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1.
Nat Commun ; 15(1): 4685, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38824129

RESUMO

Designing ultralight conductive aerogels with tailored electrical and mechanical properties is critical for various applications. Conventional approaches rely on iterative, time-consuming experiments across a vast parameter space. Herein, an integrated workflow is developed to combine collaborative robotics with machine learning to accelerate the design of conductive aerogels with programmable properties. An automated pipetting robot is operated to prepare 264 mixtures of Ti3C2Tx MXene, cellulose, gelatin, and glutaraldehyde at different ratios/loadings. After freeze-drying, the aerogels' structural integrity is evaluated to train a support vector machine classifier. Through 8 active learning cycles with data augmentation, 162 unique conductive aerogels are fabricated/characterized via robotics-automated platforms, enabling the construction of an artificial neural network prediction model. The prediction model conducts two-way design tasks: (1) predicting the aerogels' physicochemical properties from fabrication parameters and (2) automating the inverse design of aerogels for specific property requirements. The combined use of model interpretation and finite element simulations validates a pronounced correlation between aerogel density and compressive strength. The model-suggested aerogels with high conductivity, customized strength, and pressure insensitivity allow for compression-stable Joule heating for wearable thermal management.

2.
bioRxiv ; 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38463963

RESUMO

Low-abundance members of microbial communities are difficult to study in their native habitats. This includes Escherichia coli, a minor, but common inhabitant of the gastrointestinal tract and opportunistic pathogen, including of the urinary tract, where it is the primary pathogen. While multi-omic analyses have detailed critical interactions between uropathogenic Escherichia coli (UPEC) and the bladder that mediate UTI outcome, comparatively little is known about UPEC in its pre-infection reservoir, partly due to its low abundance there (<1% relative abundance). To accurately and sensitively explore the genomes and transcriptomes of diverse E. coli in gastrointestinal communities, we developed E. coli PanSelect which uses a set of probes designed to specifically recognize and capture E. coli's broad pangenome from sequencing libraries. We demonstrated the ability of E. coli PanSelect to enrich, by orders of magnitude, sequencing data from diverse E. coli using a mock community and a set of human stool samples collected as part of a cohort study investigating drivers of recurrent urinary tract infections (rUTI). Comparisons of genomes and transcriptomes between E. coli residing in the gastrointestinal tracts of women with and without a history of rUTI suggest that rUTI gut E. coli are responding to increased levels of oxygen and nitrate, suggestive of mucosal inflammation, which may have implications for recurrent disease. E. coli PanSelect is well suited for investigations of native in vivo biology of E. coli in other environments where it is at low relative abundance, and the framework described here has broad applicability to other highly diverse, low abundance organisms.

3.
Front Psychol ; 15: 1235920, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38379621

RESUMO

Background: Minority stress theory views social support as a protective factor against the effects of minority-specific stressors like internalized homophobia (IH) on mental health in sexual minority populations. However, much of the empirical validation of this theory has been conducted within predominantly White samples, resulting in a limited understanding of how the theory applies to Black sexual minority individuals. Current examinations of social support fail to capture the nuances of how Black sexual minority men may access support systems differently, resulting in a need to investigate how social support, IH, and mental health operate for Black sexual minority men. This study examined relationships between IH, depression, and different types of social support (i.e., family, friends, Black community, gay community) using a mediation model. Methods: We used data from the POWER (Promoting Our Worth Equity and Resilience) Study, which recruited Black sexual minority men at Black Pride events across six cities in the United States from 2014 to 2017, to test four mediation pathways concurrently in Stata 17. Participants (N = 4,430) completed a questionnaire assessing a variety of health and life domains, including depression symptoms, internalized homophobia, and social support. Results: IH was positively associated with depression. Lower levels of family, friend, and Black community support were all positively associated with depression symptoms. Additionally, IH was positively associated with all types of support. Finally, family, friend, and Black community support partially mediated the relationship between IH and depression. Conclusions and implications: Results suggest that the relationship between social support and depression is complex for Black sexual minority men. Findings suggest family support is an important factor for clinical intervention efforts targeting depression, and that gay community support systems should assess how their environments can better support Black sexual minority men. Overall, findings demonstrate the necessity of future examination of how social support functions differently within Black sexual minority communities.

4.
bioRxiv ; 2023 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-37786711

RESUMO

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.

