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Introduction Release of the 2015 European Society of Cardiology (ESC)/European Respiratory Society (ERS) guidelines put increased emphasis on using combination therapy, either as upfront or sequential therapy among patients with pulmonary arterial hypertension (PAH). However, with these recommendations and the therapy advances made in the last several years, little is known on the real-world treatment patterns among patients with PAH, particularly before and after publication of the 2015 ESC/ERS guidelines. Methods This was a retrospective study of adult commercial and Medicare Advantage with Part D (MAPD) enrollees with at least one claim for a PAH-related medication from January 01, 2012 to March 31, 2017, at least one medical claim with a pulmonary hypertension diagnosis, and continuous health plan enrollment at least 6 months prior to and at least 12 months following the date of the first pharmacy claim for PAH-related therapy (index date). Patients were divided into cohorts based on prescription of monotherapy or combination therapy and index date category (2012–2013, January 2014–July 2015, and August 2015–March 2017). Results Out of 1878 patients, 90.8% initiated with monotherapy and 9.2% initiated with combination therapy. The percentage of patients with index combination therapy increased from 5.7% in 2012–2013 to 13.0% in August 2015–March 2017. Patients with index combination therapy had better persistence (11.6 months versus 10.3 months) and adherence (0.95 versus 0.85). Overall, the discontinuation rate was 40% and was higher in monotherapy versus combination therapy patients (42.8% versus 12.2%). Approximately 30.2% of patients had a second regimen, of which 50% were combination regimens. The time to combination therapy initiation decreased from 10.5 months in 2012–2013 to 3.4 months in August 2015–March 2017. Conclusions The majority of patients initiated monotherapy treatment for PAH, most often a phosphodiesterase 5 inhibitor (PDE5i). Patients with upfront combination therapy increased following publication of the 2015 ESC/ERS guidelines, indicating that physicians responded to the guideline’s option of prescribing upfront combination therapy.

Vesicles that are coated by coat protein complex II (COPII) are the primary mediators of vesicular traffic from the endoplasmic reticulum to the Golgi apparatus. Secretion-associated Ras-related GTPase 1 (SAR1) is a small GTPase that is part of COPII and, upon GTP binding, recruits the other COPII proteins to the endoplasmic reticulum membrane. Mammals have two SAR1 paralogs that genetic data suggest may have distinct physiological roles, e.g. in lipoprotein secretion in the case of SAR1B. Here we identified two amino acid clusters that have conserved SAR1 paralog-specific sequences. We observed that one cluster is adjacent to the SAR1 GTP-binding pocket and alters the kinetics of GTP exchange. The other cluster is adjacent to the binding site for two COPII components, SEC31 homolog A COPII coat complex component (SEC31) and SEC23. We found that the latter cluster confers to SAR1B a binding preference for SEC23A that is stronger than that of SAR1A for SEC23A. Unlike SAR1B, SAR1A was prone to oligomerize on a membrane surface. SAR1B knockdown caused loss of lipoprotein secretion, overexpression of SAR1B but not of SAR1A could restore secretion, and a divergent cluster adjacent to the SEC31/SEC23-binding site was critical for this SAR1B function. These results highlight that small primary sequence differences between the two mammalian SAR1 paralogs lead to pronounced biochemical differences that significantly affect COPII assembly and identify a specific function for SAR1B in lipoprotein secretion, providing insights into the mechanisms of large cargo secretion that may be relevant for COPII-related diseases.

Dimethyl fumarate (DMF) is a prescribed treatment for multiple sclerosis and has also been used to treat psoriasis. The electrophilicity of DMF suggests that its immunosuppressive activity is related to the covalent modification of cysteine residues in the human proteome. Nonetheless, our understanding of the proteins modified by DMF in human immune cells and the functional consequences of these reactions remains incomplete. In this study, we report that DMF inhibits human plasmacytoid dendritic cell function through a mechanism of action that is independent of the major electrophile sensor NRF2. Using chemical proteomics, we instead identify cysteine 13 of the innate immune kinase IRAK4 as a principal cellular target of DMF. We show that DMF blocks IRAK4–MyD88 interactions and IRAK4-mediated cytokine production in a cysteine 13–dependent manner. Our studies thus identify a proteomic hotspot for DMF action that constitutes a druggable protein–protein interface crucial for initiating innate immune responses.

