Applied Biochemistry and Biotechnology

Differential Production of Pigments by Halophilic Bacteria Under the Effect of Salt and Evaluation of Their Antioxidant Activity Abstract Microorganisms that survive in the high salt environment have been shown to be a potential source for metabolites with pharmaceutical importance. In the present study, we have investigated the effect of 5 and 10% (w/v) NaCl on growth, biochemical changes, and metabolite production in seven moderately halophilic bacteria isolated from the salterns/mangrove area of South India. Metabolite production by Bacillus VITPS3 increased by 3.18-fold in the presence of 10% (w/v) NaCl concentration. Total phenolic and flavonoid content increased in Bacillus VITPS5 (11.3-fold) and Planococcus maritimus VITP21 (5.99-fold) whereas β-carotene content was less at higher NaCl concentrations. VITP21 and VITPS5, in response to NaCl, produced metabolites with higher (6.72- and 4.91-fold) DPPH and ABTS radical scavenging activity. UV/visible spectrophotometry of the extracts confirmed the presence of flavonoids, phenolics, and related compounds. 1H-NMR spectra indicated substantial changes in the metabolite production in response to salt concentration. Principal component analysis (PCA) revealed that VITP21 extracts exhibited the highest antioxidant activity compared with other extracts. The present study presents the first report on the comparative analysis of pigment production by moderate halophilic bacteria, in response to the effect of salt and their relation to radical scavenging property.

Radiolabeling of Biogenic Magnetic Nanoparticles with Rhenium-188 as a Novel Agent for Targeted Radiotherapy Abstract Use of nanoparticles as carriers of anticancer drugs is a suitable way for targeted drug delivery and reduction of the side effects. This research focuses on a novel drug carrier for therapeutic goals by the bacterial magnetic nanoparticles (magnetosomes). The unique characteristics of magnetosomes make them ideal nanobiotechnological materials. In this study, magnetic nanoparticles of Alphaproteobacterium MTB-KTN90 were labeled with the radioisotope rhenium-188 and optimized the factors affecting the labeling efficiency. The results showed that the labeling efficiency of magnetosomes with rhenium-188 was more than 96%. The optimum concentration of bacterial nanoparticles was 133 mg/ml and the best time for maximum efficiency labeling was 60 min. The labeling stability showed that the 188Re-nanoparticle complexes have good stability in 29 h. The results of magnetic nanoparticles bacterial cytotoxicity on cancer cells AsPC1 did not show significant toxicity to concentration of 100 μg/μl. Finally, the biogenic magnetic nanoparticles labeled with rhenium-188 can be introduced as a valuable candidate for the targeted therapy of tumor with reducing radiation to surrounding healthy tissues.

Elucidation of the Molecular Characteristics of Wild-Type and ALS-Linked Mutant SOD1 Using the NanoLuc Complementation Reporter System Abstract Previously, we evaluated human SOD1 (hSOD1) dimerization in living cells using the NanoLuc complementation reporter system and found that homodimerization of G85R and G93A mutant SOD1 was lower than that of wild-type hSOD1. Since these assays were performed only using N-terminal NanoBiT-tagged hSOD1 constructs in our previous study, we constructed additional hSOD1 genes with NanoBiT-tags at the C-terminus and evaluated the NanoBiT luciferase activities. Among the tested combinations, the luciferase activity in cells expressing NanoBiT-tagged wild-type hSOD1 was higher than that in cells expressing G85R or G93A mutant hSOD1. The NanoBiT luciferase activities were detected both inside and outside of cells; however, the extracellular luciferase activities were minimally dampened by treatment with brefeldin A, which inhibits canonical ER–Golgi transport. In addition to studies on the homodimerization of SOD1, we investigated the interaction between hSOD1 and three chaperone proteins, copper chaperone for SOD1 (CCS), FKBP, and GRP78. The NanoBiT luciferase activities in cells expressing NanoBiT-tagged SOD1 and CCS were relatively high, but weak signals were also observed in cells expressing SOD1 together with FKBP or GRP78. These luciferase activities were different between wild-type and mutant hSOD1. Finally, we investigated the effects of two selenocompounds, ebselen and Se-methylselenocysteine, on SOD1 dimerization and found that ebselen increased the NanoBiT luciferase activity in cells expressing wild-type and mutant hSOD1. In conclusion, we show the differential molecular characteristics of wild-type and mutant hSOD1 in live cells by transfection with NanoBiT-tagged hSOD1 and chaperone genes and demonstrate that this assay might be useful for the development and re-evaluation of chemical compounds modulating the SOD1 conformation.

