Nanoparticle-assisted laser desorption/ionization mass spectrometry: novel sample preparation methods and nanoparticle screening for plant metabolite imaging

The main goal of the presented research is development of nanoparticle based matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). This dissertation includes the application of previously developed data acquisition methods, development of novel sample preparation methods, application and comparison of novel nanoparticle matrices, and comparison of two nanoparticle matrix application methods for MALDI-MS and MALDI-MS imaging. Chapter 1 presents background information about MALDI-MS and a general workflow for MALDI-MS imaging experiments. Chapter 6 provides a summary of the presented work and possible future directions. Chapter 2 describes a “multiplex” MALDI-MS imaging technique applied to study latent human fingerprints. In this study, we applied previously developed multiplex data acquisition techniques for the detection of endogenous and exogenous compounds with structural information in a single MALDI-MS imaging experiment. This study also depicts the importance of the sublimation matrix application method for analysis of very delicate samples such as latent fingerprints. Chapter 3 presents the development of novel sample preparation methods for nanoparticle-assisted laser desorption/ionization-mass spectrometry -mass spectrometry (NALDI-MS) imaging of metabolites in plant tissues. In this chapter, we developed a novel fracturing method for MALDI-MS/ NALDI-MS imaging of rice leaf to study a plant-pathogen interactions. We successfully detected and imaged diterpenoids produced against the Xanthomonas oryzae pv. oryzae infection in rice leaves. Chapter 4 is focused on novel nanoparticle (NP) matrix development for NALDI-MS of low-molecular weight plant metabolites. This large-scale NP screening was inspired by the success of the metabolite detection/imaging in Chapter 3. In this work, we studied three different classes of NPs for desorption/ionization of a wide variety of plant metabolites and sought to shed some light on the NALDI mechanism. Chapter 5 is a further extension of the study in Chapter 4, in which we compare two NP application methods for efficiency of desorption/ionization of plant metabolites and homogeneity of application. We compare application of a nanoparticle suspension by oscillating capillary nebulizer and solvent-free sputter coating of metal NPs. The work presented in Chapters 4 and 5 will be helpful for development of novel matrices for analysis and imaging of low-molecular weight metabolites that are difficult to ionize using traditional MALDI-MS matrices

A clinical study on etiology, prognosis, outcome and role of endoscopy in upper gastrointestinal bleed in a tertiary care center

Upper gastrointestinal bleed is defined as bleeding proximal to the ligament of Treitz. The aim of this study was to know the cause of upper GI bleed, prognosis of the patients and role of upper gastrointestinal endoscopy (UGIE) in the management of upper gastrointestinal bleed (UGIB). A study of 140 cases was carried out in the Shree Krishna Hospital and Pramukh Swami Medical College, Karamsad in India between January 2014 and June 2015. All patients were selected by the detailed history and physical examination. Patients with signs and symptoms suggestive of upper GI bleeding such as hematemesis, melena, blood in the nasogastric tubes, and profuse hematochezia were included in the study. Endoscopy was performed in all patients. Rockall scoring system was used to predict the mortality in patients with upper GI bleeding. We use descriptive statistics for analysis. It was found that upper GI bleed was more common in males than females, and was more prevalent in elderly individuals. The most common symptom was found to be hematemesis followed by abdominal pain. The most common cause was portal hypertension, which has a direct correlation with alcohol addiction. UGIE has both diagnostic as well as therapeutic role in UGIB. This study showed that upper GI bleeding was more common in male patients with the most common cause being portal hypertension. We observed that Mallory-Weiss tear had a particular association with NSAIDs. In our study, the Rockall scoring system was seen to predict the mortality in patients with upper GI bleeding. Endoscopy was both diagnostic and therapeutic and endoscopic variceal ligation (EVL/Glue) was performed for esophageal and/fundic varices and adrenaline injection for peptic ulcer bleeding and Mallory-Weiss tear.Keywords: Upper GI bleeding; Endoscopy; Endoscopic variceal ligation; Mallory-Weiss tea