Several new medications for pulmonary arterial hypertension (PAH) have recently been introduced; however, current real-world data regarding US patients with PAH are limited. We conducted a retrospective administrative claims study to examine PAH treatment patterns and summarize healthcare utilization and costs among patients with newly diagnosed PAH treated in US clinical practice. Patients newly treated for PAH from 1 January 2010 to 31 March 2015 were followed for ≥12 months. Patient characteristics, treatment patterns, healthcare resource utilization, and costs were described. Adherence (proportion of days covered), persistence (months until therapy discontinuation/modification), and the probability of continuing the index regimen were analyzed by index regimen cohort (monotherapy versus combination therapy). Of 1637 eligible patients, 93.8% initiated treatment with monotherapy and 6.2% with combination therapy. The most common index regimen was phosphodiesterase type 5 inhibitor (PDE-5I) monotherapy (70.0% of patients). A total of 581 patients (35.5%) modified their index regimen during the study. Most patients (55.4%) who began combination therapy did so on or within six months of the index date. Endothelin receptor agonists (ERAs) and combination therapies were associated with higher adherence than PDE-5Is and monotherapies, respectively. Healthcare utilization was substantial across the study population, with costs in the combination therapy cohort more than doubling from baseline to follow-up. The majority of patients were treated with monotherapies (most often, PDE-5Is), despite combination therapies and ERAs being associated with higher medication adherence. Index regimen adjustments occurred early and in a substantial proportion of patients, suggesting that inadequate clinical response to monotherapies may not be uncommon.

The Zika virus was largely unknown to many health care systems before the outbreak of 2015. The unique public health threat posed by the Zika virus and the evolving understanding of its pathology required continuous communication between a health care delivery system and a local public health department. By leveraging an existing relationship, NYC Health+Hospitals worked closely with New York City Department of Health and Mental Hygiene to ensure that Zika-related processes and procedures within NYC Health+Hospitals facilities aligned with the most current Zika virus guidance. Support given by the public health department included prenatal clinical and laboratory support and the sharing of data on NYC Health+Hospitals Zika virus screening and testing rates, thus enabling this health care delivery system to make informed decisions and practices. The close coordination, collaboration, and communication between the health care delivery system and the local public health department examined in this article demonstrate the importance of working together to combat a complex public health emergency and how this relationship can serve as a guide for other jurisdictions to optimize collaboration between external partners during major outbreaks, emerging threats, and disasters that affect public health. (Disaster Med Public Health Preparedness. 2018;12:689-691)

Introduction Despite concerns over the appropriateness and quality of care provided in the intensive care unit (ICU) at the end of life, the number of elderly patients who receive critical care is increasing. Despite this, many physicians have doubts as to whether elderly patients are good candidates for ICU care because of the apparently poor outcome during and after critical care in this population. The objective of the present study was to describe the clinical characteristics and outcome of a geriatric population admitted to the ICU. Materials and methods A single-center, prospective, observational study was carried out among geriatric patients, aged 75 years or older, admitted to ICU. Results A total of 71 patients were admitted to ICU during the study period. Their mean age was 83 years (range 75–98 years), with a mean Acute Physiology and Chronic Health Evaluation-II score of 21.8 (range 8–39) on admission to ICU. A total of 48 patients (68%) required mechanical ventilation, and 39 (55%) received at least one vasoactive drug. The mean ICU length of stay was 4.6 days (range 1–18 days), and it was similar for ICU survivors and non-survivors (4.7 vs 4.5). A total of 14 patients (19.7%) were admitted after cardiac arrest, and eight (57.1%) of them died in ICU. A total of 28 patients (39.4%) died in the hospital, and 18 (25.4%) died in ICU. Conclusion Advanced age, critical illness, cardiopulmonary resuscitation, and needs for mechanical ventilation and/or vasopressor therapy are independent risk factors associated with adverse outcome in elderly patients admitted to ICU. Alternatives for ICU admission should be considered in geriatric patients with severe critical illnesses. Geriatr Gerontol Int 2015; 15: 889–894.

