The smallest functional antibody fragment: Ultralong CDR H3 antibody knob regions potently neutralize SARS-CoV-2.

Cows produce antibodies with a disulfide-bonded antigen-binding domain embedded within ultralong heavy chain third complementarity determining regions. This "knob" domain is analogous to natural cysteine-rich peptides such as knottins in that it is small and stable but can accommodate diverse loops and disulfide bonding patterns. We immunized cattle with SARS-CoV-2 spike and found ultralong CDR H3 antibodies that could neutralize several viral variants at picomolar IC50 potencies in vitro and could protect from disease in vivo. The independent CDR H3 peptide knobs were expressed and maintained the properties of the parent antibodies. The knob interaction with SARS-CoV-2 spike was revealed by electron microscopy, X-ray crystallography, NMR spectroscopy, and mass spectrometry and established ultralong CDR H3-derived knobs as the smallest known recombinant independent antigen-binding fragment. Unlike other vertebrate antibody fragments, these knobs are not reliant on the immunoglobulin domain and have potential as a new class of therapeutics.

Germline-Encoded Positional Cysteine Polymorphisms Enhance Diversity in Antibody Ultralong CDR H3 Regions.

Ab "ultralong" third H chain complementarity-determining regions (CDR H3) appear unique to bovine Abs and may enable binding to difficult epitopes that shorter CDR H3 regions cannot easily access. Diversity is concentrated in the "knob" domain of the CDR H3, which is encoded by the DH gene segment and sits atop a ß-ribbon "stalk" that protrudes far from the Ab surface. Knob region cysteine content is quite diverse in terms of total number of cysteines, sequence position, and disulfide bond pattern formation. We investigated the role of germline cysteines in production of a diverse CDR H3 structural repertoire. The relationship between DH polymorphisms and deletions relative to germline at the nucleotide level, as well as diversity in cysteine and disulfide bond content at the structural level, was ascertained. Structural diversity is formed through (1) DH polymorphisms with altered cysteine positions, (2) DH deletions, and (3) new cysteines that arise through somatic hypermutation that form new, unique disulfide bonds to alter the knob structure. Thus, a combination of mechanisms at both the germline and somatic immunogenetic levels results in diversity in knob region cysteine content, contributing to remarkable complexity in knob region disulfide patterns, loops, and Ag binding surface.

Phenylalanine hydroxylase deficiency treatment and management: A systematic evidence review of the American College of Medical Genetics and Genomics (ACMG).

PURPOSE: Elevated serum phenylalanine (Phe) levels due to biallelic pathogenic variants in phenylalanine hydroxylase (PAH) may cause neurodevelopmental disorders or birth defects from maternal phenylketonuria. New Phe reduction treatments have been approved in the last decade, but uncertainty on the optimal lifespan goal Phe levels for patients with PAH deficiency remains. METHODS: We searched Medline and Embase for evidence of treatment concerning PAH deficiency up to September 28, 2021. Risk of bias was evaluated based on study design. Random-effects meta-analyses were performed to compare IQ, gestational outcomes, and offspring outcomes based on Phe ≤ 360 µmol/L vs > 360 µmol/L and reported as odds ratio and 95% CI. Remaining results were narratively synthesized. RESULTS: A total of 350 studies were included. Risk of bias was moderate. Lower Phe was consistently associated with better outcomes. Achieving Phe ≤ 360 µmol/L before conception substantially lowered the risk of negative effect to offspring in pregnant individuals (odds ratio = 0.07, 95% CI = 0.04-0.14; P < .0001). Adverse events due to pharmacologic treatment were common, but medication reduced Phe levels, enabling dietary liberalization. CONCLUSIONS: Reduction of Phe levels to ≤360 µmol/L through diet or medication represents effective interventions to treat PAH deficiency.

Systematic evidence-based review: The application of noninvasive prenatal screening using cell-free DNA in general-risk pregnancies.

