Model-directed generation of CRISPR-Cas13a guide RNAs designs artificial sequences that improve nucleic acid detection.

Generating maximally-fit biological sequences has the potential to transform CRISPR guide RNA design as it has other areas of biomedicine. Here, we introduce model-directed exploration algorithms (MEAs) for designing maximally-fit, artificial CRISPR-Cas13a guides-with multiple mismatches to any natural sequence-that are tailored for desired properties around nucleic acid diagnostics. We find that MEA-designed guides offer more sensitive detection of diverse pathogens and discrimination of pathogen variants compared to guides derived directly from natural sequences, and illuminate interpretable design principles that broaden Cas13a targeting.

Simplified Cas13-based assays for the fast identification of SARS-CoV-2 and its variants.

The widespread transmission and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) call for rapid nucleic acid diagnostics that are easy to use outside of centralized clinical laboratories. Here we report the development and performance benchmarking of Cas13-based nucleic acid assays leveraging lyophilised reagents and fast sample inactivation at ambient temperature. The assays, which we named SHINEv.2 (for 'streamlined highlighting of infections to navigate epidemics, version 2'), simplify the previously reported RNA-extraction-free SHINEv.1 technology by eliminating heating steps and the need for cold storage of the reagents. SHINEv.2 detected SARS-CoV-2 in nasopharyngeal samples with 90.5% sensitivity and 100% specificity (benchmarked against the reverse transcription quantitative polymerase chain reaction) in less than 90 min, using lateral-flow technology and incubation in a heat block at 37 °C. SHINEv.2 also allows for the visual discrimination of the Alpha, Beta, Gamma, Delta and Omicron SARS-CoV-2 variants, and can be run without performance losses by using body heat. Accurate, easy-to-use and equipment-free nucleic acid assays could facilitate wider testing for SARS-CoV-2 and other pathogens in point-of-care and at-home settings.

Designing sensitive viral diagnostics with machine learning.

Design of nucleic acid-based viral diagnostics typically follows heuristic rules and, to contend with viral variation, focuses on a genome's conserved regions. A design process could, instead, directly optimize diagnostic effectiveness using a learned model of sensitivity for targets and their variants. Toward that goal, we screen 19,209 diagnostic-target pairs, concentrated on CRISPR-based diagnostics, and train a deep neural network to accurately predict diagnostic readout. We join this model with combinatorial optimization to maximize sensitivity over the full spectrum of a virus's genomic variation. We introduce Activity-informed Design with All-inclusive Patrolling of Targets (ADAPT), a system for automated design, and use it to design diagnostics for 1,933 vertebrate-infecting viral species within 2 hours for most species and within 24 hours for all but three. We experimentally show that ADAPT's designs are sensitive and specific to the lineage level and permit lower limits of detection, across a virus's variation, than the outputs of standard design techniques. Our strategy could facilitate a proactive resource of assays for detecting pathogens.

Equipment-free detection of SARS-CoV-2 and Variants of Concern using Cas13.

The COVID-19 pandemic, and the recent rise and widespread transmission of SARS-CoV-2 Variants of Concern (VOCs), have demonstrated the need for ubiquitous nucleic acid testing outside of centralized clinical laboratories. Here, we develop SHINEv2, a Cas13-based nucleic acid diagnostic that combines quick and ambient temperature sample processing and lyophilized reagents to greatly simplify the test procedure and assay distribution. We benchmarked a SHINEv2 assay for SARS-CoV-2 detection against state-of-the-art antigen-capture tests using 96 patient samples, demonstrating 50-fold greater sensitivity and 100% specificity. We designed SHINEv2 assays for discriminating the Alpha, Beta, Gamma and Delta VOCs, which can be read out visually using lateral flow technology. We further demonstrate that our assays can be performed without any equipment in less than 90 minutes. SHINEv2 represents an important advance towards rapid nucleic acid tests that can be performed in any location.

A community-based qualitative study on the experience and understandings of intimate partner violence and HIV vulnerability from the perspectives of female sex workers and male intimate partners in North Karnataka state, India.

BACKGROUND: Research has increasingly documented the important role that violence by clients and the police play in exacerbating HIV vulnerability for women in sex work. However few studies have examined violence in the intimate relationships of women in sex work, or drawn on community partnerships to explore the social dynamics involved. A community-based participatory research study was undertaken by community and academic partners leading intimate partner violence (IPV) and HIV prevention programs in Bagalkot district, Karnataka state, India. The purpose was to explore the experience and understandings of intimate partner violence and HIV/AIDS among women in sex work and their intimate partners in Bagalkot that would inform both theory and practice. METHODS: A community-based, interpretive qualitative methodology was used. Data was collected between July and October 2014 through in-depth interviews with 38 participants, including 10 couples, 13 individual female sex workers, and 5 individual male intimate partners. Purposive sampling was done to maximize variation on socio-demographic characteristics. Thematic content analysis was conducted through coding and categorization for each interview question in NVivo 10.0, followed by collaborative analysis to answer the research questions. RESULTS: The results showed that an array of interrelated, multi-level factors underlay the widespread acceptance and perpetuation of violence and lack of condom use in participants' intimate relationships. These included individual expectations that justified violence and reflected societal gender norms, compounded by stigma, legal and economic constraints relating to sex work. The results demonstrate that structural vulnerability to IPV and HIV must be addressed not only on the individual and relationship levels to resolve relevant triggers of violence and lack of condom use, but also the societal-level to address gender norms and socio-economic constraints among women in sex work and their partners. CONCLUSION: The study contributes to a better understanding on the interplay of individual agency and structural forces at a time when researchers and program planners are increasingly pondering how best to address complex and intersecting social and health issues. Ongoing research should assess the generalizability of the results and the effectiveness of structural interventions aiming to reduce IPV and HIV vulnerability in other contexts.

