The United States Needs A Better Testing Playbook For Future Public Health Emergencies.

The absence of a comprehensive national playbook for developing and deploying testing has hindered the United States' ability to rapidly suppress recent biological emergencies (for example, the COVID-19 pandemic and outbreaks of mpox). We describe here the Testing Playbook for Biological Emergencies, a national testing playbook we developed. It includes a set of decisions and actions for US officials to take at specific times during infectious disease emergencies to implement testing rapidly and to ensure that available testing meets clinical and public health needs. Although the United States had multiple plans at the federal level for responding to pandemic threats, US leaders were unable to quickly and efficiently operationalize those plans to deploy different types of tests during the COVID-19 pandemic in 2020-21, and again during the US mpox outbreak in 2022. The playbook fills a critical gap by providing the necessary specific and adaptable guidance for decision makers to meet this need.

Effects of clinical covariates on serum miRNA expression among women without ovarian cancer.

BACKGROUND: Serum microRNAs (miRNAs) are potential biomarkers for ovarian cancer; however, many factors may influence miRNA expression. To understand potential confounders in miRNA analysis, we examined how sociodemographic factors and comorbidities, including known ovarian cancer risk factors, influence serum miRNA levels in women without ovarian cancer. METHODS: Data from 1,576 women from the Mass General Brigham Biobank collected between 2012 and 2019, excluding subjects previously or subsequently diagnosed with ovarian cancer, were examined. Using a focused panel of 179 miRNA probes optimized for serum profiling, miRNA expression was measured by flow cytometry using the Abcam Fireplex® assay and correlated with subjects' electronic medical records. RESULTS: The study population broadly reflected the New England population. The median age of subjects was 49 years, 34% were current or prior smokers, 33% were obese (BMI >30kg/m2), 49% were postmenopausal, and 11% had undergone prior bilateral oophorectomy. Significant differences in miRNA expression were observed among ovarian risk factors such as age, obesity, menopause, BRCA1 or BRCA2 germline mutations or breast cancer in family history. Additionally, miRNA expression was significantly altered by prior bilateral oophorectomy, hypertension, and hypercholesterolemia. Other variables, such as smoking, parity, age at menarche, hormonal replacement therapy, oral contraception, breast, endometrial, or colon cancer, and diabetes were not associated with significant changes in the panel when corrected for multiple testing. CONCLUSIONS: Serum miRNA expression patterns are significantly affected by patient demographics, exposure history, and medical comorbidities. IMPACT: Understanding confounders in serum miRNA expression is important for refining clinical assays for cancer screening.

Differences in Serum miRNA Profiles by Race, Ethnicity, and Socioeconomic Status: Implications for Developing an Equitable Ovarian Cancer Screening Test.

Serum miRNAs are promising biomarkers for several clinical conditions, including ovarian cancer. To inform equitable implementation of these tests, we investigated the effects of race, ethnicity, and socioeconomic status on serum miRNA profiles. Serum samples from a large institutional biobank were analyzed using a custom panel of 179 miRNA species highly expressed in human serum, measured using the Abcam Fireplex assay via flow cytometry. Data were log-transformed prior to analysis. Differences in miRNA by race and ethnicity were assessed using logistic regression. Pairwise t tests analyzed racial and ethnic differences among eight miRNAs previously associated with ovarian cancer risk. Pearson correlations determined the relationship between mean miRNA expression and the social deprivation index (SDI) for Massachusetts residents. Of 1,586 patients (76.9% white, non-Hispanic), compared with white, non-Hispanic patients, those from other racial and ethnic groups were younger (41.9 years ± 13.2 vs. 51.3 ± 15.1, P < 0.01) and had fewer comorbidities (3.5 comorbidities ± 2.7 vs. 4.6 ± 2.8, P < 0.01). On logistic regression, miRNAs predicted race and ethnicity at an AUC of 0.69 (95% confidence interval, 0.66-0.72), which remained consistent when stratified by most comorbidities. Among eight miRNAs previously associated with ovarian cancer risk, seven significantly varied by race and ethnicity (all P < 0.01). There were no significant differences in SDI for any of these eight miRNAs. miRNA expression is significantly influenced by race and ethnicity, which remained consistent after controlling for confounders. Understanding baseline differences in biomarker test characteristics prior to clinical implementation is essential to ensure instruments perform comparably across diverse populations. PREVENTION RELEVANCE: This study aimed to understand factors affecting miRNA expression, to ensure we create equitable screening tests for ovarian cancer that perform well in diverse populations. The goal is to ensure that we are detecting ovarian cancer cases earlier (secondary prevention) in women of all races, ethnic backgrounds, and socioeconomic means.

