (>15). Areas under the curve were calculated for each ovarian response group. RESULTS: Overall, 1248 patients were enrolled. The AMH concentration had a strong positive correlation with oocyte yield (Spearman's rho = 0.74, P < 0.001). Areas under the curve (95% CI) for AMH predicting ovarian response were 0.85 (0.83 to 0.88) for low and 0.89 (0.87 to 0.91) for high response. Optimal serum AMH cut-offs for predicting a low and high response using the Elecsys AMH immunoassay were 6.4 pmol/l (0.89 ng/ml) and 14.2 pmol/l (1.99 ng/ml), respectively. Multivariable regression analysis showed that 47% (R2 = 0.470) of variation in ovarian response could be attributed to AMH alone, increasing to 50.9% (R2 = 0.509) with the addition of age, body weight, and total dose of gonadotrophin. CONCLUSION: Ovarian response and oocyte yield after stimulation in a GnRH antagonist cycle can be predicted with high accuracy using a single determination of serum AMH before ovarian stimulation.

General health in a cohort of children conceived after assisted reproductive technology in the United Kingdom: a population-based record-linkage study.

BACKGROUND: Assisted reproductive technology use is increasing annually; however, data on long-term child health outcomes including hospital admissions are limited. OBJECTIVE: This study aimed to examine the potential effects of assisted reproductive technology on any and cause-specific hospital admissions unrelated to perinatal diagnoses. STUDY DESIGN: This was a population-based record-linkage study that included a previously established cohort of children born after assisted reproductive technology in the United Kingdom between 1997 and 2009 (n=63,877), their naturally conceived siblings (n=11,343), and matched naturally conceived population controls (n=127,544) linked to their postnatal health outcomes up to March 31, 2016 to provide robust risk estimates of the potential effects of assisted reproductive technology on any and cause-specific hospital admissions unrelated to perinatal diagnoses. In addition, comparison of hospital admissions by type of treatment was made. Cox regression was used to estimate the risk of hospital admission, and negative binomial regression was used to compare the number of hospital admissions per year. RESULTS: This study had 1.6 million person-years of follow-up (mean, 12.9 years; range, 0-19 years), and the mean age at the time of first hospital admission was 6.5 years (range, 0-19 years). Singletons born after assisted reproductive technology had increased risk of any hospital admission compared with naturally conceived population controls (hazard ratio, 1.08; 95% confidence interval, 1.05-1.10) but not naturally conceived siblings (hazard ratio, 1.01; 95% confidence interval, 0.94-1.09). We observed increased risk of diagnoses related to neoplasms and diseases of the respiratory, musculoskeletal, digestive, and genitourinary systems, and lower risk of injury, poisoning, and consequences of external causes compared with naturally conceived population controls. Children born after intracytoplasmic sperm injection had a lower risk of hospital admission compared with those born after in vitro fertilization, although no such differences were observed between children born after fresh embryo transfers and those born after frozen embryo transfers. CONCLUSION: Children born after assisted reproductive technology had greater numbers of hospital admissions compared with naturally conceived population controls. Attenuation of these differences in relation to their naturally conceived siblings suggested that this could be partially attributed to the influence of parental subfertility on child health, increased parental concerns, and an actual increase in morbidity in children born after assisted conception.

Beyond the Umbrella: A Systematic Review of the Interventions for the Prevention of and Reduction in the Incidence and Severity of Ovarian Hyperstimulation Syndrome in Patients Who Undergo In Vitro Fertilization Treatments.

Ovarian hyperstimulation syndrome (OHSS) is the main severe complication of ovarian stimulation for in vitro fertilization (IVF) cycles. The aim of the current study was to identify the interventions for the prevention of and reduction in the incidence and severity of OHSS in patients who undergo IVF not included in systematic reviews with meta-analyses of randomized controlled trials (RCTs) and assess and grade their efficacy and evidence base. The best available evidence for each specific intervention was identified, analyzed in terms of safety/efficacy ratio and risk of bias, and graded using the Oxford Centre for Evidence-Based Medicine (CEBM) hierarchy of evidence. A total of 15 interventions to prevent OHSS were included in the final analysis. In the IVF population not at a high risk for OHSS, follitropin delta for ovarian stimulation may reduce the incidence of early OHSS and/or preventive interventions for early OHSS. In high-risk patients, inositol pretreatment, ovulation triggering with low doses of urinary hCG, and the luteal phase administration of a GnRH antagonist may reduce OHSS risk. In conclusion, even if not supported by systematic reviews with homogeneity of the RCTs, several treatments/strategies to reduce the incidence and severity of OHSS have been shown to be promising.