Pursuing Authenticity From Process to Outcome in a Community-Based Participatory Research Study of Intimate Partner Violence and HIV Vulnerability in North Karnataka, India.

Community-based participatory research has been seen to hold great promise by researchers aiming to bridge research and action in global health programs and practice. However, there is still much debate around whether achieving authenticity in terms of in-depth collaboration between community and academic partners is possible while pursuing academic expectations for quality. This article describes the community-based methodology for a qualitative study to explore intimate partner violence and HIV/AIDS among women in sex work, or female sex workers, and their male partners in Karnataka, South India. Developed through collaborative processes, the study methodology followed an interpretive approach to qualitative inquiry, with three key components including long-term partnerships, knowledge exchange, and orientation toward action. We then discuss lessons learned on how to pursue authenticity in terms of truly collaborative processes with inherent value that also contribute to, rather than hinder, the instrumental goal of enhancing the quality and relevance of the research outcomes.

Particulate matter pollution in the coal-producing regions of the Appalachian Mountains: Integrated ground-based measurements and satellite analysis.

This study integrates the relationship between measured surface concentrations of particulate matter 10 µm or less in diameter (PM10), satellite-derived aerosol optical depth (AOD), and meteorology in Roda, Virginia, during 2008. A multiple regression model was developed to predict the concentrations of particles 2.5 µm or less in diameter (PM2.5) at an additional location in the Appalachia region, Bristol, TN. The model was developed by combining AOD retrievals from Moderate Resolution Imaging Spectro-radiometer (MODIS) sensor on board the EOS Terra and Aqua Satellites with the surface meteorological observations. The multiple regression model predicted PM2.5 (r2 = 0.62), and the two-variable (AOD-PM2.5) model predicted PM2.5 (r2 = 0.4). The developed model was validated using particulate matter recordings and meteorology observations from another location in the Appalachia region, Hazard, Kentucky. The model was extrapolated to the Roda, VA, sampling site to predict PM2.5 mass concentrations. We used 10 km x 10 km resolution MODIS 550 nm AOD to predict ground level PM2.5. For the relevant period in 2008, in Roda, VA, the predicted PM2.5 mass concentration is 9.11 ± 5.16 µg m-3 (mean ± 1SD). IMPLICATIONS: This is the first study that couples ground-based Particulate Matter measurements with satellite retrievals to predict surface air pollution at Roda, Virginia. Roda is representative of the Appalachian communities that are commonly located in narrow valleys, or "hollows," where homes are placed directly along the roads in a region of active mountaintop mining operations. Our study suggests that proximity to heavy coal truck traffic subjects these communities to chronic exposure to coal dust and leads us to conclude that there is an urgent need for new regulations to address the primary sources of this particulate matter.

Understanding the role of peer group membership in reducing HIV-related risk and vulnerability among female sex workers in Karnataka, India.

In Karnataka state, South India, we analyzed the role of membership in peer groups in reducing HIV-related risk and vulnerability among female sex workers (FSWs). Data from three surveys conducted in Karnataka, a behavioral tracking survey and two rounds of integrated biological and behavioral assessments (IBBAs), were analyzed. Using propensity score matching, we examined the impact of group membership on selected outcomes, including condom use, experience of violence, access to entitlements, and the prevalence of sexually transmitted infections, including HIV infection. Focus group discussions were conducted with the FSWs to better understand their perceptions regarding membership in peer groups. Peer group members participating in the IBBAs had a lower prevalence of gonorrhea and/or chlamydia (5.2 vs 9.6%, p<0.001), and of syphilis (8.2 vs 10.3%, p<0.05), compared to non-members. The average treatment effect for selected outcome measures, from the propensity score matching, showed that FSWs who were members of any peer group reported significantly less experience of violence in the past six months, were less likely to have bribed police to avoid trouble in the past six months, and were more likely to have obtained at least one formal identification document in the past five years, compared to non-members. In focus group discussions, group members indicated that they had more confidence in dealing with situations of forced sex and violence. Including community mobilization and peer group formation in the context of HIV prevention programing can reduce HIV-related risk and vulnerability among FSWs.

Hide depilation and feather disintegration studies with keratinolytic serine protease from a novel Bacillus subtilis isolate.

Keratinases play an important role in biotechnological applications such as improvement of feather meal, enzymatic dehairing and production of amino acids or peptides from high molecular weight substrates. Bacillus subtilis P13, isolated from Vajreshwari hot spring (45-50 degrees C) near Mumbai, India, produces a neutral serine protease and has an optimum temperature of 65 degrees C. This enzyme preparation was keratinolytic in nature and could disintegrate whole chicken feathers, except for the remnants of shafts. The enzyme preparation also exhibited depilation of goat hides with the recovery of intact animal hair. The enzyme preparation could release peptides from ground feathers and bring about their weight reduction; however, similar action on hair was relatively weak. A single major PMSF-sensitive protease band could be detected upon zymogram analysis, indicating that a single enzyme may be responsible for feather degradation and hide depilation. The importance of these findings in the biotechnological application for feather and leather industries is discussed.