The biosecurity benefits of genetic engineering attribution.

Biology can be misused, and the risk of this causing widespread harm increases in step with the rapid march of technological progress. A key security challenge involves attribution: determining, in the wake of a human-caused biological event, who was responsible. Recent scientific developments have demonstrated a capability for detecting whether an organism involved in such an event has been genetically modified and, if modified, to infer from its genetic sequence its likely lab of origin. We believe this technique could be developed into powerful forensic tools to aid the attribution of outbreaks caused by genetically engineered pathogens, and thus protect against the potential misuse of synthetic biology.

The value proposition of the Global Health Security Index.

Infectious disease outbreaks pose major threats to human health and security. Countries with robust capacities for preventing, detecting and responding to outbreaks can avert many of the social, political, economic and health system costs of such crises. The Global Health Security Index (GHS Index)-the first comprehensive assessment and benchmarking of health security and related capabilities across 195 countries-recently found that no country is sufficiently prepared for epidemics or pandemics. The GHS Index can help health security stakeholders identify areas of weakness, as well as opportunities to collaborate across sectors, collectively strengthen health systems and achieve shared public health goals. Some scholars have recently offered constructive critiques of the GHS Index's approach to scoring and ranking countries; its weighting of select indicators; its emphasis on transparency; its focus on biosecurity and biosafety capacities; and divergence between select country scores and corresponding COVID-19-associated caseloads, morbidity, and mortality. Here, we (1) describe the practical value of the GHS Index; (2) present potential use cases to help policymakers and practitioners maximise the utility of the tool; (3) discuss the importance of scoring and ranking; (4) describe the robust methodology underpinning country scores and ranks; (5) highlight the GHS Index's emphasis on transparency and (6) articulate caveats for users wishing to use GHS Index data in health security research, policymaking and practice.

Accelerating Action in Global Health Security: Global Biosecurity Dialogue as a Model for Advancing the Global Health Security Agenda.

Biosecurity and biosafety measures are designed to mitigate intentional and accidental biological risks that pose potentially catastrophic consequences to a country's health system, security, and political and economic stability. Unfortunately, biosecurity and biosafety are often under-prioritized nationally, regionally, and globally. Security leaders often deemphasize accidental and deliberate biological threats relative to other challenges to peace and security. Given emerging biological risks, including those associated with rapid technological advances and terrorist and state interest in weapons of mass destruction, biosecurity deserves stronger emphasis in health and security fora. The Global Biosecurity Dialogue (GBD) was initiated to align national and regional donor initiatives toward a common set of measurable targets. The GBD was launched by the Nuclear Threat Initiative (NTI), with support from Global Affairs Canada's Weapons Threat Reduction Program and the Open Philanthropy Project, and in coordination with the government of The Netherlands as the 2018-19 Chair of the Global Health Security Agenda (GHSA) Action Package Prevent-3 (APP3) on Biosafety and Biosecurity. The GBD provides a multisectoral forum for sharing models, enabling new actions to achieve biosecurity-related targets, and promoting biosecurity as an integral component of health security. The GBD has contributed to new national and continent-wide actions, including the African Union and Africa Centres for Disease Control and Prevention's new regional Initiative to Strengthen Biosafety and Biosecurity in Africa. Here we present the GBD as a model for catalyzing action within APP3. We describe how the benefits of this approach could expand to other GHSA Action Packages and international health security initiatives.

Enterococcus faecalis Enhances Expression and Activity of the Enterohemorrhagic Escherichia coli Type III Secretion System.

