Structure of the SARS-CoV-2 Frameshift Stimulatory Element with an Upstream Multibranch Loop.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) frameshift stimulatory element (FSE) is necessary for programmed -1 ribosomal frameshifting (-1 PRF) and optimized viral efficacy. The FSE has an abundance of context-dependent alternate conformations, but two of the structures most crucial to -1 PRF are an attenuator hairpin and a three-stem H-type pseudoknot structure. A crystal structure of the pseudoknot alone features three RNA stems in a helically stacked linear structure, whereas a 6.9 Å cryo-EM structure including the upstream heptameric slippery site resulted in a bend between two stems. Our previous research alluded to an extended upstream multibranch loop that includes both the attenuator hairpin and the slippery site-a conformation not previously modeled. We aim to provide further context to the SARS-CoV-2 FSE via computational and medium resolution cryo-EM approaches, by presenting a 6.1 Å cryo-EM structure featuring a linear pseudoknot structure and a dynamic upstream multibranch loop.

Identifying Operational Challenges and Solutions During the COVID-19 Response Among US Public Health Laboratories.

CONTEXT: The ability to diagnose and screen for infection is an important component of the US COVID-19 response and is facilitated by public health laboratories (PHLs). Anecdotal media reports and limited case studies have described some of the challenges faced by PHLs during the pandemic, particularly initial challenges related to developing and deploying tests to PHLs, but there has not been a systematic evaluation of the experience of PHLs during the pandemic. OBJECTIVE: To document challenges and lessons learned experienced by local and state PHLs during the COVID-19 pandemic to support generation of best practices for current and future similar emergencies. DESIGN, SETTING, AND PARTICIPANTS: From February to June 2021, researchers conducted 24 interviews with 68 leaders and staff representing 28 local and state PHLs across 27 states. Thematic analysis of interview content documented operational challenges and any identified solutions or preventive measures used or proposed. MAIN OUTCOME MEASURES: Analysis identified the following themes regarding challenges faced among PHLs: strategic decision making and determining the mandate of the PHL; political interference by jurisdictional leadership; federal mismanagement of the emergency; regulatory challenges; managing partnerships with other laboratories; acquisition of appropriate supplies; insufficient information systems; acquiring and retaining workforce; and difficulty accessing sufficient funding. RESULTS: Within the identified themes, key informants provided further elaboration regarding how PHLs experienced, evaded, or solved these challenges. In addition, PHLs described how challenges evolved throughout the course of the COVID-19 pandemic and made proposals regarding how challenges could be prevented or further addressed in the future by laboratories or other decision makers and stakeholders. CONCLUSIONS: While fellow laboratories and political leadership may gain inspiration from creative solutions employed by PHLs, recognition of long-standing gaps related to funding, laboratory workforce, and consideration of laboratory needs in preparedness policies must be addressed for future large-scale outbreaks.

Symptoms of Depression, Anxiety, Post-Traumatic Stress Disorder, and Suicidal Ideation Among State, Tribal, Local, and Territorial Public Health Workers During the COVID-19 Pandemic - United States, March-April 2021.

Increases in mental health conditions have been documented among the general population and health care workers since the start of the COVID-19 pandemic (1-3). Public health workers might be at similar risk for negative mental health consequences because of the prolonged demand for responding to the pandemic and for implementing an unprecedented vaccination campaign. The extent of mental health conditions among public health workers during the COVID-19 pandemic, however, is uncertain. A 2014 survey estimated that there were nearly 250,000 state and local public health workers in the United States (4). To evaluate mental health conditions among these workers, a nonprobability-based online survey was conducted during March 29-April 16, 2021, to assess symptoms of depression, anxiety, post-traumatic stress disorder (PTSD), and suicidal ideation among public health workers in state, tribal, local, and territorial public health departments. Among 26,174 respondents, 52.8% reported symptoms of at least one mental health condition in the preceding 2 weeks, including depression (30.8%), anxiety (30.3%), PTSD (36.8%), or suicidal ideation (8.4%). The highest prevalence of symptoms of a mental health condition was among respondents aged ≤29 years (range = 13.6%-47.4%) and transgender or nonbinary persons (i.e., those who identified as neither male nor female) of all ages (range = 30.4%-65.5%). Public health workers who reported being unable to take time off from work were more likely to report adverse mental health symptoms. Severity of symptoms increased with increasing weekly work hours and percentage of work time dedicated to COVID-19 response activities. Implementing prevention and control practices that eliminate, reduce, and manage factors that cause or contribute to public health workers' poor mental health might improve mental health outcomes during emergencies.

