Development of a novel Pichia pastoris expression platform via genomic integration of lipase gene for sustained release of methanol from methyloleate.

A novel Lip+ Pichia pastoris expression platform was developed by integrating lipase Lip2 from Yarrowia lipolytica under constitutive Glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Effective expression of reporter protein amylase from Bacillus licheniformis was achieved utilizing methyloleate in Lip+Amy+host. Lipase hydrolyzed methyloleate into methanol that sustained PAOX1 induction, and oleic acid, which was readily utilized as a carbon source. The protein expression achieved in presence of methyloleate was comparable to methanol-induced cells, along with an increase in productive biomass. In Lip+Amy+ host, total amylase production of 220.9 ± 13 U/mg biomass was achieved at 96 h using methyloleate supplemented every 24 h. While 206.0 ± 17 U/mg biomass was obtained at 108 h in an Amy+ host induced with methanol every 12 h. Further, lipase expression neither affected growth nor added additional burden on the cellular machinery and no oleic acid accumulation was observed at any time point due to its emulsification and efficient utilization by lipase positive host. Similar results obtained with the second reporter protein γ-cyclodextrin glycosyltransferase (CGTase) from Evansella caseinilytica validated the platform. An alternate lipase Lip11 from Y. lipolytica was also employed in developing a Lip+ host to validate disparity between lipase background and PAOX1 induction in presence of methyloleate.

Pulmonary Manifestations in Patients of Rheumatoid Arthritis and its Correlation with Severity of Disease.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic inflammatory disease of unknown etiology marked by symmetric, peripheral polyarthritis. RA has a prevalence of 1-2% in the general adult population. The mortality rate in patients with RA increases during the course of the disease, with a tendency to accelerate after 15 yearsAim: To study the pulmonary manifestations and their severity using [Disease Activity Score (DAS)-28 score] in patients of RAMaterials and methods: Present study was conducted in the Department of Medicine, Sardar Patel Medical College and Associated Group of Hospitals Bikaner, Bikaner, Rajasthan, India, on 100 patients. This study was a cross-sectional, observational study conducted over 1 year. Consecutive cases of RA patients attending the outpatient department or admitted to the medicine wards were selected according to the inclusion and exclusion criteriaResults: Pulmonary manifestation was present in a total of 38% of cases, while the remaining 62% of cases had no pulmonary manifestation. The presence of comorbidity and C-reactive protein (CRP) was significantly associated with pulmonary manifestation in RA patients. On high-resolution computed tomography (HRCT), the most common finding was interstitial lung disease (ILD) (60.5%), with usual interstitial pneumonia (UIP) as the most common pattern. On performing a pulmonary function test (PFT), 33 patients (86.84%) had an abnormal result, with restrictive as the most common patternConclusion: The patients of RA, especially those with advanced age, long duration of disease, male sex, and associated comorbidity, should be screened for pulmonary complications of RA using X-ray chest and PFT, supplemented by HRCT chest wherever required.

Acaricide resistance in Rhipicephalus microplus collected from selected districts of Madhya Pradesh, Uttar Pradesh and Punjab states of India.

The isolates of Rhipicephalus microplus collected from Madhya Pradesh (MP), Punjab (PJB) and Uttar Pradesh (UP) states of India were characterized using laboratory standardized adult immersion test (AIT) against macrocyclic lactone (ivermectin), synthetic pyrethroids (cypermethrin and deltamethrin), organophosphates (coumaphos and diazinon) and phenylpyrazole compounds (fipronil). Out of the six isolates tested, five isolates except MTH were resistant to deltamethrin and cypermethrin at level II with RF ranging from 16.4 to 24.02 and 7.05 to 13.2, respectively. In case of organophosphates, coumaphos was less effective showing resistance level II (RF 8.52-11.2) in all the six populations compared with diazinon to which three isolates (MHW, RWA and AGS) were resistant at level II. Except MTH, other five isolates were categorized at level I with RF ranging from 1.53 to 3.02 against ivermectin. The phenylpyrazole compound however was found effective, and none of the isolates could survive at a discriminating concentration. The possible strategy for the management of multi-acaricide-resistant ticks in the surveyed districts was discussed in the present study.

Cell surface expression of γ-CGTase from Evansella caseinilytica on E. coli: Application in the enzymatic conversion of starch to γ-cyclodextrin.

