Computational design of fully artificial peptides is extensively researched by material scientists and engineers for the construction of novel nanostructures and biomaterials. Such design has yielded a peptide-based building block or bundlemer, a coiled coil peptide assembly that undergoes further physical-covalent interactions to form 1D, 2D and, potentially, 3D hierarchical assemblies and displays targeted and biomimetic material properties. Recombinant expression is a convenient, flexible tool to synthesize such artificial and modified peptides in large quantities while also enabling economical synthesis of isotopically labeled peptides and longer protein-like artificial peptides. This report describes the protocol for recombinant expression of a 31-amino acid, computationally designed bundlemer-forming peptide in Escherichia coli. Peptide yields of 10 mgs per liter of media were achieved which highlights complementary advantages of recombinant expression technique relative to conventional laboratory-scale synthesis, such as solid-phase peptide synthesis.

The bacteriolysin lysostaphin (Lst) and endolysin PlyPH are potent modular lytic enzymes with activity against clinically-relevant Gram-positive Staphylococcus aureus and Bacillus cereus, respectively. Both enzymes possess an N-terminal catalytic domain and C-terminal binding domain, with the latter conferring significant enzyme specificity. Lst and PlyPH show reduced activity in the presence of bacterial growth-supporting conditions, such as complex media. Here, we hypothesize that Lst and PlyPH bind poorly to their targets in growth media, which may influence their use in antimicrobial applications in the food industry, as therapeutics, and for control of microbial communities. To this end, binding of isolated Lst and PlyPH binding domains to target bacteria was quantified in the presence of three increasingly complex media - phosphate buffered saline (PBS), defined growth medium (AAM) and undefined complex medium (TSB) by surface plasmon resonance (SPR) and flow cytometry. Evaluation of binding kinetics by SPR demonstrated that PlyPH binding was particularly sensitive to medium composition, with 8-fold lower association and 3.4-fold lower dissociation rate constants to B. cereus in TSB compared to PBS. Flow cytometry studies indicated a decrease in the binding-dependent fluorescent populations of S. aureus and B. cereus, for lysostaphin binding domain and PlyPH binding domain, respectively, in TSB compared to PBS. Enzyme binding behavior was consistent with the enzymes' catalytic activity in the three media, thereby suggesting that compromised enzyme binding could be responsible for poor activity in more complex growth media.

Lycopersicon esculentum L., also known as tomato, is an important industrial plant due to its products which worth billions of dollars annually, besides its nutritional value and health benefits. In this study, we investigated the two-dimensional protein expression profiles in drought tolerant mutant plants derived from industrial 5MX12956 tomato variety by Cs-137 gamma radiation source induced mutations. Drought tolerance of mutants were evaluated and confirmed by in vivo and in vitro methods. Eleven drought responsive protein spots were identified by two-dimensional electrophoresis and MALDI-TOF-MS. Identified proteins which presented differential expression under drought conditions were clustered under six distinct groups based on their cellular functions. These clusters are ATP and carbohydrate metabolism, mRNA processing and protein phosphorylation, oxidation reduction and stress response, signaling and supporting cytoskeleton. Our results contributed proteomic data to drought tolerance of our tomato mutants which were originated from drought susceptible 5MX12956 variety. They may also facilitate basis for future investigations into the genetic and physiological aspects of this tolerance.

Soil salinity is a serious abiotic stress worldwide. Pecan plants (Carya illinoensis K. Koch) have been suggested for cultivation in soils with high levels of salinity owing to their huge demand. To understand the potential molecular mechanisms in pecan in response to salt stress, RNA-sequencing technology was used to compare the transcriptomes of pecan plants treated with 0, 0.3 %, or 0.6 % NaCl solutions. The results indicated that 170,086 unigenes were obtained from pecan leaf cDNA samples. Based on the assembled de novo transcriptome, 53, 535, and 7358 differentially expressed genes (DEGs) were detected between untreated and salt-treated leaves at 8, 24, and 48 h, respectively. Because of the large number of DEGs across different contrasts, a Gene Set Enrichment Analysis was selected to identify gene pathways associated with salt treatment. A total of 1858 DEGs were enriched in 66 gene sets, including 22 up-regulated and 47 down-regulated gene sets in the salt treatment groups, compared with those in the control groups. The up-regulated gene sets were mainly involved in the response to salicylic acid; the regulation of the jasmonic acid-mediated signalling pathway during the short-term treatment (8 h); and the cellular response to hypoxia, cellular respiration, and RNA modification during the long-term treatment (24-48 h). The down-regulated gene sets were predominately associated with photosynthesis, water transport, and the metabolic biosynthetic process under salt stress. Genes related to the Really Interesting New Gene superfamily protein and F-box domain protein in the ubiquitin-dependent degradation pathway were significantly up-regulated or down-regulated in different periods of the regulating process. Overall, these results not only enrich genomic resources but also provide insights into the molecular mechanism in pecan under salt stress.

