Mitochondrial sequencing identifies long noncoding RNA features that promote binding to PNPase.

Extranuclear localization of long noncoding RNAs (lncRNAs) is poorly understood. Based on machine learning evaluations, we propose a lncRNA-mitochondrial interaction pathway where polynucleotide phosphorylase (PNPase), through domains that provide specificity for primary sequence and secondary structure, binds nuclear-encoded lncRNAs to facilitate mitochondrial import. Using FVB/NJ mouse and human cardiac tissues, RNA from isolated subcellular compartments (cytoplasmic and mitochondrial) and cross-linked immunoprecipitate (CLIP) with PNPase within the mitochondrion were sequenced on the Illumina HiSeq and MiSeq, respectively. lncRNA sequence and structure were evaluated through supervised [classification and regression trees (CART) and support vector machines (SVM)] machine learning algorithms. In HL-1 cells, quantitative PCR of PNPase CLIP knockout mutants (KH and S1) was performed. In vitro fluorescence assays assessed PNPase RNA binding capacity and verified with PNPase CLIP. One hundred twelve (mouse) and 1,548 (human) lncRNAs were identified in the mitochondrion with Malat1 being the most abundant. Most noncoding RNAs binding PNPase were lncRNAs, including Malat1. lncRNA fragments bound to PNPase compared against randomly generated sequences of similar length showed stratification with SVM and CART algorithms. The lncRNAs bound to PNPase were used to create a criterion for binding, with experimental validation revealing increased binding affinity of RNA designed to bind PNPase compared to control RNA. The binding of lncRNAs to PNPase was decreased through the knockout of RNA binding domains KH and S1. In conclusion, sequence and secondary structural features identified by machine learning enhance the likelihood of nuclear-encoded lncRNAs binding to PNPase and undergoing import into the mitochondrion.NEW & NOTEWORTHY Long noncoding RNAs (lncRNAs) are relatively novel RNAs with increasingly prominent roles in regulating genetic expression, mainly in the nucleus but more recently in regions such as the mitochondrion. This study explores how lncRNAs interact with polynucleotide phosphorylase (PNPase), a protein that regulates RNA import into the mitochondrion. Machine learning identified several RNA structural features that improved lncRNA binding to PNPase, which may be useful in targeting RNA therapeutics to the mitochondrion.

The critical care literature 2023.

The number of critically ill patients that present to emergency departments across the world continues to rise. In fact, the proportion of critically ill patients in emergency departments is now higher than pre-COVID-19 pandemic levels. [1] The emergency physician (EP) is typically the first physician to evaluate and resuscitate the critically ill patient. Given the continued shortage of intensive care unit (ICU) beds, persistent staff shortages, and overall inefficient hospital throughput, EPs are often tasked with providing intensive care to these patients long beyond the initial resuscitation phase. Prolonged boarding of critically ill patients in the ED is associated with increased ICU and hospital length of stay, increased adverse events, ED staff burnout, decreased patient and family satisfaction, and, most importantly, increased mortality. [2-5]. As such, it is imperative for the EP to be knowledgeable about recent literature in resuscitation and critical care medicine, so that critically ill ED patients can continue to receive the best, most up-to-date evidence-based care. This review summarizes important articles published in 2023 that pertain to the resuscitation and management of select critically ill ED patients. Topics included in this article include cardiac arrest, post-cardiac arrest care, septic shock, rapid sequence intubation, severe pneumonia, transfusions, trauma, and critical procedures.

The critical care literature 2022.

