Functional Segregation of Overlapping Genes in HIV.

Overlapping genes pose an evolutionary dilemma as one DNA sequence evolves under the selection pressures of multiple proteins. Here, we perform systematic statistical and mutational analyses of the overlapping HIV-1 genes tat and rev and engineer exhaustive libraries of non-overlapped viruses to perform deep mutational scanning of each gene independently. We find a "segregated" organization in which overlapped sites encode functional residues of one gene or the other, but never both. Furthermore, this organization eliminates unfit genotypes, providing a fitness advantage to the population. Our comprehensive analysis reveals the extraordinary manner in which HIV minimizes the constraint of overlapping genes and repurposes that constraint to its own advantage. Thus, overlaps are not just consequences of evolutionary constraints, but rather can provide population fitness advantages.

A Quantitative Genetic Interaction Map of HIV Infection.

We have developed a platform for quantitative genetic interaction mapping using viral infectivity as a functional readout and constructed a viral host-dependency epistasis map (vE-MAP) of 356 human genes linked to HIV function, comprising >63,000 pairwise genetic perturbations. The vE-MAP provides an expansive view of the genetic dependencies underlying HIV infection and can be used to identify drug targets and study viral mutations. We found that the RNA deadenylase complex, CNOT, is a central player in the vE-MAP and show that knockout of CNOT1, 10, and 11 suppressed HIV infection in primary T cells by upregulating innate immunity pathways. This phenotype was rescued by deletion of IRF7, a transcription factor regulating interferon-stimulated genes, revealing a previously unrecognized host signaling pathway involved in HIV infection. The vE-MAP represents a generic platform that can be used to study the global effects of how different pathogens hijack and rewire the host during infection.

Considerations for using potential surrogate endpoints in cancer screening trials.

The requirement of large-scale expensive cancer screening trials spanning decades creates considerable barriers to the development, commercialisation, and implementation of novel screening tests. One way to address these problems is to use surrogate endpoints for the ultimate endpoint of interest, cancer mortality, at an earlier timepoint. This Review aims to highlight the issues underlying the choice and use of surrogate endpoints for cancer screening trials, to propose criteria for when and how we might use such endpoints, and to suggest possible candidates. We present the current landscape and challenges, and discuss lessons and shortcomings from the therapeutic trial setting. It is hugely challenging to validate a surrogate endpoint, even with carefully designed clinical studies. Nevertheless, we consider whether there are candidates that might satisfy the requirements defined by research and regulatory bodies.

Amniocentesis in pregnancies at or beyond 24 weeks: An international multicenter study.

BACKGROUND: Amniocentesis for genetic diagnosis is most commonly done between 15 and 22 weeks of gestation, but can be performed at later gestational ages. The safety and genetic diagnostic accuracy of amniocentesis have been well-established through numerous large-scale, multicenter studies for procedures before 24 weeks, but comprehensive data on late amniocentesis remain sparse. OBJECTIVES: To evaluate the indications, diagnostic yield, safety, and maternal and fetal outcomes associated with amniocentesis performed at or beyond 24 weeks of gestation. STUDY DESIGN: We conducted an international, multicenter retrospective cohort study examining pregnant individuals who underwent amniocentesis for prenatal diagnostic testing at gestational ages between 24w0d and 36w6d. The study, spanning from 2011 to 2022, involved nine referral centers. We included singleton or twin pregnancies with documented outcomes, excluding cases where other invasive procedures were performed during pregnancy or if amniocentesis was conducted for obstetric indications. We analyzed indications for late amniocentesis, types of genetic tests performed, their results, and the diagnostic yield, along with pregnancy outcomes and post-procedure complications. RESULTS: Of the 752 pregnant individuals included in our study, late amniocentesis was primarily performed for the prenatal diagnosis of structural anomalies (91.6%), followed by suspected fetal infection (2.3%) and high-risk findings from cell-free DNA screening (1.9%). The median gestational age at the time of the procedure was 28w5d, and 98.3% of pregnant individuals received results of genetic testing before birth or pregnancy termination. The diagnostic yield was 22.9%, and a diagnosis was made 2.4 times more often for fetuses with anomalies in multiple organ systems (36.4%) compared to those with anomalies in a single organ system (15.3%). Additionally, the diagnostic yield varied depending on the specific organ system involved, with the highest yield for musculoskeletal anomalies (36.7%) and hydrops fetalis (36.4%) when a single organ system or entity was affected. The most prevalent genetic diagnoses were aneuploidies (46.8%), followed by copy number variants (26.3%) and monogenic disorders (22.2%). The median gestational age at delivery was 38w3d, with an average of 59 days between the procedure and delivery date. The overall complication rate within two weeks post-procedure was 1.2%. We found no significant difference in the rate of preterm delivery between pregnant individuals undergoing amniocentesis between 24-28 weeks and those between 28-32 weeks, reinforcing the procedure's safety across these gestational periods. CONCLUSIONS: Late amniocentesis, at or after 24 weeks gestation, especially for pregnancies complicated by multiple congenital anomalies, has a high diagnostic yield and a low complication rate, underscoring its clinical utility. It provides pregnant individuals and their providers with a comprehensive diagnostic evaluation and results before delivery, enabling informed counseling and optimized perinatal and neonatal care planning.

