In-spray Supercharging of Peptides and Proteins in Electrospray Ionization Mass Spectrometry

Although electrospray ionization mass spectrometry (ESI-MS) has revolutionized biological analysis with its unique ability to analyze biomolecules, a major hurdle remains in the loss of low-abundance ions in complex biological matrices. This limits the amount of information obtained from MS analysis. Some of these ions are considered potential biomarkers for diseases such as cancer. In particular, any improvement to the detection of such ions enables cancer to be diagnosed at an earlier stage, when the tumor is smaller and has not yet metastasized. Motivated by the abovementioned challenge and building on prior research, this Ph.D. project aimed to facilitate improved detection of low-abundance peptides and proteins in complex biological samples in mass spectrometry-based proteomics. This aim was pursued through the application of supercharging reagents in nanoelectrospray ionization mass spectrometry, which is a topic of ongoing interest in both bottom-up and top-down workflows. Over the past decade, the inclusion of certain organic compounds as solution additives in electrospray ionization mass spectrometry analysis has been shown to enhance the charging of biomolecules and increase ion abundances, leading to improved ion detection. In this thesis, we report the application of cyclic alkyl carbonates in nanoelectrospray ionization mass spectrometry for bioanalytical purposes, encompassing the analysis of various peptide and protein solutions. The studies focused on investigating a series of cyclic alkyl carbonate solution additives in bottom-up proteomics, comparing their performance with conventional supercharging additives. The findings highlighted the most effective additive for improving peptide identification in nanoelectrospray ionization mass spectrometry analysis, aiding further research on complex protein mixtures in next chapters. The study was extended to liquid chromatography-mass spectrometry (nano-LC-MS) workflow. Two chapters focus on exploring and developing two distinct platforms to integrate the cyclic carbonate supercharging additive into the analysis. The rationale was to find methods to introduce the additive into the analyte solutions after chromatographic separation and just prior to the electrospray process. One platform used a sheath-liquid system, while the other employed dual-emitter electrospray ionization. A specific achievement in our work is that the newly introduced supercharger outperforms common superchargers, and it also is compatible with sheath liquid systems, unlike some conventional superchargers such as DMSO. The optimized dual-emitter method for introducing cyclic alkyl carbonate supercharging additives was employed in bottom-up proteomics studies. This supercharged analysis of intact proteins demonstrated effectiveness in enhancing protein charging and also reducing non-volatile salts interference in MS analysis. The latter is particularly valuable for the detection and analysis of low-abundance proteins in solutions derived from biological sources. This study presents a viable method for static nanoelectrospray ionization mass spectrometry analysis of intact proteins under challenging sample conditions. Furthermore, it lays the groundwork for future advancements in single-cell proteomics, where in-spray supercharging techniques could enable the detailed analysis of proteins at the single-cell level, offering new insights into cellular functions as well as facilitating early diagnosis

Effectiveness of iron therapy on breath holding spells in the children

The pathophysiology and mechanism of Breath-Holding Spells (BHS) remain controversial, and the relationship between BHS and anemia has not been clarified, although iron supplementation appears to be effective in many patients. We aimed to assess the probable relation of iron level with initiation of these spells in children.Materials & MethodsOverall, 42 children with a diagnosis of BHS, aged between 6 months to 2 yr were enrolled during Mar 2015 to Dec 2016 at Rasht 17th Shahrivar Hospital, Rasht, northern Iran. Ferrous sulfate solution prescribed 6 mg/kg/d, 3 times daily, for all of cases, regardless of their iron levels, and the response to the treatment was evaluated.ResultsTwenty-five patients were boys (59.52%). The mean age for all patients was 11.71±4.63 months. Positive family history detected in 33.33%; iron deficiency anemia in 21.42%, depletion of iron stores in 52.38%, and normal iron status in 26.19% of cases. Simple spells showed significantly higher mean of Hb in comparison with severe spells (P=0.008); also increased number of spells per month significantly decreased the mean of Hb (P=0.007). Mean frequency of spells was 40.14±47.08 before and 11.14±31.10 after iron therapy, per month (P<0.0001). Overall, 32 patients (76.19%) had complete control of spells, 7 patients (16.66%) partial, 2 cases (4.76%) weak, and 1 child (2.38%) no response after iron therapy.ConclusionIron deficiency anemia may have an important role in BHS, and treatment of anemia may decrease number of the spells

Antibacterial Efficacy of Calcium Hydroxide and Chlorhexidine Mixture for Treatment of Teeth with Primary Endodontic Lesions: A Randomized Clinical Trial