The 57-amino acid Kunitz Protease Inhibitor (KPI) domain of Protease Nexin 2 inhibits Factor XIa (FXIa) and other proteases. We previously fused KPI to human serum albumin (KPIHSA). KPIHSA inhibits coagulation Factor XIa (FXIa) 6-fold more rapidly than plasmin. We screened a bacterial expression library of KPI variants randomized at M17, and selected M17D as having the highest anti-FXIa: antiplasmin activity ratio. Expressed as HSA fusion proteins in Pichia pastoris, KPIHSA and KPI(M17D)HSA inhibited FXIa indistinguishably (Ki 9 nM) but KPI(M17D)HSA lacked detectable antiplasmin activity. Purified variant and wild-type KPIHSA were expressed and injected into mice with ferric chloride-treated carotid arteries, with or without systemic administration of tissue plasminogen activator (Tenecteplase, TNKase). The time to arterial occlusion (TTO) or reperfusion (TTR) was assessed by Doppler ultrasound. TTR did not differ between mice treated with TNKase alone or with TNKase supplemented with 38 mg/kg KPI(M17D)HSA but was significantly prolonged to >60 min in all mice treated with TNKase and 38 mg/kg KPIHSA. TTO was significantly but equally prolonged by either 38 mg/kg KPIHSA or KPI(M17D)HSA versus vehicle controls. The antiplasmin activity of KPI is relevant in vivo but its elimination did not enhance counter-thrombosis by KPI.

High Fischer ratio oligopeptides have better conditioning effects on chronic diseases caused by long-term sub-health. At present, the enzymatic method for producing high Fischer ratio oligopeptides has a low yield, complicated purification, and a high cost. The use of exopeptidases with specific catalytic activity for aromatic amino acids in the preparation of high Fischer ratio oligopeptides is an important means to solve this problem. The carboxypeptidase from Aspergillus niger M00988 was cloned, which has good specificity for hydrophobic amino acids. Mutations at important substrate binding sites 135, 160, and 206 were performed to study important factors affecting the enzyme-specific recognition of aromatic groups. The results showed that the steric hindrance of amino acid residues at position 135 and the effects of positions 160 and 206 on the binding force of the enzyme to the substrate have important effects on the specific recognition of aromatic groups by the enzyme. Therefore, the S135 G, Y160S, and Y206S mutant enzymes have good application prospects in the preparation of high Fischer ratio oligopeptides with Chlorella powder. The obtained oligopeptides' Fischer ratio reached 31.45, 38.42, and 36.54, respectively. Compared with the original enzyme, the Fischer ratio increased by 2.58 %, 25.31 %, and 19.18 %, respectively.

The bio-production of theanine is currently of significant interest due to its wide applications in food and healthcare products. Gamma glutamyl transferase (GGT) has been widely applied in L-theanine synthesis, but L-theanine yields remain prohibitively low for commercial production. In this study, a robust high-throughput screening process for isolating GGT mutants was developed through a combination of error-prone PCR techniques and a colorimetric reaction. The co-expression of PrsA lipoprotein enhances the secretion of GGT, thus GGT could be obtained quickly and easily without crushing cells. Random mutations on ggt genes were introduced by using error-prone PCR kits to build a large mutant library. A colorless compound generated by the reaction between NH4+ (released from L-theanine synthesis) and OPA was measured quantitatively by UV/visible spectroscopy when mixed with TCA and DMSO. Approximately 30 positive clones with improved color formation on the 96-well plates were identified, and mutants T413P and T463S with more than by 30 % higher transpeptidation activity versus the original GGT were isolated. To improve the operational stability and economical use, mutant GGT was immobilized on a prepared oxidized cellulose nanofiber membrane. The remaining activity of immobilized GGT was 88 % versus 72 % of free enzyme over 15 h. A fed-batch conversion was performed with the immobilized GGT, and over 70 g/L L-theanine could be accumulated within 18 h after feeding twice. Versus other studies, this is one of the best L-theanine synthesis systems using immobilized GGT.

