Molecular Mechanism of V(D)J Recombination from Synaptic RAG1-RAG2 Complex Structures.

Diverse repertoires of antigen-receptor genes that result from combinatorial splicing of coding segments by V(D)J recombination are hallmarks of vertebrate immunity. The (RAG1-RAG2)2 recombinase (RAG) recognizes recombination signal sequences (RSSs) containing a heptamer, a spacer of 12 or 23 base pairs, and a nonamer (12-RSS or 23-RSS) and introduces precise breaks at RSS-coding segment junctions. RAG forms synaptic complexes only with one 12-RSS and one 23-RSS, a dogma known as the 12/23 rule that governs the recombination fidelity. We report cryo-electron microscopy structures of synaptic RAG complexes at up to 3.4 Å resolution, which reveal a closed conformation with base flipping and base-specific recognition of RSSs. Distortion at RSS-coding segment junctions and base flipping in coding segments uncover the two-metal-ion catalytic mechanism. Induced asymmetry involving tilting of the nonamer-binding domain dimer of RAG1 upon binding of HMGB1-bent 12-RSS or 23-RSS underlies the molecular mechanism for the 12/23 rule.

Continuous glucose monitoring in pregnancies with type 1 diabetes: small increases in time-in-range improve maternal and perinatal outcomes.

BACKGROUND: Continuous glucose monitors provide detailed information regarding glycemic control in pregnant patients with type 1 diabetes. Little data have been published examining the association between continuous glucose monitor parameters and perinatal outcomes among gravidas with type 1 diabetes using continuous glucose monitors. OBJECTIVE: This study aimed to examine the association between perinatal outcomes and time-in-range as assessed by continuous glucose monitors used in pregnant individuals with type 1 diabetes. We hypothesized that higher time-in-range would be associated with lower risk of adverse perinatal outcomes. STUDY DESIGN: This multicenter retrospective cohort study included all gravidas with type 1 diabetes using continuous glucose monitors who delivered from 2020 to 2022 at 5 University of California sites. Only those with continuous glucose monitor target range set to 70 to 140 mg/dL (±10 mg/dL) were included. Time-in-range (%) was recorded at 12, 16, 20, 24, 28, and 32 weeks. The primary maternal and neonatal outcomes were preeclampsia and large for gestational age, defined as birthweight ≥95th percentile. Kruskal-Wallis tests were used to compare median time-in-range between those with and without the primary outcomes. Log-binomial regression was used to obtain risk ratios, with adjustment for microvascular disease and years with type 1 diabetes. RESULTS: A total of 91 patients were included. Most used an insulin pump (81%) and did not have diabetic microvascular disease (72%). Median time since diagnosis of type 1 diabetes was 16 years, and median periconception hemoglobin A1c was 6.7%. Compared with those with preeclampsia, normotensive gravidas had significantly higher time-in-range at nearly every time point. A similar pattern was observed for those with normal-birthweight infants compared with large-for-gestational-age infants. On adjusted analyses, every 5-unit increase in time-in-range at 12 weeks was associated with 45% and 46% reductions in the risks of preeclampsia and large for gestational age, respectively (preeclampsia: adjusted risk ratio, 0.55; 95% confidence interval, 0.30-0.99; large for gestational age: adjusted risk ratio, 0.54; 95% confidence interval, 0.29-0.99). CONCLUSION: Higher time-in-range is associated with lower risk of preeclampsia and large for gestational age. This association is observed early in gestation, when each 5-unit increase in time-in-range is associated with ∼50% reduction in the risk of these complications. These findings can be used to counsel patients regarding the risk of pregnancy complications at specific time-in-range values, and to encourage patients that even small improvements in time-in-range can have significant impact on pregnancy outcomes. Larger studies are needed to further explore these findings and to identify optimal time-in-range to reduce perinatal complication rates.

X-ray and cryo-EM structures of the mitochondrial calcium uniporter.