Dendritic cells (DCs) can take up antigen via a number of routes. Of these, several studies have shown antigen uptake via the CD40 receptor generates some of the strongest CD8+ T cell responses. At the same time, stimulation through CD40 activates DCs for cross-presentation and heightened antigen presentation. Consequently, it is desirable to combine the CD40 targeting of antigen and a CD40 stimulant into a single vaccine formulation. When the CD40 stimulant is CD40 ligand (CD40L, CD154), it is important to use a multi-trimer complex that clusters CD40 in the DC membrane, thereby engaging downstream signaling pathways. Such a multi-trimer form of CD40L can be provided by fusing the extracellular domain of CD40L with the body of surfactant protein D (SPD), a self-assembling 12-chain soluble molecule with 4 trimeric “arms.” When an antigen sequence is included within the SPD arm, the entire protein complex is termed “Multi-trimer AntiGen Adjuvant Vaccine (Vax)” or “MagaVax.” Studies in mice have shown that MagaVax elicits very high levels of antigen-specific CD8+ T cells with high TCR avidity and functional activity. MagaVax can be effectively delivered as a DNA vaccine i.m. and even more effectively using an adenoviral (Ad5) vector. For a MagaVax encoding HIV-1 Gag, vaccination with Ad5-MagaVax-Gag led to complete protection (“sterilizing immunity”) from Vaccinia-Gag challenge (i.e., 7 log reduction of tissue virus). Thus far, MagaVax formulations have been made for HIV-1 Gag, Ebola, malaria, hepatitis B virus (HBV), and tumor-specific neoantigens. In all of these cases, there may be a clinical benefit for a vaccine that elicits very strong CD8+ T cell responses.

Pulmonary arterial hypertension (PAH) is a rare, debilitating and rapidly progressive disease. Although there have been important medical advances in PAH management, the search for a cure continues. Despite an increased understanding of the disease, data on the wider effect of PAH on patients and carers, beyond the clinical symptoms, are still limited. In order to explore this, a large-scale international survey investigated four key areas affected by PAH (physical and practical, emotional, social, and information needs) and provides new insight into patients' and carers' experiences of living with the disease. The results from the survey highlight not only the limited ability of patients to carry out everyday tasks, but also the financial impact and social isolation experienced by both patients and carers. The study confirmed that a decline in a patient's World Health Organization functional class, which indicates an increase in clinical severity of the disease, is associated with greater limitations. Results from the survey demonstrate the need for multidisciplinary PAH management and a comprehensive standard of care to assess and improve all aspects of well-being for both patients and carers. In addition, they underline the need for updated PAH guidelines that address these needs.

Patients with pulmonary arterial hypertension (PAH) are at increased morbidity and mortality risk when facing the need to undergo surgical interventions. The most common complications include those arising from right ventricular (RV) failure and respiratory failure—not surprising given the complex cardiopulmonary pathophysiology of this disease. While data are limited regarding the optimal preoperative approach to these patients, it is imperative to focus on the following key components: ensuring or establishing the patient's World Health Organization (WHO) classification, pulmonary hypertension (PH) group or subgroup; assessing the status and stability of RV function; optimizing the treatment regimen; and communicating a management plan for intra- and perioperative management to all members of the interdisciplinary clinical team. This article will focus on each of these steps in the preoperative algorithm, highlighting the need for further studies in this area.

Pulmonary arterial hypertension (PAH) is a progressive and presently incurable disease resulting in distressing and debilitating symptoms for patients, including exertional dyspnea, fatigue, chest pain/discomfort, and feeling dizzy or lightheaded.12 These symptoms cause profound functional limitations that often require patients to make significant lifestyle changes and cope with formidable psychological challenges as they face this progressive disease. While the introduction of new treatments over the past decade has expanded options for improving functional exercise capacity,3 we still have limited insight into the nature of the psychological challenges facing patients and the secondary impact on their caregivers.