5.
Front Psychiatry ; 14: 1205581, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37547195

RESUMO

Background: Queer youth experience high rates of depression and suicidality. These disparities stem from stigma-based stressors, including internalized stigma (i.e., negative social views that minoritized individuals internalize about their own identity). Given the importance of this factor in understanding mental health disparities among queer youth, we completed a systematic review and meta-analysis examining the relationships between internalized stigma and outcomes of depression and suicide risk (i.e., suicidal ideation, non-suicidal self-injury, and suicidal behavior). Methods: We followed the PRISMA standards. Six bibliographic databases were searched for studies in the United States from September 2008 to March 2022. Dual independent screening of search results was performed based on a priori inclusion criteria. Results: A total of 22 studies were included for data extraction and review. Most studies examined general internalized homophobia, with few examining internalized biphobia or transphobia. Many studies examined depression as an outcome, few studies examined suicidal ideation or behavior, and no studies examined non-suicidal self-injury. Meta-analyses model results show the association between general internalized queer stigma and depressive symptoms ranged r = 0.19, 95% CI [0.14, 0.25] to r = 0.24, 95% CI [0.19, 0.29], the latter reflecting more uniform measures of depression. The association between internalized transphobia and depressive outcomes was small and positive (r = 0.21, 95% CI [-0.24, 0.67]). General internalized queer stigma and suicidal ideation had a very weak positive association (r = 0.07, 95% CI [-0.27, 0.41]) and an even smaller, weaker positive association with suicide attempt (r = 0.02, 95% CI [0.01, 0.03]). Conclusion: Implications for clinical practice, policy, and future research are discussed.

6.
Subst Abuse ; 17: 11782218231181274, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37342586

RESUMO

Lesbian, gay, bisexual, and transgender (LGBT) individuals have a high prevalence of substance use disorders (SUDs) and experience unique barriers to treatment. Little is known about the characteristics of SUD treatment facilities providing LGBT-tailored programs at the outpatient and residential levels of care. The purpose of this study is to examine the availability of LGBT-tailored programs in outpatient and residential SUD treatment facilities. Using the National Survey of Substance Abuse Treatment Services 2020, we conducted logistic regression to examine facility characteristics, including ownership, pay assistance, region, outreach, and telehealth services, associated with having an LGBT-tailored program among SUD treatment facilities. Outpatient facilities that were for-profit, had pay assistance, had community outreach services, and provided telemedicine/telehealth were more likely to have an LGBT-tailored program. Those that were government-owned, in the Midwest, and that accepted Medicaid were less likely to have an LGBT-tailored program. Residential facilities that were in the West, for-profit, and had community outreach services were more likely to have an LGBT-tailored program. This study offers a national examination of the availability of LGBT-tailored programs in SUD treatment facilities. Differences in availability based on ownership, region, pay assistance, and outreach highlight potential gaps in treatment availability.

7.
Trauma Violence Abuse ; 24(4): 2196-2209, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-35465778

RESUMO

Although there has been increased attention to campus sexual and relationship violence (SRV) because of Title IX and the #MeToo movement, much of that attention has focused on victimization of cisgender heterosexual women. This scoping review uncovers information from empirical studies on what is known about LGBTQ+ (e.g., lesbian, gay, bisexual, transgender, queer, and nonbinary) students' experiences of campus SRV. Using rigorous scoping review methods (i.e., searches of 15 databases, searches of expert websites, hand searching, reference harvesting, and forward citation chaining), we identified 60 documents published since 2000 that contained findings from empirical studies related to LGBTQ+ students and SRV on U.S. college and university campuses. Through content analysis, we summarized findings around five key themes: (1) extent and types of victimization, (2) negative outcomes, (3) knowledge of and attitudes about SRV, (4) perspectives on SRV services and prevention education programs, and (5) recommendations from study authors based on their findings. Implications for research, practice, and policy based on these findings are discussed.


Assuntos
Homossexualidade Feminina , Minorias Sexuais e de Gênero , Humanos , Feminino , Comportamento Sexual , Violência , Estudantes
8.
Am Surg ; 89(11): 4715-4719, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36169356