Mitochondrial calcium uptake is critical for regulating ATP production, intracellular calcium signalling, and cell death. This uptake is mediated by a highly selective calcium channel called the mitochondrial calcium uniporter (MCU). Here, we determined the structures of the pore-forming MCU proteins from two fungi by X-ray crystallography and single-particle cryo-electron microscopy. The stoichiometry, overall architecture, and individual subunit structure differed markedly from those described in the recent nuclear magnetic resonance structure of Caenorhabditis elegans MCU. We observed a dimer-of-dimer architecture across species and chemical environments, which was corroborated by biochemical experiments. Structural analyses and functional characterization uncovered the roles of key residues in the pore. These results reveal a new ion channel architecture, provide insights into calcium coordination, selectivity and conduction, and establish a structural framework for understanding the mechanism of mitochondrial calcium uniporter function.

Cryo-EM structure of the protein-conducting ERAD channel Hrd1 in complex with Hrd3.

Misfolded endoplasmic reticulum proteins are retro-translocated through the membrane into the cytosol, where they are poly-ubiquitinated, extracted from the membrane, and degraded by the proteasome-a pathway termed endoplasmic reticulum-associated protein degradation (ERAD). Proteins with misfolded domains in the endoplasmic reticulum lumen or membrane are discarded through the ERAD-L and ERAD-M pathways, respectively. In Saccharomyces cerevisiae, both pathways require the ubiquitin ligase Hrd1, a multi-spanning membrane protein with a cytosolic RING finger domain. Hrd1 is the crucial membrane component for retro-translocation, but it is unclear whether it forms a protein-conducting channel. Here we present a cryo-electron microscopy structure of S. cerevisiae Hrd1 in complex with its endoplasmic reticulum luminal binding partner, Hrd3. Hrd1 forms a dimer within the membrane with one or two Hrd3 molecules associated at its luminal side. Each Hrd1 molecule has eight transmembrane segments, five of which form an aqueous cavity extending from the cytosol almost to the endoplasmic reticulum lumen, while a segment of the neighbouring Hrd1 molecule forms a lateral seal. The aqueous cavity and lateral gate are reminiscent of features of protein-conducting conduits that facilitate polypeptide movement in the opposite direction-from the cytosol into or across membranes. Our results suggest that Hrd1 forms a retro-translocation channel for the movement of misfolded polypeptides through the endoplasmic reticulum membrane.

Increase in the Number of Pediatric New-Onset Diabetes and Diabetic Ketoacidosis Cases During the COVID-19 Pandemic.

OBJECTIVE: Infection with SARS-CoV-2 induces a proinflammatory state that causes hyperglycemia and may precipitate diabetic ketoacidosis (DKA) in patients with known or new-onset diabetes. We examined the trends in new-onset diabetes and DKA prior to and following the onset of the COVID-19 pandemic. METHODS: This single-center retrospective observational study included pediatric patients (aged 0 to <18 years) hospitalized with new-onset type 1 diabetes or type 2 diabetes (T2D) before (March 1, 2018, to February 29, 2020) and after (March 1, 2020 to December 31, 2020) the pandemic onset. Demographic, anthropometrics, laboratory and clinical data, and outcomes were obtained. RESULTS: Among 615 children admitted with new-onset diabetes during the entire study period, 401 were admitted before the pandemic onset, and 214 were admitted after the pandemic onset. Children admitted with new-onset diabetes in the postpandemic period were significantly more likely to present with DKA (odds ratio, 1.76; 95% confidence interval, 1.24-2.52) than in the prepandemic phase. Children with DKA after the pandemic onset had higher lengths of hospitalization and were significantly more likely to experience severe DKA (odds ratio, 2.17; 95% confidence interval, 1.34-3.52). A higher proportion of children with DKA admitted to the pediatric intensive care unit required oxygen support after the pandemic onset than before the pandemic onset (8.85% vs 1.92%). Most cases of T2D with DKA occurred following the onset of the pandemic (62.5%). CONCLUSION: A significant increase in T2D cases occurred following the onset of the COVID-19 pandemic with a greater risk of DKA and severe ketoacidosis. Racial disparity was evident with a higher proportion of Black and American Indian children presenting with ketoacidosis following the pandemic onset.