Coat protein II (COPII)-coated vesicles transport proteins and lipids from the endoplasmic reticulum to the Golgi. Crucial for the initiation of COPII coat assembly is Sec12, a guanine nucleotide exchange factor responsible for activating the small G protein Sar1. Once activated, Sar1/GTP binds to endoplasmic reticulum membranes and recruits COPII coat components (Sec23/24 and Sec13/31). Here, we report the 1.36 Å resolution crystal structure of the catalytically active, 38-kDa cytoplasmic portion of Saccharomyces cerevisiae Sec12. Sec12 adopts a β propeller fold. Conserved residues cluster around a loop we term the "K loop," which extends from the N-terminal propeller blade. Structure-guided site-directed mutagenesis, in conjunction with in vitro and in vivo functional studies, reveals that this region of Sec12 is catalytically essential, presumably because it makes direct contact with Sar1. Strikingly, the crystal structure also reveals that a single potassium ion stabilizes the K loop; bound potassium is, moreover, essential for optimum guanine nucleotide exchange activity in vitro. Thus, our results reveal a novel role for a potassium-stabilized loop in catalyzing guanine nucleotide exchange.

Blunting immunopathology without abolishing host defense is the foundation for safe and effective modulation of infectious and autoimmune diseases. Sphingosine 1-phosphate receptor 1 (S1PR1) agonists are effective in treating infectious and multiple autoimmune pathologies; however, mechanisms underlying their clinical efficacy are yet to be fully elucidated. Here, we uncover an unexpected mechanism of convergence between S1PR1 and interferon alpha receptor 1 (IFNAR1) signaling pathways. Activation of S1PR1 signaling by pharmacological tools or endogenous ligand sphingosine-1 phosphate (S1P) inhibits type 1 IFN responses that exacerbate numerous pathogenic conditions. Mechanistically, S1PR1 selectively suppresses the type I IFN autoamplification loop in plasmacytoid dendritic cells (pDCs), a specialized DC subset, for robust type I IFN release. S1PR1 agonist suppression is pertussis toxin-resistant, but inhibited by an S1PR1 C-terminal–derived transactivating transcriptional activator (Tat)-fusion peptide that blocks receptor internalization. S1PR1 agonist treatment accelerates turnover of IFNAR1, suppresses signal transducer and activator of transcription 1 (STAT1) phosphorylation, and down-modulates total STAT1 levels, thereby inactivating the autoamplification loop. Inhibition of S1P-S1PR1 signaling in vivo using the selective antagonist Ex26 significantly elevates IFN-α production in response to CpG-A. Thus, multiple lines of evidence demonstrate that S1PR1 signaling sets the sensitivity of pDC amplification of IFN responses, thereby blunting pathogenic immune responses. These data illustrate a lipid G-protein coupled receptor (GPCR)-IFNAR1 regulatory loop that balances effective and detrimental immune responses and elevated endogenous S1PR1 signaling. This mechanism will likely be advantageous in individuals subject to a range of inflammatory conditions.

Pulmonary hypertension in the setting of parenchymal lung disease and conditions associated with chronic hypoxemia is commonly encountered in clinical practice and may adversely affect patients' function and mortality. Diagnosis of this subgroup of pulmonary hypertension has evolved but still requires right heart catheterization for confirmation. The primary treatment goal is optimization of the underlying parenchymal lung or hypoxemia-associated condition prior to consideration of pharmacologic therapy. Limited published experience with pulmonary hypertension-specific medications for treatment of WHO Group 3 pulmonary hypertension suggests symptomatic and functional benefit in selected individuals. The potential for worsening ventilation–perfusion matching must be considered in these cases, however, since there is a paucity of data regarding the optimal approach to treatment selection. Ongoing medication trials and further investigation of mechanisms of hypoxic pulmonary vasoconstriction provide hope for these patients who in the past often had only lung transplantation as a potential treatment option.

Secretory proteins are transported to the Golgi complex in vesicles that bud from the endoplasmic reticulum. The cytoplasmic coat protein complex II (COPII) is responsible for cargo sorting and vesicle morphogenesis. COPII was first described in Saccharomyces cerevisiae, but its basic function is conserved throughout all eukaryotes. Nevertheless, the COPII coat has adapted to the higher complexity of mammalian physiology, achieving more sophisticated levels of secretory regulation. In this review we cover aspects of mammalian COPII-mediated regulation of secretion, in particular related to the function of COPII paralogues, the spatial organization of cargo export and the role of accessory proteins.

