Production of multiple xylanolytic and cellulolytic enzymes by thermophilic fungus Myceliophthora sp. IMI 387099

This study reports the production of xylanolytic and cellulolytic enzymes by a thermophilic fungal isolate Myceliophthora sp. using a cheap medium containing rice straw and chemically defined basal medium under solid-state culture. A combination of one factor at a time approach followed by response surface methodology using Box-Behnken design of experiments resulted in 2.5, 1.25, 1.28 and 4.23 fold increase in xylanase, endoglucanase, beta-glucosidase and FPase activity, respectively. The zymograms developed against IEF gels showed that multiple isoforms of xylanase (5), endoglucanase (4) and beta-glucosidase (2) were produced under optimized culture conditions. Moreover, thiol containing serine proteases produced during the growth of the culture had no role in the post-translational modification of these xylanases.     

Purification and characterization of two endoglucanases from Melanocarpus sp. MTCC 3922

This study reports the purification and characterization of endoglucanases (EG I and EG II) from a newly isolated thermophilic fungus, Melanocarpus sp. MTCC 3922. The molecular weight of EG I and EG II as with SDS-PAGE and pI were approximately 40 and 50 kDa, and approximately 4.0 and 3.6, respectively. EG I and EG II were optimally active at 50 and 70 degrees C, and pH 6.0 and 5.0, respectively. EG I was active over a broad range of pH (5.0-7.0), whereas, loss of activity was observed as the temperature was increased from 50 to 80 degrees C. However, EG II was active over pH 4.0-6.0 and temperature 40-80 degrees C. The presence of mercaptoethanol and SDS inhibited the EG I activity but showed no negative effect on EG II. Both the endoglucanases showed higher activity against barley-beta-glucan as compared to CMC. Km values of EG I and EG II for barley-beta-glucan were lower than CMC. Turn over number (K(cat)) and catalytic efficiency (K(cat)/Km) values of both the endoglucanases were higher with barley-beta-glucan as substrate than CMC. EG I showed affinity for Avicel indicating the presence of cellulose binding domains (CBD) whereas, EG II was found to lack CBD.     

Is One Sputum Specimen as Good as Two during Follow-Up Cultures for Monitoring Multi Drug Resistant Tuberculosis Patients in India?

Background

In India, the Revised National Tuberculosis Control Programme (RNTCP) has adopted the strategy of examining two specimens during follow-up culture examinations to monitor the treatment response of multi-drug resistant tuberculosis (MDR-TB) patients.

Objectives

To determine the incremental yield of the second sputum specimen during follow-up culture examinations among patients with MDR-TB and the effect on case management on changing from two to one specimen follow-up strategy.

Methods

A cross sectional record review of MDR-TB patients registered during 2008–09 under RNTCP was undertaken in three MDR-TB treatment sites of India.

Results

Of 1721 pairs of follow-up sputum culture examinations done among 220 MDR-TB patients, 451(26%) were positive with either of the two specimens; 29(1.7%) were culture positive only on the second specimen indicating the incremental yield. To detect one additional culture positive result on the second specimen, 59 specimens needed to be processed. If we had examined only one specimen, we would have missed 29 culture-positive results. By current RNTCP guidelines, however, a single specimen policy would have altered case management in only 3(0.2%) instances, where patients would have missed a one month extension of the intensive phase of MDR-TB treatment. There is no meaningful advantage in using two specimens for the monitoring of MDR-TB patients. A single specimen policy could be safely implemented with negligible clinical effect on MDR-TB patients and favourable resource implications for RNTCP.     

Prevalence of Pulmonary Tuberculosis among Adults in a Rural Sub-District of South India

Background

We conducted a survey to estimate point prevalence of bacteriologically positive pulmonary TB (PTB) in a rural area in South India, implementing TB program DOTS strategy since 2002.

Methods

Survey was conducted among persons ≥15 years of age in fifteen clusters selected by simple random sampling; each consisting of 5–12 villages. Persons having symptoms suggestive of PTB or history of anti-TB treatment (ATT) were eligible for sputum examination by smear microscopy for Acid Fast Bacilli and culture for Mycobacterium tuberculosis; two sputum samples were collected from each eligible person.Persons with one or both sputum specimen positive on microscopy and/or culture were labeled suffering from PTB. Prevalence was estimated after imputing missing values to correct for bias introduced by incompleteness of data.In six clusters, registered persons were also screened by X-ray chest. Persons with any abnormal shadow on X-ray were eligible for sputum examination in addition to those with symptoms and ATT. Multiplication factor calculated as ratio of prevalence while using both screening tools to prevalence using symptoms screening alone was applied to entire study population to estimate prevalence corrected for non-screening by X-ray.