Introduction: This study compared the root canal microbial count of necrotic teeth after irrigation with 6% sodium hypochlorite (NaOCl) (single session treatment) and two-session root canal treatment with two-week application of calcium hydroxide (CH) mixed with 0.2% chlorhexidine (CHX) as intracanal medicament. Methods and Materials: In this randomized clinical trial, single-rooted necrotic teeth were divided into two groups. Root canal was irrigated with 2 mL of 6% NaOCl in one group, and a mixture of 0.2% CHX and CH powder as an intracanal medicament for two weeks, in the other group. Root canal samples were obtained before and after the intervention and number of colony forming units (CFUs) was counted in each phase. Results: The reduction of Enterococcus faecalis CFU was not significantly different between the two groups (P=0.233) but the CFU of aerobic and anaerobic bacteria was significantly lower in CH+CHX group (P<0.001). Conclusion: Two-week application of CH+CHX caused significant reduced the aerobic, anaerobic and E. faecalis colony counts. Thus, it may be beneficial to carry out primary root canal treatment of necrotic teeth with endodontic lesions in two sessions with intracanal medicaments to achieve predictable results.Keywords: Calcium Hydroxide; Chlorhexidine; Endodontic Treatment; Periapical Abscess; Root Canal Therap

Efficacy of a Novel Rotary System in Reduction of Intracanal Bacteria: An in Vitro Study

Introduction: This in vitro study aimed to assess the effectiveness of a single-file rotary system (OneShape) in reduction of intracanal bacteria. Methods and Materials: Eighty one single-rooted mandibular first premolars with single canals were used. Six samples were selected as aseptic control group. Seventy five remaining specimens were infected by Enterococcus faecalis and incubated for 72 h at 37°C. Then they were randomly divided into three groups (n=25). In each group, cleaning and shaping procedures were done using either two conventional rotary systems (ProTaper and iRace), or the single-file system (OneShape). Microbial samples from the intracanal environment were taken by paper points in two steps, before and after instrumentation. Then, they were diluted and plated in blood agar. In order to compare bacterial reduction and turbidity, the Kruskal-Wallis test was used followed by the Chi-Square and Mann-Whitney tests for pairwise comparison. The level of significance was set at 0.05. Results: The control group didn’t show any bacterial growth. The pre- and post-instrumentation samples were significantly different between three groups (P=0.02). Hence, there was no significant differences between turbidity of samples (P>0.05). Conclusion: OneShape system is efficient in bacterial reduction. In this regard ProTaper is the most effective system in intracanal bacterial reduction followed by iRaCe and OneShape, respectively.Keywords: Enterococcus faecalis; iRace; OneShape; ProTape

Subjective Wearing Experience of Lehfilcon A Among Satisfied Comfilcon A Toric Lens Wearers

Jay Mashouf,1 Keith Wan,1 Brad Hall2 1Scripps Optometric Group, San Diego, CA, USA; 2Sengi, Penniac, NB, CanadaCorrespondence: Jay Mashouf, Scripps Optometric Group, 10549 Scripps Poway Pkwy, Ste. G, San Diego, CA, 92131, USA, Tel +1-858-530-2800, Email [email protected]: To evaluate the subjective wearing experience of lehfilcon A toric lenses among subjects who were already satisfied with their comfilcon A toric lenses.Methods: This was an open-label, single arm study of currently satisfied comfilcon A soft toric contact lens (Biofinity Toric, CooperVision, Pleasanton, CA) wearers that were refit with lehfilcon A soft toric lenses (Total30® for Astigmatism; Alcon Vision LLC, Fort Worth, TX). Subjects were administered a questionnaire about satisfaction with comfort, visual performance, end of day comfort, and ease of handling with lehfilcon A toric lenses.Results: A total of 40 subjects completed the study. After one month of wear the median and interquartile range (IQR) was 8 (2) for overall satisfaction with lehfilcon A comfort, 8 (1.25) for visual performance, 7 (3.25) for end of day comfort, 9 (2) for ease of handling, and finally 8 (2) for overall satisfaction.Conclusion: A high proportion of subjects were satisfied with the subjective wearing experience with lehfilcon A toric lenses, including satisfaction with comfort, visual performance, end of day comfort, and ease of handling.Plain Language Summary: There are many different types of contact lens materials and replacement schedules for patients to suit individual needs. Despite the diversity of options, contact lens dropout continues to be high, likely influenced by discomfort and poor vision. A new frequent replacement toric contact lens is available that may offer good subjective comfort for patients. However, there is minimal data on the performance of this lens. The purpose of this study was to evaluate the subjective wearing experience with the new contact lens among patients who were already satisfied with their current toric contact lenses. The results of this study suggest that a high proportion of patients were satisfied with the subjective wearing experience with the new toric contact lens, including satisfaction with comfort, visual performance, end of day comfort, and ease of handling.Keywords: comfort, toric, contact lens, lehfilcon A, Total30 for Astigmatis

