Enhancing the sequence coverage of nanodiamond-extracted early secretory proteins from the Mycobacterium tuberculosis complex.

The unambiguous identification of protein species requires high sequence coverage. In this study, we successfully improved the sequence coverage of early secretory 10 kDa cell filtrate protein (CFP-10) and 6 kDa early secretory antigenic target (ESAT-6) proteins from the Mycobacterium tuberculosis complex (MTC) in broth culture media with the use of the 4-chloro-α-cyanocinnamic acid (Cl-CCA) matrix. Conventional matrices, α-cyano-hydroxy-cinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), were also used for comparison. After nanodiamond (ND) extraction, the sequence coverage of the CFP-10 protein was 87% when CHCA and DHB matrices were used, and the ESAT-6 protein was not detected. On the other hand, the sequence coverage for ND-extracted CFP-10 and ESAT-6 could reach 94% and 100%, respectively, when the Cl-CCA matrix was used and with the removal of interference from bovine serum albumin (BSA) protein and α-crystallin (ACR) protein. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was also adopted to analyze the protein mass spectra. A total of 6 prominent ion signals were observed, including ESAT-6 protein peaks at mass-to-charge ratios (m/z) of ∼7931, ∼7974, ∼9768, and ∼9813 and CFP-10 protein peaks at m/z of ∼10 100 and ∼10 660. The ESAT-6 ion signals were always detected concurrently with CFP-10 ion signals, but CFP-10 ion signals could be detected alone without the ESAT-6 ion signals. Furthermore, the newly found ESAT-6 peaks were also confirmed using a Mag-Beads-Protein G kit with an ESAT-6 antibody to capture the ESAT-6 protein, which was also consistent with the sequence coverage analysis.

Development of a linear ion trap mass spectrometer capable of analyzing megadalton MALDI ions.

Detecting megadalton matrix-assisted laser desorption/ionization (MALDI) ions in an ion trap mass spectrometer is a technical challenge. In this study, megadalton protein and polymer ions were successfully measured by MALDI linear ion trap mass spectrometer (LIT-MS) for the first time. The LIT-MS is comprised of a Thermo linear ion trap mass analyzer and a highly sensitive charge-sensing particle detector (CSPD). A newly designed radio frequency (rf) scan mode with dipolar resonance ejection techniques is proposed to extend the mass range of LIT-MS up to one million Thomson (Th). We analyze high mass ions with mass-to charge (m/z) ratios ranging from 100 kTh to 1 MTh, including thyroglobulin, alpha-2-macroglobulin, immunoglobulins (e.g., IgG and IgM), and polymer (∼ 940 kTh) ions. Besides, it is also very challenging for ion trap mass spectrometry to detect megadalton ions at low concentrations. By adopting high affinity carboxylated/oxidized detonation nanodiamonds (oxDNDs) to enrich IgM molecules and form antibody-nanodiamond conjugates, we have successfully reached âˆ¼ 5 nM (5 µg/mL) concentration which is better than that by the other techniques.

Ionization of Submicrometer-Sized Particles by Laser-Induced Radiofrequency Plasma for Mass Spectrometric Analysis.

A laser-induced rf plasma (LIRFP) ion source was developed to ionize submicrometer-sized particles for the first time. The LIRFP ion source can increase the charge of those particles to several thousand charges via charge exchange reactions so that those particles can be trapped and analyzed with a charge detection quadrupole ion trap-mass spectrometer (CD QIT-MS). Different reagent gases for charge exchange reaction were investigated, viz. argon, nitrogen, oxygen, methane, helium, krypton, xenon, argon/methane (with ratios of 10:1 and 2:1), argon/nitrogen (with a ratio of 1:1), nitrogen/oxygen (10:1), krypton/methane (10:1), and air. The average charge of 0.75 µm polystyrene particles could reach 1631 using an argon/methane mixture with a ratio of ∼10:1. The average charges for freeze-dried Escherichia coli EC11303, Escherichia coli strain W, and Staphylococcus aureus were 842, 1112, and 971, respectively, with a mass-to-charge ratio ( m/ z) range from 107 to 108; and the average masses were 3.5 × 1010 Da, 6.0 × 1010 Da, and 5.6 × 1010 Da, respectively. The average mass and charge of the vaccinia virus were ∼9.1 × 109 Da and ∼708 with a m/ z of ∼107. This LIRFP CD QIT-MS method was rapid with only 20 min for each sample measurement.

Forced dried droplet method for MALDI sample preparation.