Results

Of 71,874 residents ≥15 years of age, 63,362 (88.2%) were screened for symptoms and ATT. Of them, 5120 (8.1%) - 4681 (7.4%) with symptoms and an additional 439 (0.7%) with ATT were eligible for sputum examination. Spot specimen were collected from 4850 (94.7%) and early morning sputum specimens from 4719 (92.2%). Using symptom screening alone, prevalence of smear, culture and bacteriologically positive PTB in persons ≥15 years of age was 83 (CI: 57–109), 152 (CI: 108–197) and 196 (CI :145–246) per 100,000 population respectively. Prevalence corrected for non-screening by X-ray was 108 (CI: 82–134), 198 (CI: 153–243) and 254 (CI: 204–301) respectively.

Conclusion

Observed prevalence suggests further strengthening of TB control program.     

TRFLP analysis reveals that fungi rather than bacteria are associated with premature yeast flocculation in brewing

Premature yeast flocculation (PYF) is a sporadic fermentation problem in the brewing industry that results in incomplete yeast utilization of fermentable sugars in wort. Culture-independent, PCR-based fingerprinting techniques were applied in this study to identify the associations between the occurrence of the PYF problem during brewery fermentation with barley malt-associated microbial communities (both bacteria and fungi). Striking differences in the microbial DNA fingerprint patterns for fungi between PYF positive (PYF +ve) and negative (PYF ?ve) barley malts were observed using the terminal restriction fragment length polymorphism (TRFLP) technique. The presence of terminal restriction fragments (TRFs) of 360–460 bp size range, for fungal HaeIII restriction enzyme-derived TRFLP profiles appeared to vary substantially between PYF +ve and PYF ?ve samples. The source of the barley malt did not influence the fungal taxa implicated in PYF. TRFLP analysis indicates bacterial taxa are unlikely to be important in causing PYF. Virtual digestion of fungal sequences tentatively linked HaeIII TRFs in the 360–460 bp size range to a diverse range of yeast/yeast-like species. Findings from this study suggest that direct monitoring of barley malt samples using molecular methods could potentially be an efficient and viable alternative for monitoring PYF during brewery fermentations.     

Akt‐ and Erk‐mediated regulation of proliferation and differentiation during PDGFRβ‐induced MSC self‐renewal

Understanding the mechanisms that direct mesenchymal stem cell (MSC) self‐renewal fate decisions is a key to most tissue regenerative approaches. The aim of this study here was to investigate the mechanisms of action of platelet‐derived growth factor receptor β (PDGFRβ) signalling on MSC proliferation and differentiation. MSC were cultured and stimulated with PDGF‐BB together with inhibitors of second messenger pathways. Cell proliferation was assessed using ethynyl‐2′‐deoxyuridine and phosphorylation status of signalling molecules assessed by Western Blots. To assess differentiation potentials, cells were transferred to adipogenic or osteogenic media, and differentiation assessed by expression of differentiation association genes by qRT‐PCR, and by long‐term culture assays. Our results showed that distinct pathways with opposing actions were activated by PDGF. PI3K/Akt signalling was the main contributor to MSC proliferation in response to activation of PDGFRβ. We also demonstrate a negative feedback mechanism between PI3K/Akt and PDGFR‐β expression. In addition, PI3K/Akt downstream signal cascades, mTOR and its associated proteins p70S6K and 4E‐BP1 were involved. These pathways induced the expression of cyclin D1, cyclin D3 and CDK6 to promote cell cycle progression and MSC proliferation. In contrast, activation of Erk by PDGFRβ signalling potently inhibited the adipocytic differentiation of MSCs by blocking PPARγ and CEBPα expression. The data suggest that PDGFRβ‐induced Akt and Erk pathways regulate opposing fate decisions of proliferation and differentiation to promote MSC self‐renewal. Thus, activation of multiple intracellular cascades is required for successful and sustainable MSC self‐renewal strategies.     

Regulation of cellulase production in two thermophilic fungi Melanocarpus sp. MTCC 3922 and Scytalidium thermophilum MTCC 4520

This paper reports regulation of cellulase production in two thermophilic fungi, Melanocarpus sp. MTCC 3922 and Scytalidium thermophilum MTCC 4520. The expression of endoglucanase (EG), avicel adsorbable endoglucanase (AAEG) and β-glucosidase in both fungi was inducible. Of the different carbon sources tested, rice straw induced maximal levels of cellulase in both fungi. While, the addition of fructose (1%, w/v) to the carboxymethylcellulose (CMC) medium resulted in two-fold increase in endoglucanase production in Melanocarpus sp., however, the addition of ethanol (1%, v/v) resulted in eight-fold-increased expression of endoglucanase in S. thermophilum. The expression profiles of different components of cellulase complex were shown to be co-regulated in S. thermophilum but independently regulated in Melanocarpus sp.     

