The Glycan Hole Area of HIV-1 Envelope Trimers Contributes Prominently to the Induction of Autologous Neutralization.

The human immunodeficiency virus type 1 (HIV-1) trimeric envelope glycoprotein (Env) is heavily glycosylated, creating a dense glycan shield that protects the underlying peptidic surface from antibody recognition. The absence of conserved glycans, due to missing potential N-linked glycosylation sites (PNGS), can result in strain-specific, autologous neutralizing antibody (NAb) responses. Here, we sought to gain a deeper understanding of the autologous neutralization by introducing holes in the otherwise dense glycan shields of the AMC011 and AMC016 SOSIP trimers. Specifically, when we knocked out the N130 and N289 glycans, which are absent from the well-characterized B41 SOSIP trimer, we observed stronger autologous NAb responses. We also analyzed the highly variable NAb responses induced in rabbits by diverse SOSIP trimers from subtypes A, B, and C. Statistical analysis, using linear regression, revealed that the cumulative area exposed on a trimer by glycan holes correlates with the magnitude of the autologous NAb response. IMPORTANCE Forty years after the first description of HIV-1, the search for a protective vaccine is still ongoing. The sole target for antibodies that can neutralize the virus are the trimeric envelope glycoproteins (Envs) located on the viral surface. The glycoprotein surface is covered with glycans that shield off the underlying protein components from recognition by the immune system. However, the Env trimers of some viral strains have holes in the glycan shield. Immunized animals developed antibodies against such glycan holes. These antibodies are generally strain specific. Here, we sought to gain a deeper understanding of what drives these specific immune responses. First, we show that strain-specific neutralizing antibody responses can be increased by creating artificial holes in the glycan shield. Second, when studying a diverse set of Env trimers with different characteristics, we found that the surface area of the glycan holes contributes prominently to the induction of strain-specific neutralizing antibodies.

OUTRIDER: A Statistical Method for Detecting Aberrantly Expressed Genes in RNA Sequencing Data.

RNA sequencing (RNA-seq) is gaining popularity as a complementary assay to genome sequencing for precisely identifying the molecular causes of rare disorders. A powerful approach is to identify aberrant gene expression levels as potential pathogenic events. However, existing methods for detecting aberrant read counts in RNA-seq data either lack assessments of statistical significance, so that establishing cutoffs is arbitrary, or rely on subjective manual corrections for confounders. Here, we describe OUTRIDER (Outlier in RNA-Seq Finder), an algorithm developed to address these issues. The algorithm uses an autoencoder to model read-count expectations according to the gene covariation resulting from technical, environmental, or common genetic variations. Given these expectations, the RNA-seq read counts are assumed to follow a negative binomial distribution with a gene-specific dispersion. Outliers are then identified as read counts that significantly deviate from this distribution. The model is automatically fitted to achieve the best recall of artificially corrupted data. Precision-recall analyses using simulated outlier read counts demonstrated the importance of controlling for covariation and significance-based thresholds. OUTRIDER is open source and includes functions for filtering out genes not expressed in a dataset, for identifying outlier samples with too many aberrantly expressed genes, and for detecting aberrant gene expression on the basis of false-discovery-rate-adjusted p values. Overall, OUTRIDER provides an end-to-end solution for identifying aberrantly expressed genes and is suitable for use by rare-disease diagnostic platforms.

Systematic Engineering of a Protein Nanocage for High-Yield, Site-Specific Modification.

Site-specific protein modification is a widely used strategy to attach drugs, imaging agents, or other useful small molecules to protein carriers. N-terminal modification is particularly useful as a high-yielding, site-selective modification strategy that can be compatible with a wide array of proteins. However, this modification strategy is incompatible with proteins with buried or sterically hindered N termini, such as virus-like particles (VLPs) composed of the well-studied MS2 bacteriophage coat protein. To assess VLPs with improved compatibility with these techniques, we generated a targeted library based on the MS2-derived protein cage with N-terminal proline residues followed by three variable positions. We subjected the library to assembly, heat, and chemical selections, and we identified variants that were modified in high yield with no reduction in thermostability. Positive charge adjacent to the native N terminus is surprisingly beneficial for successful extension, and over 50% of the highest performing variants contained positive charge at this position. Taken together, these studies described nonintuitive design rules governing N-terminal extensions and identified successful extensions with high modification potential.

