Phosphorothioate RNA Analysis by NETD Tandem Mass Spectrometry.

Therapeutic RNAs are routinely modified during their synthesis to ensure proper drug uptake, stability, and efficacy. Phosphorothioate (PS) RNA, molecules in which one or more backbone phosphates are modified with a sulfur atom in place of standard nonbridging oxygen, is one of the most common modifications because of ease of synthesis and pharmacokinetic benefits. Quality assessment of RNA synthesis, including modification incorporation, is essential for drug selectivity and performance, and the synthetic nature of the PS linkage incorporation often reveals impurities. Here, we present a comprehensive analysis of PS RNA via tandem mass spectrometry (MS). We show that activated ion-negative electron transfer dissociation MS/MS is especially useful in diagnosing PS incorporation, producing diagnostic a- and z-type ions at PS linkage sites, beyond the standard d- and w-type ions. Analysis using resonant and beam-type collision-based activation reveals that, overall, more intense sequence ions and base-loss ions result when a PS modification is present. Furthermore, we report increased detection of b- and x-type product ions at sites of PS incorporation, in addition to the standard c- and y-type ions. This work reveals that the gas-phase chemical stability afforded by sulfur alters RNA dissociation and necessitates inclusion of additional product ions for MS/MS of PS RNA.

Direct detection of OXA-48-like carbapenemase variants with and without co-expression of an extended-spectrum ß-lactamase from bacterial cell lysates using mass spectrometry.

INTRODUCTION: Antibiotic-resistant Gram-negative bacteria are of a growing concern globally, especially those producing enzymes conferring resistance. OXA-48-like carbapenemases hydrolyze most ß-lactam antibiotics, with typically low-level hydrolysis of carbapenems, but have limited effect on broad-spectrum cephalosporins. These are frequently co-expressed with extended spectrum ß-lactamases, especially CTX-M-15, which typically shows high level resistance to broad-spectrum cephalosporins, yet is carbapenem susceptible. The combined resistance profile makes the need for successful detection of these specific resistance determinants imperative for effective antibiotic therapy. OBJECTIVES: The objective of this study is to detect and identify OXA-48-like and CTX-M-15 enzymes using mass spectrometry, and to subsequently develop a method for detection of both enzyme types in combination with liquid chromatography. METHODS: Cells grown in either broth or on agar were harvested, lysed, and, in some cases buffer-exchanged. Lysates produced from bacterial cells were separated and analyzed via liquid chromatography with mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS). RESULTS: The intact proteins of OXA-48, OXA-181, and OXA-232 (collectively OXA-48-like herein) and CTX-M-15 were characterized and detected. Acceptance criteria based on sequence-informative fragments from each protein group were established as confirmatory markers for the presence of the protein(s). A total of 25 isolates were successfully tested for OXA-48 like (2), CTX-M-15 (3), or expression of both (7) enzymes. Thirteen isolates served as negative controls. CONCLUSIONS: Here we present a method for the direct and independent detection of both OXA-48-like carbapenemases and CTX-M-15 ß-lactamases using LC-MS/MS. The added sensitivity of MS/MS allows for simultaneous detection of at least two co-eluting, co-isolated and co-fragmented proteins from a single mass spectrum.

Rapid MRSA detection via tandem mass spectrometry of the intact 80 kDa PBP2a resistance protein.

Treatment of antibiotic-resistant infections is dependent on the detection of specific bacterial genes or proteins in clinical assays. Identification of methicillin-resistant Staphylococcus aureus (MRSA) is often accomplished through the detection of penicillin-binding protein 2a (PBP2a). With greater dependence on mass spectrometry (MS)-based bacterial identification, complementary efforts to detect resistance have been hindered by the complexity of those proteins responsible. Initial characterization of PBP2a indicates the presence of glycan modifications. To simplify detection, we demonstrate a proof-of-concept tandem MS approach involving the generation of N-terminal PBP2a peptide-like fragments and detection of unique product ions during top-down proteomic sample analyses. This approach was implemented for two PBP2a variants, PBP2amecA and PBP2amecC, and was accurate across a representative panel of MRSA strains with different genetic backgrounds. Additionally, PBP2amecA was successfully detected from clinical isolates using a five-minute liquid chromatographic separation and implementation of this MS detection strategy. Our results highlight the capability of direct MS-based resistance marker detection and potential advantages for implementing these approaches in clinical diagnostics.