The number of critically ill patients that present to emergency departments across the world has risen steadily for nearly two decades. Despite a decrease in initial emergency department (ED) volumes early in the COVID-19 pandemic, the proportion of critically ill patients is now higher than pre-pandemic levels [1]. The emergency physician (EP) is often the first physician to evaluate and resuscitate a critically ill patient. In addition, EPs are frequently tasked with providing critical care long beyond the initial resuscitation. Prolonged boarding of critically ill patients in the ED is associated with increased duration of mechanical ventilation, increased intensive care unit (ICU) length of stay, increased hospital length of stay, increased medication-related adverse events, and increased in-hospital, 30-day, and 90-day mortality [2-4]. Given the continued increase in critically ill patients along with the increases in boarding critically ill patients in the ED, it is imperative for the EP to be knowledgeable about recent literature in resuscitation and critical care medicine, so that critically ill patients continue to receive evidence-based care. This review summarizes important articles published in 2022 that pertain to the resuscitation and management of select critically ill ED patients. These articles have been selected based on the authors review of key critical care, resuscitation, emergency medicine, and medicine journals and their opinion of the importance of study findings as it pertains to the care of the critically ill ED patient. Topics covered in this article include cardiac arrest, post-cardiac arrest care, rapid sequence intubation, mechanical ventilation, fluid resuscitation, and sepsis.

Evaluating Antibody Mediated Protection against Alpha, Beta, and Delta SARS-CoV-2 Variants of Concern in K18-hACE2 Transgenic Mice.

SARS-CoV-2 variants of concern (VoC) are impacting responses to the COVID-19 pandemic. Here, we utilized passive immunization using human convalescent plasma (HCP) obtained from a critically ill COVID-19 patient in the early pandemic to study the efficacy of polyclonal antibodies generated to ancestral SARS-CoV-2 against the Alpha, Beta, and Delta VoC in the K18 human angiotensin converting enzyme 2 (hACE2) transgenic mouse model. HCP protected mice from challenge with the original WA-1 SARS-CoV-2 strain; however, only partially protected mice challenged with the Alpha VoC (60% survival) and failed to save Beta challenged mice from succumbing to disease. HCP treatment groups had elevated receptor binding domain (RBD) and nucleocapsid IgG titers in the serum; however, Beta VoC viral RNA burden in the lung and brain was not decreased due to HCP treatment. While mice could be protected from WA-1 or Alpha challenge with a single dose of HCP, six doses of HCP could not decrease mortality of Delta challenged mice. Overall, these data demonstrate that VoC have enhanced immune evasion and this work underscores the need for in vivo models to evaluate future emerging strains. IMPORTANCE Emerging SARS-CoV-2 VoC are posing new problems regarding vaccine and monoclonal antibody efficacy. To better understand immune evasion tactics of the VoC, we utilized passive immunization to study the effect of early-pandemic SARS-CoV-2 HCP against, Alpha, Beta, and Delta VoC. We observed that HCP from a human infected with the original SARS-CoV-2 was unable to control lethality of Alpha, Beta, or Delta VoC in the K18-hACE2 transgenic mouse model of SARS-CoV-2 infection. Our findings demonstrate that passive immunization can be used as a model to evaluate immune evasion of emerging VoC strains.

Purification processes of live virus vaccine candidates expressed in adherent Vero cell lines via multimodal chromatography in flowthrough mode.

Live virus vaccine (LVV) purification, employing chromatography, can be challenged by low binding capacities and elution yields. Alternatively, processes relying solely on enzymatic digestion steps and size-based membrane separations can be limited by suboptimal reduction of process related impurities and poorly scalable unit operations. Here, we demonstrate that the combination of flowthrough mode chromatography and an ultrafiltration/diafiltration (UF/DF) unit operation delivers a purification process for two different LVV candidates, V590 and Measles, expressed in adherent Vero cells. For V590, chromatography with mixed mode cation exchange resins returned final product yields of ∼50% and logarithmic reduction values (LRVs) of 1.7->3.4 and 2.5-3.0 for host cell DNA (hcDNA) and host cell proteins (HCPs), respectively. For Measles, chromatography with mixed mode anion exchange resins returned final product yields of ∼50% and LRVs of 1.6 and 2.2 for hcDNA and HCPs, respectively. For both V590 and Measles processing, the employed resins cleared a key HCP, fibronectin, which could foul the UF/DF unit operation, and thusly enabling it to further reduce HCPs and to formulate the final LVV products. This integrated purification process utilizes the complementary action of the two unit operations and its applicability across LVVs supports its consideration for their processing.