Symptoms of Depression, Anxiety, Post-Traumatic Stress Disorder, and Suicidal Ideation Among State, Tribal, Local, and Territorial Public Health Workers During the COVID-19 Pandemic - United States, March-April 2021.

Increases in mental health conditions have been documented among the general population and health care workers since the start of the COVID-19 pandemic (1-3). Public health workers might be at similar risk for negative mental health consequences because of the prolonged demand for responding to the pandemic and for implementing an unprecedented vaccination campaign. The extent of mental health conditions among public health workers during the COVID-19 pandemic, however, is uncertain. A 2014 survey estimated that there were nearly 250,000 state and local public health workers in the United States (4). To evaluate mental health conditions among these workers, a nonprobability-based online survey was conducted during March 29-April 16, 2021, to assess symptoms of depression, anxiety, post-traumatic stress disorder (PTSD), and suicidal ideation among public health workers in state, tribal, local, and territorial public health departments. Among 26,174 respondents, 53.0% reported symptoms of at least one mental health condition in the preceding 2 weeks, including depression (32.0%), anxiety (30.3%), PTSD (36.8%), or suicidal ideation (8.4%). The highest prevalence of symptoms of a mental health condition was among respondents aged ≤29 years (range = 13.6%-47.4%) and transgender or nonbinary persons (i.e., those who identified as neither male nor female) of all ages (range = 30.4%-65.5%). Public health workers who reported being unable to take time off from work were more likely to report adverse mental health symptoms. Severity of symptoms increased with increasing weekly work hours and percentage of work time dedicated to COVID-19 response activities. Implementing prevention and control practices that eliminate, reduce, and manage factors that cause or contribute to public health workers' poor mental health might improve mental health outcomes during emergencies.

Creating a Blueprint for the Future: Lessons Learned From Public Health Laboratories in the COVID-19 Response.

Public health laboratories have played a central role in the US response to COVID-19. Since the earliest days, myriad issues have impeded the laboratory community's ability to keep pace with the overwhelming demand for effective tests. In this article, the Association of Public Health Laboratories and a subset of its members examine the response to date and evaluate lessons learned from 4 main categories: testing surges, supplies, staffing, and regulations and policy. Within these categories, the authors offer recommendations intended both to improve the ongoing COVID-19 response and to strengthen planning for future outbreaks.

Modeling rectal cancer to advance neoadjuvant precision therapy.

Progress in rectal cancer therapy has been hindered by the lack of effective disease-specific preclinical models that account for the unique molecular profile and biology of rectal cancer. Thus, we developed complementary patient-derived xenograft (PDX) and subsequent in vitro tumor organoid (PDTO) platforms established from preneoadjuvant therapy rectal cancer specimens to advance personalized care for rectal cancer patients. Multiple endoscopic samples were obtained from 26 Stages 2 and 3 rectal cancer patients prior to receiving 5FU/RT and implanted subcutaneously into NSG mice to generate 15 subcutaneous PDXs. Second passaged xenografts demonstrated 100% correlation with the corresponding human cancer histology with maintained mutational profiles. Individual rectal cancer PDXs reproduced the 5FU/RT response observed in the corresponding human cancers. Similarly, rectal cancer PDTOs reproduced significant heterogeneity in cellular morphology and architecture. PDTO in vitro 5FU/RT treatment response replicated the clinical 5FU/RT neoadjuvant therapy pathologic response observed in the corresponding patient tumors (p < 0.05). The addition of cetuximab to the 5FU/RT regiment was significantly more sensitive in the rectal cancer PDX and PDTOs with wild-type KRAS compared to mutated KRAS (p < 0.05). Considering the close relationship between the patient's cancer and the corresponding PDX/PDTO, rectal cancer patient-derived research platforms represent powerful translational research resources as population-based tools for biomarker discovery and experimental therapy testing. In addition, our findings suggest that cetuximab may enhance RT effectiveness by improved patient selection based on mutational profile in addition to KRAS or by developing a protocol using PDTOs to identify sensitive patients.

miR-145 Antagonizes SNAI1-Mediated Stemness and Radiation Resistance in Colorectal Cancer.