The limited availability of primary human β-cells/islets and their quality (due to donor diversity) restrict the development of in vitro models for diabetes research. Human induced pluripotent stem cells (hiPSCs) may be a promising cell-source for diabetes studies, anti-diabetic drug screening and personalized therapies. However, achieving levels of maturity/functionality that are comparable to the in vivo situation and islets rebuilt from iPSCs is still challenging. Here, we compare and discuss two strategies for culturing human pancreatic β-cells derived from hiPSCs in microfluidic biochips. First, we confirmed that the protocol in conventional Petri 2D monolayer led to insulin, PDX1 and MAFA positive staining, to C-Peptide productive cells, and to tissue responsive to high/low glucose and GLP1 stimulation. This protocol and its subsequent modifications (including extracellular matrix coating, cell adhesion time, cell inoculation density, flow rate) was not successful in the 2D biochip culture. We proposed a second strategy using 3D spheroids created from honeycomb static cultures. Spheroids in static experiments carried out over 14 days demonstrated that they expressed high levels of β-cell markers (INS mRNA) and higher α-cell markers (GCG mRNA and glucagon positive staining), when compared to Petri 2D cultures. Furthermore, the 3D spheroids were specifically able to secrete insulin in response to both high/low glucose stimulation and GLP1 exposure. The spheroids were successfully inoculated into biochips and maintained for 10 days in perfusion. The 3D biochip cultures increased mRNA levels of GCG and maintained high levels of β-cell markers and responsiveness to both high/low glucose and GLP1 stimulation. Finally, C-peptide and insulin secretion were higher in biochips when compared to static spheroids. These results illustrate the promising potential for hiPSCs derived β-cells and their spheroid-based pancreas-on-chip model for pancreatic disease/diabetes modeling and anti-diabetic drug screening.

Soluble expression of recombinant proteins in E. coli is often done by translocation of the product across the inner membrane (IM) into the periplasm, where it is retained by the outer membrane (OM). While the integrity of the IM is strongly coupled to viability and impurity release, a decrease in OM integrity (corresponding to increased "leakiness") leads to accumulation of product in the extracellular space, strongly impacting the downstream process. Whether leakiness is desired or not, differential monitoring and control of IM and OM integrity are necessary for an efficient E. coli bioprocess in compliance with the guidelines of Quality by Design and Process Analytical Technology. In this review, we give an overview of relevant monitoring tools, summarize the research on factors affecting E. coli membrane integrity and provide a brief discussion on how the available monitoring technology can be implemented in real-time control of E. coli cultivations.

Salinization is a worldwide environmental problem, which is negatively impacting crop yield and thus posing a threat to the world's food security. Considering the rising threat of salinity, it is need of time, to understand the salt tolerant mechanism in plants and find avenues for the development of salinity resistant plants. Several plants tolerate salinity in a different manner, thereby halophytes and glycophytes evolved altered mechanisms to counter the stress. Therefore, in this review article, physiological, metabolic, and molecular aspects of the plant adaptation to salt stress have been discussed. The conventional breeding techniques for developing salt tolerant plants has not been much successful, due to its multigenic trait. The inflow of data from plant sequencing projects and annotation of genes led to the identification of many putative genes having a role in salt stress. The bioinformatics tools provided preliminary information and were helpful for making salt stress-specific databases. The microRNA identification and characterization led to unraveling the finer intricacies of the network. The transgenic approach finally paved a way for overexpressing some important genes viz. DREB, MYB, COMT, SOS, PKE, NHX, etc. conferred salt stress tolerance. In this review, we tried to show the effect of salinity on plants, considering ion homeostasis, antioxidant defense response, proteins involved, possible utilization of transgenic plants, and bioinformatics for coping with this stress factor. An overview of previous studies related to salt stress is presented in order to assist researchers in providing a potential solution for this increasing environmental threat.