Feasibility, Acceptability, and Preliminary Efficacy of an Intensive Clinic-Based Intervention for Children With Poorly Controlled Type 1 Diabetes.

OBJECTIVE: To evaluate the feasibility, acceptability, and preliminary efficacy of a team-based intervention for youth with type 1 diabetes (T1D) with suboptimal glycemia, as detected based on the measurement of hemoglobin A1C (HbA1C). METHODS: Forty participants with T1D for >1 year and an HbA1C level of ≥9.5% (80 mmol/mol) enrolled for a multidisciplinary intervention that included pediatric endocrinologists, pediatric psychologists, and a certified diabetes care and education specialist (CDCES). The CDCES-integrated medical management, while reinforcing physical, emotional, and behavioral health, connected with families to set and monitor goals and reviewed medication adjustments. The feasibility was assessed based on enrollment targets; acceptability based on retention rates; and preliminary efficacy based on changes in HbA1C levels, quality of life, diabetes-related strengths and resilience, hospital admissions, emergency room visits, and missed school days. RESULTS: Of 43 patients and families approached, 40 agreed to participate, 36 completed the 4-month intervention, and 31 completed full 8 months of follow-up data collection. The CDCES coach averaged 6.8 contacts per participant during the 8-month study period. The HbA1C level reduced significantly from baseline to 4 months (12.1% ± 1.6% to 11.0% ± 1.9%, P = .001) and was sustained at 8 months (10.7% ± 1.9%, P < .001). The participants reported significant increases in diabetes-specific quality of life (P < .05) and diabetes-related strength and resilience (P = .003). The missed school days reduced from 7.23 ± 7.5 days to 1.55 ± 1.9 days (P < .001), and the diabetes-related hospitalizations decreased from 0.4 ± 0.6 to 0.1 ± 0.3 (P = .009). CONCLUSION: Preliminary data suggest that a multidisciplinary intervention leveraging a team-based approach with a physician, psychologist, and CDCES can support improvements in glycemic control and psychosocial outcomes among youth with T1D with an HbA1C level above the target.

Safety and interim survival data after intracranial administration of M032, a genetically engineered oncolytic HSV-1 expressing IL-12, in pet dogs with sporadic gliomas.

OBJECTIVE: The diagnosis of glioma remains disheartening in the clinical realm. While a multitude of studies and trials have shown promise, improvements in overall survival have been disappointing. Modeling these tumors in the laboratory setting has become increasingly challenging, given their complex in situ behavior and interactions for therapeutic evasion. Dogs, particularly brachycephalic breeds, are known to spontaneously develop gliomas that resemble human gliomas both clinically and pathophysiologically, making canines with sporadic tumors promising candidates for study. Typically, survival among these dogs is approximately 2 months with palliation alone. METHODS: The authors have completed the first stage of a unique phase I dose-escalating canine clinical trial in which the safety and tolerability of M032, a nonneurovirulent oncolytic herpes simplex virus-1 vector genetically engineered to express interleukin-12, are being studied in pet dogs with gliomas undergoing maximum safe tumor resection and inoculation of the cavity with the viral infusate. RESULTS: Twenty-five canine patients were enrolled between January 2018 and August 2020. One patient was electively withdrawn from the trial by its owner, and 3 did not receive the virus. For the 21 dogs that remained, 13 had high-grade gliomas, 5 had low-grade gliomas, and 3 were undetermined. According to histopathological analysis, 62% of the tumors were oligodendrogliomas. At the time of this report, the median overall survival from the date of treatment was 151 days (± 78 days). No significant adverse events attributable to M032 or dose-limiting toxicities have been observed to date. CONCLUSIONS: In this largest study of oncolytic viral therapy for canine brain tumors to date, treatment with M032 did not cause harm and the combination of surgery and oncolytic viral therapy may have contributed to prolonged survival in pet dogs with spontaneous gliomas. Forthcoming in-depth radiographic, immunohistochemical, and genetic analyses will afford a more advanced understanding of how this treatment impacts these tumors and the immune system. Our goal is to utilize these findings bitranslationally to inform human studies and refine therapies that will improve outcomes in both humans and pet dogs with gliomas.

