Naturally Occurring Off-Switches for CRISPR-Cas9.

CRISPR-Cas9 technology would be enhanced by the ability to inhibit Cas9 function spatially, temporally, or conditionally. Previously, we discovered small proteins encoded by bacteriophages that inhibit the CRISPR-Cas systems of their host bacteria. These "anti-CRISPRs" were specific to type I CRISPR-Cas systems that do not employ the Cas9 protein. We posited that nature would also yield Cas9 inhibitors in response to the evolutionary arms race between bacteriophages and their hosts. Here, we report the discovery of three distinct families of anti-CRISPRs that specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis. We show that these proteins bind directly to N. meningitidis Cas9 (NmeCas9) and can be used as potent inhibitors of genome editing by this system in human cells. These anti-CRISPR proteins now enable "off-switches" for CRISPR-Cas9 activity and provide a genetically encodable means to inhibit CRISPR-Cas9 genome editing in eukaryotes. VIDEO ABSTRACT.

A phage-encoded anti-activator inhibits quorum sensing in Pseudomonas aeruginosa.

The arms race between bacteria and phages has led to the evolution of diverse anti-phage defenses, several of which are controlled by quorum-sensing pathways. In this work, we characterize a quorum-sensing anti-activator protein, Aqs1, found in Pseudomonas phage DMS3. We show that Aqs1 inhibits LasR, the master regulator of quorum sensing, and present the crystal structure of the Aqs1-LasR complex. The 69-residue Aqs1 protein also inhibits PilB, the type IV pilus assembly ATPase protein, which blocks superinfection by phages that require the pilus for infection. This study highlights the remarkable ability of small phage proteins to bind multiple host proteins and disrupt key biological pathways. As quorum sensing influences various anti-phage defenses, Aqs1 provides a mechanism by which infecting phages might simultaneously dampen multiple defenses. Because quorum-sensing systems are broadly distributed across bacteria, this mechanism of phage counter-defense may play an important role in phage-host evolutionary dynamics.

Reverse vaccinology-based identification of a novel surface lipoprotein that is an effective vaccine antigen against bovine infections caused by Pasteurella multocida.

Pasteurella multocida can infect a multitude of wild and domesticated animals, with infections in cattle resulting in hemorrhagic septicemia (HS) or contributing to bovine respiratory disease (BRD) complex. Current cattle vaccines against P. multocida consist of inactivated bacteria, which only offer limited and serogroup specific protection. Here, we describe a newly identified surface lipoprotein, PmSLP, that is present in nearly all annotated P. multocida strains isolated from cattle. Bovine associated variants span three of the four identified phylogenetic clusters, with PmSLP-1 and PmSLP-2 being restricted to BRD associated isolates and PmSLP-3 being restricted to isolates associated with HS. Recombinantly expressed, soluble PmSLP-1 (BRD-PmSLP) and PmSLP-3 (HS-PmSLP) vaccines were both able to provide full protection in a mouse sepsis model against the matched P. multocida strain, however no cross-protection and minimal serum IgG cross-reactivity was identified. Full protection against both challenge strains was achieved with a bivalent vaccine containing both BRD-PmSLP and HS-PmSLP, with serum IgG from immunized mice being highly reactive to both variants. Year-long stability studies with lyophilized antigen stored under various temperatures show no appreciable difference in biophysical properties or loss of efficacy in the mouse challenge model. PmSLP-1 and PmSLP-3 vaccines were each evaluated for immunogenicity in two independent cattle trials involving animals of different age ranges and breeds. In all four trials, vaccination with PmSLP resulted in an increase in antigen specific serum IgG over baseline. In a blinded cattle challenge study with a recently isolated HS strain, the matched HS-PmSLP vaccine showed strong efficacy (75-87.5% survival compared to 0% in the control group). Together, these data suggest that cattle vaccines composed of PmSLP antigens can be a practical and effective solution for preventing HS and BRD related P. multocida infections.

Prevalence of Slam-dependent hemophilins in Gram-negative bacteria.