A Comparison of Four Treatments for Generalized Convulsive Status Epilepticus

ABSTRACT Background and Methods Although generalized convulsive status epilepticus is a life-threatening emergency, the best initial drug treatment is uncertain. We conducted a five-year randomized, doubleblind, multicenter trial of four intravenous regimens: diazepam (0.15 mg per kilogram of body weight) followed by phenytoin (18 mg per kilogram), lorazepam (0.1 mg per kilogram), phenobarbital (15 mg per kilogram), and phenytoin (18 mg per kilogram). Patients were classified as having either overt generalized status epilepticus (defined as easily visible generalized convulsions) or subtle status epilepticus (indicated by coma and ictal discharges on the electroencephalogram, with or without subtle convulsive movements such as rhythmic muscle twitches or tonic eye deviation). Treatment was considered successful when all motor and electroencephalographic seizure activity ceased within 20 minutes after the beginning of the drug infusion and there was no return of seizure activity during the next 40 minutes. Analyses were performed with data on only the 518 patients with verified generalized convulsive status epilepticus as well as with data on all 570 patients who were enrolled. Results Three hundred eighty-four patients had a verified diagnosis of overt generalized convulsive status epilepticus. In this group, lorazepam was successful in 64.9 percent of those assigned to receive it, phenobarbital in 58.2 percent, diazepam and phenytoin in 55.8 percent, and phenytoin in 43.6 percent (P=0.02 for the overall comparison among the four groups). Lorazepam was significantly superior to phenytoin in a pairwise comparison (P=0.002). Among the 134 patients with a verified diagnosis of subtle generalized convulsive status epilepticus, no significant differences among the treatments were detected (range of success rates, 7.7 to 24.2 percent). In an intention-to-treat analysis, the differences among treatment groups were not significant, either among the patients with overt status epilepticus (P=0.12) or among those with subtle status epilepticus (P=0.91). There were no differences among the treatments with respect to recurrence during the 12- hour study period, the incidence of adverse reactions, or the outcome at 30 days. Conclusions As initial intravenous treatment for overt generalized convulsive status epilepticus, lorazepam is more effective than phenytoin. Although lorazepam is no more efficacious than phenobarbital or diazepam and phenytoin, it is easier to use. (N Engl J Med 1998;339:792-8.

Genetic variability of hepatitis C virus before and after combined therapy of interferon plus ribavirin

We present an analysis of the selective forces acting on two hepatitis C virus genome regions previously postulated to be involved in the viral response to combined antiviral therapy. One includes the three hypervariable regions in the envelope E2 glycoprotein, and the other encompasses the PKR binding domain and the V3 domain in the NS5A region. We used a cohort of 22 non-responder patients to combined therapy (interferon alpha-2a plus ribavirin) for which samples were obtained before initiation of therapy and after 6 or/and 12 months of treatment. A range of 25-100 clones per patient, genome region and time sample were sequenced. These were used to detect general patterns of adaptation, to identify particular adaptation mechanisms and to analyze the patterns of evolutionary change in both genome regions. These analyses failed to detect a common adaptive mechanism for the lack of response to antiviral treatment in these patients. On the contrary, a wide range of situations were observed, from patients showing no positively selected sites to others with many, and with completely different topologies in the reconstructed phylogenetic trees. Altogether, these results suggest that viral strategies to evade selection pressure from the immune system and antiviral therapies do not result from a single mechanism and they are likely based on a range of different alternatives, in which several different changes, or their combination, along the HCV genome confer viruses the ability to overcome strong selective [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]

Cell Culture Replication of a Genotype 1b Hepatitis C Virus Isolate Cloned from a Patient Who Underwent Liver Transplantation

The introduction of the genotype 2a isolate JFH1 was a major breakthrough in the field of hepatitis C virus (HCV), allowing researchers to study the complete life cycle of the virus in cell culture. However, fully competent culture systems encompassing the most therapeutically relevant HCV genotypes are still lacking, especially for the highly drug-resistant genotype 1b. For most isolated HCV clones, efficient replication in cultured hepatoma cells requires the introduction of replication-enhancing mutations. However, such mutations may interfere with viral assembly, as occurs in the case of the genotype 1b isolate Con1. In this study, we show that a clinical serum carrying a genotype 1b virus with an exceptionally high viral load was able to infect Huh7.5 cells. Similar to previous reports, inoculation of Huh7.5 cells by natural virus is very inefficient compared to infection by cell culture HCV. A consensus sequence of a new genotype 1b HCV isolate was cloned from the clinical serum (designated Barcelona HCV1), and then subjected to replication studies. This virus replicated poorly in a transient fashion in Huh7.5 cells after electroporation with in vitro transcribed RNA. Nonetheless, approximately 3 weeks post electroporation and thereafter, core protein-positive cells were detected by immunofluorescence. Surprisingly, small amounts of core protein were also measurable in the supernatant of electroporated cells, suggesting that HCV particles might be assembled and released. Our findings not only enhance the current method of cloning in vitro HCV replication-competent isolates, but also offer valuable insights for the realization of fully competent culture systems for HCV