Multicancer early detection test: Preclinical, translational, and clinical evidence-generation plan and provocative questions.

Minimally invasive molecular biomarkers have been applied to the early detection of multiple cancers in large scale case-control and cohort studies. These demonstrations of feasibility herald the potential for permanent transformation of current cancer screening paradigms. This commentary discusses the major opportunities and challenges facing the preclinical development and clinical validation of multicancer early detection test strategies. From a diverse set of early detection research perspectives, the authors recommend specific approaches and highlight important questions for future investigation.

Delivering on the promise of early detection with liquid biopsies.

Liquid biopsy approaches are relatively well developed for cancer therapy monitoring and disease relapse, but they also have incredible potential in the cancer early detection and screening field. There are, however, several challenges to overcome before this potential can be met. Research in this area needs to be cohesive and, as a driver of research, Cancer Research UK is in an ideal position to enable this.

CBFß stabilizes HIV Vif to counteract APOBEC3 at the expense of RUNX1 target gene expression.

The HIV-1 accessory protein Vif hijacks a cellular Cullin-RING ubiquitin ligase, CRL5, to promote degradation of the APOBEC3 (A3) family of restriction factors. Recently, the cellular transcription cofactor CBFß was shown to form a complex with CRL5-Vif and to be essential for A3 degradation and viral infectivity. We now demonstrate that CBFß is required for assembling a well-ordered CRL5-Vif complex by inhibiting Vif oligomerization and by activating CRL5-Vif via direct interaction. The CRL5-Vif-CBFß holoenzyme forms a well-defined heterohexamer, indicating that Vif simultaneously hijacks CRL5 and CBFß. Heterodimers of CBFß and RUNX transcription factors contribute toward the regulation of genes, including those with immune system functions. We show that binding of Vif to CBFß is mutually exclusive with RUNX heterodimerization and impacts the expression of genes whose regulatory domains are associated with RUNX1. Our results provide a mechanism by which a pathogen with limited coding capacity uses one factor to hijack multiple host pathways.

Identification and Optimization of Thienopyridine Carboxamides as Inhibitors of HIV Regulatory Complexes.

Viral regulatory complexes perform critical functions during virus replication and are important targets for therapeutic intervention. In HIV, the Tat and Rev proteins form complexes with multiple viral and cellular factors to direct transcription and export of the viral RNA. These complexes are composed of many proteins and are dynamic, making them difficult to fully recapitulate in vitro Therefore, we developed a cell-based reporter assay to monitor the assembly of viral complexes for inhibitor screening. We screened a small-molecule library and identified multiple hits that inhibit the activity of the viral complexes. A subsequent chemistry effort was focused on a thieno[2,3-b]pyridine scaffold, examples of which inhibited HIV replication and the emergence from viral latency. Notable aspects of the effort to determine the structure-activity relationship (SAR) include migration to the regioisomeric thieno[2,3-c]pyridine ring system and the identification of analogs with single-digit nanomolar activity in both reporter and HIV infectivity assays, an improvement of >100-fold in potency over the original hits. These results validate the screening strategy employed and reveal a promising lead series for the development of a new class of HIV therapeutics.