Enzymatic Modification of N-Terminal Proline Residues Using Phenol Derivatives.

A convenient enzymatic strategy is reported for the modification of proline residues in the N-terminal positions of proteins. Using a tyrosinase enzyme isolated from Agaricus bisporus (abTYR), phenols and catechols are oxidized to highly reactive o-quinone intermediates that then couple to N-terminal proline residues in high yield. Key advantages of this bioconjugation method include (1) the use of air-stable precursors that can be prepared on large scale if needed, (2) mild reaction conditions, including low temperatures, (3) the targeting of native functional groups that can be introduced readily on most proteins, and (4) the use of molecular oxygen as the sole oxidant. This coupling strategy was successfully demonstrated for the attachment of a variety of phenol-derivatized cargo molecules to a series of protein substrates, including self-assembled viral capsids, enzymes, and a chitin binding domain (CBD). The ability of the CBD to bind to the surfaces of yeast cells was found to be unperturbed by this modification reaction.

The influence of incontinence pads moisture at the loaded skin interface.

AIM: Prolonged mechanical loading on soft tissues adjacent to bony prominences can lead to pressure ulcers. The presence of moisture at the skin interface will lower the tolerance to load. Absorbent pads manage moisture in individuals with incontinence, although their role in maintaining skin health is unknown. The present study investigated the effects of moist incontinence pads on skin physiology after periods of mechanical loading. MATERIAL AND METHODS: Twelve healthy participants were recruited to evaluate a single incontinence pad design under three moisture conditions: 0% (dry), 50% and 100% fluid capacity. For each pad condition, pressure (9 kPa) or pressure in combination with shear (3 N) was applied to the sacrum, followed by a period of off-loading. Measures included trans-epidermal water loss (TEWL) and inflammatory biomarkers sampled at the skin interface. RESULTS: Results revealed no change in TEWL in the loaded dry pad condition. By contrast, when the pads contained moisture, significant increases in TEWL were observed. These increases were reversed during off-loading. Inflammatory biomarkers, specifically IL-1α/total protein ratio, were up-regulated during dry pad loading, which recovered during off-loading. Loaded moist pads caused a significant increase in biomarkers, which remained elevated throughout the test period. CONCLUSION: The study revealed a marked compromise to stratum corneum integrity when the skin was exposed to moist incontinence pads in combination with mechanical loads. These physiological changes were largely reversed during off-loading. Incontinence pads provided some protection in the dry state, although more research is required to determine optimal clinical guidance for their use.

Substrate Sorting by a Supercharged Nanoreactor.

Compartmentalization of proteases enables spatially and temporally controlled protein degradation in cells. Here we show that an engineered lumazine synthase protein cage, which possesses a negatively supercharged lumen, can exploit electrostatic effects to sort substrates for an encapsulated protease. This proteasome-like nanoreactor preferentially cleaves positively charged polypeptides over both anionic and zwitterionic substrates, inverting the inherent substrate specificity of the guest enzyme approximately 480 fold. Our results suggest that supercharged nanochambers could provide a simple and potentially general means of conferring substrate specificity to diverse encapsulated catalysts.

Biallelic Mutations in NBAS Cause Recurrent Acute Liver Failure with Onset in Infancy.

Acute liver failure (ALF) in infancy and childhood is a life-threatening emergency. Few conditions are known to cause recurrent acute liver failure (RALF), and in about 50% of cases, the underlying molecular cause remains unresolved. Exome sequencing in five unrelated individuals with fever-dependent RALF revealed biallelic mutations in NBAS. Subsequent Sanger sequencing of NBAS in 15 additional unrelated individuals with RALF or ALF identified compound heterozygous mutations in an additional six individuals from five families. Immunoblot analysis of mutant fibroblasts showed reduced protein levels of NBAS and its proposed interaction partner p31, both involved in retrograde transport between endoplasmic reticulum and Golgi. We recommend NBAS analysis in individuals with acute infantile liver failure, especially if triggered by fever.