Induction therapy with alemtuzumab, followed by lower than conventional intensity post-transplant immunosuppression (eg, tacrolimus monotherapy), has been associated with reduced morbidity and mortality in abdominal and heart transplantation. We examined 5-year outcomes in lung recipients receiving alemtuzumab in conjunction with reduced-intensity post-transplant immunosuppression (early lower-dose tacrolimus; lower-dose steroids, with or without mycophenolate mofetil), compared with lung recipients receiving other induction agents or no induction in association with post-transplant immunosuppression.A retrospective analysis was performed using prospectively collected data from a single-site clinical database of 336 lung recipients (aged ≥ 18) who received allografts between 1998 and 2005, classified by induction type: alemtuzumab, 127; Thymoglobulin, 43; daclizumab, 73; and none, 93. Survival analyses examined patient and graft survival, and freedom from acute cellular rejection (ACR), lymphocytic bronchiolitis, obliterative bronchiolitis (OB), bronchiolitis obliterans syndrome (BOS), and post-transplant lymphoproliferative disorder (PTLD).Five-year patient and graft survival differed by group (p = 0.046, p = 0.038, respectively). Alemtuzumab patient/graft survival rates were 59%/59%. Survival rates were 60%/44% for Thymoglobulin, 47%/46% for no induction, and 44%/41% for daclizumab. Freedom from ACR, lymphocytic bronchiolitis, OB, and BOS differed by group (all values, p0.008); alemtuzumab recipients showed greater 5-year freedom from each outcome (30%/82%/86%/54%) than Thymoglobulin (20%/54%/62%/27%), daclizumab (19%/55%/70%/43%), and no-induction groups (18%/70%/69%/46%). The groups did not differ in PTLD rates (≥ 94% free of PTLD at 5 years; p = 0.864). Effects were unchanged after controlling for potential covariates.Alemtuzumab induction may be associated with improved outcomes in lung transplantation. Randomized controlled trials are needed to establish any effects of this agent.

Shahid Husain, MD, MS, Martha L. Mooney, MD, MS, FACP, Lara Danziger-Isakov, MD, MPH, Frauke Mattner, MD, PhD, Nina Singh, MD, Robin Avery, MD, FIDSA, Michael Ison, MD, MS, Atul Humar, MD, MSc, Robert F. Padera, MD, PhD, Leo P. Lawler, MD, FRCR, Andy Fisher, PhD, FRCP, Richard J. Drew, MD, Kate F. Gould, MBBS, MRCP, FRCP, Amparo Sole, MD, PhD, Sean Studer, MD, MSc, Patricia Munoz, MD, Lianne G. Singer, MD, FRCPC, and Margaret Hannan, MD, FRCP, FRCPath, for the ISHLT Infectious Diseases Council Working Group on Definitions From the Division of Infectious Diseases, Transplant Infectious Diseases, University Health Network, University of Toronto, Toronto, Ontario, Canada; Eastern Virginia Medical School, Sentara Norfolk Transplant Center, Norfolk, Virginia; Center for Pediatric Infectious Diseases, Department of Infectious Disease, Medicine Institute, The Cleveland Clinic, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio; Infection Control and Hospital Epidemiology, Institute for Medical Microbiology, Hannover Medical School, Hannover, Germany; Division of Infectious Diseases, Veteran Affairs Hospital, University of Pittsburgh, Pittsburgh, Pennsylvania; Divisions of Infectious Diseases and Organ Transplantation, Northwestern University, Feinberg School of Medicine, Chicago, Illinois; Department of Medicine, Division of Infectious Diseases, University of Alberta, Edmonton, Alberta, Canada; Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts; Respiratory Transplant Medicine, Newcastle University, Institute of Transplantation, Freeman Hospital, Newcastle Upon Tyne, UK; Mater Misericordiae University Hospital, Dublin, Ireland; Health Protection Agency Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle, UK; Hospital Universitario La Fe, Valencia, Spain; Division of Pulmonary & Critical Care, Newark Beth Israel Medical Center, Newark, New Jersey; and Clinical Microbiology and Infectious Diseases, Hospital General Universitario Gregorio Maranon, Universidad Complutense, Madrid, Spain.