A simplified (modified) Duke Activity Status Index (M-DASI) to characterise functional capacity: A secondary analysis of the Measurement of Exercise Tolerance before Surgery (METS) study

Background Accurate assessment of functional capacity, a predictor of postoperative morbidity and mortality, is essential to improving surgical planning and outcomes. We assessed if all 12 items of the Duke Activity Status Index (DASI) were equally important in reflecting exercise capacity. Methods In this secondary cross-sectional analysis of the international, multicentre Measurement of Exercise Tolerance before Surgery (METS) study, we assessed cardiopulmonary exercise testing and DASI data from 1455 participants. Multivariable regression analyses were used to revise the DASI model in predicting an anaerobic threshold (AT) >11 ml kg −1 min −1 and peak oxygen consumption (VO 2 peak) >16 ml kg −1 min −1, cut-points that represent a reduced risk of postoperative complications. Results Five questions were identified to have dominance in predicting AT>11 ml kg −1 min −1 and VO 2 peak>16 ml.kg −1min −1. These items were included in the M-DASI-5Q and retained utility in predicting AT>11 ml.kg −1.min −1 (area under the receiver-operating-characteristic [AUROC]-AT: M-DASI-5Q=0.67 vs original 12-question DASI=0.66) and VO 2 peak (AUROC-VO2 peak: M-DASI-5Q 0.73 vs original 12-question DASI 0.71). Conversely, in a sensitivity analysis we removed one potentially sensitive question related to the ability to have sexual relations, and the ability of the remaining four questions (M-DASI-4Q) to predict an adequate functional threshold remained no worse than the original 12-question DASI model. Adding a dynamic component to the M-DASI-4Q by assessing the chronotropic response to exercise improved its ability to discriminate between those with VO 2 peak>16 ml.kg −1.min −1 and VO 2 peak<16 ml.kg −1.min −1. Conclusions The M-DASI provides a simple screening tool for further preoperative evaluation, including with cardiopulmonary exercise testing, to guide perioperative management

Integration of the Duke Activity Status Index into preoperative risk evaluation: a multicentre prospective cohort study.

BACKGROUND: The Duke Activity Status Index (DASI) questionnaire might help incorporate self-reported functional capacity into preoperative risk assessment. Nonetheless, prognostically important thresholds in DASI scores remain unclear. We conducted a nested cohort analysis of the Measurement of Exercise Tolerance before Surgery (METS) study to characterise the association of preoperative DASI scores with postoperative death or complications. METHODS: The analysis included 1546 participants (≥40 yr of age) at an elevated cardiac risk who had inpatient noncardiac surgery. The primary outcome was 30-day death or myocardial injury. The secondary outcomes were 30-day death or myocardial infarction, in-hospital moderate-to-severe complications, and 1 yr death or new disability. Multivariable logistic regression modelling was used to characterise the adjusted association of preoperative DASI scores with outcomes. RESULTS: The DASI score had non-linear associations with outcomes. Self-reported functional capacity better than a DASI score of 34 was associated with reduced odds of 30-day death or myocardial injury (odds ratio: 0.97 per 1 point increase above 34; 95% confidence interval [CI]: 0.96-0.99) and 1 yr death or new disability (odds ratio: 0.96 per 1 point increase above 34; 95% CI: 0.92-0.99). Self-reported functional capacity worse than a DASI score of 34 was associated with increased odds of 30-day death or myocardial infarction (odds ratio: 1.05 per 1 point decrease below 34; 95% CI: 1.00-1.09), and moderate-to-severe complications (odds ratio: 1.03 per 1 point decrease below 34; 95% CI: 1.01-1.05). CONCLUSIONS: A DASI score of 34 represents a threshold for identifying patients at risk for myocardial injury, myocardial infarction, moderate-to-severe complications, and new disability