The gut microbiota can significantly impact invading pathogens and the disease they cause; however, many of the mechanisms that dictate commensal-pathogen interactions remain unclear. Enterohemorrhagic Escherichia coli (EHEC) is a potentially lethal human intestinal pathogen that uses microbiota-derived molecules as cues to efficiently regulate virulence factor expression. Here, we investigate the interaction between EHEC and Enterococcus faecalis, a common human gut commensal, and show that E. faecalis affects both expression and activity of the EHEC type III secretion system (T3SS) via two distinct mechanisms. First, in the presence of E. faecalis there is increased transcription of genes encoding the EHEC T3SS. This leads to increased effector translocation and ultimately greater numbers of pedestals formed on host cells. The same effect was observed with several strains of enterococci, suggesting that it is a general characteristic of this group. In a mechanism separate from E. faecalis-induced transcription of the T3SS, we report that an E. faecalis-secreted protease, GelE, cleaves a critical structural component of the EHEC T3SS, EspB. Our data suggest that this cleavage actually increases effector translocation by the T3SS, supporting a model where EspB proteolysis promotes maximum T3SS activity. Finally, we report that treatment of EHEC with E. faecalis-conditioned cell-free medium is insufficient to induce increased T3SS expression, suggesting that this effect relies on cell contact between E. faecalis and EHEC. This work demonstrates a complex interaction between a human commensal and pathogen that impacts both expression and function of a critical virulence factor.IMPORTANCE This work reveals a complex and multifaceted interaction between a human gut commensal, Enterococcus faecalis, and a pathogen, enterohemorrhagic E. coli We demonstrate that E. faecalis enhances expression of the enterohemorrhagic E. coli type III secretion system and that this effect likely depends on cell contact between the commensal and the pathogen. Additionally, the GelE protease secreted by E. faecalis cleaves a critical structural component of the EHEC type III secretion system. In agreement with previous studies, we find that this cleavage actually increases effector protein delivery into host cells by the secretion system. This work demonstrates that commensal bacteria can significantly shape expression and activity of pathogen virulence factors, which may ultimately shape the progression of disease.

Establishing a theoretical foundation for measuring global health security: a scoping review.

BACKGROUND: Since the 2014-2016 West Africa Ebola epidemic, the concept of measuring health security capacity has become increasingly important within the broader context of health systems-strengthening, enhancing responses to public health emergencies, and reducing global catastrophic biological risks. Efforts to regularly and sustainably track the evolution of health security capabilities and capacities over time - while also accounting for political, social, and environmental risks - could help countries progress toward eliminating sources of health insecurity. We sought to aggregate evidence-based principles that capture a country's baseline public health and healthcare capabilities, its health security system performance before and during infectious disease crises, and its broader social, political, security, and ecological risk environments. METHODS: We conducted a scoping review of English-language scholarly and gray literature to identify evidence- and practice-based indicators and proxies for measuring health security at the country level over time. We then used a qualitative coding framework to identify recurrent themes in the literature and synthesize foundational principles for measuring global health security. Documents reviewed included English-language literature published after 2001 until the end of the research period-September 2017-to ensure relevance to the current global health security landscape; literature examining acute infectious disease threats with potential for transnational spread; and literature addressing global health security efforts at the country level. RESULTS: We synthesized four foundational principles for measuring global health security: measurement requires assessment of existing capacities, as well as efforts to build core public health, healthcare, and biosecurity capabilities; assessments of national programs and efforts to mitigate a critical subset of priority threats could inform efforts to generate useful metrics for global health security; there are measurable enabling factors facilitating health security-strengthening efforts; and finally, measurement requires consideration of social, political, and ecological risk environments. CONCLUSION: The themes identified in this review could inform efforts to systematically assess the impacts and effectiveness of activities undertaken to strengthen global health security.

Microbiota and Pathogen Proteases Modulate Type III Secretion Activity in Enterohemorrhagic Escherichia coli.

