Characterization of Bacteria by Particle Beam Mass Spectrometry

A technique is described for detecting and characterizing bacteria on a single-particle basis by mass spectrometry. The method involves generation of a particle beam of single whole cells which are rapidly volatilized and ionized in vacuum in the ion source of a quadrupole mass spectrometer. The particle beam can be generated, with minimal sample handling, from a naturally occurring aerosol or from a solution of bacteria that can be dispersed as an aerosol. The mass spectrum is generated by successively measuring the average intensities of different mass peaks. The average intensity is obtained by measuring the ion intensity distribution at the particular mass (m/e) for ion pulses from more than 1,000 bacteria particles. Bacillus cereus, Bacillus subtilis, and Pseudomonas putida samples were analyzed to test the capability of the instrument for differentiating among species of bacteria. Significant ion-intensity information was produced over the m/e range of 50 to 300, an improvement over previous pyrolysis-mass spectrometry results. The complex mass spectra contained a few unique peaks which could be used for the differentiation of the bacteria. A statistical analysis of the variations in peak intensities among the three bacteria provided a quantitative measure of the reproducibility of the instrument and its ability to differentiate among bacteria. The technique could lead to a new rapid method for the analysis of microorganisms and could be used for the detection of airborne pathogens on a continuous, real-time basis.     

Morphological and molecular screening of rice germplasm lines for low soil P tolerance

Phosphorus (P) is an essential macronutrient to all crops including rice and it plays a key role in various plant activities and development. Low availability of P in the soils negatively, influences rice crop growth and causes significant yield loss. In the present study, we characterized a set of 56 germplasm lines for their tolerance to low soil P by screening them at low soil P and optimum soil P levels along with low soil P tolerant and sensitive check varieties. These lines were genotyped for the presence/absence of tolerant allele with respect to the major low soil P tolerance QTL, Pup1, using a set of locus specific PCR-based markers, viz., K46-1, K46-2, K52 and K46CG-1. High genetic variability was observed for various traits associated with low soil P tolerance. The yield parameters from normal and low soil P conditions were used to calculate stress tolerance indices and classify the genotypes according to their tolerance level. Out of the total germplasm lines screened, 15 lines were found to be tolerant to low soil P condition based on the yield reduction in comparison to the tolerant check, but most of them harbored the complete or partial Pup1 locus. Interestingly, two tolerant germplasm lines, IC216831 and IC216903 were observed to be completely devoid of Pup1 and hence they can be explored for new loci underlying low soil P tolerance.

     

Augmentation of Autoantibodies by Helicobacter pylori in Parkinson’s Disease Patients May Be Linked to Greater Severity

Parkinson''s disease (PD) is the second most common chronic and progressive neurodegenerative disorder. Its etiology remains elusive and at present only symptomatic treatments exists. Helicobacter pylori chronically colonizes the gastric mucosa of more than half of the global human population. Interestingly, H. pylori positivity has been found to be associated with greater of PD motor severity. In order to investigate the underlying cause of this association, the Sengenics Immunome protein array, which enables simultaneous screening for autoantibodies against 1636 human proteins, was used to screen the serum of 30 H. pylori-seropositive PD patients (case) and 30 age- and gender-matched H. pylori-seronegative PD patients (control) in this study. In total, 13 significant autoantibodies were identified and ranked, with 8 up-regulated and 5 down-regulated in the case group. Among autoantibodies found to be elevated in H. pylori-seropositive PD were included antibodies that recognize Nuclear factor I subtype A (NFIA), Platelet-derived growth factor B (PDGFB) and Eukaryotic translation initiation factor 4A3 (eIFA3). The presence of elevated autoantibodies against proteins essential for normal neurological functions suggest that immunomodulatory properties of H. pylori may explain the association between H. pylori positivity and greater PD motor severity.     

Alpha(1)-antitrypsin deficiency,liver disease and emphysema

alpha(1)-Antitrypsin is a member of the serine proteinase inhibitor (serpin) superfamily and a potent inhibitor of neutrophil elastase. The most important deficiency variant of alpha(1)-antitrypsin arises from the Z mutation (Glu342Lys). This mutation perturbs the protein's tertiary structure to promote a precise, sequential intermolecular linkage that results in polymer formation. These polymers accumulate within the endoplasmic reticulum of the hepatocyte forming inclusion bodies that are associated with neonatal hepatitis, juvenile cirrhosis and adult hepatocellular carcinoma. The resultant secretory defect leads to plasma deficiency of alpha(1)-antitrypsin. This exposes lung tissue to uncontrolled proteolytic attack from neutrophil elastase, culminating in alveolar destruction. Thus, the Z alpha(1)-antitrypsin homozygote is predisposed to developing early onset basal, panacinar emphysema. In this review, we summarise the current understanding of the pathobiology of alpha(1)-antitrypsin deficiency and the associated liver cirrhosis and emphysema. We show how this knowledge has led to the development of novel therapeutic approaches to treat this condition.     