Bicelles Rich in both Sphingolipids and Cholesterol and Their Use in Studies of Membrane Proteins.

How the distinctive lipid composition of mammalian plasma membranes impacts membrane protein structure is largely unexplored, partly because of the dearth of isotropic model membrane systems that contain abundant sphingolipids and cholesterol. This gap is addressed by showing that sphingomyelin and cholesterol-rich (SCOR) lipid mixtures with phosphatidylcholine can be cosolubilized by n-dodecyl-ß-melibioside to form bicelles. Small-angle X-ray and neutron scattering, as well as cryo-electron microscopy, demonstrate that these assemblies are stable over a wide range of conditions and exhibit the bilayered-disc morphology of ideal bicelles even at low lipid-to-detergent mole ratios. SCOR bicelles are shown to be compatible with a wide array of experimental techniques, as applied to the transmembrane human amyloid precursor C99 protein in this medium. These studies reveal an equilibrium between low-order oligomer structures that differ significantly from previous experimental structures of C99, providing an example of how ordered membranes alter membrane protein structure.

Provider Preference for Growth Charts in Tracking Children with Obesity.

A web-based survey of pediatric care providers revealed differences in their preference for clinical charts that monitor growth in children with obesity. These findings are attributed to pediatric specialty training. Very few providers believe the currently available Centers for Disease Control and Prevention 2000 body mass index-for-age charts adequately track growth in children with obesity.

Epicardial adipose thickness in youth with type 1 diabetes.

BACKGROUND AND OBJECTIVE: Epicardial adipose thickness (EAT) is increased in adults with type 1 diabetes (T1D) and is thought to contribute to cardiovascular disease (CVD) in this population. Given that CVD risk factors emerge early in life, the purpose of this study was to identify whether EAT is increased in pediatric patients with T1D compared with non-diabetic controls. METHODS: Anthropometric data, blood pressure (BP), and EAT were evaluated in 20 youth with T1D and 20 age, sex, and body mass index (BMI) matched healthy controls between the ages of 5 and 18 years. RESULTS: EAT was 18.5% higher among youth with T1D compared to healthy controls (1.65 ± 0.44 mm vs 1.37 ± 0.27 mm, P = .02). In the entire cohort, EAT was correlated with age (r = 0.71, P < .001), BMI (r = .69, P < .001), waist circumference (r = 0.60, P < .001), systolic BP (r = .34, P = .03), and diastolic BP (r = 0.41, P = .009). Among youth with T1D, there were no significant correlations between EAT and HbA1c (r = -0.16, P = .50), insulin dose (r = .09, P = .71), or duration of disease (r = 0.06, P = .82). CONCLUSIONS: Youth with T1D exhibited significantly higher EAT compared to controls. Increased EAT was associated with adiposity and BP, but not duration of disease, insulin dose, or glycemic control. Increased EAT may represent a pathophysiologic mechanism leading to premature CVD in pediatric patients with T1D.

Effect of severe obesity in childhood and adolescence on risk of type 2 diabetes in youth and early adulthood in an American Indian population.