Small regulatory RNAs: from bench to bedside - a keystone symposia meeting report.

The Keystone Symposium 'Small Regulatory RNAs: From Bench to Bedside' was held in Santa Fe, New Mexico from May 1-4, 2022. The symposium was organized by Frank J. Slack, Jörg Vogel, Ivan Martinez and Karyn Schmidt, and brought together scientists working in noncoding RNA biology, therapeutics, and technologies to address mechanistic questions about small regulatory RNAs and facilitate translation of these findings into clinical applications. The conference addressed four specific aims: Aim 1. Focus on the exciting biology of small regulatory RNAs, highlighting the best current research into the role that small RNAs play in fundamental biological processes; Aim 2. Focus on the latest efforts to harness the power of these RNAs as agents in the fight against disease and provide the basic understanding that will drive the invention of powerful clinical tools; Aim 3. Attract leaders from both academia and industry working in small RNAs to one place for critical discussions that will advance the field and accelerate the bench to bedside use of this technology; Aim 4. Provide a stimulating environment where students, postdoctoral researchers and junior investigators, along with scientists from Biotechnology and Pharmaceutical companies specializing in small regulatory RNAs, can present and discuss their research with the best minds in the field.

The critical care literature 2021.

An emergency physician (EP) is often the first provider to evaluate, resuscitate, and manage a critically ill patient. Over the past two decades, the annual hours of critical care delivered in emergency departments across the United States has dramatically increased. During the period from 2006 to 2014, the extent of critical care provided in the emergency department (ED) to critically ill patients increased approximately 80%. During the same time period, the number of intubated patients cared for in the ED increased by approximately 16%. In addition to seeing more critically ill patients, EPs are often tasked with providing critical care long beyond the initial resuscitation period. Prolonged ED boarding times for critically ill patients is associated with increased duration of mechanical ventilation, increased intensive care unit (ICU) length of stay, increased hospital length of stay, increased medication-related adverse events, and increased in-hospital, 30-day, and 90-day mortality. As a result, it is imperative for the EP to be knowledgeable about recent developments in resuscitation and critical care medicine, so that the critically ill ED patient care receive current evidence-based care. These articles have been selected based on the authors review of key critical care, resuscitation, emergency medicine, and medicine journals and their opinion of the importance of study findings as it pertains to the care of the critically ill ED patient. Topics covered in this article include cardiac arrest, post-cardiac arrest care, rapid sequence intubation, mechanical ventilation, fluid resuscitation, cardiogenic shock, transfusions, and sepsis.

In situ real time monitoring of emulsification and homogenization processes for vaccine adjuvants.

Adjuvants are commonly employed to enhance the efficacy of a vaccine and thereby increase the resulting immune response in a patient. The activity and effectiveness of emulsion-based adjuvants has been heavily studied throughout pharmaceuticals; however, there exists a lack in research which monitors the formation of a stable emulsion in real time. Process analytical technology (PAT) provides a solution to meet this need. PAT involves the collection of in situ data, thereby providing real time information about the monitored process as well as increasing understanding of that process. Here, three separate PAT tools - optical particle imaging, in situ particle analysis, and Raman spectroscopy - were used to monitor two key steps involved in the formation of a stable emulsion product, emulsification and homogenization, as well as perform a stability assessment. The obtained results provided new insights-particle size decreases during emulsification and homogenization, and molecular changes do not occur during either the emulsification or homogenization steps. Further, the stability assessment indicated that the coarse emulsion product obtained from the emulsification step is stable over the course of 24 hours when mixed. To the best of our knowledge, this is the first report of an analytical methodology for in situ, real time analysis of emulsification and homogenization processes for vaccine adjuvants. Using our proposed analytical methodology, an improved understanding of emulsion-based vaccine adjuvants can now be achieved, ultimately impacting the ability to develop and deliver successful pharmaceuticals.