Towards clinical application of RayStretch for heterogeneity corrections in LDR permanent 125-I prostate brachytherapy

Purpose: RayStretch is a simple algorithm proposed for heterogeneity corrections in low-dose-rate brachytherapy. It is built on top of TG-43 consensus data, and it has been validated with Monte Carlo (MC) simulations. In this study, we take a real clinical prostate implant with 71 125I seeds as reference and we apply RayStretch to analyze its performance in worst-case scenarios. Methods and Materials: To do so, we design two cases where large calcifications are located in the prostate lobules. RayStretch resilience under various calcification density values is also explored. Comparisons against MC calculations are performed. Results: Dose-volume histogram-related parameters like prostate D90, rectum D2cc, or urethra D10 obtained with RayStretch agree within a few percent with the detailed MC results for all cases considered. Conclusions: The robustness and compatibility of RayStretch with commercial treatment planning systems indicate its applicability in clinical practice for dosimetric corrections in prostate calcifications. Its use during intraoperative ultrasound planning is foreseen

Serum and Cerebrospinal Fluid Lactate Dehydrogenase in Children with Febrile Convulsions

  Objective Tissue damage caused by febrile convulsion has not still been proved or refuted completely. Given the fact that lactate dehydrogenase as an intracellular enzyme can be increased due to tissue damage, we decided to evaluate serum and cerebrospinal fluid lactate dehydrogenase in children with febrile convulsion. Materials & Methods This is a cross-sectional study on 166 children aged 6-24 month, in three groups of simple febrile convulsion (n=56), complex febrile convulsion (n=27) with 3 different subgroups (recurrence in 24 hours, duration >15 minutes, and with focal components), and control (n=83). Patients’ serum and cerebrospinal fluid specimens were collected after meeting the inclusion criteria. Demographic information was documented and patients’ serum and cerebrospinal fluid lactate dehydrogenase and glucose were measured. Data were analyzed using SPSS software. Result The mean serum lactate dehydrogenase in simple febrile convulsion, complex febrile convulsion, and controls were 501.57± 143.70, 553.07±160.22, and 505.87±98.73 U/L, respectively. The mean cerebrospinal fluid lactate dehydrogenase in simple, complex febrile convulsion, and control groups were 22.58±11.92, 29.48±18.18, and 21.56±17.32 U/L, respectively. Only cerebrospinal fluid lactate dehydrogenase difference between complex febrile convulsion and control group (p=0.039) (In the duration >15 minutes subgroup and controls, p=0.028) was statisticallysignificant. There was a significantThere was a significant difference between sex and serum lactate dehydrogenase in the same subgroup of complex group (p=0.012). Conclusion Complex febrile convulsion may lead to increase of lactate dehydrogenase in cns of CNS cellular damag

The phenotypic and molecular analysis of the production of broad-spectrum beta lactamases (ESBLs) among strains of Escherichia coli isolated from food in Hamedan

Introduction One of the reasons for drug resistance in Escherichiacoli isolates is the production of broad-spectrum beta-lactamases. The widespread use of antibiotics in the agricultural and dairy industry has led to raisingin antibiotic resistance. Therefore, the aim of this study was to investigate the phenotypic and molecular ESBLs production among E. coli isolated from food. Materials and Methods This descriptive cross-sectional study was carried out by 93 E. coli isolated from food and poultry in Hamadan in 2017. Then, the microbial susceptibility of the beta-lactamase producing isolates was determined using the (Combined teat CT) method and according to CLSI (2015) guidelines. Also, molecular identification of genes producing ESBLs (blaSHV, blaCTX-1, and blaTEM-1) was performed by PCR method. Results Evaluation of microbial susceptibility showed that the highest antibiotic resistance was observed for nalidixic acid (%88.4), ampicillin (%76.8), tetracycline (%82.8), and sulfomethoxazole (%67). Resistance to ceftazidime, Cefoxitin, aztreonam, cefotaxime was not observed in this study. The genotypic study by PCR method showed that the frequency of blaSHV, blaCTX-1, and blaTEM-1genes in E. coli isolated from food (%5.37), (%19.35) and (%29.03), respectively. Conclusion E.coli isolated from food showed high resistance to antibiotics such as ampicillin, sulfomethoxazole, and tetracycline. On the other hand, the PCR method is more sensitive than the culture method