RESUMO

BACKGROUND: Injured patients in hemorrhagic shock have a survival benefit with massive transfusion protocol (MTP). While there are many published studies on the transfusion management of massively bleeding patients, the risk of alloimmunization in patients that have received products during an MTP activation is relatively unknown. Therefore, we sought to determine the frequency of new antibody formation in MTP patients that received blood products from an uncrossmatched megapack. MATERIALS AND METHODS: We conducted a retrospective data review of patients who underwent an MTP activation for trauma resuscitation between May 2014 and July 2020. Data were collected from patients who met the following criteria: MTP was activated, the patients received at least one unit of packed red blood cells, one unit of fresh frozen plasma, one unit of platelets, and had a repeat type and screen within 6 weeks of transfusion. These inclusion criteria resulted in 28 patients over the 6-year timeframe. RESULTS: Overall, the risk of alloimmunization secondary to MTP is 3.6% in our trauma patient population. The newly developed antibodies post-MTP are considered clinically significant, meaning they can cause hemolysis if exposed to donor red blood cells containing those antigens. DISCUSSION: Blood products should be given preferentially over crystalloids to acutely bleeding patients to prevent ischemic injury during an MTP activation despite the risk of alloimmunization. In our single-institution study, the alloimmunization rate in massive transfusions where patients receive uncrossmatched red blood cells is similar to those receiving crossmatched red blood cells.


Assuntos
Formação de Anticorpos , Ferimentos e Lesões , Humanos , Estudos Retrospectivos , Incidência , Transfusão de Sangue/métodos , Hemorragia , Ressuscitação/métodos , Centros de Traumatologia
9.
Int J Lang Commun Disord ; 58(3): 892-909, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36541222

RESUMO

BACKGROUND & AIM: To assess consonant proficiency and velopharyngeal function in 10-year-old children born with unilateral cleft lip and palate (UCLP) within the Scandcleft project. METHODS & PROCEDURES: Three parallel group, randomized, clinical trials were undertaken as an international multicentre study by nine cleft teams in five countries. Three different surgical protocols for primary palate repair (Arm B-Lip and soft palate closure at 3-4 months, hard palate closure at 36 months, Arm C-Lip closure at 3-4 months, hard and soft palate closure at 12 months, and Arm D-Lip closure at 3-4 months combined with a single-layer closure of the hard palate using a vomer flap, soft palate closure at 12 months) were tested against a common procedure (Arm A-Lip and soft palate closure at 3-4 months followed by hard palate closure at 12 months) in the total cohort of 431 children born with a non-syndromic UCLP. Speech audio and video recordings of 399 children were available and perceptually analysed. Percentage of consonants correct (PCC) from a naming test, an overall rating of velopharyngeal competence (VPC) (VPC-Rate), and a composite measure (VPC-Sum) were reported. OUTCOMES & RESULTS: The mean levels of consonant proficiency (PCC score) in the trial arms were 86-92% and between 58% and 83% of the children had VPC (VPC-Sum). Only 50-73% of the participants had a consonant proficiency level with their peers. Girls performed better throughout. Long delay of the hard palate repair (Arm B) indicated lower PCC and simultaneous hard and soft palate closure higher (Arm C). However, the proportion of participants with primary VPC (not including velopharyngeal surgeries) was highest in Arm B (68%) and lowest in Arm C (47%). CONCLUSIONS & IMPLICATIONS: The speech outcome in terms of PCC and VPC was low across the trials. The different protocols had their pros and cons and there is no obvious evidence to recommend any of the protocols as superior. Aspects other than primary surgical method, such as time after velopharyngeal surgery, surgical experience, hearing level, language difficulties and speech therapy, need to be thoroughly reviewed for a better understanding of what has affected speech outcome at 10 years. WHAT THIS PAPER ADDS: What is already known on the subject Speech outcomes at 10 years of age in children treated for UCLP are sparse and contradictory. Previous studies have examined speech outcomes and the relationship with surgical intervention in 5-year-olds. What this study adds to the existing knowledge Speech outcomes based on standardized assessment in a large group of 10-year-old children born with UCLP and surgically treated according to different protocols are presented. While speech therapy had been provided, a large proportion of the children across treatment protocols still needed further speech therapy. What are the potential or actual clinical implications of this work? Aspects other than surgery and speech function might add to the understanding of what affects speech outcome. Effective speech therapy should be available for children in addition to primary surgical repair of the cleft and secondary surgeries if needed.