Positive Correlation Between Somatic Mutations in RAS Gene and Colorectal Cancer in Telangana Population: Hospital-Based Study in a Cosmopolitan City Abstract Colorectal cancer (CRC) ranks among the most prevalent cancer types in both men and women. Screening of RAS (Kirsten rat sarcoma viral oncogene homolog (KRAS), neuro-blastoma RAS viral oncogene homolog (NRAS), and v-raf murine sarcoma viral oncogene homolog B1 (BRAF)) somatic mutations is necessary prior to considering anti-epidermal growth factor receptor (EGFR) therapies in CRC patients. Next-generation sequencing studies have confirmed that RAS gene panels could be used while developing treatment strategies for patients with CRC. The present study explored genetic mutations in KRAS, NRAS, and BRAF in CRC patients in the Telangana state of India. Patients with confirmed CRC (n = 100) who visited the Apollo hospitals were evaluated. Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissues, and pyrosequencing analysis was performed. Patient DNA samples were screened for 54 different KRAS, NRAS, and BRAF mutations, which revealed 34 somatic mutations. Exon 11 of BRAF possessed 4 mutations with highest individuals documented with G469A mutation. Pyrosequencing, a reliable method for analyzing somatic mutations present in RAS, could aid in taking treatment decisions for patients with CRC.

Achieving Maximal Production of Fusaricidins from Paenibacillus kribbensis CU01 via Continuous Fermentation Abstract In this study, we investigated the potential of Paenibacillus kribbensis CU01 in producing fusaricidin, a strong antifungal substance, via optimization of metal ions and carbon and nitrogen source, and continuous fermentation. In the cultivation of a 2-l batch, maximal production of fusaricidins (581 mg l−1) was achieved in a modified M9 medium containing metal ions, 10 g l−1 glucose, and 1 g l−1 ammonium chloride. Most of glucose was consumed at a rate of 0.74 g l−1 h−1 within 24 h and fusaricidin production began 15 h after batch cultivation. Continuous fermentation was performed using a 7-l fermenter with 2-l working volume of modified M9 medium containing 10 g l−1 glucose, 1 × 10−3 M FeSO4, and 1 × 10−6 M MnCl2. After 24 h of the start of cultivation, fresh M9 medium was continuously supplied at a flow rate of 2.5 ml min−1, and simultaneously, the same amount of cell culture broth was removed. In a continuous system, the highest fusaricidin concentration (579 mg l−1) was obtained using a dilution rate of 0.075 h−1 with an average productivity of 10.4 mg l−1 h−1 for 24 to 72 h of incubation. Based on these results, it was found that fusaricidin production using P. kribbens CU01 strain increased by at least 28 times the values reported in previous studies.

Prediction of Skin Disease Using Ensemble Data Mining Techniques and Feature Selection Method—a Comparative Study Abstract Nowadays, skin disease is a major problem among peoples worldwide. Different machine learning techniques are applied to predict the various classes of skin disease. In this research paper, we have applied six different machine learning algorithm to categorize different classes of skin disease using three ensemble techniques and then a feature selection method to compare the results obtained from different machine learning techniques. In the proposed study, we present a new method, which applies six different data mining classification techniques and then developed an ensemble approach using bagging, AdaBoost, and gradient boosting classifiers techniques to predict the different classes of skin disease. Further, the feature importance method is used to select important 15 features which play a major role in prediction. A subset of the original dataset is obtained after selecting only 15 features to compare the results of used six machine learning techniques and ensemble approach as on the whole dataset. The ensemble method used on skin disease dataset is compared with the new subset of the original dataset obtained from feature selection method. The outcome shows that the dermatological prediction accuracy of the test dataset is increased compared with an individual classifier and a better accuracy is obtained as compared with subset obtained from feature selection method. The ensemble method and feature selection used on dermatology datasets give better performance as compared with individual classifier algorithms. Ensemble method gives more accurate and effective skin disease prediction.

