Activity-based RNA-modifying enzyme probing reveals DUS3L-mediated dihydrouridylation.

Epitranscriptomic RNA modifications can regulate RNA activity; however, there remains a major gap in our understanding of the RNA chemistry present in biological systems. Here we develop RNA-mediated activity-based protein profiling (RNABPP), a chemoproteomic strategy that relies on metabolic RNA labeling, mRNA interactome capture and quantitative proteomics, to investigate RNA-modifying enzymes in human cells. RNABPP with 5-fluoropyrimidines allowed us to profile 5-methylcytidine (m5C) and 5-methyluridine (m5U) methyltransferases. Further, we uncover a new mechanism-based crosslink between 5-fluorouridine (5-FUrd)-modified RNA and the dihydrouridine synthase (DUS) homolog DUS3L. We investigate the mechanism of crosslinking and use quantitative nucleoside liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and 5-FUrd-based crosslinking and immunoprecipitation (CLIP) sequencing to map DUS3L-dependent dihydrouridine (DHU) modifications across the transcriptome. Finally, we show that DUS3L-knockout (KO) cells have compromised protein translation rates and impaired cellular proliferation. Taken together, our work provides a general approach for profiling RNA-modifying enzyme activity in living cells and reveals new pathways for epitranscriptomic RNA regulation.

Analysis of Host Responses to Hepatitis B and Delta Viral Infections in a Micro-scalable Hepatic Co-culture System.

Hepatitis B virus (HBV) remains a major global health problem with 257 million chronically infected individuals worldwide, of whom approximately 20 million are co-infected with hepatitis delta virus (HDV). Progress toward a better understanding of the complex interplay between these two viruses and the development of novel therapies have been hampered by the scarcity of suitable cell culture models that mimic the natural environment of the liver. Here, we established HBV and HBV/HDV co-infections and super-infections in self-assembling co-cultured primary human hepatocytes (SACC-PHHs) for up to 28 days in a 384-well format and highlight the suitability of this platform for high-throughput drug testing. We performed RNA sequencing at days 8 and 28 on SACC-PHHs, either HBV mono-infected or HBV/HDV co-infected. Our transcriptomic analysis demonstrates that hepatocytes in SACC-PHHs maintain a mature hepatic phenotype over time, regardless of infection condition. We confirm that HBV is a stealth virus, as it does not induce a strong innate immune response; rather, oxidative phosphorylation and extracellular matrix-receptor interactions are dysregulated to create an environment that promotes persistence. Notably, HDV co-infection also did not lead to statistically significant transcriptional changes across multiple donors and replicates. The lack of innate immune activation is not due to SACC-PHHs being impaired in their ability to induce interferon stimulated genes (ISGs). Rather, polyinosinic:polycytidylic acid exposure activates ISGs, and this stimulation significantly inhibits HBV infection, yet only minimally affects the ability of HDV to infect and persist. Conclusion: These data demonstrate that the SACC-PHH system is a versatile platform for studying HBV/HDV co-infections and holds promise for performing chemical library screens and improving our understanding of the host response to such infections.

In Vitro Selection with a Site-Specifically Modified RNA Library Reveals the Binding Preferences of N6-Methyladenosine Reader Proteins.

Epitranscriptomic RNA modifications can serve as recognition elements for the recruitment of effector proteins (i.e., "readers") to modified transcripts. While these interactions play an important role in mRNA regulation, there is a major gap in our understanding of the sequence determinants critical for the binding of readers to modified sequence motifs. Here, we develop a high-throughput platform, relying upon in vitro selection with a site-specifically modified random sequence RNA library and next-generation sequencing, to profile the binding specificity of RNA modification reader proteins. We apply our approach to interrogate the effect of sequence context on the interactions of YTH-domain proteins with N6-methyladenosine (m6A)-modified RNA. We find that while the in vitro binding preferences of YTHDC1 strongly overlap with the well-characterized DR(m6A)CH motif, the related YTH-domain proteins YTHDF1 and YTHDF2 can bind tightly to noncanonical m6A-containing sequences. Our results reveal the principles underlying substrate selection by m6A reader proteins and provide a powerful approach for investigating protein-modified RNA interactions in an unbiased manner.