A forced dried droplet method (FDD) is developed to overcome the drawbacks of the conventional dried-droplet (DD) method for matrix assisted laser desorption/ionization (MALDI) sample preparation. The crystals produced by the DD method are heterogeneous and irregularly distributed, and thus many methods have tried to solve the problems. However, most of them spend more time or need additional instruments to generate homogeneous microcrystals. The FDD sample preparation method can produce uniform microcrystals with homogeneous size distribution in few minutes without additional instruments. Stirring the sample spot solution (an agitation process) with a pipette tip can change the crystal size distribution which is observed by the microscope. Mass spectrometric analysis shows that the smaller the crystal size is, the better the ion signal intensity is. The formation of microcrystals can be explained with the effective rate of secondary nucleation. The relative standard deviation (RSD) of the FDD method is ∼16% which is comparable to the two-layer (TL) method and is better than the DD method.

Bioinformatics characterization of envelope glycoprotein from Kyasanur Forest disease virus.

BACKGROUND & OBJECTIVES: Kyasanur Forest disease (KFD) is a febrile illness characterized by haemorrhages and caused by KFD virus (KFDV), which belongs to the Flaviviridae family. It is reported to be an endemic disease in Shimoga district of Karnataka State, India, especially in forested and adjoining areas. Several outbreaks have been reported in newer areas, which raised queries regarding the changing nature of structural proteins if any. The objective of the study was to investigate amino acid composition and antigenic variability if any, among the envelope glycoprotein (E-proteins) from old and new strains of KFDV. METHODS: Bioinformatic tools and techniques were used to predict B-cell epitopes and three-dimensional structures and to compare envelope glycoprotein (E-proteins) between the old strains of KFDV and those from emerging outbreaks till 2015. RESULTS: The strain from recent outbreak in Thirthahalli, Karnataka State (2014), was similar to the older strain of KFDV (99.2%). Although mutations existed in strains from 2015 in Kerala KFD sequences, these did not alter the epitopes. INTERPRETATION & CONCLUSIONS: The study revealed that though mutations existed, there were no drastic changes in the structure or antigenicity of the E-proteins from recent outbreaks. Hence, no correlation could be established between the mutations and detection in new geographical areas. It seems that KFDV must be present earlier also in many States and due to availability of testing system and alertness coming into notice now.

Differences in Type I interferon response in human lung epithelial cells infected by highly pathogenic H5N1 and low pathogenic H11N1 avian influenza viruses.

Influenza A virus infection induces type I interferons (IFNs α/ß) which activate host antiviral responses through a cascade of IFN signaling events. Herein, we compared highly pathogenic H5N1 and low pathogenic H11N1 avian influenza viruses isolated from India, for their replication kinetics and ability to induce IFN-ß and interferon-stimulating genes (ISGs). The H5N1 virus showed a higher replication rate and induced less IFN-ß and ISGs compared to the H11N1 virus when grown in the human lung epithelial A549 cells, reflecting the generation of differential innate immune responses during infection by these viruses. The non-structural 1 (NS1) protein, a major IFN-antagonist, known to help the virus in evading host innate immune response was compared from both the strains using bioinformatics tools. Analyses revealed differences in the composition of the NS1 proteins from the two strains that may have an impact on the modulation of the innate immune response. Intriguingly, H5N1 virus attenuated IFN-ß response in a non-NS1 manner, suggesting the possible involvement of other viral proteins (PB2, PA, PB1/PB1-F2) of H5N1 in synergy with NS1. Preliminary analyses of the above proteins of the two strains by sequence comparison show differences in charged residues. The insight gained will be useful in designing experimental studies to elucidate a probable role of the polymerase protein(s) in association with NS1 in inhibiting the IFN signaling and understanding the molecular mechanism governing the difference.

High Mass Ion Detection with Charge Detector Coupled to Rectilinear Ion Trap Mass Spectrometer.

Conventional linear ion trap mass analyzers (LIT-MS) provide high ion capacity and show their MS n ability; however, the detection of high mass ions is still challenging because LIT-MS with secondary electron detectors (SED) cannot detect high mass ions. To detect high mass ions, we coupled a charge detector (CD) to a rectilinear ion trap mass spectrometer (RIT-MS). Immunoglobulin G ions (m/z ~150,000) are measured successfully with controlled ion kinetic energy. In addition, when mass-to-charge (m/z) ratios of singly charged ions exceed 10 kTh, the detection efficiency of CD is found to be greater than that of SED. The CD can be coupled to LIT-MS to extend the detection mass range and provide the potential to perform MS n of high mass ions inside the ion trap. Graphical Abstract ᅟ.

Incidence of Postoperative Pain after Single Visit and Two Visit Root Canal Therapy: A Randomized Controlled Trial.