Mitigating exhalation puffs during oxygen therapy for respiratory disease.

We investigate the dispersal of exhalations corresponding to a patient experiencing shortness of breath while being treated for a respiratory disease with oxygen therapy. Respiration through a nasal cannula and a simple O2 mask is studied using a supine manikin equipped with a controllable mechanical lung by measuring aerosol density and flow with direct imaging. Exhalation puffs are observed to travel 0.35 ± 0.02 m upward while wearing a nasal cannula, and 0.29 ± 0.02 m laterally through a simple O2 mask, posing a higher direct exposure risk to caregivers. The aerosol-laden air flows were found to concentrate in narrow conical regions through both devices at several times their concentration level compared with a uniform spreading at the same distance. We test a mitigation strategy by placing a surgical mask loosely over the tested devices. The mask is demonstrated to alleviate exposure by deflecting the exhalations from being launched directly above a supine patient. The surgical mask is found to essentially eliminate the concentrated aerosol regions above the patient over the entire oxygenation rates used in treatment in both devices.

Patient-Centered Care and Autonomy: Shared Decision-Making in Practice and a Suggestion for Practical Application in the Critically Ill.

Decision-making for the hospitalized dying and critically ill is often characterized by an understanding of autonomy that leads to clinical care and outcomes that are antithetical to patients' preferences around suffering and quality of life. A better understanding of autonomy will facilitate the ultimate goal of a patient-centered approach and ensure compassionate, high-quality care that respects our patients' values. We reviewed the medical literature and our experiences through the ethics service, palliative care service, and critical care service of a large community teaching hospital. The cumulative experience of a senior intensivist was filtered through the lens of a medical ethicist and the palliative care team. The practical application of patient-centered care was discerned from these interactions. We determined that a clearer understanding of patient-centeredness would improve the experience and outcomes of care for our patients as well as our adherence to ethical practice. The practical applications of autonomy and patient-centered care were evaluated by the authors through clinical interactions on the wards to ascertain problems in understanding their meaning. Clarification of autonomy and patient-centeredness is provided using specific examples to enhance understanding and application of these principles in patient-centered care.

Direct detection of intact Klebsiella pneumoniae carbapenemase variants from cell lysates: Identification, characterization and clinical implications.

INTRODUCTION: Carbapenemase-producing organisms (CPOs) are a growing threat to human health. Among the enzymes conferring antibiotic resistance produced by these organisms, Klebsiella pneumoniae carbapenemase (KPC) is considered to be a growing global health threat. Reliable and specific detection of this antibiotic resistance-causing enzyme is critical both for effective therapy and to mitigate further spread. OBJECTIVES: The objective of this study is to develop an intact protein mass spectrometry-based method for detection and differentiation of clinically-relevant KPC variants directly from bacterial cell lysates. The method should be specific for any variant expressed in multiple bacterial species, limit false positive results and be rapid in nature to directly influence clinical outcomes. METHODS: Lysates obtained directly from bacterial colonies were used for intact protein detection using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Bottom-up and top-down proteomic methods were used to characterize the KPC protein targets of interest. Comparisons between KPC-producing and KPC-non-producing isolates from a wide variety of species were also performed. RESULTS: Characterization of the mature KPC protein revealed an unexpected signal peptide cleavage site preceding an AXA signal peptide motif, modifying the molecular weight (MW) of the mature protein. Taking the additional AXA residues into account allowed for direct detection of the intact protein using top-down proteomic methods. Further validation was performed by transforming a KPC-harboring plasmid into a negative control strain, followed by MS detection of the KPC variant from the transformed cell line. Application of this approach to clearly identify clinically-relevant variants among several species is presented for KPC-2, KPC-3, KPC-4 and KPC-5. CONCLUSION: Direct detection of these enzymes contributes to the understanding of occurrence and spread of these antibiotic-resistant organisms. The ability to detect intact KPC variants via a simple LC-MS/MS approach could have a direct and positive impact on clinical therapy, by providing both direction for epidemiological tracking and appropriate therapy.