Effect of minichromosome maintenance protein 2 deficiency on the locations of DNA replication origins.

Minichromosome maintenance (MCM) proteins are loaded onto chromatin during G1-phase and define potential locations of DNA replication initiation. MCM protein deficiency results in genome instability and high rates of cancer in mouse models. Here we develop a method of nascent strand capture and release and show that MCM2 deficiency reduces DNA replication initiation in gene-rich regions of the genome. DNA structural properties are shown to correlate with sequence motifs associated with replication origins and with locations that are preferentially affected by MCM2 deficiency. Reduced nascent strand density correlates with sites of recurrent focal CNVs in tumors arising in MCM2-deficient mice, consistent with a direct relationship between sites of reduced DNA replication initiation and genetic damage. Between 10% and 90% of human tumors, depending on type, carry heterozygous loss or mutation of one or more MCM2-7 genes, which is expected to compromise DNA replication origin licensing and result in elevated rates of genome damage at a subset of gene-rich locations.

Whole-genome sequencing identifies genomic heterogeneity at a nucleotide and chromosomal level in bladder cancer.

Using complete genome analysis, we sequenced five bladder tumors accrued from patients with muscle-invasive transitional cell carcinoma of the urinary bladder (TCC-UB) and identified a spectrum of genomic aberrations. In three tumors, complex genotype changes were noted. All three had tumor protein p53 mutations and a relatively large number of single-nucleotide variants (SNVs; average of 11.2 per megabase), structural variants (SVs; average of 46), or both. This group was best characterized by chromothripsis and the presence of subclonal populations of neoplastic cells or intratumoral mutational heterogeneity. Here, we provide evidence that the process of chromothripsis in TCC-UB is mediated by nonhomologous end-joining using kilobase, rather than megabase, fragments of DNA, which we refer to as "stitchers," to repair this process. We postulate that a potential unifying theme among tumors with the more complex genotype group is a defective replication-licensing complex. A second group (two bladder tumors) had no chromothripsis, and a simpler genotype, WT tumor protein p53, had relatively few SNVs (average of 5.9 per megabase) and only a single SV. There was no evidence of a subclonal population of neoplastic cells. In this group, we used a preclinical model of bladder carcinoma cell lines to study a unique SV (translocation and amplification) of the gene glutamate receptor ionotropic N-methyl D-aspertate as a potential new therapeutic target in bladder cancer.

Domain movements of the enhancer-dependent sigma factor drive DNA delivery into the RNA polymerase active site: insights from single molecule studies.

Recognition of bacterial promoters is regulated by two distinct classes of sequence-specific sigma factors, σ(70) or σ(54), that differ both in their primary sequence and in the requirement of the latter for activation via enhancer-bound upstream activators. The σ(54) version controls gene expression in response to stress, often mediating pathogenicity. Its activator proteins are members of the AAA+ superfamily and use adenosine triphosphate (ATP) hydrolysis to remodel initially auto-inhibited holoenzyme promoter complexes. We have mapped this remodeling using single-molecule fluorescence spectroscopy. Initial remodeling is nucleotide-independent and driven by binding both ssDNA during promoter melting and activator. However, DNA loading into the RNA polymerase active site depends on co-operative ATP hydrolysis by the activator. Although the coupled promoter recognition and melting steps may be conserved between σ(70) and σ(54), the domain movements of the latter have evolved to require an activator ATPase.

Chiropractic care and public health: answering difficult questions about safety, care through the lifespan, and community action.