The critical care literature 2020.

Given the dramatic increase in critically ill patients who present to the emergency department for care, along with the persistence of boarding of critically ill patients, it is imperative for the emergency physician to be knowledgeable about recent developments in resuscitation and critical care medicine. This review summarizes important articles published in 2020 that pertain to the resuscitation and care of select critically ill patients. These articles have been selected based on the authors annual review of key critical care, emergency medicine and medicine journals and their opinion of the importance of study findings as it pertains to the care of critically ill ED patients. Several key findings from the studies discussed in this paper include the administration of dexamethasone to patients with COVID-19 infection who require mechanical ventilation or supplemental oxygen, the use of lower levels of positive end-expiratory pressure for patients without acute respiratory distress syndrome, and early initiation of extracorporeal membrane oxygenation for out-of-hospital cardiac arrest patients with refractory ventricular fibrillation if resources are available. Furthermore, the emergency physician should not administer tranexamic acid to patients with acute gastrointestinal bleeding or administer the combination of vitamin C, thiamine, and hydrocortisone for patients with septic shock. Finally, the emergency physician should titrate vasopressor medications to more closely match a patient's chronic perfusion pressure rather than target a mean arterial blood pressure of 65 mmHg for all critically ill patients.

The critical care literature 2019.

An emergency physician (EP) is often the first health care provider to evaluate, resuscitate, and manage a critically ill patient. In recent years, the annual hours of critical care delivered in emergency departments across the United States has steadily increased. From 2006 to 2014, emergency department (ED) visits for critically ill patients increased approximately 80%. In addition to seeing more critically ill patients, EPs are often tasked with providing critical care long beyond the initial resuscitation period. In fact, more than 50% of ED patients that require admission to the ICU remain in the ED for more than 6 h. Prolonged ED wait times for critically ill patients to be transferred to the ICU is associated with increased hospital, 30-day, and 90-day mortality. It is during these early hours of critical illness, while the patient is in the ED, where lives can be saved or lost. Therefore, it is important for the EP to be knowledgeable about recent developments in resuscitation and critical care medicine. This review summarizes important articles published in 2019 pertaining to the resuscitation and care of select critically ill patients. We chose these articles based on our opinion of the importance of the study findings and their application to emergency medicine. The following topics are covered: sepsis, rapid sequence intubation, mechanical ventilation, neurocritical care, post-cardiac arrest care, and ED-based ICUs.

Clinical Practice Statement: What is the Emergency Department Management of Patients with Angioedema Secondary to an ACE-Inhibitor?

BACKGROUND: Angioedema is a complication that has been reported in up to 1.0% of individuals taking angiotensin-converting enzyme inhibitors (ACE-Is). Importantly, the onset of angioedema can occur anywhere from hours to several years after initiation of therapy with ACE-Is. Although most cases of ACE-I-induced angioedema (ACE-I-AE) are self-limiting, a major clinical concern is development of airway compromise, which can potentially require emergent airway management. The underlying pathophysiology of ACE-I-AE is incompletely understood, but is considered to be due in large part to excess bradykinin. Numerous medications have been proposed for the treatment of ACE-I-AE. This article is an update to the 2011 Clinical Practice Committee (CPC) statement from the American Academy of Emergency Medicine. METHODS: A literature search in PubMed was performed with search terms angioedema and ACE inhibitors from August 1, 2012 to May 13, 2019. Following CPC guidelines, articles written in English were identified and then underwent a structured review for evaluation. RESULTS: The search parameters resulted in 323 articles. The abstracts of these articles were assessed independently by the reviewers, who determined there were 63 articles that were specific to ACE-I-AE, of which 46 were deemed appropriate for grading in the final focused review. CONCLUSIONS: The primary focus for the treatment of ACE-I-AE is airway management. In the absence of high-quality evidence, no specific medication therapy is recommended for its treatment. If, however, the treating physician feels the patient's presentation is more typical of an acute allergic reaction or anaphylaxis, it may be appropriate to treat for those conditions. Any patient with suspected ACE-I-AE should immediately discontinue that medication.

