Small sequence variations between two mammalian paralogs of the small GTPase SAR1 underlie functional differences in coat protein complex II assembly.

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 Disrupts Human Innate Immune Signaling by Targeting the IRAK4-MyD88 Complex.

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.

S1PR1-mediated IFNAR1 degradation modulates plasmacytoid dendritic cell interferon-α autoamplification.

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.

The structure of Sec12 implicates potassium ion coordination in Sar1 activation.

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 ß 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.

Impact of the COVID-19 pandemic on care disruptions, outcomes, and costs in patients receiving pulmonary arterial hypertension-specific therapy in the United States of America: An observational study.

Regular expert follow-up, risk assessment, and early therapeutic intervention minimize worsening of pulmonary arterial hypertension (PAH). COVID-19 lockdown measures were challenging for chronic disease management. This retrospective, longitudinal analysis used US claims data (January 12, 2016 to September 11, 2021) for patients treated with PAH-specific medication to compare in-person outpatient and specialist visits, telemedicine visits, and PAH-related tests during 6-month assessment periods pre- and immediately post-COVID-19. Hospitalizations, costs, and outcomes were compared in patients with and without care disruptions (no in-person or telemedicine outpatient visits in immediate post-COVID-19 period). Patients in the immediate post-COVID-19 (N = 599) versus the pre-COVID-19 period (N = 598) had fewer in-person outpatient visits (mean 1.27 vs. 2.12) and in-person specialist visits (pulmonologist, 22.9% vs. 37.0% of patients; cardiologist, 27.5% vs. 33.8%); and more telemedicine visits (mean 0.45 vs. 0.02). In the immediate post-COVID-19 period, patients were less likely to have a PAH-related test versus the pre-COVID-19 period (incidence rate ratio: 0.700; 95% confidence interval: 0.615-0.797), including electrocardiograms (41.7% vs. 54.2%) and 6-minute walk distance tests (16.2% vs. 24.9%). In the immediate post-COVID-19 period, 48 patients had care disruptions and, in the following year, required more hospital days than those without care disruptions (N = 240) (median 10 vs. 5 days in total) and had higher overall hospitalization costs (median US$34,755 vs. US$20,090). Our findings support the need for minimizing care disruptions to potentially avoid incremental post-disruption healthcare utilization and costs among patients with serious chronic diseases such as PAH.

Retrospective Database Analysis of Treatment Patterns Among Patients with Pulmonary Arterial Hypertension.

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.

Palliative care referrals after lung transplantation in major transplant centers in the United States.

OBJECTIVE: Although lung transplantation is a widely used treatment modality for patients with end-stage lung disease, its long-term outcomes are limited. Including palliative approaches in the care of lung transplant recipients may be beneficial; however, systematic information regarding the utilization of palliative care services for lung recipients is lacking. DESIGN AND SETTING: Of the 27 transplant centers meeting the inclusion criteria (an annual lung transplant volume >or=15 for the past 5 years and the availability of palliative care or pain services at the center), 74 clinicians representing either the transplant or palliative care program from 18 centers completed surveys. RESULTS: Both transplant and palliative care clinician respondents strongly favored the idea of integrating palliative care into lung transplant care. However, the number of palliative care referrals made during the last year was low (

CD28 down-regulation on CD4 T cells is a marker for graft dysfunction in lung transplant recipients.

RATIONALE: Repeated antigen-driven proliferations cause CD28 on T cells to down-regulate. We hypothesized that alloantigen-induced proliferations could cause CD28 down-regulation in lung transplant recipients. OBJECTIVES: To ascertain if CD28 down-regulation on CD4 T cells associated with manifestations of allograft dysfunction in lung transplant recipients. METHODS: Peripheral blood CD4 T cells from 65 recipients were analyzed by flow cytometry, cytokine multiplex and proliferative assays, and correlated with clinical events. MEASUREMENTS AND MAIN RESULTS: Findings that CD28 was present on less than 90% of total CD4 T cells were predominantly seen among the recipients with bronchiolitis obliterans syndrome (specificity = 88%). Perforin and granzyme B were produced by >50% of the CD4(+)CD28(null) cells, but less than 6% of autologous CD4(+)CD28(+) cells (P < 0.006). CD4(+)CD28(null) cells also had increased productions of proinflammatory cytokines, but less frequently expressed regulatory T-cell marker FoxP3 (2.1 +/- 1.3%), compared with autologous CD4(+)CD28(+) (9.5 +/- 1.4; P = 0.01). Cyclosporine A (100 ng/ml) inhibited proliferation of CD4(+)CD28(null) cells by 33 +/- 11% versus 68 +/- 12% inhibition of CD4(+)CD28(+) (P = 0.025). FEV(1) fell 6 months later (0.35 +/- 0.04 L) in recipients with CD4(+)CD28(+)/CD4(total) less than 90% (CD28% Low) compared with 0.08 +/- 0.08 L among CD4(+)CD28(+)/CD4(total) (CD28% High) greater than 90% (CD28% High) recipients (P = 0.013). Two-year freedom from death or retransplantation in CD28% Low recipients was 32 +/- 10% versus 78 +/- 6% among the CD28% High subjects (P < 0.0001). CONCLUSIONS: CD28 down-regulation on CD4 cells is associated with bronchiolitis obliterans syndrome and poor outcomes in lung transplantation recipients. CD4(+)CD28(null) cells have unusual, potentially pathogenic characteristics, and could be important in the progression of allograft dysfunction. These findings may illuminate a novel paradigm of transplantation immunopathogenesis, and suggest that CD28 measurements could identify recipients at risk for clinical deteriorations.