The purpose of this collaborative summary is to document current chiropractic involvement in the public health movement, reflect on social ecological levels of influence as a profession, and summarize the relationship of chiropractic to the current public health topics of: safety, health issues through the lifespan, and effective participation in community health issues. The questions that are addressed include: Is spinal manipulative therapy for neck and low-back pain a public health problem? What is the role of chiropractic care in prevention or reduction of musculoskeletal injuries in children? What ways can doctors of chiropractic stay updated on evidence-based information about vaccines and immunization throughout the lifespan? Can smoking cessation be a prevention strategy for back pain? Does chiropractic have relevance within the VA Health Care System for chronic pain and comorbid disorders? How can chiropractic use cognitive behavioral therapy to address chronic low back pain as a public health problem? What opportunities exist for doctors of chiropractic to more effectively serve the aging population? What is the role of ethics and the contribution of the chiropractic profession to public health? What public health roles can chiropractic interns perform for underserved communities in a collaborative environment? Can the chiropractic profession contribute to community health? What opportunities do doctors of chiropractic have to be involved in health care reform in the areas of prevention and public health? What role do citizen-doctors of chiropractic have in organizing community action on health-related matters? How can our future chiropractic graduates become socially responsible agents of change?

Phenotype-Guided Comparative Genomics Identifies the Complete Transport Pathway of the Antimicrobial Lasso Peptide Ubonodin in Burkholderia.

New antibiotics are needed as bacterial infections continue to be a leading cause of death, but efforts to develop compounds with promising antibacterial activity are hindered by a poor understanding of─and limited strategies for elucidating─their modes of action. We recently discovered a novel lasso peptide, ubonodin, that is active against opportunistic human lung pathogens from the Burkholderia cepacia complex (Bcc). Ubonodin inhibits RNA polymerase, but only select strains were susceptible, indicating that having a conserved cellular target does not guarantee activity. Given the cytoplasmic target, we hypothesized that cellular uptake of ubonodin determines susceptibility. Although Bcc strains harbor numerous nutrient uptake systems, these organisms lack close homologues of the single known lasso peptide membrane receptor, FhuA. Thus, a straightforward homology-driven approach failed to uncover the identity of the ubonodin transporter(s). Here, we used phenotype-guided comparative genomics to identify genes uniquely associated with ubonodin-susceptible Bcc strains, leading to the identification of PupB as the ubonodin outer membrane (OM) receptor in Burkholderia. The loss of PupB renders B. cepacia resistant to ubonodin, whereas expressing PupB sensitizes a resistant strain. We also examine how a conserved iron-regulated transcriptional pathway controls PupB to further tune ubonodin susceptibility. PupB is only the second lasso peptide OM receptor to be uncovered and the first outside of enterobacteria. Finally, we elucidate the full transport pathway for ubonodin by identifying its inner membrane receptor YddA in Burkholderia. Our work provides a complete picture of the mode of action of ubonodin and establishes a general framework for deciphering the transport pathways of other natural products with cytoplasmic targets.

COVID-19 Infectious Disease Prevention and Mitigation Practices by Chiropractic Physicians and Licensed Massage Therapists in Mississippi: A Needs Assessment to Inform Health Education and Promotion.

Objective: The purpose of this study was to assess self-reported infection prevention processes and their effect on businesses of chiropractic doctors (DCs) and licensed massage therapists (LMTs) in Mississippi during the COVID-19 pandemic. Methods: We developed a survey that was electronically delivered to all licensed DCs and LMTs in Mississippi between August and September 2020. Assessments were made using Qualtrics software, with data management and subsequent analysis including Pearson's χ2 test. Results: Responses were based on 32 of 323 DCs and 69 of 934 LMTs that were still seeing patients through the pandemic (n = 101, response rate 8%). The DC and LMT practitioners (94%) used treatment table and/or surface sanitizing (91.8%) and hand washing and/or sanitizing (89.8%) between all patients. Female practitioners reported practicing handwashing for at least 20 seconds, whereas male practitioners reported practicing handwashing for at least 15 seconds (P < .001). DCs were more likely to report using gloves for personal protective equipment, and LMTs were more likely to report using face masks (P < .001). Other COVID-19 procedures included limiting practice to acute care (82.5%), checking all patient temperatures (62.9%), sign-in and wait in the car (53.2% LMT vs 6.5% DC, P < .001), and prohibiting all nonpatient visitors (87.7% LMTs vs 9.4% DCs, P < .001). DCs (96.9%) and LMTs (89.9%) reported making referrals for COVID-19 testing or treatment when indicated. LMTs (82.3%) reported seeing fewer patients (P = .03), and older practitioners reported the most economic impact (P = .003) by the pandemic. Patient concerns and LMTs needing more time to perform infection control (P = .04) were reasons cited by practitioners for the reduced number of visits seen. Conclusion: Most respondents had moderate to high compliance with guidelines on recommended infection prevention processes during fall 2020 of the COVID-19 pandemic. This assessment of compliance may be used to help guide future health education and promotion research of disease prevention and mitigation as well as physical and economic burdens faced by DCs and LMTs in Mississippi during a pandemic.