Epithelial-to-mesenchymal transition (EMT) has been closely linked with therapy resistance and cancer stem cells (CSCs). However, EMT pathways have proven challenging to therapeutically target. MicroRNA 145 (miR-145) targets multiple stem cell transcription factors and its expression is inversely correlated with EMT. Therefore, we hypothesized that miR-145 represents a therapeutic target to reverse snail family transcriptional repressor 1 (SNAI1)-mediated stemness and radiation resistance (RT). Stable expression of SNAI1 in DLD1 and HCT116 cells (DLD1-SNAI1; HCT116-SNAI1) increased expression of Nanog and decreased miR-145 expression compared to control cells. Using a miR-145 luciferase reporter assay, we determined that ectopic SNAI1 expression significantly repressed the miR-145 promoter. DLD1-SNAI1 and HCT116-SNAI1 cells demonstrated decreased RT sensitivity and, conversely, miR-145 replacement significantly enhanced RT sensitivity. Of the five parental colon cancer cell lines, SW620 cells demonstrated relatively high endogenous SNAI1 and low miR-145 levels. In the SW620 cells, miR-145 replacement decreased CSC-related transcription factor expression, spheroid formation, and radiation resistance. In rectal cancer patient-derived xenografts, CSC identified by EpCAM+/aldehyde dehydrogenase (ALDH)+ demonstrated high expression of SNAI1, c-Myc, and Nanog compared with non-CSCs (EpCAM+/ALDH-). Conversely, patient-derived CSCs demonstrated low miR-145 expression levels relative to non-CSCs. These results suggest that the SNAI1:miR-145 pathway represents a novel therapeutic target in colorectal cancer to overcome RT resistance.

Diversity, Productivity, and Stability of an Industrial Microbial Ecosystem.

Managing ecosystems to maintain biodiversity may be one approach to ensuring their dynamic stability, productivity, and delivery of vital services. The applicability of this approach to industrial ecosystems that harness the metabolic activities of microbes has been proposed but has never been tested at relevant scales. We used a tag-sequencing approach with bacterial small subunit rRNA (16S) genes and eukaryotic internal transcribed spacer 2 (ITS2) to measuring the taxonomic composition and diversity of bacteria and eukaryotes in an open pond managed for bioenergy production by microalgae over a year. Periods of high eukaryotic diversity were associated with high and more-stable biomass productivity. In addition, bacterial diversity and eukaryotic diversity were inversely correlated over time, possibly due to their opposite responses to temperature. The results indicate that maintaining diverse communities may be essential to engineering stable and productive bioenergy ecosystems using microorganisms.

Regulation of NF-κB signaling by oxidized glycerophospholipid and IL-1ß induced miRs-21-3p and -27a-5p in human aortic endothelial cells.

Exposure of endothelial cells (ECs) to agents such as oxidized glycerophospholipids (oxGPs) and cytokines, known to accumulate in atherosclerotic lesions, perturbs the expression of hundreds of genes in ECs involved in inflammatory and other biological processes. We hypothesized that microRNAs (miRNAs) are involved in regulating the inflammatory response in human aortic endothelial cells (HAECs) in response to oxGPs and interleukin 1ß (IL-1ß). Using next-generation sequencing and RT-quantitative PCR, we characterized the profile of expressed miRNAs in HAECs pre- and postexposure to oxGPs. Using this data, we identified miR-21-3p and miR-27a-5p to be induced 3- to 4-fold in response to oxGP and IL-1ß treatment compared with control treatment. Transient overexpression of miR-21-3p and miR-27a-5p resulted in the downregulation of 1,253 genes with 922 genes overlapping between the two miRNAs. Gene Ontology functional enrichment analysis predicted that the two miRNAs were involved in the regulation of nuclear factor κB (NF-κB) signaling. Overexpression of these two miRNAs leads to changes in p65 nuclear translocation. Using 3' untranslated region luciferase assay, we identified 20 genes within the NF-κB signaling cascade as putative targets of miRs-21-3p and -27a-5p, implicating these two miRNAs as modulators of NF-κB signaling in ECs.