Treatment patterns, healthcare resource utilization, and healthcare costs among patients with pulmonary arterial hypertension in a real-world US database.

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.

Malignancy in lung transplantation: biomarkers, gender differences, and consideration of a systems biology approach.

We present here new molecular tools such as gene expression microarrays as promising biomarkers for diagnosis and prognosis in lung cancer as well as for an early diagnosis of malignancy after lung transplantation. Data regarding gender differences in survival, recently collected in a single-institution study, may allow better targeting of immunosuppression goals to maintain effectiveness while minimizing cancer development and progression. Combining these differing types of data using an integrative approach that is the central theme of systems biology may allow us to provide powerful and more individualized risks of cancer after lung transplantation.

Gender differences in survival after lung transplant: implications for cancer etiology.

Predictors of survival after lung transplant were analyzed in a large cohort of 990 lung transplanted patients from a single center. The overall survival was 41.6%, (41.5% in males, and 41.8% in females), the average length of the follow up was 45.84+/-51.98 months (range 0-282.47 months). Females tend to live longer than males: 50.75+/-55.41 months versus 40.64+/-47.60 months, respectively. Males had a risk of dying during the follow up that was 1.18 (95% CI 1.01-1.40) relative to females, after adjusting for ethnicity, age, smoking status, diagnosis and donor characteristics. Females who had at least one full term pregnancy during their life had better survival rates than females who had no full term pregnancies. Our results of a better survival after lung transplant in females, and among them in those who had at least a full term pregnancy support the hypothesis of a hormonal contribution to survival and of the development of immunotolerance after pregnancy. This model could be useful for understanding the role of immunity in cancer development.

Course of illness after the onset of chronic rejection in lung transplant recipients.

BACKGROUND: Despite the overall negative impact of chronic rejection on quality of life and survival after lung transplant, the specific clinical indicators of deterioration have not been identified. OBJECTIVES: To describe the course of illness after the onset of chronic rejection, including demographic and transplant variables, morbidity, mortality, health resource utilization, and end-of-life care, and to identify clinical indicators of deterioration in health and limited survival after the onset of chronic rejection. METHODS: The medical records of 311 recipients of lung transplants between 1998 and 2004 were reviewed retrospectively to identify 60 recipients who experienced chronic rejection. RESULTS: Median survival after chronic rejection was 31.34 months. Time to rejection (mean, 26.05 months; SD, 16.85) was significantly correlated with overall survival without need of a retransplant (r = 0.64; P < .001). The earlier the onset of chronic rejection or the need for oxygen at home, the shorter was the period of survival after chronic rejection and the more frequent were hospital and intensive care unit admissions and prolonged stays. Of the 26 recipients who died, 65% died at the transplant center, and all but 1 died in the intensive care unit; 3 died after multiple attempts of cardiopulmonary resuscitation; life support was ultimately withdrawn in 69%. CONCLUSIONS: Lung transplant recipients who experience chronic graft rejection have high rates of morbidity, mortality, and health resource utilization; however, the course of illness after chronic rejection is highly variable.

Zika Virus Preparedness and Response Efforts Through the Collaboration Between a Health Care Delivery System and a Local Public Health Department.

ABSTRACTThe 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).

Binding of mRNA to the bacterial translation initiation complex.

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.

Simkania negevensis in bronchoalveolar lavage of lung transplant recipients: a possible association with acute rejection.

BACKGROUND: Simkania negevensis is a novel organism closely related to chlamydiae. The organism has been associated with community acquired pneumonia and acute exacerbation of chronic obstructive pulmonary disease. The prevalence and pathogenic potential of S. negevensis is not known in lung transplant recipients. METHODS: In this multicenter study comparative analysis of bronchoalveolar lavage (BAL) in lung transplants (Tx) and kidney Tx, immunocompromised and nasopharyngeal (NP) washes of immunocompetent patients was done. The BAL specimens were tested by nested polymerase chain reaction (PCR) for C. pneumoniae and S. negevensis. Selected S. negevensis positive PCR cases were confirmed by culture. RESULTS: In the initial 41 BAL samples S. negevensis was detected in 97.5% (40/41) of lung transplant recipients as compared to 14.1% (1/7) in other organ transplant recipients (P<0.0001). In the sequential samples of 19 lung transplant recipients, 59% (24/41) had concomitant positive PCR and rejection as compared to 30% (3/10) who had negative PCR but had rejection (P=0.16). S. negevensis infection had hazard ratio of 3.29 (95% CI: 0.73-14.76; P=0.11) for developing acute rejection. CONCLUSION: S. negevensis is highly prevalent in liver Tx recipients and may be associated with acute rejection.

