Design and Selection of Heterodimerizing Helical Hairpins for Synthetic Biology.

Synthetic biology applications would benefit from protein modules of reduced complexity that function orthogonally to cellular components. As many subcellular processes depend on peptide-protein or protein-protein interactions, de novo designed polypeptides that can bring together other proteins controllably are particularly useful. Thanks to established sequence-to-structure relationships, helical bundles provide good starting points for such designs. Typically, however, such designs are tested in vitro and function in cells is not guaranteed. Here, we describe the design, characterization, and application of de novo helical hairpins that heterodimerize to form 4-helix bundles in cells. Starting from a rationally designed homodimer, we construct a library of helical hairpins and identify complementary pairs using bimolecular fluorescence complementation in E. coli. We characterize some of the pairs using biophysics and X-ray crystallography to confirm heterodimeric 4-helix bundles. Finally, we demonstrate the function of an exemplar pair in regulating transcription in both E. coli and mammalian cells.

Combined Use of RT-qPCR and NGS for Identification and Surveillance of SARS-CoV-2 Variants of Concern in Residual Clinical Laboratory Samples in Miami-Dade County, Florida.

Over the course of the COVID-19 pandemic, SARS-CoV-2 variants of concern (VOCs) with increased transmissibility and immune escape capabilities, such as Delta and Omicron, have triggered waves of new COVID-19 infections worldwide, and Omicron subvariants continue to represent a global health concern. Tracking the prevalence and dynamics of VOCs has clinical and epidemiological significance and is essential for modeling the progression and evolution of the COVID-19 pandemic. Next generation sequencing (NGS) is recognized as the gold standard for genomic characterization of SARS-CoV-2 variants, but it is labor and cost intensive and not amenable to rapid lineage identification. Here we describe a two-pronged approach for rapid, cost-effective surveillance of SARS-CoV-2 VOCs by combining reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and periodic NGS with the ARTIC sequencing method. Variant surveillance by RT-qPCR included the commercially available TaqPath COVID-19 Combo Kit to track S-gene target failure (SGTF) associated with the spike protein deletion H69-V70, as well as two internally designed and validated RT-qPCR assays targeting two N-terminal-domain (NTD) spike gene deletions, NTD156-7 and NTD25-7. The NTD156-7 RT-qPCR assay facilitated tracking of the Delta variant, while the NTD25-7 RT-qPCR assay was used for tracking Omicron variants, including the BA.2, BA.4, and BA.5 lineages. In silico validation of the NTD156-7 and NTD25-7 primers and probes compared with publicly available SARS-CoV-2 genome databases showed low variability in regions corresponding to oligonucleotide binding sites. Similarly, in vitro validation with NGS-confirmed samples showed excellent correlation. RT-qPCR assays allow for near-real-time monitoring of circulating and emerging variants allowing for ongoing surveillance of variant dynamics in a local population. By performing periodic sequencing of variant surveillance by RT-qPCR methods, we were able to provide ongoing validation of the results obtained by RT-qPCR screening. Rapid SARS-CoV-2 variant identification and surveillance by this combined approach served to inform clinical decisions in a timely manner and permitted better utilization of sequencing resources.

Predicting soft tissue thicknesses overlying the iliac crests and greater trochanters of younger and older adults.

Soft tissues overlying the hip play a critical role in protecting against fractures during fall-related hip impacts. Consequently, the development of an efficient and cost-effective method for estimating hip soft tissue thicknesses in living people may prove to be valuable for assessing an individual's injury risk and need to adopt preventative measures. The present study used multiple linear stepwise regression to generate prediction equations from participant characteristics (i.e., height, sex) and anthropometric measurements of the pelvis, trunk, and thigh to estimate soft tissue thickness at the iliac crests (IC) and greater trochanters (GT) in younger (16-35 years of age: 37 males, 37 females) and older (36-65 years of age: 38 males, 38 females) adults. Equations were validated against soft tissue thicknesses measured from full body Dual-energy X-ray Absorptiometry scans of independent samples (younger: 13 males, 13 females; older: 13 males, 12 females). Younger adult prediction equations exhibited adjusted R2 values ranging from 0.704 to 0.791, with more explained variance for soft tissue thicknesses at the GT than the IC; corresponding values for the older adult equations were higher overall and ranged from 0.819 to 0.852. Predicted and actual soft tissue thicknesses were significantly correlated for both the younger (R2 = 0.466 to 0.738) and older (R2 = 0.842 to 0.848) adults, averaging ≤ 0.75cm of error. This research demonstrates that soft tissue thicknesses overlying the GT and IC can be accurately predicted from equations using anthropometric measurements. These equations can be used by clinicians to identify individuals at higher risk of hip fractures who may benefit from the use of preventative measures.