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.

Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific T cell engagers.

Adoptive cell therapy (ACT) utilizing γδ T cells is becoming a promising option for the treatment of cancer, because it offers an off-the-shelf allogeneic product that is safe, potent, and clinically effective. Approaches to engineer or enhance immune-competent cells for ACT, like expression of chimeric antigen receptors (CARs) or combination treatments with bispecific T cell engagers, have improved the specificity and cytotoxic potential of ACTs and have shown great promise in preclinical and clinical settings. Here, we test whether electroporation of γδ T cells with CAR or secreted bispecific T cell engager (sBite) mRNA is an effective approach to improve the cytotoxicity of γδ T cells. Using a CD19-specific CAR, approximately 60% of γδ T cells are modified after mRNA electroporation and these cells show potent anticancer activity in vitro and in vivo against two CD19-positive cancer cell lines. In addition, expression and secretion of a CD19 sBite enhances γδ T cell cytotoxicity, both in vitro and in vivo, and promotes killing of target cells by modified and unmodified γδ T cells. Taken together, we show that transient transfection of γδ T cells with CAR or sBite mRNA by electroporation can be an effective treatment platform as a cancer therapeutic.

Critical evaluation of an autologous peripheral blood mononuclear cell-based humanized cancer model.

The use of humanized mouse models for oncology is rapidly expanding. Autologous patient-derived systems are particularly attractive as they can model the human cancer's heterogeneity and immune microenvironment. In this study, we developed an autologous humanized mouse cancer model by engrafting NSG mice with patient-derived xenografts and infused matched peripheral blood mononuclear cells (PBMCs). We first defined the time course of xenogeneic graft-versus-host-disease (xGVHD) and determined that only minimal xGVHD was observed for up to 8 weeks. Next, colorectal and pancreatic cancer patient-derived xenograft bearing NSG mice were infused with 5x106 human PBMCS for development of the humanized cancer models (iPDX). Early after infusion of human PBMCs, iPDX mice demonstrated engraftment of human CD4+ and CD8+ T cells in the blood of both colorectal and pancreatic cancer patient-derived models that persisted for up to 8 weeks. At the end of the experiment, iPDX xenografts maintained the features of the primary human tumor including tumor grade and cell type. The iPDX tumors demonstrated infiltration of human CD3+ cells with high PD-1 expression although we observed significant intra and inter- model variability. In summary, the iPDX models reproduced key features of the corresponding human tumor. The observed variability and high PD-1 expression are important considerations that need to be addressed in order to develop a reproducible model system.

COVID-19 Pandemic-Related Impacts on Newborn Screening Public Health Surveillance.

Newborn screening (NBS) is an essential public health service that performs screening to identify those newborns at increased risk for a panel of disorders, most of which are genetic. The goal of screening is to link those newborns at the highest risk to timely intervention and potentially life-saving treatment. The global COVID-19 pandemic led to disruptions within the United States public health system, revealing implications for the continuity of newborn screening laboratories and follow-up operations. The impacts of COVID-19 across different states at various time points meant that NBS programs impacted by the pandemic later could benefit from the immediate experiences of the earlier impacted programs. This article will review the collection, analysis, and dissemination of information during the COVID-19 pandemic facilitated by a national, centralized technical assistance and resource center for NBS programs.

Structural basis of mismatch recognition by a SARS-CoV-2 proofreading enzyme.

Coronavirus 3'-to-5' exoribonuclease (ExoN), residing in the nonstructural protein (nsp) 10­nsp14 complex, boosts replication fidelity by proofreading RNA synthesis and is critical for the virus life cycle. ExoN also recognizes and excises nucleotide analog inhibitors incorporated into the nascent RNA, undermining the effectiveness of nucleotide analog­based antivirals. Here we present cryo­electron microscopy structures of both wild-type and mutant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nsp10-nsp14 in complex with an RNA substrate bearing a 3'-end mismatch at resolutions ranging from 2.5 to 3.9 angstroms. The structures reveal the molecular determinants of ExoN substrate specificity and offer insight into the molecular mechanisms of mismatch correction during coronavirus RNA synthesis. Our findings provide guidance for rational design of improved anticoronavirus therapies.

