Evaluation of freeze-dried human sera as a novel approach for ATR-FTIR spectroscopic analysis as compared to conventionally used thin dry film sera Abstract Objective Evaluation of the diagnostic potential of freeze dried sera in comparison to thin dry film analysis for recording ATR-FTIR spectra. Results For this purpose, we compared our novel sample preparation technique i.e. freeze dried with conventional technique i.e. thin dry film sera. Using both methods ATR-FTIR spectra were recorded from Salmonella Typhi infected and healthy control human sera samples. When PCA was applied PC1 scores showed more inter-class variation among infected and healthy controls when freeze dried sample was used (90 %) as compared to thin dry film method (46 %). Conclusions Potential of ATR-FTIR for discrimination of bio-molecules between two classes of samples is enhanced when freeze dried sera instead of thin dry film method is used.

Comparison of two commonly used methods for stimulating T cells Abstract Objective Although several methods have been reported and used for in vitro T cell amplification, there are no consistent reports on the optimal stimulation conditions and the characterization of these stimulated T cells. The current study aimed to determine the optimal conditions for efficient T cell amplification by two commonly used methods involving CD3/CD28 antibody and phytohemagglutinin (PHA), respectively. Results Orthogonal design and CCK8 assay showed that 5 μg/mL CD3, 5 μg/mL CD28, and 100 ng/mL IL2 for the first method and 50 μg/mL PHA for the second method was optimal for T cell stimulation. Flow cytometry demonstrated that the percentage of CD8+ in the stimulated groups significantly increased, while the percentage of CD4+/CD8+ was significantly decreased compared with the unstimulated group. The percentage of CD4+ showed no significant difference among the three groups. Notably, there was no significant difference between the two stimulated groups. In addition, the percentage of apoptotic cells was significantly increased in the stimulated groups compared with the unstimulated group, but showed no remarkable difference between the PHA and CD3/CD28 stimulation groups. Glycolysis analysis showed that the glycolytic capacity and glycolytic reserve were both significantly increased in the PHA and CD3/CD28 groups compared with the unstimulated group, with no significant difference noted between the stimulated groups. Conclusions Although both stimulation methods showed similar efficacies, we suggest the PHA method might be better considering its easy application and cost-effective nature.

Epigenetic modification enhances ergot alkaloid production of Claviceps purpurea Abstract Objective To enhance ergot alkaloid production of Claviceps purpurea Cp-1 strain by epigenetic modification approach. Results The chemical epigenetic modifiers were screened to promote ergot alkaloid production of the Cp-1 strain. The histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) was found to significantly enhance the alkaloid productivity of the strain. Particularly, the titers of total ergot alkaloids were gradually increased with the increase of SAHA concentration in the fermentation medium, and the highest production of ergot alkaloids could be achieved at the concentration of 500 μM SAHA. Specially, the titers of ergometrine and total ergot alkaloids were as high as 95.4 mg/L and 179.7 mg/L, respectively, which were twice of those of the control. Furthermore, the mRNA expression levels of the most functional genes in the ergot alkaloid synthesis (EAS) gene cluster were up-regulated under SAHA treatment. It was proposed that SAHA might increase histone acetylation in the EAS gene cluster region in the chromosome, which would loosen the chromosome structure, and subsequently up-regulate the mRNA expression levels of genes involved in the biosynthesis of ergot alkaloids, thereby resulting in the markedly increase in the production of ergot alkaloids. Conclusions The ergot alkaloid production by the C. purpurea Cp-1 strain can be effectively increased by the application of histone deacetylase inhibitor. Our work provides a reference for using the chemical epigenetic modifiers to improve SM production in other fungi.

Immobilization of lactoperoxidase on Fe 3 O 4 magnetic nanoparticles with improved stability Abstract Objective The study aimed to develop a facile and effectual method to increase the stability of lactoperoxidase (LPO) by using its immobilization on Fe3O4 magnetic nanoparticles (Fe3O4 MNPs). Results The successful immobilization of LPO on Fe3O4 MNPs was confirmed by using Fourier transform infrared spectroscopy (FT-IR) and field emission scanning electron microscopy (FE-SEM). The Km values of free LPO and LPO immobilized on Fe3O4 were 53.19, 72.46 mM and their Vmax values were 0.629, 0.576 µmol/mL min respectively. The overall results indicated that the stability of the immobilized LPO was significantly improved compared to free LPO. The LPO immobilized on Fe3O4 (LPO– Fe3O4) retained 28% of the initial activity within 30 days at 25 °C whereas the free enzyme lost its activity after 7 days at the same temperature. Moreover, evaluation of the thermal stability of LPO at 75 °C determined the conservation of 19% of the initial activity of LPO in the LPO– Fe3O4 sample after 60 min whereas the free enzyme lost its activity after 5 min. Conclusions According to the present results, Fe3O4 magnetic nanoparticles are suitable for the immobilization of LPO.