Iron acquisition systems are crucial for pathogen growth and survival in iron-limiting host environments. To overcome nutritional immunity, bacterial pathogens evolved to use diverse mechanisms to acquire iron. Here, we examine a heme acquisition system that utilizes hemophores called hemophilins which are also referred to as HphAs in several Gram-negative bacteria. In this study, we report three new HphA structures from Stenotrophomonas maltophilia, Vibrio harveyi, and Haemophilus parainfluenzae. Structural determination of HphAs revealed an N-terminal clamp-like domain that binds heme and a C-terminal eight-stranded ß-barrel domain that shares the same architecture as the Slam-dependent Neisserial surface lipoproteins. The genetic organization of HphAs consists of genes encoding a Slam homolog and a TonB-dependent receptor (TBDR). We investigated the Slam-HphA system in the native organism or the reconstituted system in Escherichia coli cells and found that the efficient secretion of HphA depends on Slam. The TBDR also played an important role in heme uptake and conferred specificity for its cognate HphA. Furthermore, bioinformatic analysis of HphA homologs revealed that HphAs are conserved in the alpha, beta, and gammaproteobacteria. Together, these results show that the Slam-dependent HphA-type hemophores are prevalent in Gram-negative bacteria and further expand the role of Slams in transporting soluble proteins. IMPORTANCE: This paper describes the structure and function of a family of Slam (Type IX secretion System) secreted hemophores that bacteria use to uptake heme (iron) while establishing an infection. Using structure-based bioinformatics analysis to define the diversity and prevalence of this heme acquisition pathway, we discovered that a large portion of gammaproteobacterial harbors this system. As organisms, including Acinetobacter baumannii, utilize this system to facilitate survival during host invasion, the identification of this heme acquisition system in bacteria species is valuable information and may represent a target for antimicrobials.

Test and Treat for Prediabetes: A Review of the Health Effects of Prediabetes and the Role of Screening and Prevention.

The term prediabetes describes blood glucose levels above the normal range but below the threshold to diagnose type 2 diabetes. Several population health initiatives encourage a test and treat approach for prediabetes. In this approach, screening and identification of individuals with prediabetes should be followed by prompt referral to structured lifestyle modification programs or pharmacologic interventions that have been shown to prevent or delay the progression to type 2 diabetes in clinical trials. Here we provide a critical review of evidence for this test and treat approach by examining health outcomes associated with prediabetes and the availability and effectiveness of lifestyle modification approaches that target prediabetes. We also describe current limitations to the reach and uptake of evidence-based treatment options for prediabetes. Finally, we highlight lessons learned from identifying and labeling other preconditions to consider challenges and opportunities that may arise with increasing awareness of prediabetes as part of routine preventive care.

A Culturally Adapted, Telehealth, Community Health Worker Intervention on Blood Pressure Control among South Asian Immigrants with Type II Diabetes: Results from the DREAM Atlanta Intervention.

BACKGROUND: South Asians face a high prevalence of type II diabetes (DMII) and comorbid hypertension (HTN). Community health worker (CHW) interventions have the potential to improve chronic disease outcomes, yet few have been tailored to South Asian populations in the United States. OBJECTIVE: To test the effectiveness of an evidence-based CHW-led and culturally-tailored HTN and DMII management program for South Asian adults with diabetes and comorbid uncontrolled HTN (systolic blood pressure (SBP) > 130 mmHg or diastolic blood pressure (DBP) > 80 mmHg). DESIGN: Randomized-controlled Trial. PARTICIPANTS: South Asian adults with DMII and comorbid HTN. INTERVENTION: The Diabetes Research, Education, and Action for Minorities (DREAM) Atlanta intervention was a CHW telehealth intervention designed to improve blood pressure (BP). The treatment group received five virtual group-based health education sessions, an action plan, and follow-up calls to assess goal setting activities. The control group received only the first session. Main Measures included: feasibility, improvement in BP control, and decreases in SBP, DBP, weight, and hemoglobin A1c (HbA1c). KEY RESULTS: A total of 190 South Asian adults were randomized (97 to the treatment group and 93 to the control group); 94% of treatment group participants completed all 5 telehealth sessions. At endpoint, BP control increased 33.7% (95% CI: 22.5, 44.9, p < 0.001) in the treatment group and 16.5% (95%: 6.2, 26.8, p = 0.003) in the control group; the adjusted intervention effect was 1.8 (95% CI: 1.0, 3.2, p = 0.055). Mean weight decreased by 4.8 pounds (95% CI: -8.2, -1.4, p = 0.006) in the treatment group, and the adjusted intervention effect was -5.2 (95% CI: -9.0, -1.4, p = 0.007. The intervention had an overall retention of 95%. CONCLUSIONS: A culturally-tailored, CHW-led telehealth intervention is feasible and can improve BP control among South Asian Americans with DMII. GOV REGISTRATION: NCT04263311.