Pain Outcomes with an Elliptical Regimen (POWER) Study: Identifying the Proper Dosage of Exercise for Therapeutic Effect in Persons with Chronic Back Pain.

BACKGROUND CONTEXT: Exercise therapy for low back pain has long been prescribed as one of the initial remedies for back pain. Traditional therapy is completed under a therapist's supervision and consists of lumbar stabilization, aerobic exercise and stretching exercises. Recent studies have explored treating back pain with aerobic exercise such as walking which can be done anywhere and without supervision which is lower cost and easily administered. PURPOSE: To assess a therapeutic dosage of aerobic exercise that is associated with pain reduction in persons experiencing low back pain. STUDY DESIGN: Case series. PARTICIPANT DESCRIPTION: Sixteen patients entered the study and twelve patients completed the study (mean ± SD: age 51 ± 11 years; weight 89.2 ± 16 kg). Subjects were included if they were ages 18-65, had chronic back pain lasting for more than 3 months and a score of greater than 30% on the Oswestry Low Back Disability Questionnaire. METHODS: Subjects underwent a six-week exercise program using the elliptical trainer three times each week. Exercise duration was steadily increased each week for the length of the study. The total cumulative amount of work that coincided with significant reductions in chronic low back pain was then identified. RESULTS: At 4 weeks, pain scores were significantly reduced from baseline (3.2 vs 4.7, p<0.0001). This significant pain reduction corresponded to an average of 30.8 Kcal/kg of body mass in cumulative work performed. Pain was significantly reduced by 21% and 32% on the Oswestry Questionnaire and the PROMIS 29 respectively. CONCLUSIONS: These pilot findings suggest that approximately 30.8 kcal/Kg of accumulated physiological work is a therapeutic "dosage" of exercise needed for significant reduction in chronic back pain. Clinicians can begin to use this benchmark for their oversight of rehabilitation programs to determine if an exercise program has been sufficiently intense and long enough in duration for managing their patients with chronic low back pain.

Non-signaling Chimeric Antigen Receptors Enhance Antigen-Directed Killing by γδ T Cells in Contrast to αß T Cells.

Chimeric antigen receptor (CAR)-modified T cells have demonstrated efficacy against B cell leukemias/lymphomas. However, redirecting CAR T cells to malignant T cells is more challenging due to product-specific cis- and trans-activation causing fratricide. Other challenges include the potential for product contamination and T cell aplasia. We expressed non-signaling CARs (NSCARs) in γδ T cells since donor-derived γδ T cells can be used to prevent product contamination, and NSCARs lack signaling/activation domains, but retain antigen-specific tumor cell-targeting capability. As a result, NSCAR targeting requires an alternative cytotoxic mechanism, which can be achieved through utilization of γδ T cells that possess major histocompatibility complex (MHC)-independent cytotoxicity. We designed two distinct NSCARs and demonstrated that they do not enhance tumor-killing by αß T cells, as predicted. However, both CD5-NSCAR- and CD19-NSCAR-modified γδ T cells enhanced cytotoxicity against T and B cell acute lymphoblastic leukemia (T-ALL and B-ALL) cell lines, respectively. CD5-NSCAR expression in γδ T cells resulted in a 60% increase in cytotoxicity of CD5-expressing T-ALL cell lines. CD19-NSCAR-modified γδ T cells exhibited a 350% increase in cytotoxicity against a CD19-expressing B-ALL cell line compared to the cytotoxicity of naive cells. NSCARs may provide a mechanism to enhance antigen-directed anti-tumor cytotoxicity of γδ T cells through the introduction of a high-affinity interaction while avoiding self-activation.

Phased Diploid Genome Sequence for the Fast-Growing Microalga Picochlorum celeri.

Picochlorum celeri is a fast-growing marine microalga with high biomass productivity. Here, we report the use of PacBio sequencing to assemble the phased diploid genome of P. celeri.