An inclusive outlook on the fate and persistence of pesticides in the environment and integrated eco-technologies for their degradation.

Intensive and inefficient exploitation of pesticides through modernized agricultural practices has caused severe pesticide contamination problems to the environment and become a crucial problem over a few decades. Due to their highly toxic and persistent properties, they affect and get accumulated in non-target organisms, including microbes, algae, invertebrates, plants as well as humans, and cause severe issues. Considering pesticide problems as a significant issue, researchers have investigated several approaches to rectify the pesticide contamination problems. Several analyses have provided an extensive discussion on pesticide degradation but using specific technology for specific pesticides. However, in the middle of this time, cleaner techniques are essential for reducing pesticide contamination problems safely and environmentally friendly. As per the research findings, no single research finding provides concrete discussion on cleaner tactics for the remediation of contaminated sites. Therefore, in this review paper, we have critically discussed cleaner options for dealing with pesticide contamination problems as well as their advantages and disadvantages have also been reviewed. As evident from the literature, microbial remediation, phytoremediation, composting, and photocatalytic degradation methods are efficient and sustainable and can be used for treatment at a large scale in engineered systems and in situ. However, more study on the bio-integrated system is required which may be more effective than existing technologies.

A multi-plex protein expression system for production of complex enzyme formulations in Trichoderma reesei.

Heterologous protein production has been challenging in the hyper-cellulolytic fungus, Trichoderma reesei as the species is known for poor transformation efficiency, low homologous recombination frequency, and marginal screening systems for the identification of successful transformants. We have applied the 2A-peptide multi-gene expression system to co-express four proteins, which include three cellulases: a cellobiohydrolase (CBH1), an endoglucanase (EG1), and a ß-D-glucosidase (BGL1), as well as the enhanced green fluorescent protein (eGFP) marker protein. We designed a new chassis vector, pTrEno-4X-2A, for this work. Expression of these cellulase enzymes was confirmed by real-time quantitative reverse transcription PCR and immunoblot analysis. The activity of each cellulase was assessed using chromogenic substrates, which confirmed the functionality of the enzymes. Expression and activity of these enzymes were proportional to the level of eGFP fluorescence, thereby validating the reliability of this screening technique. An 18-fold differencein protein expression was observed between the first and third genes within the 2A-peptide construct. The availability of this new multi-gene expression and screening tool is expected to greatly impact multi-enzyme applications, such as the production of complex commercial enzyme formulations and metabolic pathway enzymes, especially those destined for cell-free applications.

Factors regulating cellulolytic gene expression in filamentous fungi: an overview.

The growing demand for biofuels such as bioethanol has led to the need for identifying alternative feedstock instead of conventional substrates like molasses, etc. Lignocellulosic biomass is a relatively inexpensive feedstock that is available in abundance, however, its conversion to bioethanol involves a multistep process with different unit operations such as size reduction, pretreatment, saccharification, fermentation, distillation, etc. The saccharification or enzymatic hydrolysis of cellulose to glucose involves a complex family of enzymes called cellulases that are usually fungal in origin. Cellulose hydrolysis requires the synergistic action of several classes of enzymes, and achieving the optimum secretion of these simultaneously remains a challenge. The expression of fungal cellulases is controlled by an intricate network of transcription factors and sugar transporters. Several genetic engineering efforts have been undertaken to modulate the expression of cellulolytic genes, as well as their regulators. This review, therefore, focuses on the molecular mechanism of action of these transcription factors and their effect on the expression of cellulases and hemicellulases.

Generating genomic platforms to study Candida albicans pathogenesis.

The advent of the genomic era has made elucidating gene function on a large scale a pressing challenge. ORFeome collections, whereby almost all ORFs of a given species are cloned and can be subsequently leveraged in multiple functional genomic approaches, represent valuable resources toward this endeavor. Here we provide novel, genome-scale tools for the study of Candida albicans, a commensal yeast that is also responsible for frequent superficial and disseminated infections in humans. We have generated an ORFeome collection composed of 5099 ORFs cloned in a Gateway™ donor vector, representing 83% of the currently annotated coding sequences of C. albicans. Sequencing data of the cloned ORFs are available in the CandidaOrfDB database at http://candidaorfeome.eu. We also engineered 49 expression vectors with a choice of promoters, tags and selection markers and demonstrated their applicability to the study of target ORFs transferred from the C. albicans ORFeome. In addition, the use of the ORFeome in the detection of protein-protein interaction was demonstrated. Mating-compatible strains as well as Gateway™-compatible two-hybrid vectors were engineered, validated and used in a proof of concept experiment. These unique and valuable resources should greatly facilitate future functional studies in C. albicans and the elucidation of mechanisms that underlie its pathogenicity.