Modulation of various host cellular machinery during COVID-19 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) emerged in December 2019, causing a range of respiratory infections from mild to severe. This resulted in the ongoing global COVID-19 pandemic, which has had a significant impact on public health. The World Health Organization declared COVID-19 as a global pandemic in March 2020. Viruses are intracellular pathogens that rely on the host's machinery to establish a successful infection. They exploit the gene expression machinery of host cells to facilitate their own replication. Gaining a better understanding of gene expression modulation in SARS-CoV2 is crucial for designing and developing effective antiviral strategies. Efforts are currently underway to understand the molecular-level interaction between the host and the pathogen. In this review, we describe how SARS-CoV2 infection modulates gene expression by interfering with cellular processes, including transcription, post-transcription, translation, post-translation, epigenetic modifications as well as processing and degradation pathways. Additionally, we emphasise the therapeutic implications of these findings in the development of new therapies to treat SARS-CoV2 infection.

Cardiometabolic Risk in Asian Americans by Social Determinants of Health: Serial Cross-sectional Analyses of the NHIS, 1999-2003 to 2014-2018.

BACKGROUND: Diabetes and hypertension are common in Asian Americans and vary by subgroup. There may be further variation by social determinants of health (SDOHs), but few studies have examined this previously. OBJECTIVE: To examine the associations of SDOHs and diabetes and hypertension within and across Asian subgroups in the USA DESIGN: Series cross-sectional analyses SETTING: National Health Interview Surveys (NHIS) from 1999 to 2018 PARTICIPANTS: Asian-American adults (Chinese, Filipino, Asian Indian, and Other Asian [Korean, Vietnamese, Japanese, and other]) MEASUREMENTS: Self-reported diabetes and hypertension prevalence in pooled 5-year increments over 1999-2018 and multivariable regression models to assess the adjusted prevalence of diabetes or hypertension by poverty, marital status, education, and years in the USA, adjusting for age, sex, BMI, and health insurance status RESULTS: From 1999-2003 to 2014-2018, the age- and sex-adjusted prevalence of diabetes increased for Other Asians (absolute change: 4.6%) but not for other subgroups; age- and sex-adjusted hypertension prevalence significantly increased for Asian Indians and Other Asians (absolute change: 5-7.5%). For Filipinos, high school education or less was associated with an increase in diabetes prevalence over time (difference from 1999-2003 to 2014-2018: +6.0 (95% CI: 2.0-10.0)), while for Asian Indians, college education or higher was associated with an increase in diabetes prevalence for the same period (difference: +2.7 (95% CI: 0.01-5.4). Differences over the 2 time periods (1999-2003 and 2014-2018) show that Filipino and Other Asians, who lived in the USA for ≥10 years, increased in diabetes prevalence. Similar variations in associations of SDOHs by Asian subgroup were seen for hypertension. LIMITATIONS: Self-reported primary outcomes and multi-year data were pooled due to small sample sizes. CONCLUSIONS: The influence of SDOHs on cardiometabolic risk is not uniform among Asian Americans, implying tailored strategies may be needed for different population subgroups. PRIMARY FUNDING SOURCE: NIH.

Actinobacillus utilizes a binding protein-dependent ABC transporter to acquire the active form of vitamin B6.