From peptides to proteins: coiled-coil tetramers to single-chain 4-helix bundles.

The design of completely synthetic proteins from first principles-de novo protein design-is challenging. This is because, despite recent advances in computational protein-structure prediction and design, we do not understand fully the sequence-to-structure relationships for protein folding, assembly, and stabilization. Antiparallel 4-helix bundles are amongst the most studied scaffolds for de novo protein design. We set out to re-examine this target, and to determine clear sequence-to-structure relationships, or design rules, for the structure. Our aim was to determine a common and robust sequence background for designing multiple de novo 4-helix bundles. In turn, this could be used in chemical and synthetic biology to direct protein-protein interactions and as scaffolds for functional protein design. Our approach starts by analyzing known antiparallel 4-helix coiled-coil structures to deduce design rules. In terms of the heptad repeat, abcdefg -i.e., the sequence signature of many helical bundles-the key features that we identify are: a = Leu, d = Ile, e = Ala, g = Gln, and the use of complementary charged residues at b and c. Next, we implement these rules in the rational design of synthetic peptides to form antiparallel homo- and heterotetramers. Finally, we use the sequence of the homotetramer to derive in one step a single-chain 4-helix-bundle protein for recombinant production in E. coli. All of the assembled designs are confirmed in aqueous solution using biophysical methods, and ultimately by determining high-resolution X-ray crystal structures. Our route from peptides to proteins provides an understanding of the role of each residue in each design.

Usefulness of the Rapid Office Strain Assessment (ROSA) tool in detecting differences before and after an ergonomics intervention.

BACKGROUND: Most ergonomics studies on office workstations evaluate the effects of an intervention only by subjective measures such as musculoskeletal pain and discomfort. Limited evidence has been provided regarding risk factor reduction in office environments through standardized methods assessments. The Rapid Office Strain Assessment (ROSA) tool can provide an estimation of risk factor exposure for office workers as a means by which the outcome of interventions can be quantified. PURPOSE: The aim of the study was to evaluate if ROSA scores reflect changes in risk factors after an ergonomics intervention among office workers. METHODS: Office workers (n = 60) were divided into two groups. The experimental group received a workstation intervention and the control group received no intervention. Changes in ROSA scores were compared before and after the intervention in both groups. RESULTS: Statistically significant reductions in the ROSA final and section scores occurred after the intervention in the experimental group with (mean reduction of 2.9, 0.8 and 1.6 points for sections A, B and C, respectively). In contrast, no differences were detected in the control group (mean increase of 0.1 point for sections A and C and mean reduction of 0.1 point for Section B). CONCLUSIONS: These findings show that ROSA scores reflect changes in risk factors after an ergonomics intervention in an office environment. Consequently, this tool can be used for identifying and controlling risk factors among computer workers, before and after interventions.

Identification and optimization of a novel series of selective PIP5K inhibitors.

Phosphatidyl inositol (4,5)-bisphosphate (PI(4,5)P2) plays several key roles in human biology and the lipid kinase that produces PI(4,5)P2, PIP5K, has been hypothesized to provide a potential therapeutic target of interest in the treatment of cancers. To better understand and explore the role of PIP5K in human cancers there remains an urgent need for potent and specific PIP5K inhibitor molecules. Following a high throughput screen of the AstraZeneca collection, a novel, moderately potent and selective inhibitor of PIP5K, 1, was discovered. Detailed exploration of the SAR for this novel scaffold resulted in the considerable optimization of both potency for PIP5K, and selectivity over the closely related kinase PI3Kα, as well as identifying several opportunities for the continued optimization of drug-like properties. As a result, several high quality in vitro tool compounds were identified (8, 20 and 25) that demonstrate the desired biochemical and cellular profiles required to aid better understanding of this complex area of biology.

SARS-CoV-2 and Variant Diagnostic Testing Approaches in the United States.