Familiarity with and advocacy of Healthy People 2010 goals by Mississippi Chiropractic Association members.

OBJECTIVE: The purpose of this study was to determine the familiarity with and stated advocacy of Healthy People 2010 objectives by member doctors of the Mississippi Chiropractic Association. METHODS: Peer experts established face validity of a questionnaire regarding the Leading Health Indicators. This survey was distributed to 157 Mississippi Chiropractic Association members in 2009 during a conference and a follow-up by postal mail. RESULTS: Most doctors of chiropractic in the sample (n = 68, or 43% response) consider themselves wellness-oriented health care providers. Forty-two percent had read, 29% had not read, and another 29% were unsure whether they had read the Healthy People 2010 national objectives. Almost half (44%) strongly or somewhat agreed that their office practice reflects support for the Healthy People 2010 objective. In contrast, 27% disagree and 29% were unsure if their practice reflects the Healthy People 2010 objectives. There were differences between support and practice behaviors for some of the objectives. Chiropractors who have read the objectives tend to be more supportive of the national goals. Doctors of chiropractic in this sample are supportive of most Leading Health Indicators, and the majority reports that they incorporate these public health goals into their practices. CONCLUSION: Familiarity with reading the Health People objectives seems to be related to reported practice behaviors. There is a need to improve the percentage of practicing doctors of chiropractic who are familiar with Healthy People objectives. Future health education initiatives may assist doctors of chiropractic in further incorporating public health objectives into their practice behaviors and improving quality health care.

Filoviruses are ancient and integrated into mammalian genomes.

BACKGROUND: Hemorrhagic diseases from Ebolavirus and Marburgvirus (Filoviridae) infections can be dangerous to humans because of high fatality rates and a lack of effective treatments or vaccine. Although there is evidence that wild mammals are infected by filoviruses, the biology of host-filovirus systems is notoriously poorly understood. Specifically, identifying potential reservoir species with the expected long-term coevolutionary history of filovirus infections has been intractable. Integrated elements of filoviruses could indicate a coevolutionary history with a mammalian reservoir, but integration of nonretroviral RNA viruses is thought to be nonexistent or rare for mammalian viruses (such as filoviruses) that lack reverse transcriptase and replication inside the nucleus. Here, we provide direct evidence of integrated filovirus-like elements in mammalian genomes by sequencing across host-virus gene boundaries and carrying out phylogenetic analyses. Further we test for an association between candidate reservoir status and the integration of filoviral elements and assess the previous age estimate for filoviruses of less than 10,000 years. RESULTS: Phylogenetic and sequencing evidence from gene boundaries was consistent with integration of filoviruses in mammalian genomes. We detected integrated filovirus-like elements in the genomes of bats, rodents, shrews, tenrecs and marsupials. Moreover, some filovirus-like elements were transcribed and the detected mammalian elements were homologous to a fragment of the filovirus genome whose expression is known to interfere with the assembly of Ebolavirus. The phylogenetic evidence strongly indicated that the direction of transfer was from virus to mammal. Eutherians other than bats, rodents, and insectivores (i.e., the candidate reservoir taxa for filoviruses) were significantly underrepresented in the taxa with detected integrated filovirus-like elements. The existence of orthologous filovirus-like elements shared among mammalian genera whose divergence dates have been estimated suggests that filoviruses are at least tens of millions of years old. CONCLUSIONS: Our findings indicate that filovirus infections have been recorded as paleoviral elements in the genomes of small mammals despite extranuclear replication and a requirement for cooption of reverse transcriptase. Our results show that the mammal-filovirus association is ancient and has resulted in candidates for functional gene products (RNA or protein).