OBJECTIVES: The risk of early-onset type 2 diabetes associated with the severity of obesity in youth is not well understood. This study aims to determine metabolic alterations and type 2 diabetes risk among American Indian children who are obese or severely obese. METHODS: Incidence rates of diabetes before 20 years (youth-onset) and 45 years were computed in 2728 children who were from 5 to <10 years and 4317 adolescents who were from 10 to <18 years without diabetes examined between 1965 and 2007. Obesity was defined as age-sex-adjusted body mass index (BMI) ≥95th percentile, and its severity was quantified as the percentage of the 95th percentile (%BMIp95 ). RESULTS: In the younger cohort, 0.9% of those non-obese and 2.9% of those with 100% to <120%BMIp95 had impaired glucose tolerance (IGT) compared to 8.6% of those with ≥140%BMIp95 . In the older cohort, 2.9% of those non-obese and 9.8% of those with 100% to <120%BMIp95 had IGT compared to 13.3% of those with ≥160%BMIp95 . The incidence of youth-onset diabetes was 3.8 and 4.9/1000 person-years in the child and adolescent cohorts, respectively, and before the age of 45 was 12.3 and 16.8/1000 person-years, respectively. Incidence rates of youth-onset diabetes in those with the most severe obesity (≥140%BMIp95 ) were 2.3 to 5.1 times as high as in those with the least severe obesity (100 to <120%BMIp95 ), and for onset of diabetes before the age of 45 were 1.6 to 2.2 times as high. CONCLUSIONS: Severe obesity in an American Indian population is a major driver of type 2 diabetes developing in adolescents and young adults.

Best practices for managing large CryoEM facilities.

This paper provides an overview of the discussion and presentations from the Workshop on the Management of Large CryoEM Facilities held at the New York Structural Biology Center, New York, NY on February 6-7, 2017. A major objective of the workshop was to discuss best practices for managing cryoEM facilities. The discussions were largely focused on supporting single-particle methods for cryoEM and topics included: user access, assessing projects, workflow, sample handling, microscopy, data management and processing, and user training.

Mapping the interaction between factor VIII and von Willebrand factor by electron microscopy and mass spectrometry.

Association with the D'D3 domain of von Willebrand factor (VWF) stabilizes factor VIII (FVIII) in the circulation and maintains it at a level sufficient to prevent spontaneous bleeding. We used negative-stain electron microscopy (EM) to visualize complexes of FVIII with dimeric and monomeric forms of the D'D3 domain. The EM averages show that FVIII interacts with the D'D3 domain primarily through its C1 domain, with the C2 domain providing a secondary attachment site. Hydrogen-deuterium exchange mass spectrometry corroborated the importance of the C1 domain in D'D3 binding and implicates additional surface regions on FVIII in the interaction. Together, our results establish that the C1 domain is the major binding site on FVIII for VWF, reiterate the importance of the a3 acidic peptide in VWF binding, and suggest that the A3 and C2 domains play ancillary roles in this interaction.

Structural organization of the functional domains of Clostridium difficile toxins A and B.

Clostridium difficile toxins A and B are members of an important class of virulence factors known as large clostridial toxins (LCTs). Toxin action involves four major steps: receptor-mediated endocytosis, translocation of a catalytic glucosyltransferase domain across the membrane, release of the enzymatic moiety by autoproteolytic processing, and a glucosyltransferase-dependent inactivation of Rho family proteins. We have imaged toxin A (TcdA) and toxin B (TcdB) holotoxins by negative stain electron microscopy to show that these molecules are similar in structure. We then determined a 3D structure for TcdA and mapped the organization of its functional domains. The molecule has a "pincher-like" head corresponding to the delivery domain and two tails, long and short, corresponding to the receptor-binding and glucosyltransferase domains, respectively. A second structure, obtained at the acidic pH of an endosome, reveals a significant structural change in the delivery and glucosyltransferase domains, and thus provides a framework for understanding the molecular mechanism of LCT cellular intoxication.

Fertility sparing treatment of vaginal clear cell adenocarcinoma in a DES-Naïve young woman.