Enteric pathogens have complex interactions with the gut microbiota. Most of what is known about them has focused on microbiota-derived metabolites or small molecules that serve as nutrients and/or signals to aid in growth or transcriptionally regulate virulence gene expression. A common virulence strategy is to express a type III secretion system (T3SS), which is a molecular syringe deployed by many Gram-negative pathogens to hijack host cell function. Enterohemorrhagic Escherichiacoli (EHEC) requires its T3SS to colonize the intestinal tract and cause disease. Here we report that a prominent member of the intestinal microbiota, Bacteroides thetaiotamicron (Bt), secretes proteases that cleave the translocon of the T3SS of EHEC to enhance effector translocation into host cells. This is in contrast from an endogenous protease from EHEC itself (namely, EspP) that cleaves the translocon protein EspB in a different site to limit effector translocation. The EspB protein forms the T3SS pore in mammalian cells, and pore proteins are conserved in the T3SSs from several pathogens. This is the first demonstration of a commensal species directly processing a pathogen's T3SS, posing a new paradigm for how the microbiota can influence the severity of disease caused by bacterial pathogens. Because T3SSs are employed by many pathogens, this phenomenon has broad implications to commensal-pathogen relationships.IMPORTANCE The gut microbiota is usually regarded as providing colonization resistance against enteric pathogens. However, some pathogens evolved to thrive with the aid of certain members of the microbiota. Several Gram-negative bacteria employ type three secretion systems (T3SSs), which are molecular syringes that deliver effector proteins to host cells, hijacking host cell function. Here we show that the T3SS of enterohemorrhagic E. coli (EHEC) is cleaved by self and microbiota-derived proteases. Self-cleavage limits effector translocation, while cleavage by the microbiota member Bacteroides thetaiotamicron (Bt) exacerbates effector translocation and lesion formation on epithelial cells.

The QseG Lipoprotein Impacts the Virulence of Enterohemorrhagic Escherichia coli and Citrobacter rodentium and Regulates Flagellar Phase Variation in Salmonella enterica Serovar Typhimurium.

The QseEF histidine kinase/response regulator system modulates expression of enterohemorrhagic Escherichia coli (EHEC) and Salmonella enterica serovar Typhimurium virulence genes in response to the host neurotransmitters epinephrine and norepinephrine. qseG, which encodes an outer membrane lipoprotein, is cotranscribed with qseEF in these enteric pathogens, but there is little knowledge of its role in virulence. Here, we found that in EHEC QseG interacts with the type III secretion system (T3SS) gate protein SepL and modulates the kinetics of attaching and effacing (AE) lesion formation on tissue-cultured cells. Moreover, an EHEC ΔqseG mutant had reduced intestinal colonization in an infant rabbit model. Additionally, in Citrobacter rodentium, an AE lesion-forming pathogen like EHEC, QseG is required for full virulence in a mouse model. In S Typhimurium, we found that QseG regulates the phase switch between the two flagellin types, FliC and FljB. In an S Typhimurium ΔqseG mutant, the phase-variable promoter for fljB is preferentially switched into the "on" position, leading to overproduction of this phase two flagellin. In infection of tissue-cultured cells, the S Typhimurium ΔqseG mutant provokes increased inflammatory cytokine production versus the wild type; in vivo, in a murine infection model, the ΔqseG strain caused a more severe inflammatory response and was attenuated versus the wild-type strain. Collectively, our findings demonstrate that QseG is important for full virulence in several enteric pathogens and controls flagellar phase variation in S Typhimurium, and they highlight both the complexity and conservation of the regulatory networks that control the virulence of enteric pathogens.

Resolving Subcellular miRNA Trafficking and Turnover at Single-Molecule Resolution.

Regulation of microRNA (miRNA) localization and stability is critical for their extensive cytoplasmic RNA silencing activity and emerging nuclear functions. Here, we have developed single-molecule fluorescence-based tools to assess the subcellular trafficking, integrity, and activity of miRNAs. We find that seed-matched RNA targets protect miRNAs against degradation and enhance their nuclear retention. While target-stabilized, functional, cytoplasmic miRNAs reside in high-molecular-weight complexes, nuclear miRNAs, as well as cytoplasmic miRNAs targeted by complementary anti-miRNAs, are sequestered stably within significantly lower-molecular-weight complexes and rendered repression incompetent. miRNA stability and activity depend on Argonaute protein abundance, whereas miRNA strand selection, unwinding, and nuclear retention depend on Argonaute identity. Taken together, our results show that miRNA degradation competes with Argonaute loading and target binding to control subcellular miRNA abundance for gene silencing surveillance. Probing single cells for miRNA activity, trafficking, and metabolism promises to facilitate screening for effective miRNA mimics and anti-miRNA drugs.