Patients with symptoms and signs of stroke presenting to a rural chiropractic practice.

OBJECTIVE: The purpose of this article was to present a retrospective case series of patients with symptoms and signs suggesting a stroke or prodrome and discuss the potential for health education and promotion initiatives in chiropractic that might affect this serious disease. METHODS: A database and hand search of records from a rural Mississippi chiropractic practice was performed to identify all cases of patients presenting with symptoms and signs of stroke during the past 4 years, as well as stroke mortality among active patients during the same period. RESULTS: Six subjects met inclusion criteria of about 500 active patients during any given year of the 4-year study period, including patients who had stroke symptoms or signs (n = 4) or who died as a result of stroke without presenting to the office (n = 2). Records and magnetic resonance angiograms were obtained after referral was made for a 77-year-old white female, 71-year-old white male, 59-year-old white male, and 24-year-old Hispanic female. Three of the 4 surviving patients were eventually diagnosed with stroke, and one with seizure disorder. Common presenting complaints were unilateral arm weakness and slurred speech; other complaints included episodic loss of vision, dysphagia, dysphonia, and same-sided leg weakness. CONCLUSIONS: Patients with symptoms and signs of stroke may infrequently present to chiropractic physicians for evaluation and treatment. Prevention, screening, early identification of stroke symptoms and signs, and referral for prompt treatment are cornerstones of the national stroke policy as espoused by the Centers for Disease Control. Chiropractic physicians have opportunities for practice-related health education initiatives focusing on the role of health and wellness in stroke prevention and for prompt lifesaving referral of emergency presentations.

Full-Coverage Chiropractic in Medicare: A Proposal to Eliminate Inequities, Improve Outcomes, and Reduce Health Disparities Without Increasing Overall Program Costs.

OBJECTIVE: The purpose of this article is to discuss evidence that supports the resolution of inequities for Medicare beneficiaries who receive chiropractic care. DISCUSSION: Medicare covers necessary examinations, imaging, exercise instruction, and treatments for beneficiaries with back pain when provided by medical doctors, osteopaths, and their associated support staff such as nurse practitioners, physician assistants, clinical nurse specialists, and physical therapists. However, if the same patient with back pain presents to a chiropractor, then the only service that is covered by Medicare is manipulation of the spine. Current evidence does not support this inequity in Medicare beneficiary service coverage. There is no evidence to show an increase in serious risks associated with chiropractic treatment of neck or back pain in Medicare beneficiaries. Chiropractors support national public health goals and endorse safe, evidence-based practices. Chiropractic care for Medicare beneficiaries has been associated with enhanced clinical outcomes such as faster recovery, fewer back surgeries a year later, reduced opioid-associated disability, fewer traumatic injuries and falls, and slower declines in activities of daily living and disability over time. Further evidence points to lower costs, fewer medical physician visits for low back pain, less opioid-related expense, and less back-surgery expense with chiropractic utilization. Use is lower among vulnerable populations: seniors, lower income women, and black and Hispanic beneficiaries who may be most affected by current inequities associated with the limited coverage. In this era of evidence-based and patient-centered care, beneficiaries who receive chiropractic care are very satisfied with the care they receive. CONCLUSION: The current evidence suggests a need for change in US policy toward chiropractic in Medicare and support for HR 3654. Ending inequities by providing patients full coverage for chiropractic services has the potential to enhance care outcomes and reduce health disparities without increasing program costs.

Approaching acute pain in emergency settings; European Society for Emergency Medicine (EUSEM) guidelines-part 2: management and recommendations.