The present study aims to valorize chestnut burrs, an important lignocellulosic waste, through a biorefinery concept. A solid residue rich in glucan (41.36 ± 0.59 %) and lignin (39.06 ± 0.01 %) obtained from a previous process of pre-hydrolysis was subjected to four treatments with water or NaOH to enhance enzymatic hydrolysis. Saccharification was performed using different ratios of commercial cellulases and β-glucosidases and at controlled pH 4.8 or 6.0 (with citrate buffer) or uncontrolled pH. Carbohydrate-rich solutions with or without nutrients were used to produce bacteriocins by Lactobacillus plantarum CECT 211. The use of NaOH at high temperatures (120 and 130 °C) was the most suitable treatment to improve saccharification. Regarding the production of bacteriocins, the best result was obtained using the enzymatic solution obtained at controlled pH 6.0, supplemented with MRS broth nutrients (except glucose). Thus, the concentrations of bacteriocins obtained in this culture medium (9.21 BU/mL) was 1.22 and 1.98 times higher than those obtained in the nutrient supplemented medium buffered at pH 4.8 (7.56 BU/mL) and in the commercial MRS broth (4.65 BU/mL), respectively. These results highlight the feasibility of the technology developed in this work.

The acetone-butanol-ethanol (ABE) fermentation by solventogenic clostridia has a long history of industrial butanol production. The Clostridium beijerinckii mutant BA101 has been widely studied for ABE fermentation owing to its enhanced butanol production capacity. Here, we characterized the BA101 mutant under controlled environmental conditions in parallel with the parental strain C. beijerinckii NCIMB 8052. To investigate the correlation between phenotype and genotype, we carried out the genome sequencing of BA101. Through comparative genomic analysis, several mutations in the genes encoding transcriptional regulator, sensor kinase, and phosphatase were identified in the BA101 genome as well as other sibling mutants. Among them, the SNP in the Cbei_3078 gene encoding PAS/PAC sensor hybrid histidine kinase was unique to the BA101 strain. The identified mutations relevant to the observed physiological behaviors of BA101 could be potential genetic targets for rational engineering of solventogenic clostridia toward desired phenotypes.

Sixty-seven yeast strains were isolated from castor beans then their endogenous lipids were stained by Nile Red (NR) fluorescence dye, and flow cytometry was used to obtain a strain with a high relative mean fluorescence intensity (MFI) value. The highest MFI value was obtained for strain CM33, which produced a maximum lipid content of 20.8 % dry cell weight (DCW). Based on the sequence of the ITS-5.8S-ITS rDNA and D1/D2 26S rDNA regions, CM33 showed 99 % identity with Rhodotorula paludigena. The potential of CM33 to assimilate various carbon sources was examined by growth on minimal media using glucose, glycerol, sucrose or xylose. CM33 was grown in glucose-based medium for 96 h and exhibited a maximum lipid content of 23.9 % DCW. Furthermore, when cells were cultured on molasses waste, their biomass, lipid content and lipid concentration reached 16.5 g/L, 37.1 % DCW and 6.1 g/L, respectively. These results demonstrated the potential of R. paludigena CM33 to contribute to a value-added carbon chain by converting renewable waste materials for biolipid production.

Non-proteinogenic trans-4-hydroxy-l-proline (t4HYP), a crucial naturally occurred amino acid, is present in most organisms. t4HYP is a regio- and stereo-selectively hydroxylated product of l-proline and a valuable building block for pharmaceutically important intermediates/ingredients synthesis. Microbial production of t4HYP has aroused extensive investigations because of its low-cost and environmentally benign features. Herein, we reported metabolic engineering of endogenous l-proline biosynthetic pathway to enhance t4HYP production in trace l-proline-producing Escherichia coli BL21(DE3) (21-S0). The genes responsible for by-product formation from l-proline, pyruvate, acetyl-CoA, and isocitrate in the biosynthetic network of 21-S0 were knocked out to channel the metabolic flux towards l-proline biosynthesis. PdhR was knocked out to remove its negative regulation and aceK was deleted to ensure isocitrate dehydrogenase's activity and to increase NADPH/NADP+ level. The other genes for l-proline biosynthesis were enhanced by integration of strong promoters and 5'-untranslated regions. The resulting engineered E. coli strains 21-S1 ∼ 21-S9 harboring a codon-optimized proline 4-hydroxylase-encoding gene (P4H) were grown and fermented. A titer of 4.82 g/L of t4HYP production in 21-S6 overexpressing P4H was obtained at conical flask level, comparing with the starting 21-S0 (26 mg/L). The present work paves an efficient metabolic engineering way for higher t4HYP production in E. coli.