The Zymoseptoria tritici ORFeome: A Functional Genomics Community Resource.

Libraries of protein-encoding sequences can be generated by identification of open reading frames (ORFs) from a genome of choice that are then assembled into collections of plasmids termed ORFeome libraries. These represent powerful resources to facilitate functional genomic characterization of genes and their encoded products. Here, we report the generation of an ORFeome for Zymoseptoria tritici, which causes the most serious disease of wheat in temperate regions of the world. We screened the genome of strain IP0323 for high confidence gene models, identifying 4,075 candidates from 10,933 predicted genes. These were amplified from genomic DNA, were cloned into the Gateway entry vector pDONR207, and were sequenced, providing a total of 3,022 quality-controlled plasmids. The ORFeome includes genes predicted to encode effectors (n = 410) and secondary metabolite biosynthetic proteins (n = 171) in addition to genes residing at dispensable chromosomes (n = 122) or those that are preferentially expressed during plant infection (n = 527). The ORFeome plasmid library is compatible with our previously developed suite of Gateway destination vectors, which have various combinations of promoters, selection markers, and epitope tags. The Z. tritici ORFeome constitutes a powerful resource for functional genomics and offers unparalleled opportunities to understand the biology of Z. tritici.[Formula: see text] Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Engineering cellulases for conversion of lignocellulosic biomass.

Lignocellulosic biomass is a renewable source of energy, chemicals and materials. Many applications of this resource require the depolymerization of one or more of its polymeric constituents. Efficient enzymatic depolymerization of cellulose to glucose by cellulases and accessory enzymes such as lytic polysaccharide monooxygenases is a prerequisite for economically viable exploitation of this biomass. Microbes produce a remarkably diverse range of cellulases, which consist of glycoside hydrolase (GH) catalytic domains and, although not in all cases, substrate-binding carbohydrate-binding modules (CBMs). As enzymes are a considerable cost factor, there is great interest in finding or engineering improved and robust cellulases, with higher activity and stability, easy expression, and minimal product inhibition. This review addresses relevant engineering targets for cellulases, discusses a few notable cellulase engineering studies of the past decades and provides an overview of recent work in the field.

Functional Characterization of a Novel Oxidative Stress Protection Protein in the Pathogenic Yeast Candida glabrata.

Candida species are important pathogens of humans and the fourth most commonly isolated pathogen from nosocomial blood stream infections. Although Candida albicans is the principle causative agent of invasive candidosis, the incidence of Candida glabrata infections has rapidly grown. The reason for this increase is not fully understood, but it is clear that the species has a higher innate tolerance to commonly administered azole antifungals, in addition to being highly tolerant to stresses especially oxidative stress. Taking the approach that using the model organism, Saccharomyces cerevisiae, with its intrinsic sensitivity to oxidative stress, we hypothesized that by expressing mediators of stress resistance from C. glabrata in S. cerevisiae, it would result in induced resistance. To test this we transformed, en-masse, the C. glabrata ORFeome library into S. cerevisiae. This resulted in 1,500 stress resistant colonies and the recovered plasmids of 118 ORFs. Sequencing of these plasmids revealed a total of 16 different C. glabrata ORFs. The recovery of genes encoding known stress protectant proteins such as GPD1, GPD2 and TRX3 was predicted and validated the integrity of the screen. Through this screen we identified a C. glabrata unique ORF that confers oxidative stress resistance. We set to characterise this gene herein, examining expression in oxidative stress sensitive strains, comet assays to measure DNA damage and synthetic genetic array analysis to identify genetic interaction maps in the presence and absence of oxidative stress.

Phylogenetics-based identification and characterization of a superior 2,3-butanediol dehydrogenase for Zymomonas mobilis expression.