Purpose of Review Given the rapid development of diagnostic approaches to test for and diagnose infection with SARS-CoV-2 and its associated variants including Omicron (B.1.1.529), many options are available to diagnose infection. Multiple established diagnostic companies are now providing testing platforms whereas initially, testing was being performed with simple PCR-based tests using standard laboratory reagents. Recent Findings Additional testing platforms continue to be developed, including those to detect specific variants, but challenges with testing, including obtaining testing reagents and other related supplies, are frequently encountered. With time, the testing supply chain has improved, and more established companies are providing materials to support these testing efforts. In the United States (U.S.), the need for rapid assay development and subsequent approval through the attainment of emergency use authorization (EUA) has superseded the traditional arduous diagnostic testing approval workflow mandated by the FDA. Through these efforts, the U.S. has been able to continue to significantly increase its testing capabilities to address this pandemic; however, challenges still remain due to the diversity of the performance characteristics of tests being utilized and newly discovered viral variants. Summary This review provides an overview of the current diagnostic testing landscape, with pertinent information related to SARS-CoV-2 virology, variants and antibody responses that are available to diagnose infection in the U.S.

Diagnostic Testing for SARS-CoV-2 Infection.

PURPOSE OF REVIEW: Given the rapid development of diagnostic approaches to test for and diagnose infection with SARS-CoV-2, many options are available to assess infection. Multiple established diagnostic companies are now providing testing platforms whereas initially, testing was being performed with simple PCR-based tests using standard laboratory reagents. RECENT FINDINGS: Additional testing platforms continue to be developed but challenges with testing, including obtaining testing reagents and other related supplies, are frequently encountered. With time, the testing supply chain will improve and more companies will be providing materials to support these testing efforts. In the USA, the need for rapid assay development and subsequent approval through attainment of emergency use authorization (EUA) has superseded the traditional arduous diagnostic testing approval workflow mandated by the FDA. It is anticipated that the USA will be able to continue to significantly increase its testing capabilities to address this pandemic; however, challenges remain due to the diversity of the performance characteristics of tests being utilized. SUMMARY: This review provides an overview of the current diagnostic testing landscape, with pertinent information related to SARS-CoV-2 virology and antibody responses, that is available to diagnose infection.

Three-dimensional video analysis of helmet-to-ground impacts in North American youth football.

This study presents a video analysis of helmet-to-ground impacts in youth football (≤14 years). A total of 21 non-injurious helmet-to-ground impact cases were assessed from game video of two age divisions (9-12 years: n = 9; 13-14 years: n = 12) using a novel multi-camera videogrammetry approach. Descriptive parameters related to the game situation and impact mechanisms were documented. Motion analysis software was used to manually track and compute three-dimensional helmet kinematics and uncertainty of the motion tracking analysis was assessed. Overall, the impact cases primarily followed a body-to-body, body-to-ground, helmet-to-ground contact progression. Impact locations on the helmet were mostly distributed across the rear and side helmet shell. The resultant pre-impact velocities for these cases averaged 4.04 ± 1.24 m/s at an angle of -49.6° to the ground. The average resultant impact-induced change in helmet velocity was 3.32 ± 1.14 m/s; the time interval associated with the duration of helmet-to-ground contact was approximately 0.06 s. The average maximum uncertainty (±) error of the position coordinates from the helmet tracking was 1.5 ± 0.3 cm. In summary, this video-based methodology can effectively be used to quantify helmet impact velocities and locations in youth football games. To date, the acquisition of such information has largely been limited to professional football game footage. Therefore, the data reported here may help inform the development of more representative assessment methods for youth-specific helmet test standards.

Query-guided protein-protein interaction inhibitor discovery.

Protein-protein interactions (PPIs) are central to biological mechanisms, and can serve as compelling targets for drug discovery. Yet, the discovery of small molecule inhibitors of PPIs remains challenging given the large and typically shallow topography of the interacting protein surfaces. Here, we describe a general approach to the discovery of orthosteric PPI inhibitors that mimic specific secondary protein structures. Initially, hot residues at protein-protein interfaces are identified in silico or from experimental data, and incorporated into secondary structure-based queries. Virtual libraries of small molecules are then shape-matched against the queries, and promising ligands docked to target proteins. The approach is exemplified experimentally using two unrelated PPIs that are mediated by an α-helix (p53/hDM2) and a ß-strand (GKAP/SHANK1-PDZ). In each case, selective PPI inhibitors are discovered with low µM activity as determined by a combination of fluorescence anisotropy and 1H-15N HSQC experiments. In addition, hit expansion yields a series of PPI inhibitors with defined structure-activity relationships. It is envisaged that the generality of the approach will enable discovery of inhibitors of a wide range of unrelated secondary structure-mediated PPIs.

Recovery does not always signal the end of the battle: A case of post SARS-CoV-2 multisystem inflammatory syndrome in an adult.