Bilateral Bilothorax: An Unusual Cause of Bilateral Exudative Pleural Effusion.

Bilothorax is an uncommon cause of exudative pleural effusion; the majority of reported cases are right-sided while a bilateral presentation is extremely rare. The majority of cases are secondary to biliary obstruction, an extension of hepatic infections, and iatrogenic complications following percutaneous procedures or surgical interventions. Imaging studies and a diagnostic pleural tap can confirm the diagnosis. Early recognition and complete drainage are important to prevent life-threatening complications, including empyema formation. We present a case of a 71-year-old female who developed bilateral bilothorax as a complication of gallstone pancreatitis.

Pressure Injuries at Intensive Care Unit Admission as a Prognostic Indicator of Patient Outcomes.

BACKGROUND: Pressure injuries, also known as pressure ulcers, are a serious complication of immobility. Patients should be thoroughly examined for pressure injuries when admitted to the intensive care unit to optimize treatment. Whether community-acquired pressure injuries correlate with poor hospital outcomes among critically ill patients is understudied. OBJECTIVES: To determine whether pressure injuries present upon admission to the intensive care unit can serve as a predictive marker for longer hospitalization and increased mortality. METHODS: This study retrospectively analyzed admissions of adult patients to a 24-bed medical-surgical intensive care unit in a large level I trauma center in the northeast United States from 2010 to 2012. The association of pressure injuries with mortality and length of stay was assessed, using multivariable logistic regression and generalized linear models, adjusted for age, comorbidities, Acute Physiology and Chronic Health Evaluation III score, and other patient characteristics. RESULTS: Among 2723 patients, 180 (6.6%) had a pressure injury at admission. Patients with a pressure injury had longer mean unadjusted stay (15.6 vs 10.5 days; P < .001) and higher in-hospital mortality rate (32.2% vs 18.3%; P < .001) than did patients without a pressure injury at admission. After multivariable adjustment, pressure injuries were associated with a mean increase in length of stay of 3.1 days (95% CI 1.5-4.7; P < .001). Pressure injuries were not associated with mortality after adjusting for the Acute Physiology and Chronic Health Evaluation III score, but they may serve as a marker for increased risk of mortality if an Acute Physiology and Chronic Health Evaluation III score is unavailable. CONCLUSION: Pressure injuries present at admission to the intensive care unit are an objective, easy-to-identify finding associated with longer stays. Pressure injuries might have a modest association with higher risk of mortality.

Value, Chronic Critical Illness, and Choosing Wisely.

Management of limited health-care resources has been of growing concern. Stewardship of health-care dollars and avoidance of low-value care is being increasingly recognized as a matter that affects all practitioners. This review aims to examine a particular pathological state with multifactorial origins: chronic critical illness (CCI). This condition exerts a large toll on society as well as individual patients and their families. Here, we offer a brief review as to the incidence/prevalence of CCI and suggestions for prevention. Emphasis should be placed on the importance of early, open communication among physicians and patients about their end-of-life decisions and advanced directives, so that decisions can be made wisely and with the patient's best interests in mind.

Tissue Edema, Fluid Balance, and Patient Outcomes in Severe Sepsis: An Organ Systems Review.