Soluble expression of enzymes inside the cell is a prerequisite for the successful biotransformation of valuable products. Some key enzymes involved in biotransformation processes, however, are hardly expressed in their soluble forms. Here, we propose an inducible plasmid display, which is a molecular evolution strategy coupled with a high-throughput screening and/or selection method, as a simple and powerful tool for improving the solubility of target enzymes. Specifically, the Oct-1 DNA-binding domain and intein (i.e., auto-processing domain) were employed as anchoring and protein trans-splicing motifs to develop the system, in which the probability of protein trans-splicing is dependent on the soluble property of target proteins. The applicability of inducible plasmid display was investigated using an α-1,2-fucosyltransferase (FucT2) from Helicobacter pylori, a highly insoluble and unstable enzyme in the cytoplasmic space of Escherichia coli, as a model protein. One round of the overall inducible plasmid display process, which consists of in vivo production of FucT2 mutants and in vitro screening, enabled soluble expression of FucT2 and selection of plasmids containing the corresponding genetic information. The inducible plasmid display developed in this study will contribute to the rapid and efficient screening and/or selection of soluble proteins.

Dehydroquinase or 3-dehydroquinate dehydratase (DHQD) reversibly cleaves 3-dehydroquinate to form 3-dehydroshikimate. Here, we describe the functional and structural features of a cold active type II 3-dehydroquinate dehydratase from the psychrophilic yeast, Glaciozyma antarctica PI12 (GaDHQD). Functional studies showed that the enzyme was active at low temperatures (10-30 °C), but displayed maximal activity at 40 °C. Yet the enzyme was stable over a wide range of temperatures (10-70 °C) and between pH 6.0-10.0 with an optimum pH of 8.0. Interestingly, the enzyme was highly thermo-tolerant, denaturing only at approximately 84 °C. Three-dimensional structure analyses showed that the G. antarctica dehydroquinase (GaDHQD) possesses psychrophilic features in comparison with its mesophilic and thermophilic counterparts such as higher numbers of non-polar residues on the surface, lower numbers of arginine and higher numbers of glycine-residues with lower numbers of hydrophobic interactions. On the other hand, GaDHQD shares some traits (i.e. total number of hydrogen bonds, number of proline residues and overall folding) with its mesophilic and thermophilic counterparts. Combined, these features contribute synergistically towards the enzyme's ability to function at both low and high temperatures.

The nitrogen-fixing α-proteobacterium Sinorhizobium meliloti genome codifies at least 50 response regulator (RR) proteins mediating different and, in many cases, unknown processes. RR-mutant library screening allowed us to identify genes potentially implicated in survival to acid conditions. actJ mutation resulted in a strain with reduced growth rate under mildly acidic conditions as well as a lower capacity to tolerate a sudden shift to lethal acidic conditions compared with the parental strain. Mutation of the downstream gene actK, which encodes for a histidine kinase, showed a similar phenotype in acidic environments suggesting a functional two-component system. Interestingly, even though nodulation kinetics, quantity, and macroscopic morphology of Medicago sativa nodules were not affected in actJ and actK mutants, ActK was required to express the wild-type nitrogen fixation phenotype and ActJK was necessary for full bacteroid development and nodule occupancy. The actJK regulatory system presented here provides insights into an evolutionary process in rhizobium adaptation to acidic environments and suggests that actJK-controlled functions are crucial for optimal symbiosis development.