Assuntos
Fenda Labial , Fissura Palatina , Insuficiência Velofaríngea , Criança , Feminino , Humanos , Pré-Escolar , Fissura Palatina/cirurgia , Fissura Palatina/complicações , Fenda Labial/cirurgia , Fenda Labial/complicações , Fala , Resultado do Tratamento , Ensaios Clínicos Controlados Aleatórios como Assunto , Palato Duro , Insuficiência Velofaríngea/cirurgia , Insuficiência Velofaríngea/complicações
10.
Int J Pediatr ; 2022: 4423558, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36119548

RESUMO

Objective: To examine admission trends, complications, and costs for inpatient infantile hemangioma (IH) associated with propranolol therapy utilizing the Pediatric Health Information System (PHIS) database. Study Design. A retrospective cohort study was completed using the PHIS database. The PHIS database was queried from 2008 to 2020 for children without cardiac disease and between the ages of three weeks and one year who were admitted with a diagnosis of IH and administered propranolol. Admissions were trended annually and by geographic region. Primary outcomes were length of stay (LOS), readmission, mortality, propranolol-related complications, and costs. Bivariate and multivariable analyses were employed to identify predictors of the primary outcomes. Results: A total of 2290 unique patient encounters were identified. Admissions steadily decreased after 2011, with variations by geographic region. There was no mortality and only 60 (2.6%) propranolol-related complications. African-American race (odds ratio (OR) 1.20 [95% CI: 1.02-1.41]), respiratory comorbidities (OR 2.04 [95% CI: 1.42-2.93]), neurologic conditions (OR 1.34 [95% CI: 1.09-1.59]), admission to an intensive care unit (OR 1.31 [95% CI: 1.09-1.59]), bronchospasm (OR 1.37 [95% CI: 1.22-1.55]), and hyperkalemia (OR 1.86 [95% CI: 1.08-3.20]) were associated with increased LOS. Neurologic conditions (OR 2.87 [95% CI: 1.76-4.67]) and respiratory comorbidities (OR 2.48 [CI: 1.43-4.30]) were associated with readmission. Average cost per admission was $5,158 ($3,259 to $8,560 range). Conclusion: There is an overall national decline in rate of admissions for IH propranolol therapy. Inpatient admission may be beneficial for patients with neurologic or respiratory conditions.

11.
J Neurol ; 269(8): 4089-4101, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35612658

RESUMO

Measurement of upper limb function is critical for tracking clinical severity in amyotrophic lateral sclerosis (ALS). The Amyotrophic Lateral Sclerosis Rating Scale-revised (ALSFRS-r) is the primary outcome measure utilised in clinical trials and research in ALS. This scale is limited by floor and ceiling effects within subscales, such that clinically meaningful changes for subjects are often missed, impacting upon the evaluation of new drugs and treatments. Technology has the potential to provide sensitive, objective outcome measurement. This paper is a structured review of current methods and future trends in the measurement of upper limb function with a particular focus on ALS. Technologies that have the potential to radically change the upper limb measurement field and explore the limitations of current technological sensors and solutions in terms of costs and user suitability are discussed. The field is expanding but there remains an unmet need for simple, sensitive and clinically meaningful tests of upper limb function in ALS along with identifying consensus on the direction technology must take to meet this need.


Assuntos
Esclerose Lateral Amiotrófica , Esclerose Lateral Amiotrófica/diagnóstico , Humanos , Extremidade Superior
12.
Nat Biotechnol ; 40(7): 1123-1131, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35241837

RESUMO

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.


Assuntos
Aprendizado de Máquina , Ácidos Nucleicos , Redes Neurais de Computação
13.
Molecules ; 27(3)2022 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-35164252

RESUMO

Solvents that stabilize protein structures can improve and expand their biochemical applications, particularly with the growing interest in biocatalytic-based processes. Aiming to select novel solvents for protein stabilization, we explored the effect of alkylammonium nitrate protic ionic liquids (PILs)-water mixtures with increasing cation alkyl chain length on lysozyme conformational stability. Four PILs were studied, that is, ethylammonium nitrate (EAN), butylammonium nitrate (BAN), hexylammonium nitrate (HAN), and octylammonium nitrate (OAN). The surface tension, viscosity, and density of PIL-water mixtures at low to high concentrations were firstly determined, which showed that an increasing cation alkyl chain length caused a decrease in the surface tension and density as well as an increase in viscosity for all PIL solutions. Small-angle X-ray scattering (SAXS) was used to investigate the liquid nanostructure of the PIL solutions, as well as the overall size, conformational flexibility and changes to lysozyme structure. The concentrated PILs with longer alkyl chain lengths, i.e., over 10 mol% butyl-, 5 mol% hexyl- and 1 mol% octylammonium cations, possessed liquid nanostructures. This detrimentally interfered with solvent subtraction, and the more structured PIL solutions prevented quantitative SAXS analysis of lysozyme structure. The radius of gyration (Rg) of lysozyme in the less structured aqueous PIL solutions showed little change with up to 10 mol% of PIL. Kratky plots, SREFLEX models, and FTIR data showed that the protein conformation was maintained at a low PIL concentration of 1 mol% and lower when compared with the buffer solution. However, 50 mol% EAN and 5 mol% HAN significantly increased the Rg of lysozyme, indicating unfolding and aggregation of lysozyme. The hydrophobic interaction and liquid nanostructure resulting from the increased cation alkyl chain length in HAN likely becomes critical. The impact of HAN and OAN, particularly at high concentrations, on lysozyme structure was further revealed by FTIR. This work highlights the negative effect of a long alkyl chain length and high concentration of PILs on lysozyme structural stability.