Adaptation of a MR imaging protocol into a real-time clinical biometric ultrasound protocol for persons with spinal cord injury at risk for deep tissue injury: A reliability study.

BACKGROUND: High strain in soft tissues that overly bony prominences are considered a risk factor for pressure ulcers (PUs) following spinal cord impairment (SCI) and have been computed using Finite Element methods (FEM). The aim of this study was to translate a MRI protocol into ultrasound (US) and determine between-operator reliability of expert sonographers measuring diameter of the inferior curvature of the ischial tuberosity (IT) and the thickness of the overlying soft tissue layers on able-bodied (AB) and SCI using real-time ultrasound. MATERIAL AND METHODS: Part 1: Fourteen AB participants with a mean age of 36.7 ± 12.09 years with 7 males and 7 females had their 3 soft tissue layers in loaded and unloaded sitting measured independently by 2 sonographers: tendon/muscle, skin/fat and total soft tissue and the diameter of the IT in its short and long axis. Part 2: Nineteen participants with SCI were screened, three were excluded due to abnormal skin signs, and eight participants (42%) were excluded for abnormal US signs with normal skin. Eight SCI participants with a mean age of 31.6 ± 13.6 years and all male with 4 paraplegics and 4 tetraplegics were measured by the same sonographers for skin, fat, tendon, muscle and total. Skin/fat and tendon/muscle were computed. RESULTS: AB between-operator reliability was good (ICC = 0.81-0.90) for 3 soft tissues layers in unloaded and loaded sitting and poor for both IT short and long axis (ICC = -0.028 and -0.01). SCI between-operator reliability was good in unloaded and loaded for total, muscle, fat, skin/fat, tendon/muscle (ICC = 0.75-0.97) and poor for tendon (ICC = 0.26 unloaded and ICC = -0.71 loaded) and skin (ICC = 0.37 unloaded and ICC = 0.10). CONCLUSION: A MRI protocol was successfully adapted for a reliable 3 soft tissue layer model and could be used in a 2-D FEM model designed to estimate soft tissue strain as a novel risk factor for the development of a PU.

Complex mechanical conditioning of cell-seeded agarose constructs can influence chondrocyte biosynthetic activity.

Articular cartilage with its inherently poor capacity for self-regeneration represents a primary target for tissue engineering strategies, with approaches focusing on the in vitro generation of neo-cartilage using chondrocyte-seeded 3D scaffolds subjected to mechanical conditioning. Although uniaxial compression regimens have significantly up-regulated proteoglycan synthesis, their effects on the synthesis of collagen have been modest. Articular cartilage is subjected to shear forces during joint motion. Accordingly, this study utilized an apparatus to apply biaxial loading to chondrocytes seeded within agarose constructs with endplates. The chondrocytes yielded a monotonic increase in proteoglycan synthesis both in free swelling culture up to day 8 and when the constructs were subjected to dynamic compression alone (15% amplitude at a frequency of 1 Hz for 48 h). However, when dynamic shear (10% amplitude at 1 Hz) was superimposed on dynamic compression, total collagen synthesis was also up-regulated, within 3 days of culture, without compromising proteoglycan synthesis. Histological analysis revealed marked collagen deposition around individual chondrocytes. A significant proportion (50%) of collagen was released into the culture medium, suggesting that it had only been partially synthesized in its mature state. The overall biosynthetic activity was enhanced more when the biaxial stimulation was applied in a continuous mode as opposed to intermittent loading. Results of the present study strongly suggest that proteoglycan and collagen synthesis may be triggered by uncoupled mechanosensitive cellular responses. The proposed in vitro model and the prescribed conditioning protocols demonstrated that a short pre-culture period is preferable to long free swelling culture condition as it enables a significantly higher up-regulation of collagen. Biotechnol. Bioeng. 2017;114: 1614-1625. © 2017 Wiley Periodicals, Inc.