The γ-cyclodextrin glycosyltransferase (γ-CGTase) from Evansella caseinilytica was expressed on the cell surface of E. coli using pAIDA-I autotransporter and was further utilized in the conversion of starch to γ-cyclodextrins (CDs). Maximum cyclization activity of 2.28 ± 0.46 U/g biomass was achieved after 3 h of induction using 0.1 mM IPTG at 37 ºC. Surface expression of γ-CGTase was confirmed using flow cytometry employing a FITC-conjugated anti-HIS antibody. Biochemical characterization of surface-displayed γ-CGTase revealed optima at pH 10.0 and 40 ºC along with a t1/2 of 24.75 min at 50ºC. The Km and Vmax values on soluble potato starch were 10.94 mg/ml and 4.33 µmoles min-1 g-1 DCW respectively, and the activation energy was calculated to be 89.8 kJ/mol. The surface displayed γ-CGTase was further utilized for CD production and specifically, γ-CD conversion was obtained. The maximum conversion was achieved at 50 ºC, pH 9.0 using soluble potato starch (2.5%; w/v) taking a final enzyme concentration of 0.6 U/g starch. The surface-displayed γ-CGTase was able to convert soluble potato starch (2.5%) into γ-CDs with a 72.7% specific yield and no other peaks corresponding to α- and ß-CDs were observed on HPLC. The enzyme was found to be ~100% operationally stable for up to 2 consecutive cycles of 24 h, with > 75% storage stability at - 20 ºC even after 7 days.

Gamma cyclodextrin glycosyltransferase from evansella caseinilytica: production, characterization and product specificity.

Alkalohalophilic Evansella caseinilytica produced an extracellular cyclodextrin glycosyltransferase (CGTase) with cyclization activity of 43.5 ± 4.4 U/L in M1 medium containing 1% starch and 6% NaCl in nutrient broth at 37 ºC, pH 9.0, after 48 h. This is the first report of CGTase from this bacterium. 0.1% starch was found to induce CGTase, and further optimization using one variable at a time approach followed by statistical optimization led to 5.5-fold enhancement resulting in 240.5 ± 5.46 U/L. Six parameters were identified as positive signals using Plackett-Burman (PB). Of these, yeast extract, MgSO4 and tryptone were taken further for Response Surface Methodology (RSM) by disposing beef extract and fixing starch and soya peptone. The optimized M4 medium consisted of tryptone (0.1%, w/v), yeast extract (0.25%, w/v), MgSO4 (8 mM, w/v), potato starch (0.1%, w/v) and soya peptone (0.2%, w/v). CGTase was further purified with 6.44-fold purification and 19.32% yield employing starch affinity. It was found to be monomeric, corresponding to a size of 68 kDa as estimated by SDS-PAGE and was further confirmed to be 65 kDa by size exclusion chromatography. γ-Cyclodextrins were produced as the major product with a conversion of 5% soluble starch into 20.38% γ-cyclodextrins after 24 h reaction, as determined by HPLC. Peptide fingerprint after LC-MS analysis matched with IPT/TIG domain-containing protein within the genome of E. caseinilytica. Further blastp analysis revealed the closest homology with γ-CGTase from an alkalophilic E. clarkii, thereby confirming CGTase from E. caseinilytica as γ-CGTase.

Bacterial Gamma-Glutamyl Transpeptidase, an Emerging Biocatalyst: Insights Into Structure-Function Relationship and Its Biotechnological Applications.

Gamma-glutamyl transpeptidase (GGT) enzyme is ubiquitously present in all life forms and plays a variety of roles in diverse organisms. Higher eukaryotes mainly utilize GGT for glutathione degradation, and mammalian GGTs have implications in many physiological disorders also. GGTs from unicellular prokaryotes serve different physiological functions in Gram-positive and Gram-negative bacteria. In the present review, the physiological significance of bacterial GGTs has been discussed categorizing GGTs from Gram-negative bacteria like Escherichia coli as glutathione degraders and from pathogenic species like Helicobacter pylori as virulence factors. Gram-positive bacilli, however, are considered separately as poly-γ-glutamic acid (PGA) degraders. The structure-function relationship of the GGT is also discussed mainly focusing on the crystallization of bacterial GGTs along with functional characterization of conserved regions by site-directed mutagenesis that unravels molecular aspects of autoprocessing and catalysis. Only a few crystal structures have been deciphered so far. Further, different reports on heterologous expression of bacterial GGTs in E. coli and Bacillus subtilis as hosts have been presented in a table pointing toward the lack of fermentation studies for large-scale production. Physicochemical properties of bacterial GGTs have also been described, followed by a detailed discussion on various applications of bacterial GGTs in different biotechnological sectors. This review emphasizes the potential of bacterial GGTs as an industrial biocatalyst relevant to the current switch toward green chemistry.

Obstructing mass lesion of epiglottis: it can be tubercular.

We report a case of 60-year old male who had difficulty in breathing as well as in swallowing. On examination, he was found to be having proliferative growth of epiglottis and right aryepiglottic fold mimicking neoplasm. So emergency tracheostomy was performed and biopsy taken. He was found to be having asymptomatic miliary mottling on routine x-ray chest PA view. Further on HRCT, it turned out to be lesion suggesting tubercular etiology. Histopathology (epiglottic biopsy) report confirmed the whole process as tubercular. The patient recovered promptly in due course with anti-tubercular treatment. Point remains to be seen that if we can avoid tracheostomy and its complications in such cases.

Pneumothorax: a rare presentation of pulmonary mycetoma.

Pneumothorax due to mycetoma is extremely rare and has been described only in patients undergoing intensive cytotoxic therapy for hematologic malignancies. A non-immunocompromised subject presenting with pneumothorax due to rupture of the mycetoma into the pleural cavity is being described here.