Is routine transvaginal cervical length measurement cost-effective in a population where the risk of spontaneous preterm birth is low?

INTRODUCTION: A recent meta-analysis has suggested that routine measurement of the cervical length should be performed in conjunction with the anomaly scan to identify a group of women at increased risk of preterm delivery. We decided to investigate whether this recommendation is justifiable in a population where the risk of preterm birth is low. MATERIAL AND METHODS: We reviewed 12 years of obstetric data from the Coombe Women and Infants University Hospital. Relative risks of adverse outcomes from the randomized controlled trial were applied and we extrapolated the possible numbers of women requiring intervention. We then used published neonatal data to estimate the cost of neonatal care and estimated the costs of providing the service. RESULTS: Over 12 years from 2000 until 2011, there were 94 646 singleton deliveries, 1776 happening before 34 weeks. Spontaneous onset occurred in 882 (49.7%) of this group. These 882 births were studied. If we apply the figures from a randomized controlled trial, 1609 women (1.7% from our total population) would be expected to have a cervical length 15 mm. If we gave vaginal progesterone to all women with a sonographically short cervix, we would reduce the delivery rate before 34 weeks by 27.7%. The annual costs of providing the service were estimated to be €109 249 and the cost of immediate neonatal care was estimated to be €380 514. CONCLUSION: Given the implications associated with preterm delivery, routine measurement of cervical length at the time of the anomaly scan may be justifiable from a cost point of view in a population where the risk of preterm birth is low.

Interpregnancy changes in maternal weight and body mass index.

OBJECTIVE: This longitudinal study compared changes in maternal weight and body mass index (BMI) in early pregnancy in the time interval between when a woman first attended for antenatal care with her first child and when she next attended for antenatal care. STUDY DESIGN: We studied women with a singleton pregnancy who delivered their first baby weighing ≥ 500 g in 2009 and who attended again for antenatal care with an ongoing pregnancy before January 1, 2012. Maternal weight and height were measured before 18 weeks' gestation in both pregnancies and BMI was calculated. RESULTS: Of the 3,284 primigravidas, the mean weight at the first visit in 2009 was 66.4 kg (standard deviation [SD] 12.7). The mean BMI was 24.5 kg/m(2) (SD 4.6), and 11.3% (n = 370) were obese. Of the 3,284 women, 1,220 (37.1%) re-attended for antenatal care before 2012 after sonographic confirmation of an ongoing pregnancy. Of the 1,220 women who re-attended, 788 (64.6%) had gained weight (mean 4.6 kg [SD 3.9]), 402 (33%) had lost weight (mean 3 kg [SD 2.9]), and 30 (2.4%) had maintained their weight. CONCLUSION: The birth of a first baby was associated with an increase in maternal weight in two-thirds of women when they next attended for antenatal care.

Inhibition of a NEDD8 Cascade Restores Restriction of HIV by APOBEC3G.

Cellular restriction factors help to defend humans against human immunodeficiency virus (HIV). HIV accessory proteins hijack at least three different Cullin-RING ubiquitin ligases, which must be activated by the small ubiquitin-like protein NEDD8, in order to counteract host cellular restriction factors. We found that conjugation of NEDD8 to Cullin-5 by the NEDD8-conjugating enzyme UBE2F is required for HIV Vif-mediated degradation of the host restriction factor APOBEC3G (A3G). Pharmacological inhibition of the NEDD8 E1 by MLN4924 or knockdown of either UBE2F or its RING-protein binding partner RBX2 bypasses the effect of Vif, restoring the restriction of HIV by A3G. NMR mapping and mutational analyses define specificity determinants of the UBE2F NEDD8 cascade. These studies demonstrate that disrupting host NEDD8 cascades presents a novel antiretroviral therapeutic approach enhancing the ability of the immune system to combat HIV.

