PCR amplification of shorter fragments from the devR (Rv3133c) gene significantly increases the sensitivity of tuberculosis diagnosis

This study was designed to assess the vital issue of gene target length and PCR assay performance in relation to the detection of Mycobacterium tuberculosis in clinical specimens. Two PCR assays that amplify fragments of varying lengths from the devR gene of M. tuberculosis were evaluated. Using M. tuberculosis DNA the 'short-length' PCR assay detected 250-500 genome equivalents vs. 500-1,000 genome equivalents by the 'long-length' assay. In comparison to a highly sensitive smear microscopy test (universal sample processing smear), the sensitivity of the 'short-length' assay was 97.8% vs. 69.9% of the 'long-length' assay in sputum specimens (n=506) from patients being evaluated for a possible diagnosis of tuberculosis. The 27.9% absolute increase in sensitivity was statistically significant (P<0.001). Our results indicate that in a clinical setting when all other conditions are equal, the amplification of a shorter gene fragment of devR increases the sensitivity and efficiency of the PCR assay in spite of using a single copy gene as target.     

Structure-activity relationship study between Ornithyl-Proline and Lysyl-Proline based tripeptidomimics as angiotensin-converting enzyme inhibitors

A designed library of tripeptidomimics of Ornithyl-Proline (Orn-Pro) and Lysyl-Proline (Lys-Pro) conjugated with various unnatural amino acids and carboxylic acid derived heterocyclics was synthesized and screened for possible inhibitors of angiotensin-converting enzyme (ACE). Among the tripeptidomimics 10[MTP-Orn-Pro], 11[HTP-Orn-Pro], 14[TA-Orn-Pro] and 20[BPA-Orn-Pro] showed prominent inhibition with IC50 values in micromolar concentrations. Structure-activity relationship study indicated that C3 side chain of Orn as compared to C4 side chain of Lys at P1' position was better suited to inhibit ACE, with propionic acid (C3) derived heterocyclics and unnatural amino acids.     

Production and characterization of a highly active cellobiase from Aspergillus niger grown in solid state fermentation

Summary Aspergillus niger produced extracellular cellobiase when grown on different lignocellulosic substrates in solid state fermentation. The enzyme activity and yield were variable according to the carbon source. In Vogel’s medium, the cellobiase productivity was significantly higher on wheat bran, followed by Leptochloa fusca (kallar grass) straw augmented with corn steep liquor. Maximum yield of cellobiase/g wheat bran was significantly higher than the values reported on other potent fungi, bacteria and recombinants, harboring heterologous gene for cellobiase. This enzyme in the presence and absence of Trichoderma reesei and celluloclast, saccharified the biomass and the percentage saccharification as well as glucose yield from lignocellulosic biomass was doubled in its presence. The partially purified enzyme was thermotolerant as evidenced by melting temperature, activation energy demand for active catalysis, enthalpy and entropy of activation for reversible or irreversible thermal inactivation.     

A New Antimicrobial Fatty Acid from the Calcareous Sponge Paragrantia cf. waguensis

A new acetylenic fatty acid, 1 , has been isolated from the title sponge. The structure of the molecule was elucidated to contain an enyne and a thiophene by spectroscopic methods. Compound 1 showed a weak cytotoxic effect against NBT‐T2 rat bladder epithelial cells (IC₅₀>20 μg/ml), and antimicrobial activity with minimal‐inhibitory concentrations (MIC) of 64 and 128 μg/ml against Staphylococcus aureus and Escherichia coli, respectively.     

Collection of antimicrobial peptides database and its derivatives: Applications and beyond

Collection of antimicrobial peptides (CAMP), CAMPSign, and ClassAMP are open‐access resources that have been developed to enhance research on antimicrobial peptides (AMPs). Comprehensive information on AMPs and machine learning‐based predictive models are made available for users through these resources. As of date, CAMP_R3 has 10,247 sequences, 757 structures, and 114 family‐specific signatures of AMPs along with associated tools for AMP sequence and structure analysis. CAMPSign uses family‐specific sequence conservation, in the form of patterns and hidden Markov models for identification of AMPs. ClassAMP can be used to classify AMPs as antibacterial, antifungal, or antiviral based on sequence information. Here we describe CAMP and its derivatives and illustrate, with a few examples, the contribution of these online resources to the advancement of our current understanding of AMPs.     