Background: Primary vaginal cancer is rare and comprises 1-2% of female genital tract cancers. Among the types of vaginal cancer, adenocarcinoma accounts for only 10% with the peak incidence in women less than 20 years old. Clear cell type vaginal adenocarcinoma is most associated with exposure to diethylstilbestrol (DES) in-utero. Case: We present a case of an 18-year-old nulliparous woman, DES-exposure naive, who was diagnosed with stage I clear cell vaginal adenocarcinoma during a routine pelvic exam for abnormal vaginal bleeding. She underwent a fertility-preserving radical vaginectomy and pelvic lymphadenectomy with neovagina creation and uterovaginal cervical reconstruction. She has been without disease for 28 months. Conclusion: Although rare, vaginal cancer can be diagnosed on routine women's health exams. Early screening and diagnosis allow for innovative fertility-preserving surgical approaches without compromising oncologic outcomes. To our knowledge, this is the first case of a fertility-preserving radical vaginectomy, neovagina creation using a vertical rectus abdominis myocutaneous (VRAM) flap, and uterocervicovaginal reconstruction to successfully treat early stage clear cell vaginal adenocarcinoma with surgery alone, sparing the patient from adjuvant chemotherapy or radiation.

Electronic Dashboard to Improve Outcomes in Pediatric Patients With Type 1 Diabetes Mellitus.

BACKGROUND AND OBJECTIVES: Incidence of type 1 diabetes mellitus (T1DM) is increasing, and these patients often have poor glycemic control. Electronic dashboards summating patient data have been shown to improve patient outcomes in other conditions. In addition, educating patients on T1DM has shown to improve glycated hemoglobin (A1C) levels. We hypothesized that using data from the electronic dashboard to monitor defined diabetes management activities to implement population-based interventions would improve patient outcomes. METHODS: Inclusion criteria included patients aged 0 to 18 years at Phoenix Children's Hospital with T1DM. Patient data were collected via the electronic dashboard, and both diabetes management activities (A1C, patient admissions, and visits to the emergency department) and patient outcomes (patient education, appointment compliance, follow-up after hospital admission) were analyzed. RESULTS: This study revealed that following implementation of the electronic dashboard, the percentage of patients receiving appropriate education increased from 48% to 80% (Z-score = 23.55, P < .0001), the percentage of patients attending the appropriate number of appointments increased from 50% to 68.2%, and the percentage of patients receiving follow-up care within 40 days after a hospital admission increased from 43% to 70%. The median A1C level decreased from 9.1% to 8.2% (Z-score = -6.74, P < .0001), and patient admissions and visits to the emergency department decreased by 20%. CONCLUSIONS: This study shows, with the implementation of an electronic dashboard, we were able to improve outcomes for our pediatric patients with T1DM. This tool can be used at other institutions to improve care and outcomes for pediatric patients with T1DM and other chronic conditions.

Impact of overweight and obesity on epicardial adipose tissue in children with type 1 diabetes.

OBJECTIVES: Epicardial adipose tissue (EAT) thickness, a novel marker of cardiovascular disease (CVD), is increased in children with a healthy weight and type 1 diabetes (T1D). The prevalence of obesity has increased in children with T1D and may confer additional CVD risk. The purpose of this study was to examine EAT thickness in youth with and without T1D in the setting of overweight/obesity. METHODS: Youth with overweight/obesity and T1D (n=38) or without T1D (n=34) between the ages of 6-18 years were included in this study. Echocardiogram using spectral and color flow Doppler was used to measure EAT and cardiac function. Waist circumference, blood pressure, and HbA1c, were used to calculate estimated glucose disposal rate (eGDR) to estimate insulin resistance in children with T1D. RESULTS: EAT thickness was not significantly different in youth with T1D compared to controls (2.10 ± 0.67 mm vs. 1.90 ± 0.59 mm, p=0.19). When groups were combined, EAT significantly correlated with age (r=0.449, p≤0.001), BMI (r=0.538, p≤0.001), waist circumference (r=0.552, p≤0.001), systolic BP (r=0.247, p=0.036), myocardial performance index (r=-0.287, p=0.015), ejection fraction (r=-0.442, p≤0.001), and cardiac output index (r=-0.306, p=0.009). In the group with T1D, diastolic BP (r=0.39, p=0.02) and eGDR (r=-0.48, p=0.002) correlated with EAT. CONCLUSIONS: EAT was associated with measures of adiposity and insulin resistance but does not differ by diabetes status among youth with overweight/obesity. These findings suggest that adiposity rather than glycemia is the main driver of EAT thickness among youth with T1D.