Penile compression clamps: A model of the internal mechanical state of penile soft tissues.

AIMS: Prostate cancer is the most frequently diagnosed male cancer and urinary incontinence represents a major consequence following surgery. Penile compression clamps (PCCs) which externally occlude the urethra may be used to manage the incontinence. Despite potential complication of PCCs, such as deformation-inflicted tissue damage, to date, there are no reported biomechanical criteria for design of PCCs, in terms of quantitative parameters for evaluating the safety-versus-efficacy of existing or future designs. METHODS: We developed a set of computational three-dimensional models of the penis, to which compression was applied using five generic PCC designs. The internal mechanical states of the soft tissues of the penis were then analysed using finite element simulations. RESULTS: Stresses in skin, fat, and tunica albuginea regularly exceeded 10 kPa (75 mmHg). Cuff-type and knurl-type PCCs pose the highest potential risks to tissue health with elevated tissue stresses around the entire penile perimeter (cuff) or urethral stress concentrations (knurl). The soft and contoured PCCs produced the lowest values of these mechanical parameters. CONCLUSIONS: The present study identified design characteristics, including envelopment, adaptability, and durability which provide the safest mechanical conditions in the penis and thus minimize the risk of tissue damage while still managing incontinence. Such data should help to design a safer clamp.

Negative feedback buffers effects of regulatory variants.

Mechanisms conferring robustness against regulatory variants have been controversial. Previous studies suggested widespread buffering of RNA misexpression on protein levels during translation. We do not find evidence that translational buffering is common. Instead, we find extensive buffering at the level of RNA expression, exerted through negative feedback regulation acting in trans, which reduces the effect of regulatory variants on gene expression. Our approach is based on a novel experimental design in which allelic differential expression in a yeast hybrid strain is compared to allelic differential expression in a pool of its spores. Allelic differential expression in the hybrid is due to cis-regulatory differences only. Instead, in the pool of spores allelic differential expression is not only due to cis-regulatory differences but also due to local trans effects that include negative feedback. We found that buffering through such local trans regulation is widespread, typically compensating for about 15% of cis-regulatory effects on individual genes. Negative feedback is stronger not only for essential genes, indicating its functional relevance, but also for genes with low to middle levels of expression, for which tight regulation matters most. We suggest that negative feedback is one mechanism of Waddington's canalization, facilitating the accumulation of genetic variants that might give selective advantage in different environments.

NPCC4: New York City climate risk information 2022-observations and projections.

New York City (NYC) faces many challenges in the coming decades due to climate change and its interactions with social vulnerabilities and uneven urban development patterns and processes. This New York City Panel on Climate Change (NPCC) report contributes to the Panel's mandate to advise the city on climate change and provide timely climate risk information that can inform flexible and equitable adaptation pathways that enhance resilience to climate change. This report presents up-to-date scientific information as well as updated sea level rise projections of record. We also present a new methodology related to climate extremes and describe new methods for developing the next generation of climate projections for the New York metropolitan region. Future work by the Panel should compare the temperature and precipitation projections presented in this report with a subset of models to determine the potential impact and relevance of the "hot model" problem. NPCC4 expects to establish new projections-of-record for precipitation and temperature in 2024 based on this comparison and additional analysis. Nevertheless, the temperature and precipitation projections presented in this report may be useful for NYC stakeholders in the interim as they rely on the newest generation of global climate models.

NPCC4: Climate risk and equity-advancing knowledge toward a sustainable future | Conclusions.

This chapter provides an overview of the major themes, findings, and recommendations from NPCC4. It presents summary statements from each chapter of the assessment which identify salient and pressing issues raised and provides recommendations for future research and for enhancement of climate resiliency. The chapter also outlines a set of broader recommendations for future NPCC work and identifies some key topics for the next assessment.