A Pilot Study Combining a GC-Sensor Device with a Statistical Model for the Identification of Bladder Cancer from Urine Headspace

There is a need to reduce the number of cystoscopies on patients with haematuria. Presently there are no reliable biomarkers to screen for bladder cancer. In this paper, we evaluate a new simple in–house fabricated, GC-sensor device in the diagnosis of bladder cancer based on volatiles. Sensor outputs from 98 urine samples were used to build and test diagnostic models. Samples were taken from 24 patients with transitional (urothelial) cell carcinoma (age 27-91 years, median 71 years) and 74 controls presenting with urological symptoms, but without a urological malignancy (age 29-86 years, median 64 years); results were analysed using two statistical approaches to assess the robustness of the methodology. A two-group linear discriminant analysis method using a total of 9 time points (which equates to 9 biomarkers) correctly assigned 24/24 (100%) of cancer cases and 70/74 (94.6%) controls. Under leave-one-out cross-validation 23/24 (95.8%) of cancer cases were correctly predicted with 69/74 (93.2%) of controls. For partial least squares discriminant analysis, the correct leave-one-out cross-validation prediction values were 95.8% (cancer cases) and 94.6% (controls). These data are an improvement on those reported by other groups studying headspace gases and also superior to current clinical techniques. This new device shows potential for the diagnosis of bladder cancer, but the data must be reproduced in a larger study.     

Salt-induced modulation in growth,photosynthetic capacity,proline content and ion accumulation in sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.)

Salt-induced changes in growth, photosynthetic pigments, various gas exchange characteristics, relative membrane permeability (RMP), relative water content (RWC) and ion accumulation were examined in a greenhouse experiment on eight sunflower (Helianthus annuus L.) cultivars. Sunflower cultivars, namely Hysun-33, Hysun-38, M-3260, S-278, Alstar-Rm, Nstt-160, Mehran-II and Brocar were subjected to non-stress (0 mM NaCl) or salt stress (150 mM NaCl) in sand culture. On the basis of percent reduction in shoot biomass, cvs. Hysun-38 and Nstt-160 were found to be salt tolerant, cvs. Hysun-33, M-3260, S-278 and Mehran-II moderately tolerant and Alstar-Rm and Brocar salt sensitive. Salt stress markedly reduced growth, different gas exchange characteristics such as photosynthetic rate (A), water-use efficiency (WUE) calculated as A/E, transpiration rate (E), internal CO₂ concentration (C _i) and stomatal conductance (g _s) in all cultivars. The effect of 150 mM NaCl stress was non-significant on chlorophyll a and b contents, chlorophyll a/b ratio, RWC, RMP and leaf and root Cl⁻, K⁺ and P contents; however, salt stress markedly enhanced C _i /C _a ratio, free proline content and leaf and root Na⁺ concentrations in all sunflower cultivars. Of all cultivars, cv. Hysun-38 was higher in gas exchange characteristics, RWC and proline contents as compared with the other cultivars. Overall, none of the earlier-mentioned physiological attributes except leaf K⁺/Na⁺ ratio was found to be effective in discriminating the eight sunflower cultivars as the response of each cultivar to salt stress appraised using various physiological attributes was cultivar-specific.     

Involvement of acid ceramidase in the degradation of bioactive N-acylethanolamines