The critical care literature 2018.

An emergency physician (EP) is often the first health care provider to evaluate, resuscitate, and manage a critically ill patient. In recent years, the annual hours of critical care delivered in emergency departments (EDs) across the United States increased more than 200% (Herring et al., 2013). In addition to seeing more critically ill patients, EPs are often tasked with providing critical care long beyond the initial resuscitation period. In fact, more than 50% of ED patients that require admission to the intensive care unit (ICU) remain in the ED for more than 6 h (Rose et al., 2016). Longer ED boarding times for critically ill patients is associated with a negative impact on inpatient morbidity and mortality (Mathews et al., 2018). It is during these early hours of critical illness, while the patient is in the ED, where lives can be saved or lost. Therefore, it is important for the EP to be knowledgeable about recent developments in critical care medicine. This review summarizes important articles published in 2018 pertaining to the resuscitation and care of select critically ill patients. We chose these articles based on our opinion of the importance of the study findings and their application to clinical care in the ED. The following topics are covered: cardiac arrest, post-arrest care, septic shock, rapid sequence intubation, mechanical ventilation, fluid resuscitation, and metabolic acidosis.

National Early Warning Score Is Modestly Predictive of Care Escalation after Emergency Department-to-Floor Admission.

BACKGROUND: Decompensation on the medical floor is associated with increased in-hospital mortality. OBJECTIVE: Our aim was to determine the accuracy of the National Early Warning Score (NEWS) in predicting early, unplanned escalation of care in patients admitted to the hospital from the emergency department (ED) compared to the Shock Index (SI) and the quick Sepsis-Related Organ Failure Assessment (qSOFA) score. METHODS: We conducted a retrospective cohort study of patients admitted directly from the ED to monitored or unmonitored beds (November 9, 2015 to April 30, 2018) in 3 hospitals. Interhospital transfers were excluded. Patient data, vital status, and bed assignment were extracted from the electronic medical record. Scores were calculated using the last set of vital signs prior to leaving the ED. Primary endpoint was in-hospital death or placement in an intermediate or intensive care unit within 24 h of admission from the ED. Scores were compared using the area under the receiver operating curve (AUROC). RESULTS: Of 46,018 ED admissions during the study window, 39,491 (85.8%) had complete data, of which 3.7% underwent escalation in level of care within 24 h of admission. NEWS outperformed (AUROC 0.69; 95% confidence interval [CI] 0.68-0.69) qSOFA (AUROC 0.63; 95% CI 0.62-0.63; p < 0.001) and SI (AUROC 0.60; 95% CI 0.60-0.61; p < 0.001) at predicting unplanned escalations or death at 24 h. CONCLUSIONS: This multicenter study found NEWS was superior to the qSOFA score and SI in predicting early, unplanned escalation of care for ED patients admitted to a general medical-surgical floor.

Clerkships in Emergency Medicine.

Planning for clerkships in emergency medicine (EM) can be stressful, prolonged, and challenging. Therefore, medical students should start planning for them early. In this article, we offer guidance regarding several issues pertinent to the EM clerkship, such as the best time to schedule one (or more) during medical school, the most appropriate institution or program to schedule it, the process of selecting and applying for the clerkship, and the number of EM clerkships to consider. We will explain why an EM clerkship should be scheduled between June and October and the reason that 2 EM clerkships at different sites are sufficient for the majority of students. Additionally, we emphasize that clerkships in emergency departments associated with EM residency programs or with reputations for outstanding student teaching tend to be most beneficial. Above all, students interested in EM should attempt to leave a great impression after completing their clerkships by providing stellar patient care, demonstrating enthusiasm at all times, and maintaining professionalism. In turn, they will gain knowledge and clinical experiences that should prove valuable in their future.

Angiotensin II for the emergency physician.