Current methods for the production of natural vanilla extract are long and tedious, and the efficiency of the vanillin extraction is usually conditioned by different factors during the traditional curing process (temperatures and weather conditions). As an important fraction of vanillin is present in the form of glucovanillin in green beans, endogenous β-glucosidases contribute to its hydrolysis; however, these enzymes lose efficiency during the curing process. The use of extremophilic organisms as a source of an appropriate exogenous enzyme can offer a valid alternative when producing natural vanillin. Here, a β-glucosidase from the thermo-acidophilic organism Alicyclobacillus acidiphilus (AacGH1) was cloned, expressed in E. coli BL21, and fully characterized in respect to both function and crystal structure. Notably, AacGH1 was stable at a temperature up to 50 °C and exhibited good tolerance to glucose, fructose and organic solvents, in particular it maintained full activity in the presence of up to 20 % (v/v) ethanol. The enzyme was then successfully applied to an ethanol-water (20 % (v/v)) extract of green vanilla beans and the complete hydrolysis of glucovanillin (1.7 mM) to vanillin, and other flavour compounds commonly found in vanilla, was achieved using 0.5 mg/mL of enzyme in just 15 min at 30 °C.

Large-scale cultivations of photoautotrophic microorganisms represent a very promising and potentially cost-effective alternative for climate change mitigation, when associated to the co-production of high value bioproducts, such as fatty acids and carotenoids, considering the growing demand for natural products. During microalgae cultivation, CO2 enrichment is a requirement to reach high productivities, although high CO2 levels are normally stressful to microalgae. On the other hand, cellular stress is a well reported strategy to induce carotenoid and fatty acids production. This work evaluated extracellular carotenoid production from the mangrove-isolated microalga Parachlorella kessleri cultivated under 5, 15 and 30% CO2 in stirred tank photobioreactors. In the 10th day of cultivation, CO2 supply was interrupted until the end of the cultivation (14th day), causing a stressful and imperative condition for microalgae cells to release the red pigment. Growth kinetics, physiological parameters and bioproducts production were evaluated. Growth kinetics were similar under all tested conditions and differences were not statistically significant, with the highest values of μmax, biomass concentration, lipid content and CO2 fixation rate of 0.77 d-1, 1.24 g L-1, 241 mg g-1 (dw) and 165 mg L-1 d-1, respectively. In contrast, total carotenoid concentrations varied significantly (p < 0.01), with the highest concentration of 0.030 μg mL-1 under 5% CO2. The produced red pigment presented antioxidant activity and characteristics of carotenoids confirmed by UV-vis and tandem mass spectrometry (MS/MS). The fatty acid profiles in the biomass varied in response to CO2 levels in the cultivations. In general, higher CO2 concentrations (15 and 30%) favored the production of saturated and mono-unsaturated fatty acids, suitable as biodiesel feedstock, while drastically decreased the production of the polyunsaturated.

Bacillus megaterium has become increasingly important for the biotechnological production of valuable compounds of industrial and pharmaceutical importance. Despite recent advances in rational strain design of B. megaterium, these studies have been largely impaired by the lack of molecular tools that are not state-of-the-art for comprehensive genome engineering approaches. In the current work, we describe the adaptation of the CRISPR-Cas9 vector pJOE8999 to enable efficient genome editing in B. megaterium. Crucial modifications comprise the exchange of promoter elements and associated ribosomal binding sites as well as the implementation of a 5-fluorouracil based counterselection system to facilitate proper plasmid curing. In addition, the functionality and performance of the new CRISPR-Cas9 vector pMOE was successfully evaluated by chromosomal disruption studies of the endogenous β-galactosidase gene (BMD_2126) and demonstrated an outstanding efficiency of 100 % based on combinatorial pheno- and genotype analyses. Furthermore, pMOE was applied for the genomic deletion of a steroid esterase gene (BMD_2256) that was identified among several other candidates as the gene encoding the esterase, which prevented accumulation of pharmaceutically important glucocorticoid esters. Recombinant expression of the bacterial chloramphenicol acetyltransferase 1 gene (cat1) in the resulting esterase deficient B. megaterium strain ultimately yielded C21-acetylated as well as novel C21-esterified derivates of cortisone.