Bioactive N-acylethanolamines (NAEs) include palmitoylethanolamide, oleoylethanolamide, and anandamide, which exert anti-inflammatory, anorexic, and cannabimimetic actions, respectively. The degradation of NAEs has been attributed to two hydrolases, fatty acid amide hydrolase and NAE acid amidase (NAAA). Acid ceramidase (AC) is a lysosomal enzyme that hydrolyzes ceramide (N-acylsphingosine), which resembles NAAA in structure and function. In the present study, we examined the role of AC in the degradation of NAEs. First, we demonstrated that purified recombinant human AC can hydrolyze various NAEs with lauroylethanolamide (C12:0-NAE) as the most reactive NAE substrate. We then used HEK293 cells metabolically labeled with [¹⁴C]ethanolamine, and revealed that overexpressed AC lowered the levels of ¹⁴C-labeled NAE. As analyzed with liquid chromatography-tandem mass spectrometry, AC overexpression decreased the amounts of different NAE species. Furthermore, suppression of endogenous AC in LNCaP prostate cells by siRNA increased the levels of various NAEs. Lastly, tissue homogenates from mice genetically lacking saposin D, a presumable activator protein of AC, showed much lower hydrolyzing activity for NAE as well as ceramide than the homogenates from wild-type mice. These results demonstrate the ability of AC to hydrolyze NAEs and suggest its physiological role as a third NAE hydrolase.     

A whole genome analyses of genetic variants in two Kelantan Malay individuals

BackgroundThe sequencing of two members of the Royal Kelantan Malay family genomes will provide insights on the Kelantan Malay whole genome sequences. The two Kelantan Malay genomes were analyzed for the SNP markers associated with thalassemia and Helicobacter pylori infection. Helicobacter pylori infection was reported to be low prevalence in the north-east as compared to the west coast of the Peninsular Malaysia and beta-thalassemia was known to be one of the most common inherited and genetic disorder in Malaysia.ResultBy combining SNP information from literatures, GWAS study and NCBI ClinVar, 18 unique SNPs were selected for further analysis. From these 18 SNPs, 10 SNPs came from previous study of Helicobacter pylori infection among Malay patients, 6 SNPs were from NCBI ClinVar and 2 SNPs from GWAS studies. The analysis reveals that both Royal Kelantan Malay genomes shared all the 10 SNPs identified by Maran (Single Nucleotide Polymorphims (SNPs) genotypic profiling of Malay patients with and without Helicobacter pylori infection in Kelantan, 2011) and one SNP from GWAS study. In addition, the analysis also reveals that both Royal Kelantan Malay genomes shared 3 SNP markers; HBG1 (rs1061234), HBB (rs1609812) and BCL11A (rs766432) where all three markers were associated with beta-thalassemia.ConclusionsOur findings suggest that the Royal Kelantan Malays carry the SNPs which are associated with protection to Helicobacter pylori infection. In addition they also carry SNPs which are associated with beta-thalassemia. These findings are in line with the findings by other researchers who conducted studies on thalassemia and Helicobacter pylori infection in the non-royal Malay population.     

Introgression of cotton leaf curl virus-resistant genes from Asiatic cotton (Gossypium arboreum) into upland cotton (G. hirsutum)

Cotton is under the constant threat of leaf curl virus, which is a major constraint for successful production of cotton in the Pakistan. A total of 3338 cotton genotypes belonging to different research stations were screened, but none were found to be resistant against the Burewala strain of cotton leaf curl virus (CLCuV). We explored the possibility of transferring virus-resistant genes from Gossypium arboreum (2n = 26) into G. hirsutum (2n = 52) through conventional breeding techniques. Hybridization was done manually between an artificial autotetraploid of G. arboreum and an allotetraploid G. hirsutum, under field conditions. Boll shedding was controlled by application of exogenous hormones, 50 mg/L gibberellic acid and 100 mg/L naphthalene acetic acid. Percentage pollen viability in F(1) hybrids was 1.90% in 2(G. arboreum) x G. hirsutum and 2.38% in G. hirsutum x G. arboreum. Cytological studies of young buds taken from the F(1) hybrids confirmed that they all were sterile. Resistance against CLCuV in the F(1) hybrids was assessed through grafting, using the hybrid plant as the scion; the stock was a virus susceptible cotton plant, tested under field and greenhouse conditions. All F(1) cotton hybrids showed resistance against CLCuV, indicating that it is possible to transfer resistant genes from the autotetraploid of the diploid donor specie G. arboreum into allotetraploid G. hirsutum through conventional breeding, and durable resistance against CLCuV can then be deployed in the field.