Updates in preimplantation genetic testing (PGT).

Preimplantation genetic testing (PGT) involves taking a biopsy of an early embryo created through in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI). Genetic testing is performed on the biopsy, in order to select which embryo to transfer. PGT began as an experimental procedure in the 1990s, but is now an integral part of assisted human reproduction (AHR). PGT allows for embryo selection which can reduce the risk of transmission of inherited disease and may reduce the chance of implantation failure and pregnancy loss. This is a rapidly evolving area, which raises important ethical issues. This review article aims to give a brief history of PGT, an overview of the current evidence in PGT along with highlighting exciting areas of research to advance this technology.

Uptake rates and attitudes to influenza and COVID-19 vaccination in pregnancy - a prospective cohort study.

Influenza and COVID-19 are highly prevalent RNA viruses. Pregnancy increases the frequency of severe maternal morbidity and mortality associated with these viruses. Vaccination plays an important role in protecting pregnant women and their infants from adverse outcomes. In this prospective study, we aimed to determine the vaccination uptake rate for influenza and COVID-19 in a pregnant population and to explore reasons why women remained unvaccinated. A prospective cohort study was conducted over a two-week period in December 2022 in the National Maternity Hospital, Dublin. There were 588 women surveyed over the 2-week period. Overall, 377 (57%) were vaccinated that year for seasonal influenza, a significant rise from 39% in a similar study in 2016. The majority (n = 488, 83%) of women reported receiving at least one COVID-19 vaccine. However only 132 (22%) received a COVID-19 vaccine in pregnancy, despite 76% (n = 466) stating they would be happy to receive it. Factors such as age, obesity, co-morbidities, ethnic group, and type of antenatal care received were shown to influence vaccination rates. We recommend that the importance of vaccination be stressed regularly to eligible patients at their antenatal clinic visits and where possible combining influenza/COVID-19 vaccination on the same day to improve uptake.

A roadmap to precision treatments for familial pulmonary fibrosis.

Interstitial lung diseases (ILDs) in adults and children (chILD) are a heterogeneous group of lung disorders leading to inflammation, abnormal tissue repair and scarring of the lung parenchyma often resulting in respiratory failure and death. Inherited factors directly cause, or contribute significantly to the risk of developing ILD, so called familial pulmonary fibrosis (FPF), and monogenic forms may have a poor prognosis and respond poorly to current treatments. Specific, variant-targeted or precision treatments are lacking. Clinical trials of repurposed drugs, anti-fibrotic medications and specific treatments are emerging but for many patients no interventions exist. We convened an expert working group to develop an overarching framework to address the existing research gaps in basic, translational, and clinical research and identified areas for future development of preclinical models, candidate medications and innovative clinical trials. In this Position Paper, we summarise working group discussions, recommendations, and unresolved questions concerning precision treatments for FPF.

Pregnancy outcomes following recurrent miscarriage.

BACKGROUND: Recurrent miscarriage affects 1-2% of the population, and the literature has focussed on causes, treatment, and live birth rate. AIM: This study aimed to assess the reproductive outcomes for patients who attended a specialist recurrent miscarriage clinic for investigation and treatment. METHODS: Prospective analysis of all patients who attended a recurrent miscarriage clinic from January 2014 to January 2021. RESULTS: Of the 488 patients who attended a specialist clinic, 318 had a further pregnancy with 299 included in this study. The median age was 37 years, with 55.6% having a previous live birth. The subsequent live birth rate was 75.3%, 22.0% had a further pregnancy loss, 1.7% had an ongoing pregnancy, and 1% attended another institution after the second trimester. The rate of preeclampsia was 2.2%, pregnancy-induced hypertension was 2.2%, fetal growth restriction was 5.3%, preterm birth ≤ 34 weeks was 1.8%, and preterm birth > 34 weeks < 37 weeks was 6.6%. CONCLUSION: Patients who attend a dedicated recurrent miscarriage clinic for investigation and treatment have a high live birth rate in a subsequent pregnancy. A subsequent pregnancy following recurrent pregnancy loss does not appear to be associated with an increased risk of adverse pregnancy outcomes.