Refractory hypotension is one of the most common and difficult clinical problems faced by acute care clinicians, and it poses a particularly large problem to the emergency physician when a patient in undifferentiated shock arrives in the department. Angiotensin II (Ang-2) has been previously used as a vasopressor to combat shock; the feasibility of its clinical use has been reinvigorated after approval of a human synthetic formulation of the medication by the US Food and Drug Administration in 2017 and the European Medicines Agency in 2019. A thorough literature search was completed, and in this review, we discuss the discovery and development of Ang-2, its complex mechanisms of vasoconstriction, its potential adverse effects and its potential role in clinical practice for emergency physicians.

The critical care literature 2017.

An emergency physician (EP) is often the first health care provider to evaluate, resuscitate, and manage a critically ill patient. Between 2001 and 2009, the annual hours of critical care delivered in emergency departments (EDs) across the United States increased >200% [1]! This trend has persisted since then. In addition to seeing more critically ill patients, EPs are often tasked with providing critical care long beyond the initial resuscitation period. In fact, >33% of critically ill patients who are brought to an ED remain there for >6 h [1]. Longer ED boarding times for critically ill patients have been associated with a negative impact on inpatient morbidity and mortality [2]. During these crucial early hours of illness, detrimental pathophysiologic processes begin to take hold. It is during these early hours of illness where lives can be saved, or lost. Therefore, it is important for the EP to be knowledgeable about recent developments in critical care medicine. This review summarizes important articles published in 2017 pertaining to the resuscitation and care of select critically ill patients in the ED. We chose these articles based on our opinion of the importance of the study findings and their application to clinical care. The following topics are covered: sepsis, vasolidatory shock, cardiac arrest, post-cardiac arrest care, post-intubation sedation, and pulmonary embolism.

Combined Residency Programs in Emergency Medicine.

There are currently 5 combined residencies in emergency medicine (EM), namely EM/pediatrics, EM/internal medicine, EM/internal medicine/critical care, EM/family medicine and EM/anesthesiology. These combined programs vary from 5-6 years in length. Like categorical programs, the decision to enter a 5- or 6-year program should be an informed and comprehensive decision. We describe the history and current status of the combined EM programs, discuss the process of applying to a combined EM program, describe the life of combined EM residents, and explore common career opportunities available to combined EM program graduates.

Discovery and optimization of a novel series of pyrazolyltetrahydropyran N-type calcium channel (Cav 2.2) blockers for the treatment of pain.

A novel series of pyrazolyltetrahydropyran N-type calcium channel blockers are described. Structural modifications of the series led to potent compounds in both a cell-based fluorescent calcium influx assay and a patch clamp electrophysiology assay. Representative compounds from the series were bioavailable and showed efficacy in the rat CFA and CCI models of inflammatory and neuropathic pain.

Discovery of N-arylpyrroles as agonists of GPR120 for the treatment of type II diabetes.

The discovery of a novel series of N-arylpyrroles as agonists of GPR120 (FFAR4) is discussed. One lead compound is a potent GPR120 agonist, has good selectivity for related receptor GPR40 (FFAR1), has acceptable PK properties, and is active in 2 models of Type 2 Diabetes in mice.

Discovery of novel benzo[b]thiophene tetrazoles as non-carboxylate GPR40 agonists.

GPR40 partial agonism is a promising new mechanism for the treatment of type 2 diabetes mellitus with clinical proof of concept. Most of the GPR40 agonists in the literature have a carboxylic acid functional group, which may pose a risk for idiosyncratic drug toxicity. A novel series of GPR40 agonists containing a tetrazole as a carboxylic acid bioisostere was identified. This series of compounds features a benzo[b]thiophene as the center ring, which is prone to oxidation during phase 1 metabolism. Following SAR optimization targeting GPR40 agonist activity and intrinsic clearance in microsomes (human and rat), potent and metabolically stable compounds were selected for in vivo evaluation. The compounds are efficacious at lowering blood glucose in a SD rat oGTT model.