Bacteria require high-efficiency uptake systems to survive and proliferate in nutrient-limiting environments, such as those found in host organisms. ABC transporters in the bacterial plasma membrane provide a mechanism for transport of many substrates. In this study, we examine an operon containing a periplasmic binding protein in Actinobacillus for its potential role in nutrient acquisition. The electron density map of 1.76 Å resolution obtained from the crystal structure of the periplasmic binding protein was best fit with a molecular model containing a pyridoxal-5'-phosphate (P5P/pyridoxal phosphate/the active form of vitamin B6) ligand within the protein's binding site. The identity of the P5P bound to this periplasmic binding protein was verified by isothermal titration calorimetry, microscale thermophoresis, and mass spectrometry, leading us to name the protein P5PA and the operon P5PAB. To illustrate the functional utility of this uptake system, we introduced the P5PAB operon from Actinobacillus pleuropneumoniae into an Escherichia coli K-12 strain that was devoid of a key enzyme required for P5P synthesis. The growth of this strain at low levels of P5P supports the functional role of this operon in P5P uptake. This is the first report of a dedicated P5P bacterial uptake system, but through bioinformatics, we discovered homologs mainly within pathogenic representatives of the Pasteurellaceae family, suggesting that this operon exists more widely outside the Actinobacillus genus.

The social determinants of health associated with cardiometabolic diseases among Asian American subgroups: a systematic review.

BACKGROUND: Asian Americans represent one of the fastest-growing immigrant groups in the U.S. and are at high risk for cardiometabolic diseases (CMDs), including type 2 diabetes, hypertension, coronary artery disease, and stroke. Despite the growth of Asians in the U. S, there is a gap in understanding the heterogeneity of CMDs across Asian subgroups and how these might be affected by the social determinants of health (SDOH), or the environment in which people live and work. OBJECTIVE: The purpose of this systematic review is to examine the current literature on CMDs among Asian Americans and identify the SDOH that are associated with the incidence and/or prevalence of CMDs among specific Asian subgroups. METHODS: PubMed, Embase, Web of Science were searched for articles published in Jan 2000-Nov 2020. The reproducible strategy yielded 2732 articles. The articles were reviewed based on the following inclusion criteria: (1) observational study published in the U.S., (2) adult population includes specific Asian subgroups, (3) exposures include SDOH, and (4) outcomes include a CMD, defined as type 2 diabetes, hypertension, coronary artery disease, or stroke. RESULTS: In this review, 14 studies were identified and organized into four key themes: acculturation (n = 9), socioeconomic status (SES) (n = 6), social context (n = 2), and health literacy (n = 1). The most represented Asian subgroups in the literature were Chinese, Filipino, and South Asians. Acculturation was the most described social factor in the included reviews. Seven studies found associations between higher acculturation levels and higher prevalence of CMD. However, the measure of acculturation varied by study and included various combinations of the country of birth, number of years residing in the U.S., and English proficiency. The effects of SES, measured as income level and educational attainment, varied by racial subgroups. One study found that higher levels of education were associated with CMD among South Asians. CONCLUSION: Acculturation, SES, social context, and health literacy impact the risk of CMD among Asian Americans; these vary across subgroups. Future research disentangling SDOHs on the risk of CMDs by Asian subgroup is necessary to provide better informed preventive practices and interventions.

Trends in Diet Counseling Among Stroke Versus Non-Stroke Patients: Evidence from the NAMCS, 2011-2016.

INTRODUCTION: Dietary modifications in post-stroke patients facilitated by diet counseling improves post-stroke recovery and stroke recurrence. The extent to which clinicians provide dietary counseling for these patients is unknown. METHODS: 2011 to 2016 National Ambulatory Medical Care Surveys (NAMCS) data were used to assess trends in post-stroke diet consultations by age. Multivariate logistic regression models assessed the likelihood of dietary counseling provision among patients with and without stroke. RESULTS: The proportion of patients with stroke aged 60-79 who received diet counseling decreased from 18.2% in 2011 to 5.3%, 11.9%, 8.7%, 13.4%, and 15.2% in 2012-2016, respectively. Among patients without stroke aged 60-79, diet counseling rate decreased from 12.9% in 2011 to 7%, 9.5%, 10.5%, 13.5%, and 12% in 2012-2016, respectively. Similar trends were observed among patients with and without stroke aged over 80. CONCLUSIONS: The proportion of patients with and without stroke receiving dietary counseling has remained low over the past half-decade. It is likely multifactorial- related to clinician knowledge, patients' receptiveness, and system-level factors of time and reimbursement. Future interventions should explore methods to address barriers to nutrition recommendations for post-stroke patients and patient activation to adopt dietary changes.