Frenemies: Signaling and Nutritional Integration in Pathogen-Microbiota-Host Interactions.

The mammalian gastrointestinal (GI) microbiota is highly adapted to thrive in the GI environment and performs key functions related to host nutrition, physiology, development, immunity, and behavior. Successful host-bacterial associations require chemical signaling and optimal nutrient utilization and exchange. However, this important balance can be severely disrupted by environmental stimuli, with one of the most common insults upon the microbiota being infectious diseases. Although the microbiota acts as a barrier toward enteric pathogens, many enteric pathogens exploit signals and nutrients derived from both the microbiota and host to regulate their virulence programs. Here we review several signaling and nutrient recognition systems employed by GI pathogens to regulate growth and virulence. We discuss how shifts in the microbiota composition change host susceptibility to infection and how dietary changes or manipulation of the microbiota could potentially prevent and/or ameliorate GI infections.

Colitogenic Bacteroides thetaiotaomicron Antigens Access Host Immune Cells in a Sulfatase-Dependent Manner via Outer Membrane Vesicles.

Microbes interact with the host immune system via several potential mechanisms. One essential step for each mechanism is the method by which intestinal microbes or their antigens access specific host immune cells. Using genetically susceptible mice (dnKO) that develop spontaneous, fulminant colitis, triggered by Bacteroides thetaiotaomicron (B. theta), we investigated the mechanism of intestinal microbial access under conditions that stimulate colonic inflammation. B. theta antigens localized to host immune cells through outer membrane vesicles (OMVs) that harbor bacterial sulfatase activity. We deleted the anaerobic sulfatase maturating enzyme (anSME) from B. theta, which is required for post-translational activation of all B. theta sulfatase enzymes. This bacterial mutant strain did not stimulate colitis in dnKO mice. Lastly, access of B. theta OMVs to host immune cells was sulfatase dependent. These data demonstrate that bacterial OMVs and associated enzymes promote inflammatory immune stimulation in genetically susceptible hosts.

Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism.

Yeasts, which have been a component of the human diet for at least 7,000 years, possess an elaborate cell wall α-mannan. The influence of yeast mannan on the ecology of the human microbiota is unknown. Here we show that yeast α-mannan is a viable food source for the Gram-negative bacterium Bacteroides thetaiotaomicron, a dominant member of the microbiota. Detailed biochemical analysis and targeted gene disruption studies support a model whereby limited cleavage of α-mannan on the surface generates large oligosaccharides that are subsequently depolymerized to mannose by the action of periplasmic enzymes. Co-culturing studies showed that metabolism of yeast mannan by B. thetaiotaomicron presents a 'selfish' model for the catabolism of this difficult to breakdown polysaccharide. Genomic comparison with B. thetaiotaomicron in conjunction with cell culture studies show that a cohort of highly successful members of the microbiota has evolved to consume sterically-restricted yeast glycans, an adaptation that may reflect the incorporation of eukaryotic microorganisms into the human diet.

Meeting report: SMART timing--principles of single molecule techniques course at the University of Michigan 2014.

Four days after the announcement of the 2014 Nobel Prize in Chemistry for "the development of super-resolved fluorescence microscopy" based on single molecule detection, the Single Molecule Analysis in Real-Time (SMART) Center at the University of Michigan hosted a "Principles of Single Molecule Techniques 2014" course. Through a combination of plenary lectures and an Open House at the SMART Center, the course took a snapshot of a technology with an especially broad and rapidly expanding range of applications in the biomedical and materials sciences. Highlighting the continued rapid emergence of technical and scientific advances, the course underscored just how brightly the future of the single molecule field shines.