PURPOSE: Noninvasive prenatal screening (NIPS) using cell-free DNA has been assimilated into prenatal care. Prior studies examined clinical validity and technical performance in high-risk populations. This systematic evidence review evaluates NIPS performance in a general-risk population. METHODS: Medline (PubMed) and Embase were used to identify studies examining detection of Down syndrome (T21), trisomy 18 (T18), trisomy 13 (T13), sex chromosome aneuploidies, rare autosomal trisomies, copy number variants, and maternal conditions, as well as studies assessing the psychological impact of NIPS and the rate of subsequent diagnostic testing. Random-effects meta-analyses were used to calculate pooled estimates of NIPS performance (P < .05). Heterogeneity was investigated through subgroup analyses. Risk of bias was assessed. RESULTS: A total of 87 studies met inclusion criteria. Diagnostic odds ratios were significant (P < .0001) for T21, T18, and T13 for singleton and twin pregnancies. NIPS was accurate (≥99.78%) in detecting sex chromosome aneuploidies. Performance for rare autosomal trisomies and copy number variants was variable. Use of NIPS reduced diagnostic tests by 31% to 79%. Conclusions regarding psychosocial outcomes could not be drawn owing to lack of data. Identification of maternal conditions was rare. CONCLUSION: NIPS is a highly accurate screening method for T21, T18, and T13 in both singleton and twin pregnancies.

Diversity of ACE2 and its interaction with SARS-CoV-2 receptor binding domain.

COVID-19, the clinical syndrome caused by the SARS-CoV-2 virus, has rapidly spread globally causing hundreds of millions of infections and over two million deaths. The potential animal reservoirs for SARS-CoV-2 are currently unknown, however sequence analysis has provided plausible potential candidate species. SARS-CoV-2 binds to the angiotensin I converting enzyme 2 (ACE2) to enable its entry into host cells and establish infection. We analyzed the binding surface of ACE2 from several important animal species to begin to understand the parameters for the ACE2 recognition by the SARS-CoV-2 spike protein receptor binding domain (RBD). We employed Shannon entropy analysis to determine the variability of ACE2 across its sequence and particularly in its RBD interacting region, and assessed differences between various species' ACE2 and human ACE2. Recombinant ACE2 from human, hamster, horseshoe bat, cat, ferret, and cow were evaluated for RBD binding. A gradient of binding affinities were seen where human and hamster ACE2 were similarly in the low nanomolar range, followed by cat and cow. Surprisingly, horseshoe bat (Rhinolophus sinicus) and ferret (Mustela putorius) ACE2s had poor binding activity compared with the other species' ACE2. The residue differences and binding properties between the species' variants provide a framework for understanding ACE2-RBD binding and virus tropism.

Epidemiology of High-Heel Shoe Injuries in U.S. Women: 2002 to 2012.

The purpose of the present study was to investigate the epidemiology of high-heel-related injuries among a nationally representative population of women in the United States and to analyze the demographic differences within this group. The data used in the present study were collected from the Consumer Product Safety Commission's National Electronic Injury Surveillance System. A total of 3294 injuries, representing an estimated 123,355 high-heel-related injuries, were treated in emergency departments within the United States from 2002 to 2012. The overall rate of high-heel-related injuries for the study was 7.32 per 100,000 females (95% confidence interval 7.08 to 7.56). The injury rate was greatest for young adult females, with the greatest rates observed for those aged 20 to 29 years (18.38 per 100,000 females) and those aged 30 to 39 years (11.07 per 100,000 females). The results from the present study suggest that high-heel-related injuries have nearly doubled during the 11-year period from 2002 to 2012. Injuries from high heels are differential by body region, with most injuries occurring as sprains and strains to the foot and ankle. Although high heels might be stylish, from a health standpoint, it could be worthwhile for females and those interested in wearing high heels to understand the risks of wearing high-heeled shoes and the potential harm that precarious activities in high-heeled shoes can cause. The results of the present study can be used in the development of a prospective cohort study to investigate the risk of injury from high-heeled shoes, accounting for the exposure time and studying differences in demographics (e.g., age and race).

The transitioning from trials to extended follow-up studies.

BACKGROUND: Investigators may elect to extend follow-up of participants enrolled in a randomized clinical trial after the trial comes to its planned end. The additional follow-up may be initiated to learn about longer term effects of treatments, including adverse events, costs related to treatment, or for reasons unrelated to treatment such as to observe the natural course of the disease using the established cohort from the trial. PURPOSE: We examine transitioning from trials to extended follow-up studies when the goal of additional follow-up is to observe longer term treatment effects. METHODS: We conducted a literature search in selected journals from 2000 to 2012 to identify trials that extended follow-up for the purpose of studying longer term treatment effects and extracted information on the operational and logistical issues in the transition. We also draw experience from three trials coordinated by the Johns Hopkins Coordinating Centers that made transitions to extended follow-up: the Alzheimer's Disease Anti-inflammatory Prevention Trial, Multicenter Uveitis Steroid Treatment trial, and Childhood Asthma Management Program. RESULTS: Transitions are not uncommon in multicenter clinical trials, even in trials that continued to the planned end of the trial. Transitioning usually necessitates new participant consents. If study infrastructure is not maintained during the transition, participants will be lost and re-establishing the staff and facilities will be costly. Merging data from the trial and follow-up study can be complicated by changes in data collection measures and schedules. LIMITATIONS: Our discussion and recommendations are limited to issues that we have experienced in transitions from trials to follow-up studies. DISCUSSION: We discuss issues such as maintaining funding, institutional review board and consent requirements, contacting participants, and combining data from the trial and follow-up phases. We conclude with a list of recommendations to facilitate transitions from a trial to an extended follow-up study.