Soil salinity is one of the critical issue worldwide that adversely affect soil fertility. Salt stress significantly limits crop yield and grain quality; therefore, there is an urgent need to develop a strategy to improve salt stress tolerance. In present study, we reported that rice glutaredoxin (OsGrx_C7) plays a positive response in salt induced stress. Gene expression analysis, silencing, and overexpression of OsGrx_C7 gene were used to discover the role of OsGrx_C7 in response to salt stress. Gene expression analysis suggested that OsGrx_C7 expression was induced under salt stress and ubiquitously expressed in rice including root and shoot. The silencing of osgrx_c7 gene leads to increased sensitivity to salt stress, indicating its importance in salt stress tolerance. A gain-of-function approach showed that OsGrx_C7 may act as an important determinant in salt stress, compared with WT, and revealed higher biomass accumulation, improved root and plant growth under salt stress. Under salt stress condition, OsGrx_C7 overexpressing rice plants showed lower level of lipid peroxidation and Na+/K+ ratio, while proline accumulation, soluble sugar content and GSH/GSSG ratio was higher compared to WT. Furthermore, expression analysis suggested that OsGrx_C7 acted as positive regulator of salt tolerance by reinforcing the expression of transporters (OsHKT2;1, OsHKT1;5 and OsSOS1) engaged in Na+ homeostasis in overexpressing plants. Overall our study revealed that OsGrx_C7 emerged as a key mediator in response to salt stress in rice and could be used for engineering tolerance against salt stress in rice and other crops.

The genus Thialkalivibrio plays an essential role in the biological desulfurization system. However, to date, the sulfur oxidation pathways of Thialkalivibrio are not clearly understood. Here, we performed transcriptomic analysis on Thialkalivibrio versutus D301 with either thiosulfate or chemical sulfur as the sulfur source to understand it. The results show that T. versutus D301 has a higher growth rate and sulfur oxidation activity when thiosulfate is utilized. The use of chemical sulfur as sulfur source leads to decreased expression of genes involved in carbon metabolism, ribosome synthesis and oxidative phosphorylation in T. versutus D301. Potentially due to the adsorption to sulfur particles, the genes related to flagellum assembly and motivation are significantly induced in T. versutus D301 in the presence of chemical sulfur. In the periplasm, both thiosulfate and polysulfide from the chemical sulfur are oxidized to sulfate via the similar truncated Sox system (SoxAXYZB). Then, part of polysulfide reached to cytoplasm through an unidentified route is oxidized to sulfite by the Dsr-like system. The sulfite in the cytoplasm is further catalyzed to sulfate by SoxB or SoeABC. Overall, the difference in the oxidation rates of D301 can be mainly attributed to the bioavailability of the two sulfur sources, not the sulfur oxidation pathways.

The present work reveals zinc oxide nanorods (ZnO NRs) obtained via green synthesis approach utilizing phytochemicals present in the Leea asiatica plant. The synthesized ZnO NRs was confirmed by various characterization methods like X-ray diffraction, FTIR, SEM-EDX, UV-vis, TEM and Raman spectra analysis. The obtained nanorods show less particle size with a distinctive high surface area. The photodegradation activity of obtained ZnO NRs shows 81.3 % of dye degradation at high concentration whereas 42.9 % in low concentration and also antioxidant test shows a better result for free radical scavenging. To understand the safety of the ZnO NRs zebrafish embryos was utilized as a model and found to be less toxic after assessment. Furthermore, to understand the anticancer activity of ZnO NRs it was assessed against breast cancer cell line through MTT, XTT, NRU, and LDH assay. The ZnO NRs exposed against breast cancer cell line (MCF-7) had resulted in necrosis and apoptosis which was further confirmed by genotoxicity assay. Also, the molecular mechanisms behind the necrosis and apoptosis are also discussed to show the involvement of ZnO NRs towards breast cancer cells (MCF-7). The present study on ZnO NRs gives a wide spectrum understanding of using ZnO NRs as a potential drug for human breast cancer by showing its physiological pathways as well as a novel way to synthesize ZnO NRs using Leea asiatica plant extract.