Thiophilic-interaction chromatography of enzymatically active tissue prostate-specific antigen (T-PSA) and its modulation by zinc ions

Prostate-specific antigen (PSA) is a serine protease secreted both by normal prostate glandular epithelial cells and prostate cancer cells. We explored "thiophilic-interaction chromatography" (TIC) to isolate tissue prostate-specific antigen (T-PSA) from fresh human prostate cancer tissue harvested by radical prostatectomy for the purpose to characterize T-PSA for its enzymatic activity and sensitivity to zinc ions. We have shown, for the first time, that T-PSA has strong affinity for the thiophilic gel (T-gel). The average recovery of T-PSA from T-gel is over 87%. The presence of PSA in the column eluate was confirmed by ELISA and SDS/PAGE. Western blot developed with monoclonal antibody to PSA revealed that T-PSA was predominantly in the "free" form having a molecular weight of 33 kDa. Furthermore, T-PSA was found to be enzymatically active. T-PSA was found to be less enzymatically active as compared to seminal plasma PSA. The inhibition of enzymatic activity of both f-PSA and T-PSA over a wide range of concentrations of Zn(2+) ions (10nM to 50 microM) was comparable. In contrast, the enzymatic activity of chymotrypsin, another serine-protease, was affected differently. At higher concentrations of Zn(2+) (10 microM and higher) the enzymatic activity of chymotrypsin was inhibited, whereas, at lower concentrations of Zn(2+) (5 microM and lower), the enzymatic activity was enhanced.     

Callus and cell suspension culture of <Emphasis Type="Italic">Viola odorata</Emphasis> as in vitro production platforms of known and novel cyclotides

Callus from Opuntia streptacantha (cv. Tuna loca), Opuntia megacantha (cv. Rubí reina), and Opuntia ficus-indica (cv. Rojo vigor) were exposed to jasmonic acid (JA) and abiotic stress (drought and UV light) to improve the metabolite production. The callus growth curves, phenolic acids and flavonoids content, antioxidant activity and phenylalanine ammonia lyase (PAL) activity were analyzed under normal and stress conditions. In O. streptacantha callus, the phenolics concentration increased 1.6 to 3 times times in presence of 5% PEG or after irradiation with UV light for 240 min, respectively, while flavonoids triplicate with UV light. A significant increase in antioxidant activity was observed in calli from the three Opuntia species in media with 50 µM JA. The relationships between metabolites/PAL activity, and metabolites/antioxidant activity were analyzed using a surface response methodology. Results showed that PAL activity, induced with PEG and UV, correlated with flavonoids content in O. megacantha and O. ficus-indica calli; PAL activity was related to both flavonoids and phenolics compounds in O. ficus-indica and O. megacantha calli exposed to JA, but only to flavonoids in O. streptacantha callus. In general, the JA stimulated simultaneously the metabolic pathways for phenolics and flavonoids synthesis, while abiotic stress induced mainly flavonoids route. As the stressed Opuntia calli exhibited as high antioxidant activity as cladodes, they are a promising system for research on antioxidant biosynthesis and/or to identify new compounds with antioxidant properties.     

Genetic analysis and mapping of adult plant resistance loci to leaf rust in durum wheat cultivar Bairds

Key message

New leaf rust adult plant resistance (APR) QTL QLr.cim - 6BL was mapped and confirmed the known pleotropic APR gene Lr46 effect on leaf rust in durum wheat line Bairds.

Abstract

CIMMYT-derived durum wheat line Bairds displays an adequate level of adult plant resistance (APR) to leaf rust in Mexican field environments. A recombinant inbred line (RIL) population developed from a cross of Bairds with susceptible parent Atred#1 was phenotyped for leaf rust response at Ciudad Obregon, Mexico, during 2013, 2014, 2015 and 2016 under artificially created epidemics of Puccinia triticina (Pt) race BBG/BP. The RIL population and its parents were genotyped with the 50 K diversity arrays technology (DArT) sequence system and simple sequence repeat (SSR) markers. A genetic map comprising 1150 markers was used to map the resistance loci. Four significant quantitative trait loci (QTLs) were detected on chromosomes 1BL, 2BC (centromere region), 5BL and 6BL. These QTLs, named Lr46, QLr.cim-2BC, QLr.cim-5BL and QLr.cim-6BL, respectively, explained 13.5–60.8%, 9.0–14.3%, 2.8–13.9%, and 11.6–29.4%, respectively, of leaf rust severity variation by the inclusive composite interval mapping method. All of these resistance loci were contributed by the resistant parent Bairds, except for QLr.cim-2BC, which came from susceptible parent Atred#1. Among these, the QTL on chromosome 1BL was the known pleiotropic APR gene Lr46, whereas QLr.cim-6BL, a consistently detected locus, should be a new leaf rust resistance locus in durum wheat. The mean leaf rust severity of RILs carrying all four QTLs ranged from 8.0 to 17.5%, whereas it ranged from 10.9 to 38.5% for three QTLs (Lr46 + 5BL + 6BL) derived from the resistant parent Bairds. Two RILs with four QTLs combinations can be used as sources of complex APR in durum wheat breeding.