Evaluation of two methods for detection of viable Bacillus anthracis simulant spores in maritime environmental samples.

Analytical methods exist to detect biothreat agents in environmental samples during a response to biological contamination incidents. However, the coastal zone facilities and assets of the US Coast Guard (USCG), including response boats in diverse geographical areas and maritime environmental conditions, can pose complex and unique challenges for adapting existing analytical detection methods. The traditional culture (TC) and the rapid viability polymerase chain reaction (RV-PCR) methods were evaluated for their compatibility for maritime environmental surface and grab sample analysis to detect spores of Bacillus thuringiensis subspecies kurstaki (Btk), a surrogate for Bacillus anthracis. The representative samples collected from a USCG installation included surfaces, such as aluminum on boats, nonskid tread on decks of watercraft, computer touchscreens, and concrete piers, and grab samples of boat washdown water, soil, vegetation, and gravel from surrounding areas. Replicate samples were spiked with Btk spores at two to three tenfold increasing levels and analyzed. Out of a total of 150 samples collected and analyzed, the TC method gave 10 false-positive and 19 false-negative results, while the RV-PCR method-based analysis resulted in 0 false-positive and 26 false-negative results. An abundance of microbial background and particulates in some samples interfered with true results, while both methods gave similar results for samples with low microbial background and particulates. Improved and high-throughput sample processing methods are needed for analysis of complex environmental samples.

Trypanosoma brucei Mitochondrial DNA Polymerase POLIB Contains a Novel Polymerase Domain Insertion That Confers Dominant Exonuclease Activity.

Trypanosoma brucei and related parasites contain an unusual catenated mitochondrial genome known as kinetoplast DNA (kDNA) composed of maxicircles and minicircles. The kDNA structure and replication mechanism are divergent and essential for parasite survival. POLIB is one of three Family A DNA polymerases independently essential to maintain the kDNA network. However, the division of labor among the paralogs, particularly which might be a replicative, proofreading enzyme, remains enigmatic. De novo modeling of POLIB suggested a structure that is divergent from all other Family A polymerases, in which the thumb subdomain contains a 369 amino acid insertion with homology to DEDDh DnaQ family 3'-5' exonucleases. Here we demonstrate recombinant POLIB 3'-5' exonuclease prefers DNA vs RNA substrates and degrades single- and double-stranded DNA nonprocessively. Exonuclease activity prevails over polymerase activity on DNA substrates at pH 8.0, while DNA primer extension is favored at pH 6.0. Mutations that ablate POLIB polymerase activity slow the exonuclease rate suggesting crosstalk between the domains. We show that POLIB extends an RNA primer more efficiently than a DNA primer in the presence of dNTPs but does not incorporate rNTPs efficiently using either primer. Immunoprecipitation of Pol I-like paralogs from T. brucei corroborates the pH selectivity and RNA primer preferences of POLIB and revealed that the other paralogs efficiently extend a DNA primer. The enzymatic properties of POLIB suggest this paralog is not a replicative kDNA polymerase, and the noncanonical polymerase domain provides another example of exquisite diversity among DNA polymerases for specialized function.

Ribonucleotide Misincorporation and Reverse Transcriptase Activities of Plasmodium falciparum Apicoplast DNA Polymerase.

Plasmodium falciparumis the most common and harmful causative agent of malaria worldwide. As a member of the phylum Apicomplexa, P. falciparum is characterized by the presence of a unique and essential organelle called the apicoplast. Reminiscent of an algal chloroplast, the apicoplast possesses its own genome, which is maintained by a single apicoplast DNA polymerase (apPol). Ribonucleotides misincorporated into the genome are among the most common lesions encountered by DNA polymerases, and the ability to replicate past these lesions varies widely among characterized enzymes. Here, we have investigated the ribonucleotide (rNTP) misincorporation frequency of apPol and determined its reverse transcriptase (RT) activity across templating ribonucleotides. Pre-steady-state kinetic experiments indicate that apPol does not have an unusually high discrimination between deoxy and ribonucleotides, with frequencies ranging between 104 and 106 depending on the identity of the ribonucleotide. Once incorporated into its template, apPol can replicate across ribonucleotides using its RT activity, but extension of a deoxynucleotide basepaired with the ribonucleotide is slow relative to a canonical basepair. Exonuclease assays indicate that apPol proofreads ribonucleotides an order of magnitude faster than extension, suggesting that most, but not all, misincorporated ribonucleotides will be excised. Although the components have not been identified, ribonucleotide excision repair or other tolerance mechanisms may exist in the P. falciparum apicoplast, and more targeted proteomic efforts will be needed to elucidate them.