NPCC4: Tail risk, climate drivers of extreme heat, and new methods for extreme event projections.

We summarize historic New York City (NYC) climate change trends and provide the latest scientific analyses on projected future changes based on a range of global greenhouse gas emissions scenarios. Building on previous NPCC assessment reports, we describe new methods used to develop the projections of record for sea level rise, temperature, and precipitation for NYC, across multiple emissions pathways and analyze the issue of the "hot models" associated with the 6th phase of the Coupled Model Intercomparison Project (CMIP6) and their potential impact on NYC's climate projections. We describe the state of the science on temperature variability within NYC and explain both the large-scale and regional dynamics that lead to extreme heat events, as well as the local physical drivers that lead to inequitable distributions of exposure to extreme heat. We identify three areas of tail risk and potential for its mischaracterization, including the physical processes of extreme events and the effects of a changing climate. Finally, we review opportunities for future research, with a focus on the hot model problem and the intersection of spatial resolution of projections with gaps in knowledge in the impacts of the climate signal on intraurban heat and heat exposure.

Heterologous prime-boost vaccination drives early maturation of HIV broadly neutralizing antibody precursors in humanized mice.

A protective HIV vaccine will likely need to induce broadly neutralizing antibodies (bnAbs). Vaccination with the germline-targeting immunogen eOD-GT8 60mer adjuvanted with AS01B was found to induce VRC01-class bnAb precursors in 97% of vaccine recipients in the IAVI G001 phase 1 clinical trial; however, heterologous boost immunizations with antigens more similar to the native glycoprotein will be required to induce bnAbs. Therefore, we designed core-g28v2 60mer, a nanoparticle immunogen to be used as a first boost after eOD-GT8 60mer priming. We found, using a humanized mouse model approximating human conditions of VRC01-class precursor B cell diversity, affinity, and frequency, that both protein- and mRNA-based heterologous prime-boost regimens induced VRC01-class antibodies that gained key mutations and bound to near-native HIV envelope trimers lacking the N276 glycan. We further showed that VRC01-class antibodies induced by mRNA-based regimens could neutralize pseudoviruses lacking the N276 glycan. These results demonstrated that heterologous boosting can drive maturation toward VRC01-class bnAb development and supported the initiation of the IAVI G002 phase 1 trial testing mRNA-encoded nanoparticle prime-boost regimens.

Human immunoglobulin gene allelic variation impacts germline-targeting vaccine priming.

Vaccine priming immunogens that activate germline precursors for broadly neutralizing antibodies (bnAbs) have promise for development of precision vaccines against major human pathogens. In a clinical trial of the eOD-GT8 60mer germline-targeting immunogen, higher frequencies of vaccine-induced VRC01-class bnAb-precursor B cells were observed in the high dose compared to the low dose group. Through immunoglobulin heavy chain variable (IGHV) genotyping, statistical modeling, quantification of IGHV1-2 allele usage and B cell frequencies in the naive repertoire for each trial participant, and antibody affinity analyses, we found that the difference between dose groups in VRC01-class response frequency was best explained by IGHV1-2 genotype rather than dose and was most likely due to differences in IGHV1-2 B cell frequencies for different genotypes. The results demonstrate the need to define population-level immunoglobulin allelic variations when designing germline-targeting immunogens and evaluating them in clinical trials.

Human immunoglobulin gene allelic variation impacts germline-targeting vaccine priming.

Vaccine priming immunogens that activate germline precursors for broadly neutralizing antibodies (bnAbs) have promise for development of precision vaccines against major human pathogens. In a clinical trial of the eOD-GT8 60mer germline-targeting immunogen, higher frequencies of vaccine-induced VRC01-class bnAb-precursor B cells were observed in the high dose compared to the low dose group. Through immunoglobulin heavy chain variable (IGHV) genotyping, statistical modeling, quantification of IGHV1-2 allele usage and B cell frequencies in the naive repertoire for each trial participant, and antibody affinity analyses, we found that the difference between dose groups in VRC01-class response frequency was best explained by IGHV1-2 genotype rather than dose and was most likely due to differences in IGHV1-2 B cell frequencies for different genotypes. The results demonstrate the need to define population-level immunoglobulin allelic variations when designing germline-targeting immunogens and evaluating them in clinical trials. One-Sentence Summary: Human genetic variation can modulate the strength of vaccine-induced broadly neutralizing antibody precursor B cell responses.