Diabetes Complications in Racial and Ethnic Minority Populations in the USA.

PURPOSE OF REVIEW: Racial and ethnic minority populations have a higher burden of diabetes-related complications. There have been many epidemiologic studies to better define these racial/ethnic disparities in diabetes outcomes with additional studies offering interventions to mitigate them. This narrative review highlights the epidemiologic trends in diabetes complications specific to racial and ethnic minorities and underscores differences in microvascular and macrovascular complications of diabetes, health care utilization, and diabetes prevention efforts and also reviews interventions aimed to reduce racial/ethnic disparities and their limitations. RECENT FINDINGS: While we have seen in general an overall improvement in complication rates for all people with diabetes, the disparities between Black and Hispanic compared to non-Hispanic White people with diabetes seem to persist. There is a continued need to better understand the underlying causes of and strategies to mitigate race/ethnicity disparities in diabetes complications in the USA.

Lifestyle Interventions for Diabetes Prevention in South Asians: Current Evidence and Opportunities.

PURPOSE OF REVIEW: This review summarizes the burden of diabetes in South Asian populations and reviews recent evidence for diabetes prevention through lifestyle modification among South Asians worldwide. We indicate important gaps in the current literature and point to opportunities for additional research in this area. RECENT FINDINGS: Randomized, controlled, efficacy studies and implementation research show that lifestyle intervention can be an effective, cost-effective, and feasible method for reducing diabetes risk, improving cardiometabolic health, and improving lifestyle behaviors in South Asian populations, a population at high diabetes risk and elevated rates of diabetes risk factors. Additional research is needed to address diabetes risk reduction in normal-weight South Asians and individuals with impaired fasting glucose; improve community-level implementation, individual uptake, and dissemination of proven programs; and assess long-term outcomes of interventions.

A Community Health Worker-Led Intervention to Improve Blood Pressure Control in an Immigrant Community With Comorbid Diabetes: Data From Two Randomized, Controlled Trials Conducted in 2011-2019.

Evidence-based strategies addressing comorbid hypertension and diabetes are needed among minority communities. We analyzed the outcome of blood pressure (BP) control using pooled data from two community health worker interventions in New York City conducted between 2011 and 2019, focusing on participants with comorbid hypertension and diabetes. The adjusted odds of controlled BP (< 140/90 mmHg) for the treatment group were significant compared with the control group (odds ratio = 1.4; 95% confidence interval = 1.1, 1.8). The interventions demonstrated clinically meaningful reductions in BP among participants with comorbid hypertension and diabetes.

The role of trust in the likelihood of receiving a COVID-19 vaccine: Results from a national survey.

High acceptance of coronavirus disease 2019 (COVID-19) vaccines is instrumental to ending the pandemic. Vaccine acceptance by subgroups of the population depends on their trust in COVID-19 vaccines. We surveyed a probability-based internet panel of 7832 adults from December 23, 2020-January 19, 2021 about their likelihood of getting a COVID-19 vaccine and the following domains of trust: an individual's generalized trust, trust in COVID-19 vaccine's efficacy and safety, trust in the governmental approval process and general vaccine development process for COVID-19 vaccines, trust in their physician about COVID-19, and trust in other sources about COVID-19. We included identified at-risk subgroups: healthcare workers, older adults (65-74-year-olds and ≥ 75-year-olds), frontline essential workers, other essential workers, and individuals with high-risk chronic conditions. Of 5979 respondents, only 57.4% said they were very likely or somewhat likely to get a COVID-19 vaccine. More hesitant respondents (p < 0.05) included: women, young adults (18-49 years), Blacks, individuals with lower education, those with lower income, and individuals without high-risk chronic conditions. Lack of trust in the vaccine approval and development processes explained most of the demographic variation in stated vaccination likelihood, while other domains of trust explained less variation. We conclude that hesitancy for COVID-19 vaccines is high overall and among at-risk subgroups, and hesitancy is strongly tied to trust in the vaccine approval and development processes. Building trust is critical to ending the pandemic.