FT-IR spectroscopy is a non-destructive testing technique that requires minimal sample preparation time and allows the rapid characterization of structural features of complex, polymeric material. This technique has been frequently used in the past for the detection of PHA (Polyhydroxyalkanoates) but rarely for their quantification. In this work, by using cluster and discriminant statistical analysis of FT-IR data, different models are proposed for rapid identification of PHA monomers produced under different growth conditions by bacterial strains, and for their semi quantification. The results on the ability to produce large amounts of PHA (of 21 strains) in different environmental conditions of medium, substrates and deficiency of nutrients are presented. The spectral data analysis gives qualitative and semi quantitative information about the PHA produced in the samples. Models are proposed to test a large number of cultural conditions of strains and substrates in the field of screening and for identifying best conditions of PHA production in lab scale bioreactor and on industrial scale.

Cancer immunotherapy is a promising intervention to fight against this global health problem. In particular targeting immune checkpoints, such as cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and programmed-death protein 1 (PD-1), by specific monoclonal antibodies is a current treatment for many malignances. A possible innovation in this field is based on the induction of humoral responses in the host by suppressing the effects of such immune checkpoints and as consequence favoring the activation of cellular immunity against the tumor cells. In this study, chimeric protein comprising the B subunit of Escherichia coli heat-labile enterotoxin as carrier and the extracellular domain of CTLA-4 (LTB-CTLA4) was produced in Nicotiana benthamiana by transient expression. The recombinant protein was accumulated up to 1.29 μg/g of leaves fresh weight on 4 day-post-infiltration. The integrity of the plant-made LTB-CTLA4 antigen was confirmed by western blot analysis and ELISA. Immunogenicity of the plant-made LTB-CTLA4 was assessed in BALB/c mice and the results showed that humoral responses were induced against both the LTB and CTLA-4 moieties. The plant-made LTB-CTLA4 stands as a promising candidate for the design of advanced protection studies against cancer in murine models.

Trypsin, chymotrypsin, penicillin G acylase and ficin extract have been stabilized by immobilization on glyoxyl agarose, adding different aliphatic compounds bearing a primary amine group during the immobilization: ethyl amine, butyl amine, hexyl amine (at concentrations ranging from 0 to 20 mM) and octyl amine (from 0 to 10 mM) to analyze their effects on the immobilized enzyme stability. As expected, the presence of amines reduced the intensity of the enzyme-support multipoint covalent attachment, and therefore the enzyme stability. However, it is clear that this effect is higher using octyl amine for all enzymes (in some cases the enzyme immobilized in the presence of 10 mM octyl amine was almost inactivated while the reference kept over 50 % of the initial activity). This way, it seems that the most important effect of the presence of aminated compounds came from the generation of steric hindrances to the enzyme/support multi-reaction promoted by the ammines that are interacting with the aldehyde groups. In some instances, just 1 mM of aminated compounds is enough to greatly decrease enzyme stability. The results suggested that, if the composition of the enzyme extract is unknown, to eliminate small aminated compounds may be necessary to maximize the enzyme-support reaction.

Achieving optimal productivity and desired product quality of the therapeutic monoclonal antibody (mAb) is one of the primary goals of process development. Across the various mAb programs at our company, we observed that increasing the specific productivity (qp) results in a decrease in the % galactosylation (%Gal) level on the protein. In order to gain further insight into this correlation, cells were cultured under different process conditions such as pH or media osmolality or in the presence of supplements such as sodium butyrate. A range of qp and N-glycan profiles were obtained with the greatest changes observed under high pH (lower qp, higher %Gal), higher osmolality (higher qp, lower %Gal) or sodium butyrate (moderately higher qp, moderately lower %Gal) conditions. Abundance of individual glycan species highlighted different bottlenecks in the N-glycosylation pathway depending on the treatment condition. Transcriptomics analysis was performed to identify changes in gene expression profiles that correlate with the inverse relationship between qp and %Gal. Results showed downregulation of Beta-1,4-galactosyltransferase 1 (B4GalT1), UDP-GlcNAc and Mn2+ transporter (slc35a3 and slc39a8 respectively) for the high osmolality conditions. Significant downregulation of slc39a8 (Mn2+ transporter) was observed for the sodium butyrate condition. No significant differences were observed for any of the genes in the N-glycosylation pathway under the high pH condition even though this condition showed highest %Gal. Together, data suggests that different treatments have distinct complex mechanisms by which the overall glycan levels of a mAb are influenced. Further studies based on these results will help build the knowledge necessary to design strategies to obtain the desired productivity and product quality of mAbs.