We describe a case of SARS-CoV-2 post-infectious inflammatory syndrome in an adult who presented with multiorgan failure two months following his initial diagnosis of SARS-CoV-2 infection. This case highlights clinician's early recognition of this devastating sequela and challenges in appropriate management of this patient.

The Kinase Chemogenomic Set (KCGS): An Open Science Resource for Kinase Vulnerability Identification.

We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS), current Version 1.0, is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.

Exploring the Utility and Student Perceptions of Synthetic Cadavers in an Undergraduate Human Anatomy Course.

The synthetic cadaver is a high-fidelity model intended to replace or supplement other anatomy learning modalities. Academic attainment and student perceptions were examined in an undergraduate human anatomy course using a combination of plastic models and synthetic cadavers to learn lower body anatomy ("Experimental group"), compared to a Historical group who used only plastic models. Grades on an upper body test, for which both groups used only plastic models, were compared to ensure that no academic differences existed between groups (P = 0.7653). Students in the Experimental group performed better on the lower body test for which they used both plastic models and synthetic cadavers (median = 73.8% (95% CI: 72.0%-75.0%) compared to the Historical group (70.1% (95% CI: 68.3%-70.7%), P < 0.0001); however, less than half of students (49%) attributed this to the synthetic cadavers. Students' perception of laboratory resources (P < 0.0001) and learning experience (P < 0.0001) both improved with the addition of synthetic cadavers compared to using only plastic models, and 60% of students in the Experimental group agreed that the synthetic cadavers would be a key reason that they would choose that institution for undergraduate studies. This investigation showed improved student grades when plastic models and synthetic cadavers were combined, in addition to improved student perceptions of the learning experience. Results of the student questionnaires also suggested that although synthetic cadavers carry a notable up-front cost, they may be a useful recruitment tool for institutions.

Liver transplantation as a novel strategy for resolution of congenital afibrinogenemia in a pediatric patient.

Fibrinogen replacement therapy is a treatment mainstay for patients with afibrinogenemia and significant bleeding. A male infant with congenital afibrinogenemia and several spontaneous hemarthroses commenced cryoprecipitate prophylaxis but developed severe urticarial reactions. He transitioned to a human fibrinogen concentrate (HFC) (RiaSTAP® , CSL Behring; 70 mg/kg biweekly) but continued experiencing hemarthroses (estimated annualized bleeding rate [ABR]: 5-6) and severe anaphylactic reactions, despite pre- and postinfusion medications. Following switching to a new HFC (Fibryga® , Octapharma; 50 mg/kg biweekly), ABR was 0-1 with no further infusion reactions. Aged 9 years, because of limited quality of life, development of obesity and fatty liver disease, he underwent orthotopic liver transplant (OLT) under HFC coverage. Pharmacokinetic analysis guided presurgical fibrinogen levels > 150 mg/dL. No intraoperative HFC infusions were required. Coagulation profile and fibrinogen levels remained within normal limits during and posttransplant. To our knowledge, this is the first pediatric report of afibrinogenemia successfully treated with OLT.

False-positive reverse transcriptase polymerase chain reaction screening for SARS-CoV-2 in the setting of urgent head and neck surgery and otolaryngologic emergencies during the pandemic: Clinical implications.

BACKGROUND: No reports describe falsepositive reverse transcriptase polymerase chain reaction (RT-PCR) for novel coronavirus in preoperative screening. METHODS: Preoperative patients had one or two nasopharyngeal swabs, depending on low or high risk of viral transmission. Positive tests were repeated. RESULTS: Forty-three of 52 patients required two or more preoperative tests. Four (9.3%) had discrepant results (positive/negative). One of these left the coronavirus disease (COVID) unit against medical advice despite an orbital abscess, with unknown true disease status. The remaining 3 of 42 (7.1%) had negative repeat RT-PCR. Although ultimately considered falsepositives, one was sent to a COVID unit postoperatively and two had urgent surgery delayed. Assuming negative repeat RT-PCR, clear chest imaging, and lack of subsequent symptoms represent the "gold standard," RT-PCR specificity was 0.97. CONCLUSIONS: If false positives are suspected, we recommend computed tomography (CT) of the chest and repeat RT-PCR. Validated serum immunoglobulin testing may ultimately prove useful.

Comparison of international normalized ratio determined by point-of-care to standard laboratory testing before and after reversal of heparin in cardiac surgery.