BACKGROUND: Zymomonas mobilis has recently been shown to be capable of producing the valuable platform biochemical, 2,3-butanediol (2,3-BDO). Despite this capability, the production of high titers of 2,3-BDO is restricted by several physiological parameters. One such bottleneck involves the conversion of acetoin to 2,3-BDO, a step catalyzed by 2,3-butanediol dehydrogenase (Bdh). Several Bdh enzymes have been successfully expressed in Z. mobilis, although a highly active enzyme is yet to be identified for expression in this host. Here, we report the application of a phylogenetic approach to identify and characterize a superior Bdh, followed by validation of its structural attributes using a mutagenesis approach. RESULTS: Of the 11 distinct bdh genes that were expressed in Z. mobilis, crude extracts expressing Serratia marcescens Bdh (SmBdh) were found to have the highest activity (8.89 µmol/min/mg), when compared to other Bdh enzymes (0.34-2.87 µmol/min/mg). The SmBdh crystal structure was determined through crystallization with cofactor (NAD+) and substrate (acetoin) molecules bound in the active site. Active SmBdh was shown to be a tetramer with the active site populated by a Gln247 residue contributed by the diagonally opposite subunit. SmBdh showed a more extensive supporting hydrogen-bond network in comparison to the other well-studied Bdh enzymes, which enables improved substrate positioning and substrate specificity. This protein also contains a short α6 helix, which provides more efficient entry and exit of molecules from the active site, thereby contributing to enhanced substrate turnover. Extending the α6 helix to mimic the lower activity Enterobacter cloacae (EcBdh) enzyme resulted in reduction of SmBdh function to nearly 3% of the total activity. In great contrast, reduction of the corresponding α6 helix of the EcBdh to mimic the SmBdh structure resulted in ~ 70% increase in its activity. CONCLUSIONS: This study has demonstrated that SmBdh is superior to other Bdhs for expression in Z. mobilis for 2,3-BDO production. SmBdh possesses unique structural features that confer biochemical advantage to this protein. While coordinated active site formation is a unique structural characteristic of this tetrameric complex, the smaller α6 helix and extended hydrogen network contribute towards improved activity and substrate promiscuity of the enzyme.

Engineering enhanced cellobiohydrolase activity.

Glycoside Hydrolase Family 7 cellobiohydrolases (GH7 CBHs) catalyze cellulose depolymerization in cellulolytic eukaryotes, making them key discovery and engineering targets. However, there remains a lack of robust structure-activity relationships for these industrially important cellulases. Here, we compare CBHs from Trichoderma reesei (TrCel7A) and Penicillium funiculosum (PfCel7A), which exhibit a multi-modular architecture consisting of catalytic domain (CD), carbohydrate-binding module, and linker. We show that PfCel7A exhibits 60% greater performance on biomass than TrCel7A. To understand the contribution of each domain to this improvement, we measure enzymatic activity for a library of CBH chimeras with swapped subdomains, demonstrating that the enhancement is mainly caused by PfCel7A CD. We solve the crystal structure of PfCel7A CD and use this information to create a second library of TrCel7A CD mutants, identifying a TrCel7A double mutant with near-equivalent activity to wild-type PfCel7A. Overall, these results reveal CBH regions that enable targeted activity improvements.

Controlled antibiotic-releasing Antheraea assama silk fibroin suture for infection prevention and fast wound healing.

BACKGROUND: The quest for developing silk fibroin as a biomaterial for drug release systems continues to draw research interest owing to its impressive mechanical properties as well as biocompatibility and biodegradability. The aim of this study is to develop low-temperature O2 plasma-treated muga (Antheraea assama) silk fibroin (AASF) yarn impregnated with amoxicillin trihydrate as controlled antibiotic-releasing suture (AASF/O2/AMOX) for preventing postoperative site bacterial infection and fast wound healing. METHODS: In this experimental study, AASF and AASF/O2/AMOX sutures are used to close the surgical wounds of adult male Wistar rats of 4 months old and weighing 200-230 g. RESULTS: Surface hydrophilicity induced by O2 plasma results in an increase in drug-impregnation efficiency of AASF/O2 yarn by 16.7%. In vitro drug release profiles show continuous and prolonged release of AMOX from AASF/O2/AMOX yarn up to 336 hours. In vitro hemolysis assay reveals that O2 plasma treatment and subsequent impregnation of AMOX do not affect the heertetmocompatibility of AASF yarn. The AASF/O2/AMOX yarn proves to be effective for in vitro growth inhibition of Staphylococcus aureus and Escherichia coli, whereas AASF offers no antibacterial activity against both types of bacteria. In vivo histopathology studies and colony-forming unit count data revealed accelerated wound healing activity of AASF/O2/AMOX over AASF yarn through rapid synthesis and proliferation of collagen, hair follicle, and connective tissues. CONCLUSION: Outcomes of this work clearly demonstrate the potential use of AASF/O2/AMOX yarn as a controlled antibiotic-releasing suture biomaterial for superficial surgical applications.