Bioremediation of highly toxic arsenic via carbon-fiber-assisted indirect As(III) oxidation by moderately-thermophilic, acidophilic Fe-oxidizing bacteria Abstract Objective To enable removal of highly toxic As(III) from acidic waters by inducing indirect microbial As(III) oxidation by Fe-oxidizing bacteria via carbon-assisted redox-coupling between As(III) oxidation and Fe3+ reduction. Results Carbon-fiber (CF) was shown to function as an electron-mediator to catalyze chemical (abiotic) redox-coupling between As(III) oxidation and Fe3+ reduction. Accordingly, by taking advantage of Fe3+ regeneration by Fe-oxidizing bacteria, it was possible to promote oxidative removal of As(III) as ferric arsenate at moderate temperature. This reaction can be of use under the situation where a high-temperature treatment is not immediately available. Arsenic once concentrated as ferric arsenate on carbon-fibers can be collected to undergo phase-transformation to crystalline scorodite as the next re-solubilization/re-crystallization step at a higher temperature (70 °C). Conclusions While extremely acidophilic Fe-oxidizing bacteria are widely found in nature, the As-oxidizing counterparts, especially those grown on moderately-thermophilic and mesophilic temperatures, are hardly known. In this regard, the finding of this study could make a possible introduction of the semi-passive, low-temperature As-treatment using readily available Fe-oxidizing bacteria.

Recombinant human Cyclophilin A stimulates hair follicle cells via Wnt/β-catenin signaling pathway Abstract Objectives To explore potential effects of recombinant peptidyl-prolyl isomerase human Cyclophilin A (CypA) in the growth of mouse hair and human hair follicle dermal papilla cells (HDP). Results The hair growth of 8-weeks C57BL/6 mouse was significantly stimulated by recombinant human CypA protein. CypA also promoted Human Dermal Papilla (HDP) cell growth and induced β-catenin protein through GSK3β inhibition. Conclusions The molecular chaperone CypA stimulates hair follicle and HDP cells through Wnt/β-catenin signaling pathway, suggesting that it might be a key molecule for hair loss disorder therapy.

Deletion of Gcw13 represses autophagy in Pichia pastoris cells grown in methanol medium with sufficient amino acids Abstract Objective The purpose of this article is to study the underlying cause of the induction of autophagy in Pichia pastoris cells grown in amino acid-rich methanol medium during methanol adaptation. Results Autophagy was induced in P. pastoris GS115 when cells were grown in amino acid-rich methanol medium. Transcriptome analysis revealed that genes involved in amino acid biosynthesis were upregulated. The deletion of Gcw13, a GPI-anchored protein that plays a role in the endocytosis of the general amino acid permease Gap1, resulted in the inhibition of autophagy, the activation of TORC1 and an increase in the uptake of glutamine and asparagine in methanol-grown cells. Conclusions Our results demonstrated that the autophagy induced in P. pastoris cells grown in amino acid-rich methanol medium was nitrogen source independent and may be due to a Gcw13-dependent decrease in amino acid uptake during methanol adaptation.

Evaluating aeration and stirring effects to improve itaconic acid production from glucose using Aspergillus terreus Abstract The effects of the bioreactor conditions, in particular the mode and intensity of aeration and mixing were studied on itaconic acid (IA) fermentation efficiency by Aspergillus terreus strain from glucose substrate. IA was produced in batch system by systematically varying the oxygen content of the aeration gas (from 21 to 31.5 vol% O2) and the stirring rate (from 150 to 600 rpm). The data were analyzed kinetically to characterize the behavior of the process, and besides, the performances were evaluated comparatively with the literature. It turned out that the operation of the bioreactor with either the higher inlet O2 concentration (31.5 vol% O2) or faster stirring (600 rpm) could enhance biological IA generation the most, resulting in yield and volumetric productivity of 0.31 g IA/g glucose and 0.32 g IA/g glucose and 3.15 g IA/L day and 4.26 g IA/L day, respectively. Overall, the significance of fermentation settings was shown in this work regarding IA production catalyzed by A. terreus and notable advances could be realized by adjusting the aeration and stirring towards an optimal combination. Graphic abstract

Selection of potential anti-adhesion drugs by in silico approaches targeted to ALS3 from Candida albicans Abstract Objective To select potential ligands of ALS3 for drug development with anti-adhesion and/or anti-biofilm activities. Methodology ALS3 model was considered stable by DM. The main features of protein flexibility were represented by two conformers which were used in the virtual screening. Twenty-four small molecules were selected for in vitro assays. Five of them presented the best biological activity with ability to inhibit the adhesion and C. albicans biofilm formation on abiotic surface. Results To select potential ligands of ALS3 for drug development with anti-adhesion and/or anti-biofilm activities. Conclusion In silico tools application was able to select promising compounds with anti-adhesion activity, opening a new perspective of medical device treatment.

A food-grade engineered Lactococcus lactis strain delivering Helicobacter pylori Lpp20 alleviates bacterial infection in H. pylori -challenged mice Abstract Objective To construct a food-grade bacterium producing and delivering H. pylori Lpp20 antigen and evaluate its immune efficacy against H. pylori challenges with aim to develop anti-H. pylori oral vaccines and functional foods. Results Lpp20 was expressed as a 22 kDa protein in Lactococcus lactis, constituting 11.2% of the cell lysate proteins, and recognized by mouse antisera. Mice orally gavaged with the engineered bacterium had elevated serum IgG levels and lowered urease activity of stomach following H. pylori challenges. Conclusions This study firstly reports a food-grade L. lactis strain delivering Lpp20 to mucosal immunization sites, demonstrating a novel efficient production and safe utilization mode of Lpp20, offering a promising vaccine candidate and health food sources.