Leading the pack: Best practices in comparative canine cancer genomics to inform human oncology.

Pet dogs develop spontaneous cancers at a rate estimated to be five times higher than that of humans, providing a unique opportunity to study disease biology and evaluate novel therapeutic strategies in a model system that possesses an intact immune system and mirrors key aspects of human cancer biology. Despite decades of interest, effective utilization of pet dog cancers has been hindered by a limited repertoire of necessary cellular and molecular reagents for both in vitro and in vivo studies, as well as a dearth of information regarding the genomic landscape of these cancers. Recently, many of these critical gaps have been addressed through the generation of a highly annotated canine reference genome, the creation of several tools necessary for multi-omic analysis of canine tumours, and the development of a centralized repository for key genomic and associated clinical information from canine cancer patients, the Integrated Canine Data Commons. Together, these advances have catalysed multidisciplinary efforts designed to integrate the study of pet dog cancers more effectively into the translational continuum, with the ultimate goal of improving human outcomes. The current review summarizes this recent progress and provides a guide to resources and tools available for comparative study of pet dog cancers.

Screening for Cardiovascular Disease in Pregnancy: Is There a Need?

Maternal mortality in the United States has been on the rise. Every year, about 700 women die from pregnancy-related complications. Cardiovascular disease (CVD) accounts for a large majority of pregnancy-related deaths driven by the lack of recognition and delays in diagnosis due to the overlap of normal pregnancy symptoms with those of CVD. Risk factors for CVD including race, advanced maternal age, hypertension, diabetes, obesity, socioeconomic status, and geographic region play an important role in CVD-related deaths. Several risk assessment models are available to stratify women with a known diagnosis of CVD. However, most women who die from CVD during pregnancy or the postpartum period do not have a prior diagnosis of CVD, and cardiomyopathy is an important contributor. The California Maternal Quality Care Collaborative (CMQCC) developed an algorithm to screen all pregnant and postpartum women to allow stratification into low or high risk for CVD. The algorithm has been validated in diverse patient populations. We propose universal CVD screening for all women in the antepartum and postpartum period to identify women at risk and to provide education and awareness for both patients and healthcare providers. This screening tool would work to reduce the increasing rates of severe maternal mortality and morbidity while having a significant impact on healthcare costs in the United States.

Reconstitution of Helicobacter pylori VacA toxin from purified components.

Helicobacter pylori VacA is a pore-forming toxin that causes multiple alterations in human cells and contributes to the pathogenesis of peptic ulcer disease and gastric cancer. The toxin is secreted by H. pylori as an 88 kDa monomer (p88) consisting of two domains (p33 and p55). While an X-ray crystal structure for p55 exists and p88 oligomers have been visualized by cryo-electron microscopy, a detailed analysis of p33 has been hindered by an inability to purify this domain in an active form. In this study, we expressed and purified a recombinant form of p33 under denaturing conditions and optimized conditions for the refolding of the soluble protein. We show that refolded p33 can be added to purified p55 in trans to cause vacuolation of HeLa cells and inhibition of IL-2 production by Jurkat cells, effects identical to those produced by the p88 toxin from H. pylori. The p33 protein markedly enhances the cell binding properties of p55. Size exclusion chromatography experiments suggest that p33 and p55 assemble into a complex consistent with the size of a p88 monomer. Electron microscopy of these p33/p55 complexes reveals small rod-shaped structures that can convert to oligomeric flower-shaped structures in the presence of detergent. We propose that the oligomerization observed in these experiments mimics the process by which VacA oligomerizes when in contact with membranes of host cells.