STUDY OBJECTIVE: To compare point-of-care (POC) of international normalized ratio to laboratory-derived values before and after cardiopulmonary bypass, with the primary aim of evaluating for any change in the relationship between the tests. METHODS: This is a prospective observational study with 50 patients undergoing cardiac surgery enrolled. The International normalized ratio measured at two time points, precardiopulmonary bypass and after heparin reversal with protamine using both POC i-STAT and standard laboratory analysis for both time points. A difference of 0.2 between tests at either time point was considered clinically significant based on previous literature. A paired t test was used to test for a changing or statistically significant mean difference between tests. At both time points values were categorized into absolute difference of more than 0.2 or less than 0.2, and a Fisher's exact test was used to determine if an association existed between heparin reversal and a difference more than 0.2. Bland-Altman plots were also evaluated for agreement. RESULTS: A statistically and clinically significant mean difference [0.09 vs. 0.25, difference -0.163 95% confidence interval (-0.25, -0.08), P = 0.003] was seen between the laboratory and POC tests when pre and postheparin reversal samples were compared. A significantly greater number of patients had a clinically relevant difference between the tests post compared with pre (four patients vs. 18 patients, P = 0.001). Linear regression analysis of the difference compared with the means, showed significant correlation suggesting the presence of a proportional bias (pre r = 0.488, P = <0.01, post r = 0.571, P = <0.01). CONCLUSION: Clinically significant differences exist between POC and laboratory testing of international normalized ratio after heparin reversal during cardiac surgery. ClinicalTrials.gov Identifier NCT03267823.

Rapid office strain assessment (ROSA): Cross cultural validity, reliability and structural validity of the Brazilian-Portuguese version.

Forty-three occupational health professionals (observers) and 90 workers were enrolled in this study to perform the cross-cultural adaptation of the Rapid Office Strain Assessment into Brazilian Portuguese (ROSA-Br) and evaluate its psychometric properties. After cross-cultural adaptation, the measurement properties were checked in three stages: study 1: pre-testing (27 observers rated 15 office worker videos), study 2: intra- and inter-observer reliability (26 observers rated 15 office worker videos), and study 3: validity and accuracy of ROSA-Br final scores (90 office workers). For the ROSA scores, acceptable intraclass correlation coefficients were found for 75% and 86% of the intra-observer reliability comparisons for non-trained and trained observers, respectively, and for 100% of the inter-observer reliability comparisons (0.43-0.86). For construct validity, moderate correlations were observed for 70% of the comparisons between ROSA final scores and other ergonomic instruments. Moderate accuracy was observed for a ROSA-Br final score of 6 (AUC [area under the curve] = 0.72, 0.89). Taken together, these results support the use of the ROSA-Br for ergonomic field assessments and research.

Identification and Characterization of Dual Inhibitors of the USP25/28 Deubiquitinating Enzyme Subfamily.

The ubiquitin proteasome system is widely postulated to be a new and important field of drug discovery for the future, with the ubiquitin specific proteases (USPs) representing one of the more attractive target classes within the area. Many USPs have been linked to critical axes for therapeutic intervention, and the finding that USP28 is required for c-Myc stability suggests that USP28 inhibition may represent a novel approach to targeting this so far undruggable oncogene. Here, we describe the discovery of the first reported inhibitors of USP28, which we demonstrate are able to bind to and inhibit USP28, and while displaying a dual activity against the closest homologue USP25, these inhibitors show a high degree of selectivity over other deubiquitinases (DUBs). The utility of these compounds as valuable probes to investigate and further explore cellular DUB biology is highlighted by the demonstration of target engagement against both USP25 and USP28 in cells. Furthermore, we demonstrate that these inhibitors are able to elicit modulation of both the total levels and the half-life of the c-Myc oncoprotein in cells and also induce apoptosis and loss of cell viability in a range of cancer cell lines. We however observed a narrow therapeutic index compared to a panel of tissue-matched normal cell lines. Thus, it is hoped that these probes and data presented herein will further advance our understanding of the biology and tractability of DUBs as potential future therapeutic targets.

Progress towards a public chemogenomic set for protein kinases and a call for contributions.

Protein kinases are highly tractable targets for drug discovery. However, the biological function and therapeutic potential of the majority of the 500+ human protein kinases remains unknown. We have developed physical and virtual collections of small molecule inhibitors, which we call chemogenomic sets, that are designed to inhibit the catalytic function of almost half the human protein kinases. In this manuscript we share our progress towards generation of a comprehensive kinase chemogenomic set (KCGS), release kinome profiling data of a large inhibitor set (Published Kinase Inhibitor Set 2 (PKIS2)), and outline a process through which the community can openly collaborate to create a KCGS that probes the full complement of human protein kinases.