Severe sepsis and septic shock remain among the deadliest diseases managed in the intensive care unit. Fluid resuscitation has been a mainstay of early treatment, but the deleterious effects of excessive fluid administration leading to tissue edema are becoming clearer. A positive fluid balance at 72 hours is associated with significantly increased mortality, yet ongoing fluid administration beyond a durable increase in cardiac output is common. We review the pathophysiologic and clinical data showing the negative effects of edema on pulmonary, renal, central nervous, hepatic, and cardiovascular systems. We discuss data showing increased morbidity and mortality following nonjudicious fluid administration and challenge the assumption that patients who are fluid responsive are also likely to benefit from that fluid. The distinctions between fluid requirement, responsiveness, and tolerance are central to newer concepts of resuscitation. We summarize data in each organ system showing a predictable increase in morbidity and mortality with nonbeneficial fluid administration, providing a better framework for precision in volume management of the patient with severe sepsis.

The dangers of extreme body mass index values in patients with Clostridium difficile.

PURPOSE: To examine the association between body mass index (BMI) and in-hospital mortality in patients presenting with Clostridium difficile infections in emergency department visits (ED) in the USA. Infected patients with extreme BMIs may have an elevated mortality risk, but prior studies examining this question have been too small to reach definitive conclusions. METHODS: Data were from the Nationwide Emergency Department Sample (NEDS), Healthcare Cost and Utilization Project (HCUP), Agency for Healthcare Research and Quality during 2012. NEDS records emergency department (ED) visits across the USA and provides statistical sampling weights to approximate a nationally representative sample of US hospital-based EDs. Inclusion criteria were adults age 18 or older with an ICD-9 code for C. difficile infection (008.45) and a documented body mass index ICD-9 V code (V85.x). Logistic regression was used to predict mortality after adjusting for demographic variables and chronic comorbidities defined by Elixhauser. RESULTS: A weighted sample of 22,937 ED visits met all inclusion criteria. The cohort's mean age was 66. 64.6% were female. The unadjusted mortality rate was 6.5%. Patients with a BMI < 19 kg/m2 had an adjusted odds ratio of 2.73; 95% CI (1.80, 4.16), p < 0.001 compared to patients with a BMI of 19.0-4.9 kg/m2 (the referent category). In obese patients, only BMI values >40 kg/m2 were associated with significantly greater mortality risk. CONCLUSION: Being underweight (BMI < 19) or morbidly obese (BMI > 40) was associated with increased risk of in-hospital mortality in patients presenting with C. difficile infections.

Structural Characterization of Native Proteins and Protein Complexes by Electron Ionization Dissociation-Mass Spectrometry.

Mass spectrometry (MS) has played an increasingly important role in the identification and structural and functional characterization of proteins. In particular, the use of tandem mass spectrometry has afforded one of the most versatile methods to acquire structural information for proteins and protein complexes. The unique nature of electron capture dissociation (ECD) for cleaving protein backbone bonds while preserving noncovalent interactions has made it especially suitable for the study of native protein structures. However, the intra- and intermolecular interactions stabilized by hydrogen bonds and salt bridges can hinder the separation of fragments even with preactivation, which has become particularly problematic for the study of large macromolecular proteins and protein complexes. Here, we describe the capabilities of another activation method, 30 eV electron ionization dissociation (EID), for the top-down MS characterization of native protein-ligand and protein-protein complexes. Rich structural information that cannot be delivered by ECD can be generated by EID. EID allowed for the comparison of the gas-phase and the solution-phase structural stability and unfolding process of human carbonic anhydrase I (HCA-I). In addition, the EID fragmentation patterns reflect the structural similarities and differences among apo-, Zn-, and Cu,Zn-superoxide dismutase (SOD1) dimers. In particular, the structural changes due to Cu-binding and a point mutation (G41D) were revealed by EID-MS. The performance of EID was also compared to that of 193 nm ultraviolet photodissociation (UVPD), which allowed us to explore their qualitative similarities and differences as potential valuable tools for the MS study of native proteins and protein complexes.