Assuntos
Líquidos Iônicos/química , Muramidase/química , Cátions/química , Interações Hidrofóbicas e Hidrofílicas , Conformação Proteica , Espalhamento a Baixo Ângulo , Difração de Raios X
14.
Societies (Basel) ; 12(6)2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36714171

RESUMO

In this article, we apply and combine elements from four theoretical frameworks (i.e., Minority Stress Theory, Person-in-Environment and Risk and Resilience Framework, Interpersonal-Psychological Theory of Suicide, and Intersectionality) to explain the problem of queer youth suicide through our integrated conceptual model, Queer Prevention of Youth Suicidality Model (Queer-PRYSM). The need for this conceptual model is based on the current state of the literature, including mixed empirical findings on factors related to queer youth suicidality, no scholarly consensus on specific contributing factors regarding high rates of suicidality among queer youth (including queer youth subgroups), and the absence of a unifying theory to explain the queer youth suicide risk. To address these limitations in theory, evidence, and scholarship explaining suicidality among queer youth we present our integrated model with growing, current, relevant research with queer youth. Queer-PRYSM includes minority stressors specific to queer youth, mental health problems, interpersonal-psychological factors, socioecological factors (i.e., family, school, peers, and community), and intersectionality concepts. Queer-PRYSM is essential to understanding the relationship of distal and proximal risk and protective factors in queer youth suicide and developing evidence-informed suicide preventive interventions that can be incorporated into practice, policy, and system structures.

15.
Front Psychol ; 12: 753954, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34777153

RESUMO

This paper is a systematic review and meta-analysis on sexual orientation identity development milestones among people who are lesbian, gay, bisexual, or another sexual minority identity (LGB+). Common milestones measured in the 30 studies reviewed were becoming aware of queer attractions, questioning one's sexual orientation, self-identifying as LGB+, coming out to others, engaging in sexual activity, and initiating a romantic relationship. Milestones occurred in different sequences, although attraction was almost always first, often followed by self-identification and/or sexual activity; coming out and initiating a romantic relationship often followed these milestones. Meta-analysis results showed that the mean effect sizes and 95% confidence intervals varied by milestone: attraction [M age=12.7 (10.1, 15.3)], questioning one's orientation [M age=13.2 [12.8, 13.6]), self-identifying [M age=17.8 (11.6, 24.0)], sexual activity [M age=18.1 (17.6, 18.6)], coming out [M age=19.6 (17.2, 22.0)], and romantic relationship [M age=20.9 (13.2, 28.6)]. Nonetheless, results also showed substantial heterogeneity in the mean effect sizes. Additional meta-analyses showed that milestone timing varied by sex, sexual orientation, race/ethnicity, and birth cohort. Although patterns were found in LGB+ identity development, there was considerable diversity in milestone trajectories.

16.
NPJ Vaccines ; 6(1): 77, 2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34021159

RESUMO

Antibody-dependent enhancement (ADE) is suspected to influence dengue virus (DENV) infection, but the role ADE plays in vaccination strategies incorporating live attenuated virus components is less clear. Using a heterologous prime-boost strategy in rhesus macaques, we examine the effect of priming with DENV purified inactivated vaccines (PIVs) on a tetravalent live attenuated vaccine (LAV). Sera exhibited low-level neutralizing antibodies (NAb) post PIV priming, yet moderate to high in vitro ADE activity. Following LAV administration, the PIV primed groups exhibited DENV-2 LAV peak viremias up to 1,176-fold higher than the mock primed group, and peak viremia correlated with in vitro ADE. Furthermore, PIV primed groups had more balanced and higher DENV-1-4 NAb seroconversion and titers than the mock primed group following LAV administration. These results have implications for the development of effective DENV vaccine prime-boost strategies and for our understanding of the role played by ADE in modulating DENV replication.