Second-trimester miscarriage: a review of postnatal investigations and subsequent pregnancy outcomes.

BACKGROUND: Second-trimester loss is pregnancy loss after the 12th and before the 24th completed weeks of pregnancy. This study aims to review cases of second-trimester miscarriage who attended a large maternity hospital and to examine pregnancy outcomes in this group of women. METHODS: This study is a review of cases of second-trimester miscarriage using descriptive, exploratory design, involving a retrospective chart review. RESULTS: In this study, 106 cases of second-trimester miscarriage were reviewed. The cause of the miscarriage was found in 42.5% (n = 45) of cases. The majority of women, 84.5% (n = 82) had a normal pelvic ultrasound scan and 18.3% (n = 17) of cases were diagnosed with antiphospholipid syndrome. In women who became pregnant again, 60.9% (n = 39) had a live birth. CONCLUSIONS: Establishing the cause of second-trimester miscarriage can be challenging, despite completing all recommended investigations. Outcomes in subsequent pregnancies are reassuring. This review highlights the need to undertake all recommended investigations to elicit the cause of second-trimester miscarriage and underpins the need for further research in this area.

Developing future clinical leaders in patient safety: the Irish experience.

INTRODUCTION: It is 20 years since the Institute of Medicine advocated a national approach to improve care and patient safety. Patient safety infrastructure has greatly improved in certain countries. In Ireland, patient safety infrastructure is in ongoing development. To contribute to this, the Royal College of Physicians of Ireland/International Society for Quality in Healthcare Scholar in Residence Programme was launched in 2016. This programme aims to improve patient safety and develop a movement of future clinician leaders to drive improvements in patient safety and the quality of care. METHODS: Doctors in postgraduate training complete a year-long immersive mentorship. This involves monthly group meetings with key patient safety opinion makers, one-on-one mentorship, leadership courses, conference attendance and presentations. Each scholar undertakes a quality improvement (QI) project. RESULTS: A QI project was associated with a decrease in caesarean section rates from 13.7% to 7.6% (p=0.0002) among women in spontaneous labour at term with a cephalic presentation. Other projects are ongoing. CONCLUSION: Medical error, patient safety and QI must be addressed comprehensively at both undergraduate and postgraduate level. We believe the Irish mentorship programme will help to change the paradigm and improve patient safety.

Gastric neuroendocrine tumours.

BACKGROUND: Gastric neuroendocrine tumours (NETs) are increasingly recognised, and management decisions may be difficult due to an incomplete understanding of aetiology, natural history and optimum therapy. This article presents a current understanding based on recent advances in epidemiology, classification, molecular profiling, and treatment. METHODS: Relevant medical literature was identified from searches of PubMed and references cited in appropriate articles identified. Selection of articles was based on peer review, journal and relevance. RESULTS: Gastric NETs may be divided into three clinical prognostic groups: type I is associated with autoimmune atrophic gastritis and hypergastrinaemia, type II is associated with Zollinger-Ellison syndrome, and type III lesions are gastrin-independent, have the greatest metastatic potential and poorest prognosis. There has been an increased frequency of gastric NETs reported. Management approaches have evolved in parallel with advances in endoscopic staging and surgery, as well as improved understanding of the biology and natural history of NETs. CONCLUSIONS: Gastric NETs present a spectrum of activity from indolent tumours to metastatic malignancy. Treatment decisions for patients must be individualised and are best managed by a multidisciplinary team approach. The current evidence base is limited to small series and efforts to treat patients within clinical networks of expertise are warranted.