Blastocystis sp. in Irritable Bowel Syndrome (IBS) - Detection in Stool Aspirates during Colonoscopy

Blastocystis is one of the most common gut parasites found in the intestinal tract of humans and animals. Its'' association with IBS is controversial, possibly as a result of irregular shedding of parasites in stool and variation in stool detection. We aimed to screen for Blastocystis in colonic stool aspirate samples in adult patients with and without IBS undergoing colonoscopy for various indications and measure the interleukin levels (IL-8, IL-3 and IL-5). In addition to standard stool culture techniques, polymerase chain reaction (PCR) techniques were employed to detect and subtype Blastocystis. All the serum samples collected were subjected for ELISA studies to measure the interleukin levels (IL-8, IL-3 and IL-5). Among 109 (IBS n = 35 and non-IBS n = 74) adults, direct stool examination and culture of colonic aspirates were initially negative for Blastocystis. However, PCR analysis detected Blastocystis in 6 (17%) IBS and 4 (5.5%) non-IBS patients. In the six positive IBS patients by PCR method, subtype 3 was shown to be the most predominant (3/6: 50%) followed by subtype 4 (2/6; 33.3%) and subtype 5 (1/6; 16.6%). IL-8 levels were significantly elevated in the IBS Blasto group and IBS group (p<0.05) compared to non-IBS and non-IBS Blasto group. The level of IL-3 in were seen to be significantly higher in than IBS Blasto group and IBS group (p<0.05) compared to non-IBS. Meanwhile, the IL-5 levels were significantly higher in IBS Blasto group (p<0.05) compared to non-IBS and non-IBS Blasto group. This study implicates that detecting Blastosystis by PCR method using colonic aspirate samples during colonoscopy, suggests that this may be a better method for sample collection due to the parasite’s irregular shedding in Blastocystis-infected stools. Patients with IBS infected with parasite showed an increase in the interleukin levels demonstrate that Blastocystis does have an effect in the immune system.     

6-mer peptide selectively anneals to a pathogenic serpin conformation and blocks polymerization. Implications for the prevention of Z alpha(1)-antitrypsin-related cirrhosis.

Conformational diseases such as amyloidosis, Alzheimer's disease, prion diseases, and the serpinopathies are all caused by structural rearrangements within a protein that transform it into a pathological species. These diseases are typified by the Z variant of alpha(1)-antitrypsin (E342K), which causes the retention of protein within hepatocytes as inclusion bodies that are associated with neonatal hepatitis and cirrhosis. The inclusion bodies result from the Z mutation perturbing the conformation of the protein, which facilitates a sequential interaction between the reactive center loop of one molecule and beta-sheet A of a second. Therapies to prevent liver disease must block this reactive loop-beta-sheet polymerization without interfering with other proteins of similar tertiary structure. We have used reactive loop peptides to explore the differences between the pathogenic Z and normal M alpha(1)-antitrypsin. The results show that the reactive loop is likely to be partially inserted into beta-sheet A in Z alpha(1)-antitrypsin. This conformational difference from M alpha(1)-antitrypsin was exploited with a 6-mer reactive loop peptide (FLEAIG) that selectively and stably bound Z alpha(1)-antitrypsin. The importance of this finding is that the peptide prevented the polymerization of Z alpha(1)-antitrypsin and did not significantly anneal to other proteins (such as antithrombin, alpha(1)-antichymotrypsin, and plasminogen activator inhibitor-1) with a similar tertiary structure. These findings provide a lead compound for the development of small molecule inhibitors that can be used to treat patients with Z alpha(1)-antitrypsin deficiency. Furthermore they demonstrate how a conformational disease process can be selectively inhibited with a small peptide.     

Insilico Structural 3D Modelling of Novel Cry1Ib9 and Cry3A Toxins from Local Isolates of Bacillus thuringiensis

Three-dimensional (3D) models for the 79.2 kDa activated Cry1Ib9 and 77.4 kDa activated Cry3A δ-endotoxins from Bacillus thuringiensis (Bt) native isolates that are specifically toxic to Coleopteran insect pests were constructed by utilizing homology modeling online tool. Evidences presented here, based on the identification of structural equivalent residues of Cry1Ib9 and Cry3A toxin through homology modelling indicate that, they share a common Bt toxin tridimensional structure. The main differences observed in Cry1I9 domain I at positions α2b (S56-I60), α4 (F78-l93) and additionally β0 (Q10-L12), α8a (T280-V282) were observed, in domain II at positions α9b (P333-L339), β6(T390-Q393), β7(V398-W404), β8 (V418-W425), β9 (E453-N454), β10 (S470-I479) where as in domain III the changes were observed at positions β19 (R601-F607), β20 (609-L613), β21 (S618-F627) and α11a (K655-F664), α13, α14 components present at downstream sites, where as in Cry3A main differences observed in domain I is at the position of α4 (P105-I152), α5 (Q163-A185), β1A(E190-L192), α6 (F193-Y217), Domain II is not consevered and main variations were observed at β2 (E292-L295), β3(V299-L308), β4(I340-F347), β5(D356-P368), β6(I375-T377), β7(V389-F394), β8(K398-N405), β9(Y416-Y427), β10 (T436-Y439), β12(G476-H495), β12A (M503-I504) where as in domain III main variations observed at positions of β18 (P583-I593), β19(F604-S610), β20(P611-L615), β21(N619-G626). Cry1Ib9 and Cry3A contain the most variable regions in the loops of domain II, which determine the specificity of these toxins. These are the first models of Coleopteran-active protein from native isolates of Bt and its importance can be perceived since members of this group of toxins are potentially important candidates for coleoptera insect pest control programs.