18.
Nat Commun ; 11(1): 4131, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807807

RESUMO

Recent outbreaks of viral hemorrhagic fevers (VHFs), including Ebola virus disease (EVD) and Lassa fever (LF), highlight the urgent need for sensitive, deployable tests to diagnose these devastating human diseases. Here we develop CRISPR-Cas13a-based (SHERLOCK) diagnostics targeting Ebola virus (EBOV) and Lassa virus (LASV), with both fluorescent and lateral flow readouts. We demonstrate on laboratory and clinical samples the sensitivity of these assays and the capacity of the SHERLOCK platform to handle virus-specific diagnostic challenges. We perform safety testing to demonstrate the efficacy of our HUDSON protocol in heat-inactivating VHF viruses before SHERLOCK testing, eliminating the need for an extraction. We develop a user-friendly protocol and mobile application (HandLens) to report results, facilitating SHERLOCK's use in endemic regions. Finally, we successfully deploy our tests in Sierra Leone and Nigeria in response to recent outbreaks.


Assuntos
Ebolavirus/patogenicidade , Doença pelo Vírus Ebola/diagnóstico , Febre Lassa/diagnóstico , Vírus Lassa/patogenicidade , Anticorpos Antivirais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Ebolavirus/genética , Doença pelo Vírus Ebola/virologia , Febre Lassa/virologia , Vírus Lassa/genética
19.
Nature ; 582(7811): 277-282, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32349121

RESUMO

The great majority of globally circulating pathogens go undetected, undermining patient care and hindering outbreak preparedness and response. To enable routine surveillance and comprehensive diagnostic applications, there is a need for detection technologies that can scale to test many samples1-3 while simultaneously testing for many pathogens4-6. Here, we develop Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a platform for scalable, multiplexed pathogen detection. In the CARMEN platform, nanolitre droplets containing CRISPR-based nucleic acid detection reagents7 self-organize in a microwell array8 to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combination of CARMEN and Cas13 detection (CARMEN-Cas13) enables robust testing of more than 4,500 crRNA-target pairs on a single array. Using CARMEN-Cas13, we developed a multiplexed assay that simultaneously differentiates all 169 human-associated viruses with at least 10 published genome sequences and rapidly incorporated an additional crRNA to detect the causative agent of the 2020 COVID-19 pandemic. CARMEN-Cas13 further enables comprehensive subtyping of influenza A strains and multiplexed identification of dozens of HIV drug-resistance mutations. The intrinsic multiplexing and throughput capabilities of CARMEN make it practical to scale, as miniaturization decreases reagent cost per test by more than 300-fold. Scalable, highly multiplexed CRISPR-based nucleic acid detection shifts diagnostic and surveillance efforts from targeted testing of high-priority samples to comprehensive testing of large sample sets, greatly benefiting patients and public health9-11.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Técnicas Analíticas Microfluídicas/métodos , Viroses/diagnóstico , Viroses/virologia , Animais , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Farmacorresistência Viral/genética , Genoma Viral/genética , HIV/classificação , HIV/genética , HIV/isolamento & purificação , Humanos , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Técnicas Analíticas Microfluídicas/instrumentação , RNA Guia de Cinetoplastídeos/genética , SARS-CoV-2 , Sensibilidade e Especificidade
20.
Preprint em Inglês | bioRxiv | ID: ppbiorxiv-967026

RESUMO

The emergence and outbreak of SARS-CoV-2, the causative agent of COVID-19, has rapidly become a global concern and has highlighted the need for fast, sensitive, and specific tools to surveil circulating viruses. Here we provide assay designs and experimental resources, for use with CRISPR-based nucleic acid detection, that could be valuable for ongoing surveillance. We provide assay designs for detection of 67 viral species and subspecies, including: SARS-CoV-2, phylogenetically-related viruses, and viruses with similar clinical presentation. The designs are outputs of algorithms that we are developing for rapidly designing nucleic acid detection assays that are comprehensive across genomic diversity and predicted to be highly sensitive and specific. Of our design set, we experimentally screened 4 SARS-CoV-2 designs with a CRISPR-Cas13 detection system and then extensively tested the highest-performing SARS-CoV-2 assay. We demonstrate the sensitivity and speed of this assay using synthetic targets with fluorescent and lateral flow detection. Moreover, our provided protocol can be extended for testing the other 66 provided designs. Assay designs are available at https://adapt.sabetilab.org/.

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