BACKGROUND: In Europe, healthcare systems and education, as well as the clinical care and health outcomes of patients, varies across countries. Likewise, the management of acute events for patients also differs, dependent on the emergency care setting, e.g. pre-hospital or emergency department. There are various barriers to adequate pain management and factors common to both settings including lack of knowledge and training, reluctance to give opioids, and concerns about drug-seeking behaviour or abuse. There is no single current standard of care for the treatment of pain in an emergency, with management based on severity of pain, injury and local protocols. Changing practices, attitudes and behaviour can be difficult, and improvements and interventions should be developed with barriers to pain management and the needs of the individual emergency setting in mind. METHODS: With these principles at the forefront, The European Society for Emergency Medicine (EUSEM) launched a programme-the European Pain Initiative (EPI)-with the aim of providing information, advice, and guidance on acute pain management in emergency settings. RESULTS AND CONCLUSIONS: This article provides treatment recommendations from recently developed guidelines, based on a review of the literature, current practice across Europe and the clinical expertise of the EPI advisors. The recommendations have been developed, evaluated, and refined for both adults and children (aged ≥ 1 year, ≤ 15 years), with the assumption of timely pain assessment and reassessment and the possibility to implement analgesia. To provide flexibility for use across Europe, options are provided for selection of appropriate pharmacological treatment.

Approaching acute pain in emergency settings: European Society for Emergency Medicine (EUSEM) guidelines-part 1: assessment.

Pain management is a vital component of patient care, particularly in the emergency setting. Pain can hinder the opportunities to treat and manage pain-causing conditions and remains one of the primary reasons patients seek emergency medical care, yet despite this, pain often remains under-acknowledged, under-assessed and undertreated. Despite the importance of effective management of acute pain, there are currently no well-defined emergency medicine guidelines at a European level to support healthcare professionals in achieving this goal. The European Society for Emergency Medicine (EUSEM) identified this as a significant unmet requirement, for improving day-to-day patient management and for providing guidance to trainees and non-emergency medicine physicians. Under the auspices of EUSEM, a programme-the European Pain Initiative-was launched with the aim of providing information, advice and guidance on pain management in pre-hospital and emergency department settings. Search terms were developed to search MEDLINE, Cochrane database, Google Scholar and EMBASE online databases to return English language articles published in the last 10 years. A working package of reference materials was evaluated against inclusion and exclusion criteria and levels of evidence ascribed. A short survey was developed by the European Pain Initiative Steering Committee for completion by EUSEM members to evaluate actual clinical practice. A working document of > 800 publications was identified for further review and evaluation against agreed criteria. Some further publications were included by the Steering Committee to explore older literature for long-established analgesics, or newly emergent literature that was considered important for inclusion but was identified as the guideline development was underway. This article provides the methodology used to inform the guidelines, including survey results of EUSEM members on assessment and treatment of acute pain. A companion manuscript in this issue presents an evidence-based review and recommendations for individualised evaluation of acute pain in patients in emergency settings.

European Society For Emergency Medicine position paper on emergency medical systems' response to COVID-19.

The 2019 novel coronavirus acute respiratory epidemic is creating a stressed situation in all the health systems of the affected countries. Emergency medical systems and specifically the emergency departments as the front line of the health systems are suffering from overload and severe working conditions, the risk of contagion and transmission of the health professionals adds a substantial burden to their daily work. Under the perspective of European Society For Emergency Medicine, the recommendations provided by the health authorities are reviewed focus on the emergency department's activity.

Stability and nuclear localization of yeast telomerase depend on protein components of RNase P/MRP.

RNase P and MRP are highly conserved, multi-protein/RNA complexes with essential roles in processing ribosomal and tRNAs. Three proteins found in both complexes, Pop1, Pop6, and Pop7 are also telomerase-associated. Here, we determine how temperature sensitive POP1 and POP6 alleles affect yeast telomerase. At permissive temperatures, mutant Pop1/6 have little or no effect on cell growth, global protein levels, the abundance of Est1 and Est2 (telomerase proteins), and the processing of TLC1 (telomerase RNA). However, in pop mutants, TLC1 is more abundant, telomeres are short, and TLC1 accumulates in the cytoplasm. Although Est1/2 binding to TLC1 occurs at normal levels, Est1 (and hence Est3) binding is highly unstable. We propose that Pop-mediated stabilization of Est1 binding to TLC1 is a pre-requisite for formation and nuclear localization of the telomerase holoenzyme. Furthermore, Pop proteins affect TLC1 and the RNA subunits of RNase P/MRP in very different ways.