Longitudinal Associations Between Body Mass Index During Young Adulthood, Subsequent Weight Change, and Incident Diabetes During Mid- and Older-Adulthood in Non-Hispanic White and African American Populations: The Atherosclerosis Risk in Communities Study.

Background: Excess body mass index (BMI) and weight gain are well-known risk factors for diabetes. Nevertheless, the associations of BMI and weight gain in young adulthood with subsequent diabetes in African Americans, and the standardized effects of these weight variables have not been well studied. Methods: We studied 12,672 white and African American men and women 45-64 years of age (i.e., during mid-adulthood) who participated in the Atherosclerosis Risk in Communities Study visit 1 (1987-1989), and were reexamined at three follow-up examinations. Associations between recalled BMI at age 25 (i.e., during young adulthood) and subsequent weight change with incident diabetes at ages 45 and above (i.e., during mid-adulthood to older adulthood) were examined using Cox proportional hazard models. Results: Over the 9-year follow-up, we identified 1,501 cases of incident diabetes. The incidence rates were higher among African Americans (men: 24.5 and women: 26.3 per 1,000 person-years) compared to whites (men: 16.3 and women: 10.5 per 1,000 person years). Compared to normal-weight individuals at age 25, those who were overweight or obese and those who gained more weight after age 25 had a higher risk of developing diabetes later in all four race-sex groups with the highest risk in African Americans. In the race-sex groups combined, the mutually adjusted hazard ratio for BMI at age 25 and percent weight change were 1.97 (1.79-2.17) and 2.89 (2.59-3.11), respectively, comparing the 85th to the 15th percentiles of the exposures. Conclusions: African Americans were at higher risk of diabetes than whites. Both higher BMI at age 25 and subsequent weight gain were independently associated with higher risk for diabetes in all the race-sex groups; however, overall weight gain was more potent than BMI.

Cardiometabolic Correlates of Physical Activity and Sedentary Patterns in U.S. Youth.

INTRODUCTION: Daily or weekly averages of physical activity and sedentary behavior could mask patterns of behavior throughout the week that independently affect cardiovascular health. We examined associations between day-to-day physical activity and sedentary behavior latent classes and cardiovascular disease (CVD) risk factors in U.S. youth. METHODS: Data were from 3984 youth ages 6-17 yr from the National Health and Nutrition Examination Survey (2003-2006) and from previously published accelerometry latent classes characterizing average counts per minute and percent of wear time in moderate-to-vigorous physical activity (MVPA) and sedentary behavior. Multiple linear regression was used to examine associations of the classes with waist circumference, systolic and diastolic blood pressure, HDL-C and LDL-C, triglycerides, glucose, and insulin. RESULTS: Participants spent 50.4% of the day in sedentary behavior and 5.3% of the day in moderate-to-vigorous physical activity. Average counts per minute were 516.4 for a 7-d period. Significant differences in CVD risk factors were between extreme classes with few differences observed in intermediate classes. Youth in latent class 4 (highest average counts per minute) had lower systolic blood pressure (-4.11 mm Hg, 95% confidence interval [CI] = -7.74 to -0.55), lower glucose (-4.25 mg·dL, 95% CI = -7.84 to -0.66]), and lower insulin (-6.83 µU·mL, 95% CI = -8.66 to -4.99]) compared with youth in class 1 (lowest average counts per minute). Waist circumference was lower for the least sedentary class (-2.54 cm, 95% CI = -4.90 to -0.19) compared with the most sedentary class. Some associations were attenuated when classes were adjusted for mean physical activity or sedentary level. CONCLUSIONS: There is some indication that patterns, in addition to the total amount of physical activity and sedentary behavior, may be important for cardiovascular health in youth. Longitudinal studies are needed to examine associations between physical activity and sedentary behavior patterns and changes in CVD risk factors.