INTRODUCTION: Root Canal Treatment (RCT) has become a mainstream procedure in dentistry. A successful RCT is presented by absence of clinical signs and symptoms in teeth without any radiographic evidence of periodontal involvement. Completing this procedure in one visit or multiple visits has long been a topic of discussion. AIM: To evaluate the incidence of postoperative pain after root canal therapy performed in single visit and two visits. MATERIAL AND METHODS: An unblinded/ open label randomized controlled trial was carried out in the endodontic department of the Dental Institute, where 78 patients were recruited from the regular pool of patients. A total of 66 maxillary central incisors requiring root canal therapy fulfilled the inclusion and exclusion criteria. Using simple randomization by biased coin randomization method, the selected patients were assigned into two groups: group A (n=33) and group B (n=33). Single visit root canal treatment was performed for group A and two visit root canal treatment for group B. Independent sample t-test was used for statistical analysis. RESULTS: Thirty three patients were allotted to group A where endodontic treatment was completed in single visit while 33 patients were allotted to group B where endodontic treatment was completed in two visits. One patient dropped-out from Group A. Hence in Group A, 32 patients were analysed while in Group B, 33 patients were analysed. After 6 hours, 12 hours and 24 hours of obturation, pain was significantly higher in Group B as compared to Group A. However, there was no significant difference in the pain experienced by the patients 48 hours after treatment in both the groups. CONCLUSION: Incidence of pain after endodontic treatment being performed in one-visit or two-visits is not significantly different.

Measuring masses of large biomolecules and bioparticles using mass spectrometric techniques.

Large biomolecules and bioparticles play a vital role in biology, chemistry, biomedical science and physics. Mass is a critical parameter for the characterization of large biomolecules and bioparticles. To achieve mass analysis, choosing a suitable ion source is the first step and the instruments for detecting ions, mass analyzers and detectors should also be considered. Abundant mass spectrometric techniques have been proposed to determine the masses of large biomolecules and bioparticles and these techniques can be divided into two categories. The first category measures the mass (or size) of intact particles, including single particle quadrupole ion trap mass spectrometry, cell mass spectrometry, charge detection mass spectrometry and differential mobility mass analysis; the second category aims to measure the mass and tandem mass of biomolecular ions, including quadrupole ion trap mass spectrometry, time-of-flight mass spectrometry, quadrupole orthogonal time-of-flight mass spectrometry and orbitrap mass spectrometry. Moreover, algorithms for the mass and stoichiometry assignment of electrospray mass spectra are developed to obtain accurate structure information and subunit combinations.

An in vitro comparison of bond strengths of Gutta-percha/AH Plus, Resilon/Epiphany self-etch and EndoREZ obturation system to intraradicular dentin using a push-out test design.

AIM: To evaluate and compare the push-out bond strengths of three obturation materials; Gutta-percha/AH Plus, Resilon/Epiphany self-etch (SE) and EndoREZ obturation system to intraradicular dentin. MATERIALS AND METHODS: Sixty single-canal anterior teeth were prepared and assigned to experimental groups (n = 20), designated as Group I: Gutta-percha/AH Plus, Group II: Resilon/Epiphany SE and Group III: EndoREZ sealer/EndoREZ points. After obturation, each tooth was prepared for push-out assessment with root slices of 2 mm thickness using universal testing machine. STATISTICAL ANALYSIS: Two way analysis of variance and Scheffe's test. RESULTS: Gutta-percha/AH Plus root fillings showed significantly highest bond strength. Also, root segment location did not have a significant influence on bond strength. CONCLUSION: The adhesiveness quality to root dentin promoted by newer methacrylate resin-based obturation systems like Resilon/Epihany SE and EndoREZ is compromised even when teeth with simple anatomic features were obturated under well-monitored laboratory conditions.

Morphometric and allozyme variation in Culex tritaeniorhynchus mosquito populations from India.

Four populations of Culex tritaeniorhynchus (Giles) (Diptera: Culicidae), collected from Bellary, Cuddalore, Pune, and the Microbial Containment Complex laboratory culture in India were analyzed for morphological and allozyme variation. Multivariate analysis based on eight morphological characteristics and three morphometric indices was used to investigate the morphological variations among the four populations. Principal component analysis of the data suggested that siphon, saddle, and anal gills related variables were most important. Discriminant factor analysis of morphological data revealed that the four populations form significantly different clusters which can be differentiated from each other based on siphon, saddle, and pectin teeth related variables. Allozyme electrophoresis of the four populations revealed that the mean heterozygosity per locus value had high variation, ranging from 0.0879 to 1.794. Fst values between 0 and 0.519 suggested genetic differentiation within these populations. Fis values ranged from 0 to 1 with most of the values closer to 1. The allelic frequencies and Nei's genetic identity values showed that genetic differences between populations were small, but significant. Some of the morphological and allozyme variations in the Cx. tritaeniorhynchus populations could be partly attributed to the environmental conditions. The findings suggested that transition of morphological characters and allozyme variations in Cx. tritaeniorhynchus populations seem to be consequences of influence and selection by the environmental conditions. These results indicated that populations of Cx. tritaeniorhynchus in non-endemic areas of Japanese encephalitis (JE) virus infection have higher adaptability as compared to endemic areas of JE infection.