Background Idiopathic pulmonary fibrosis (IPF) is often associated with elevations in pulmonary artery pressures. Although primary pulmonary arterial hypertension (PAH) has been associated with primary graft dysfunction (PGD), the role of secondary PAH in mediating PGD risk in patients with IPF is incompletely understood. The purpose of this study was to evaluate the relationship between mean pulmonary artery pressure (mPAP) and PGD among patients with IPF. Methods We performed a multicenter prospective cohort study of 126 lung transplant procedures performed for IPF between March 2002 and August 2007. The primary outcome was grade 3 PGD at 72 h after lung transplant. The mPAP was measured as the initial reading following insertion of the right-sided heart catheter during lung transplant. Multivariable logistic regression was used to adjust for confounding variables. Results The mPAP for patients with PGD was 38.5 ± 16.3 mm Hg vs 29.6 ± 11.5 mm Hg for patients without PGD (mean difference, 8.9 mm Hg [95% CI, 3.6-14.2]; P = .001). The increase in odds of PGD associated with each 10-mm Hg increase in mPAP was 1.64 (95% CI, 1.18-2.26; P = .003). In multivariable models, this relationship was independent of confounding by other clinical variables, although the use of cardiopulmonary bypass partially attenuated the relationship. Conclusions Higher mPAP in patients with IPF is associated with the development of PGD.

Although the literature continues to portray chronic rejection after lung transplantation as ominous with no known treatment, no studies have examined family and clinician caregivers' perceptions of the diagnosis of chronic rejection and its impact on the course of clinical care. We explored the meaning and impact of chronic rejection from the perspective of family (n=10) and clinician (n=3) caregivers. We found that family caregivers considered the onset of chronic rejection to be inevitable, irreversible, unpredictable, and going back to pretransplant. Clinicians considered chronic rejection as a harbinger of deterioration and peril and expressed trepidation about informing recipients and their family caregivers about the diagnosis. Despite the heightened caregiving duties and challenges of treating chronic rejection, its unpredictable course and the prospect of retransplant instilled hope for stabilization or cure among most clinicians and caregivers, leading them to support recipients' wishes to pursue potentially futile treatments. Until recipients were no longer competent, caregivers believed all treatment options (including retransplant) had been exhausted, or suffering was prolonged, caregivers were reluctant to halt extraordinary treatment measures. Caregivers perceived that certainty regarding poor prognosis was required for palliative care and that palliative care was end-of-life care. Consequently, trials of aggressive treatment typically precluded palliative care.

Summary Translation initiation is a key step for regulating the level of numerous proteins within the cell. In bacteria, the 30S initiation complex directly binds to the translation initiation region (TIR) of the mRNA. How the ribosomal 30S subunit assembles on highly structured TIR is not known. Using fluorescence-based experiments, we assayed 12 different mRNAs that form secondary structures with various stabilities and contain Shine-Dalgarno (SD) sequences of different strengths. A strong correlation was observed between the stability of the mRNA structure and the association and dissociation rate constants. Interestingly, in the presence of initiation factors and initiator tRNA, the association kinetics of structured mRNAs showed two distinct phases. The second phase was found to be important for unfolding structured mRNAs to form a stable 30S initiation complex. We show that unfolding of structured mRNAs requires a SD sequence, the start codon, fMet-tRNA fMet , and the GTP bound form of initiation factor 2 bound to the 30S subunit.

(A consensus report from The Pulmonary Council of the International Society for Heart and Lung Transplantation)

Despite concerns over the appropriateness and quality of care provided in the intensive care unit (ICU) at the end of life, the number of elderly patients who receive critical care is increasing. Despite this, many physicians have doubts as to whether elderly patients are good candidates for ICU care because of the apparently poor outcome during and after critical care in this population. The objective of the present study was to describe the clinical characteristics and outcome of a geriatric population admitted to the ICU.A single-center, prospective, observational study was carried out among geriatric patients, aged 75 years or older, admitted to ICU.A total of 71 patients were admitted to ICU during the study period. Their mean age was 83 years (range 75-98 years), with a mean Acute Physiology and Chronic Health Evaluation-II score of 21.8 (range 8-39) on admission to ICU. A total of 48 patients (68%) required mechanical ventilation, and 39 (55%) received at least one vasoactive drug. The mean ICU length of stay was 4.6 days (range 1-18 days), and it was similar for ICU survivors and non-survivors (4.7 vs 4.5). A total of 14 patients (19.7%) were admitted after cardiac arrest, and eight (57.1%) of them died in ICU. A total of 28 patients (39.4%) died in the hospital, and 18 (25.4%) died in ICU.Advanced age, critical illness, cardiopulmonary resuscitation, and needs for mechanical ventilation and/or vasopressor therapy are independent risk factors associated with adverse outcome in elderly patients admitted to ICU. Alternatives for ICU admission should be considered in geriatric patients with severe critical illnesses.