PENTOSAN POLYSULFATE AND VISION: Findings from an International Survey of Exposed Individuals.

PURPOSE: To investigate patient-reported visual function among individuals taking pentosan polysulfate (PPS) for interstitial cystitis. METHODS: A 27-item online survey was distributed to an international mailing list of individuals with interstitial cystitis in November 2018. Demographic characteristics, PPS exposure history, subjective visual function, and previous macular diagnoses were queried. The impact of PPS use, grouped by tertile of cumulative exposure, on visual function and macular diagnoses was assessed with multivariate logistic regression. RESULTS: The survey was completed by 912 respondents. Eight hundred and sixty-one (96.4%) were women, and the median age was 55 [interquartile range (IQR), 45-64 years]. Among PPS users, the median exposure was 547.5 g (IQR, 219-1,314 g). Respondents in the highest PPS exposure tertile were more likely to report difficulty with reading small print [adjusted odds ratio 2.29, 95% confidence interval (CI) 1.15-4.57] and to have a diagnosis of macular degeneration and/or pigmentary maculopathy (adjusted odds ratio 2.41, 95% CI 1.44-4.03) than unexposed respondents. CONCLUSION: In this large sample of individuals with interstitial cystitis, those in the highest PPS exposure category were more likely to have difficulties reading small print and to report a previous diagnosis of macular disease. Further study of objective measures of visual function in PPS users is warranted.

Overcoming the Digital Divide in the Post-COVID-19 "Reset": Enhancing Group Virtual Visits with Community Health Workers.

The COVID-19 pandemic created numerous barriers to the implementation of participant-facing research. For most, the pandemic required rapid transitioning to all virtual platforms. During this pandemic, the most vulnerable populations are at highest risk of falling through the cracks of engagement in clinical care and research. Nonetheless, we argue that we should reframe the discussion to consider how this transition may create opportunities to engage extensively to reach populations. Here, we present our experience in Atlanta (Georgia, United States) in transitioning a group visit model for South Asian immigrants to a virtual platform and the pivotal role community members in the form of community health workers can play in building capacity among participants. We provide details on how this model helped address common barriers to group visit models in clinical practice and how our community health worker team innovatively addressed the digital challenges of working with an elderly population with limited English proficiency.

PilN Binding Modulates the Structure and Binding Partners of the Pseudomonas aeruginosa Type IVa Pilus Protein PilM.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that expresses type IVa pili. The pilus assembly system, which promotes surface-associated twitching motility and virulence, is composed of inner and outer membrane subcomplexes, connected by an alignment subcomplex composed of PilMNOP. PilM binds to the N terminus of PilN, and we hypothesize that this interaction causes functionally significant structural changes in PilM. To characterize this interaction, we determined the crystal structures of PilM and a PilM chimera where PilM was fused to the first 12 residues of PilN (PilM·PilN(1-12)). Structural analysis, multiangle light scattering coupled with size exclusion chromatography, and bacterial two-hybrid data revealed that PilM forms dimers mediated by the binding of a novel conserved motif in the N terminus of PilM, and binding PilN abrogates this binding interface, resulting in PilM monomerization. Structural comparison of PilM with PilM·PilN(1-12) revealed that upon PilN binding, there is a large domain closure in PilM that alters its ATP binding site. Using biolayer interferometry, we found that the association rate of PilN with PilM is higher in the presence of ATP compared with ADP. Bacterial two-hybrid data suggested the connectivity of the cytoplasmic and inner membrane components of the type IVa pilus machinery in P. aeruginosa, with PilM binding to PilB, PilT, and PilC in addition to PilN. Pull-down experiments demonstrated direct interactions of PilM with PilB and PilT. We propose a working model in which dynamic binding of PilN facilitates functionally relevant structural changes in PilM.