Preparation of Polar-Modified Styrene-Divinylbenzene Copolymer and Its Adsorption Performance for Comprehensive Utilization of Sugarcane Bagasse Dilute-Acid Hydrolysate Abstract Lignocellulosic hydrolysate contains complex nonsugar compounds and undegraded sugars in the process of preparing platform compound levulinic acid (LA) and furfural by one-step dilute-acid hydrolysis. For efficiently and comprehensively utilizing the hydrolysate, a series of polar modified resins were synthesized for adsorption and separation of the sugarcane bagasse hydrolysate to obtain platform compounds and fermentable hydrolysate simultaneously. The adsorption capacities of LA and furfural were optimized to 85.32 mg/g and 33.55 mg/g on polar modified resin prepared with 80 wt% glycidyl methacrylate (GMA -80), which was much higher than nonpolar resin (4.16 mg/g and 16.14 mg/g). GMA-80 obtained the best comprehensive adsorption property, whose desorption rates were 99.90% and 89.86% for LA and furfural, respectively, and its regeneration performance was also excellent, indicating that the resin is a potential adsorbent and expected to be used in the separation and purification of the lignocellulosic hydrolysate.

Suspension State Promotes Drug Resistance of Breast Tumor Cells by Inducing ABCC3 Overexpression Abstract Mechanical microenvironment plays a critical role in cancer drug resistance and this study supposed that suspension state might be involved in drug resistance of breast tumor cells. The viability of cell was detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Cell cycle and apoptosis were detected by flow cytometry. Gene and protein were tested by RT-qPCR and Western blot, respectively. Drug resistance of MDA-MB-231 cells cultured for 72 h under suspension state was significantly increased. Suspension state was found to induce the overexpression of adenosine triphosphate–binding cassette subfamily C member 3 (ABCC3) in MDA-MB-231 cells. Silencing of ABCC3 significantly decreased drug resistance of suspension MDA-MB-231 cells. Moreover, suspension state was able to increase lamin A/C accumulation in MDA-MB-231 cells and lamin A/C regulated the expression of ABCC3. Moreover, lamin A/C knockdown also decreased drug resistance of suspension MDA-MB-231 cells, but the effect on drug resistance was less than that of ABCC3 knockdown. Suspension state plays a vital role in promoting drug resistance of MDA-MB-231 cells by inducing ABCC3 overexpression, and lamin A/C accumulation is associated with this process.

Enhancing Activity by Supercritical CO 2 Mediated Immobilization of Lipase on Mesocellular Foam in Preparation of Hexyl Laurate Abstract Hexyl laurate is employed in several cosmetics having great demand. It could be synthesized catalytically like a “natural” perfume using a lipase. The use of mesocellular foam silica (MCF) for immobilization of lipases could be made using supercritical CO2 as a medium to enhance its activity in comparison with the normal techniques. Three different catalysts were supported on MCF such as Candida antractica B (CALB), Amano AYS, and Porcine pancreas (PPL), and their activity was evaluated in the preparation of hexyl laurate from lauric acid and hexyl alcohol. CALB@ MCF was the best among all. A systematic study was conducted to assess the effects of different operating parameters. It was ternary complex mechanism with inhibition by hexyl alcohol. The enzyme was reusable and the process is green.

Nanotechnological Applications Hold a Pivotal Position in Boosting Stem Cells Osteogenic Activity: In Vitro and In Vivo Studies Abstract This approach was constructed to appraise the therapeutic effectiveness of a single i.v. dose of osteoblasts generated from co-culturing BM-MSCs with nano-HA, Pt-NPs, or Pt-HA-nanocomposite in osteoporotic rats. MSCs were grown, propagated in culture, and characterized. The effect of the suggested nanoplatforms on the survival, osteogenic differentiation, and mineralization of BM-MSCs was assessed by MTT assay, real-time PCR analysis, and Alizarin red S staining, respectively. Thereafter, the generated osteoblasts were employed for the treatment of ovariectomized rats. Our results revealed that the selected nanoplatforms upregulate the expression of osteogenic differentiation related genes (Runx-2 and BMP-2) significantly and enhance calcium deposition in BM-MSCs after 7 and 21 days, respectively, whereas the in vivo study validated that the infusion of the generated osteoblasts considerably downturn serum BALP, BSP, and SOST levels; upswing OSX level; and regain femur bone mineralization and histoarchitecture. Conclusively, the outcomes of this work provide scientific evidence that transplanting osteoblasts derived from differentiation of BM-MSCs in the presence of nanoplatforms in ovariectomized rats restores bone remodeling balance which constitutes a new hope for the treatment of osteoporosis.