A beginners guide to SNP calling from high-throughput DNA-sequencing data.

High-throughput DNA sequencing (HTS) is of increasing importance in the life sciences. One of its most prominent applications is the sequencing of whole genomes or targeted regions of the genome such as all exonic regions (i.e., the exome). Here, the objective is the identification of genetic variants such as single nucleotide polymorphisms (SNPs). The extraction of SNPs from the raw genetic sequences involves many processing steps and the application of a diverse set of tools. We review the essential building blocks for a pipeline that calls SNPs from raw HTS data. The pipeline includes quality control, mapping of short reads to the reference genome, visualization and post-processing of the alignment including base quality recalibration. The final steps of the pipeline include the SNP calling procedure along with filtering of SNP candidates. The steps of this pipeline are accompanied by an analysis of a publicly available whole-exome sequencing dataset. To this end, we employ several alignment programs and SNP calling routines for highlighting the fact that the choice of the tools significantly affects the final results.

Rare variants in TMEM132D in a case-control sample for panic disorder.

Genome-wide association studies have identified common variants associated with common diseases. Most variants, however, explain only a small proportion of the estimated heritability, suggesting that rare variants might contribute to a larger extent to common diseases than assumed to date. Here, we use next-generation sequencing to test whether such variants contribute to the risk for anxiety disorders by re-sequencing 40 kb including all exons of the TMEM132D locus which we have previously shown to be associated with panic disorder and anxiety severity measures. DNA from 300 patients suffering from anxiety disorders, mostly panic disorder (84.7%), and 300 healthy controls was screened for the presence of genetic variants using next-generation re-sequencing in a pooled approach. Results were verified by individual re-genotyping. We identified 371 variants of which 247 had not been reported before, including 15 novel non-synonymous variants. The majority, 76% of these variants had a minor allele frequency less than 5%. While we did not identify additional common variants in TMEM132D associated with panic disorders, we observed an overrepresentation of presumably functional coding variants in healthy controls as compared to cases as well as a higher rate of private coding variants in cases, with one non-synonymous coding variant present in four patients but not in any of the matched controls nor in over 5,500 individuals of different ethnic origins from publicly available re-sequencing datasets. Our data suggest that not only common but also putatively functional and/or rare variants within TMEM132D might contribute to the risk to develop anxiety disorders.

Synthetic Nanoclay Gels Do Not Cause Skin Irritation in Healthy Human Volunteers.

Synthetic clays are promising biomaterials for delivery of therapeutic molecules in regenerative medicine. However, before their use can be translated into clinical applications, their safety must be assessed in human volunteers. The aim of this study was to test the hypothesis that a synthetic nanoclay (LAPONITE) does not cause irritation to the human skin. To achieve this, a nanoclay gel at two different concentrations (1.5 and 3% w/v) was applied on the forearm of healthy volunteers for 24 h. 1% sodium lauryl sulfate (SLS) and 3% (w/v) polyacrylic acid were used as the positive and negative controls, respectively. The compromise in the skin barrier function was measured by trans-epidermal water loss (TEWL), erythema by spectroscopic measurements, and skin inflammatory biomarkers (IL-1α and IL-1RA) by the enzyme-linked immunosorbent assay. We found that the nanoclay caused no prolonged increase in TEWL, erythema, or induction of inflammatory cytokines. This was in contrast to 1% SLS, a known irritant, which induced significant increases in both skin erythema and TEWL. We conclude that the nanoclay is not an irritant and is thus suitable for therapeutic interventions at the skin surface.