Ribosomes are responsible for translating the information in messenger ribonucleic acids (mRNAs) to synthesise proteins that the cell needs to carry out its function. Protein synthesis begins with the 30S ribosomal subunit recruiting a mRNA with the help of an initiator transfer RNA and three initiation factors. Successful recruitment of the mRNA results in the formation of the 30S pre-initiation complex, which is followed by the joining of the 50S ribosomal subunit to form the 70S ribosome. Recent studies indicate the order in which the initiation factors bind and promote the steps in initiation complex formation. The formation of the pre-initiation/initiation complex is often the rate-limiting step during the process of translation, as it is influenced by a number of translational regulatory mechanisms. As a result, the process of initiation can play a significant role in gene expression. Key Concepts: Translation initiation is a key step for regulating gene expression. Initiation factors 1, 2 and 3 are essential proteins that promote the binding of the initiator tRNA to the 30S subunit P site and the joining of the 50S subunit. Initiation factors 1, 2 and 3 are critical for the positioning the mRNA in the correct reading frame for translation. Efficient requirement of mRNA by the 30S subunit depends on the Shine–Dalgarno sequence in the mRNA forming base pairs with the anti-Shine–Dalgarno sequence located at the 3′-end of 16S rRNA. Kinetic studies have shown the order in which initiation factors bind to the 30S subunit. Cryo-EM and crystallographic work have revealed the three-dimensional location of initiation factors on the 30S subunit and the 70S ribosome. Keywords: initiation factors; translation; Shine–Dalgarno sequence; ribosome; mRNA; tRNA

Translation initiation is a key step for regulating the synthesis of several proteins. In bacteria, translation initiation involves the interaction of the mRNA with the ribosomal small subunit. Additionally, translation initiation factors 1, 2, and 3, and the initiator tRNA, also assemble on the ribosomal small subunit and are essential for efficiently recruiting an mRNA for protein biosynthesis. In the following chapter, we describe fluorescence-based methods for studying the interaction of mRNA with the bacterial initiation complex. Model mRNAs with a covalently attached fluorescent probe showed an increase in fluorescence intensity when bound to the bacterial initiation complex. Utilizing the increase in fluorescence intensity upon mRNA binding to the bacterial initiation complex, we determined the equilibrium binding constants and the association and dissociation rate constants. These methods are important for quantitatively analyzing the effects of mRNA secondary structure and the role of the initiation factors in recruitment of mRNA by the bacterial initiation complex.

Precise and coordinated movement of the tRNA –mRNA complex within the ribosome is a fundamental step during protein biosynthesis. The molecular mechanism for this process is still poorly understood. Here we describe a new sensitive method for monitoring elongation factor G-dependent translocation of the mRNA in the ribosome. In this method, the fluorescent probe pyrene is covalently attached to the 3 0 end of a short mRNA sequence at position þ 9. Translocation of the mRNA by one codon results in a significant decrease in the fluorescence emission of pyrene and can be used to directly monitor mRNA movement using rapid kinetic methods. Importantly, this method offers the flexibility of using any tRNA or tRNA analog in order to elucidate the molecular mechanism of translocation. Our results show that the mRNA is translocated at the same rate as the tRNAs, which is consistent with the view that the movement of the tRNAs and the mRNA are coupled in the ribosome. Furthermore, an anticodon stem-loop analog of tRNA is translocated from the ribosomal A site at a rate constant that is 350-fold lower than peptidyl tRNA, indicating that the D stem, T stem and acceptor stem of A site tRNA contribute significantly to the rate of translocation. q 2003 Elsevier Science Ltd. All rights reserved

Nat. Cell Biol. 14, 20–28 (2012); published online 22 December 2011; corrected after print 6 January 2012 In the version of this review initially published online and in print, the key in figure 2 was incorrect. The grey dots should represent Sec23–24, the blue lines should represent Sec13–31 and the green dots should represent TRAPPI.