Aspergillus galactomannan antigen in the bronchoalveolar lavage fluid for the diagnosis of invasive aspergillosis in lung transplant recipients.

BACKGROUND: The clinical utility of Platelia Aspergillus enzyme immunoassay (EIA) for galactomannan (GM) antigen detection in bronchoalveolar lavage (BAL) for the diagnosis of invasive aspergillosis (IA) in lung transplant recipients is not known. METHODS: BAL fluid samples from consecutive lung transplant recipients who underwent bronchoscopy were prospectively analyzed for GM. RESULTS: A total of 333 BAL samples from 116 patients were tested. Invasive aspergillosis was documented in 5.2% (6/116) of the patients. Samples analyzed included 9 BALs from two patients with proven IA, 19 BALs from four patients with probable IA, and 305 BALs from 110 patients without IA. At the index cutoff value of > or =0.5, the sensitivity was 60%; specificity was 95%, with positive and negative likelihood ratios of 14 and 0.41, respectively. Increasing the index cutoff value to > or =1.0 yielded a sensitivity of 60%, a specificity of 98%, and the positive and negative likelihood ratios of 28 and 0.40, respectively. Two of six patients with IA receiving antifungal prophylaxis had false-negative results. CONCLUSIONS: A Platelia EIA index cut-off > or =1.0 in the BAL fluid in a lung transplant recipient with a compatible clinical illness may be considered as suggestive of IA.

Evaluation of the electronic version of the questionnaire for lung transplant patients.

UNLABELLED: Context-Recent modifications to the QLTP (Questionnaire for Lung Transplant Patients), including changing items from dichotomous to multiple dimension scaling, adding psychological symptoms, and converting to an electronic format (e-QLTP), made it necessary to reevaluate its reliability, validity, recipient satisfaction, and feasibility of administering the e-QLTP in the clinical setting. Purpose-To report the final modifications, psychometric properties, recipient satisfaction, and feasibility of administering the e-QLTP, a patient report outcome measure of symptoms and activity tolerance. Methods-Sixty lung recipients completed the original QLTP and the e-QLTP and rated their satisfaction with the e-version during a routine posttransplant evaluation; 65% (38 of 60) also completed a retest version. Correlations were computed for retest stability, concurrent validity between versions of the QLTP, and construct validity among the subscales of the e-QLTP and forced expiratory volumes in 1 second. Using the After Scenario Questionnaire, participants rated their satisfaction with the ease, amount of time, and support information when completing the e-QLTP. RESULTS: The e-QLTP and subscales were internally consistent (alpha = .73 - .90) and stable (intraclass correlations = .47 - .93). Significant correlations (P = .001) were found between the e-QLTP and the original QLTP (r = 0.53-0.56) and between the e-QLTP subscales and forced expiratory volumes in 1 second (r = 0.51 - 0.53). The overall mean satisfaction score was 1.27 (+/- 0.47). Conclusions-The e-QLTP is a reliable and valid measure of physical and psychological symptoms after lung transplantation. It is feasible to complete in the clinical setting and recipients are highly satisfied with its use. Its computerized functionality enhances assessment and management of symptoms over time.

Considerations for optimal management of patients with pulmonary arterial hypertension: a multi-stakeholder roundtable discussion.

A roundtable panel of national and regional managed care decision makers and providers met to discuss pulmonary arterial hypertension (PAH) and strategies for management. As a rare, complex disease with high economic costs and potentially devastating outcomes, PAH necessitates that managed care providers balance optimal care with efficient use of healthcare resources. PAH specialists are recognized by health plans as knowledgeable experts and integral partners in managing patients and resources. The diagnosis of PAH must be confirmed by a right heart catheterization. Available therapies are indicated almost exclusively for patients with PAH (riociguat is also indicated in chronic thromboembolic pulmonary hypertension) and target 1 of 3 pathways: endothelin receptor antagonists for the endothelin pathway; phosphodiesterase type-5 inhibitors and soluble guanylate cyclase stimulators for the nitric oxide pathway; and prostanoids as well as a prostacyclin receptor agonist for the prostacyclin pathway, with combination therapy becoming more common. Even in the modern treatment era, as shown in the REVEAL and French registries, PAH is often diagnosed years after symptoms first appear, which leads to a poor prognosis and increased burden on the healthcare system. Facilitating treatment of patients with PAH through centers of excellence, and coordinating care management between health plans and providers with evidence-based approaches can lead to both better results for patients and lower healthcare costs. When PAH experts have access to the right treatments for the right patients at the right time, they can work with insurers to improve the health of patients with PAH while helping to reduce the impact on the healthcare system.

Rapid kinetic analysis of EF-G-dependent mRNA translocation in the ribosome.

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' end of a short mRNA sequence at position +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.

Prognostic factors associated with adverse outcome among critically ill elderly patients admitted to the intensive care unit.

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.