Penicillin impregnation on oxygen plasma surface functionalized chitosan/Antheraea assama silk fibroin: Studies of antibacterial activity and antithrombogenic property.

Low temperature plasma can effectively tailor the surface properties of natural polymeric biomaterials according to the need for various biomedical applications. Non-mulberry silk, Antheraea assama silk fibroin (AASF) is a natural polymer having excellent biocompatibility and mechanical strength yet unlike mulberry silk, Bombyx mori silk fibroin, has drawn less interest in biomedical research. In the quest for developing as potential biomaterial, surface functionalization of plasma induced chitosan (Cs) grafted AASF ((AASF/O2-CS)g/O2) yarn is carried out using oxygen (O2) plasma. The (AASF/O2-CS)g/O2 yarn exhibits enhanced antithrombogenic property as well as antimicrobial activity against Gram positive (Bacillus subtilis) and Gram negative (Escherichia coli) bacteria as compared to AASF yarn. Moreover, impregnation of antibiotic drug (penicillin G sodium salt, PEN) on (AASF/O2-CS)g/O2 yarn further improves the observed properties. In-vitro hemolysis assay reveals that O2 plasma treatment and subsequent impregnation of PEN do not affect the hemocompatibility of AASF yarn. The present research findings demonstrate that plasma induced grafting of Cs followed by penicillin impregnation could significantly improve the potential applicability of AASF in the field of surgical research.

Immobilization of trypsin on plasma prepared Ag/PPAni nanocomposite film for efficient digestion of protein.

This work demonstrates the efficacy of a support matrix prepared by plasma process for trypsin immobilization without any surface activator. Plasma polymerization cum sputtering process is used to prepare the nanocomposite support matrix. Plasma sputtered silver nanoparticles (AgNPs) are uniformly embedded into plasma polymerized aniline (PPAni) film. Various characterization tools are employed to study the surface morphology, microstructure and chemical composition of the support matrices. Trypsin is immobilized onto the support matrix via the formation of covalent bond between them. Plasma generated free radicals on composite films activate the support matrix and make it efficient for increasing the tertiary enzyme stability via multipoint covalent attachment. Trypsin immobilized onto Ag/PPAni matrix has more hydrolyzing capacity of bovine serum albumin (BSA) than free trypsin as well as trypsin immobilized onto PPAni films.

Modular gene over-expression strategies for Candida albicans.

Over-expression is a valid functional genomics approach to characterise genes of unknown function on a genome-wide scale. Strains are engineered to over-express a specific gene and the resulting gain-of-function phenotype assessed. Here, we describe the strategy we are adopting to synthesise a Candida albicans ORFeome collection and the options available to create over-expressing strains from this collection.

Laparoscopic pelvic exenteration for advanced pelvic cancers: a review of 16 cases.

OBJECTIVE: The aim of this study was to retrospectively evaluate, in a series of 16 consecutive patients, the technique, feasibility and oncological safety of laparoscopic anterior exenteration for locally advanced pelvic cancers. STUDY DESIGN: Since August 2003, 16 patients with locally advanced pelvic cancer were considered. All patients were in a good general condition, in the age group of 50-60 years of which 12 had cervical carcinoma and 4 had bladder carcinoma. RESULTS: The median operative time was 180 min. The mean number of harvested pelvic iliac nodes was 14. All margins were tumor-free. The median postoperative hospital stay was 3 days. Three patients had postoperative complications; two had subacute intestinal obstruction and one had ureteric leak. The median follow-up was 15 months. CONCLUSIONS: Our results have demonstrated the feasibility and oncological safety of performing anterior exenteration laparoscopically in advanced pelvic cancer patients with acceptable morbidity. Intermediate-term follow-up validates the adequacy of this procedure.