Protein engineering of glutathione transferase for the development of optical biosensor to detect xenobiotics

Glutathione transferases (GSTs, EC. 2.5.1.18) are inducible enzymes that play essential role in detoxification and degradation of toxic compounds, including pesticides. The purpose of the present study is the development of an optical enzyme biosensor based on GSTs, for the detection and determination of pesticides in environmental samples. Protein engineering was used for the creation of a GST variant with higher selectivity towards pesticides. cDNA libraries were created from Phaseolus vulgaris and Glycine max stressed plants using degenerated primers and reverse transcription-PCR. Large diversity in GST genes was accomplished employing directed evolution through DNA shuffling of a mixture of GST genes from P. vulgaris and G. max stressed plants. The shuffled library of chimaeric GST genes was cloned in E. coli expression plasmid. Screening of the library led to the isolation of a novel GST enzyme that displays both glutathione transferase and glutathione peroxidase activities. The enzyme was purified by affinity chromatography and characterized by kinetic analysis towards 20 different substrates and 66 different pesticides. The results showed that the organoclorine insecticides and strobilurins (fungicides) are strong inhibitors of the enzyme. The specificity of the enzyme towards pesticides was further improved using site-saturation mutagenesis at position Phe117. The mutant Phe117Ile displays 5-fold higher catalytic efficiency and selectivity towards organochlorine insecticides. Therefore, the mutant GSTPhe117Ile was used for the development of an optical biosensor. The enzyme was immobilized in alkoxysilane (TEOS/PTMOS) sol-gel system in the presence of the pH indicators bromocresol purple (acidic) and phenol red (basic). The bioactive material exhibits linearity in the range of 0.625-30 μΜ α-endosulfan (pH=4-7) at 562 nm and was used for the development of an analytical method for the determination of α-endosulfan in environmental samples

Efficient Hierarchical Domain Adaptation for Pretrained Language Models

The remarkable success of large language models has been driven by dense models trained on massive unlabeled, unstructured corpora. These corpora typically contain text from diverse, heterogeneous sources, but information about the source of the text is rarely used during training. Transferring their knowledge to a target domain is typically done by continuing training in-domain. In this paper, we introduce a method to permit domain adaptation to many diverse domains using a computationally efficient adapter approach. Our method is based on the observation that textual domains are partially overlapping, and we represent domains as a hierarchical tree structure where each node in the tree is associated with a set of adapter weights. When combined with a frozen pretrained language model, this approach enables parameter sharing among related domains, while avoiding negative interference between unrelated ones. Experimental results with GPT-2 and a large fraction of the 100 most represented websites in C4 show across-the-board improvements in-domain. We additionally provide an inference time algorithm for a held-out domain and show that averaging over multiple paths through the tree enables further gains in generalization, while adding only a marginal cost to inference.Comment: NAACL 2022 accepted paper camera ready versio

Cloning and Characterization of a Biotic-Stress-Inducible Glutathione Transferase from Phaseolus vulgaris

Glutathione transferases (GSTs, EC 2.5.1.18) are ubiquitous proteins in plants that play important roles in stress tolerance and in the detoxification of toxic chemicals and metabolites. In this study, we systematically examined the catalytic diversification of a GST isoenzyme from Phaseolus vulgaris (PvGST) which is induced under biotic stress treatment (Uromyces appendiculatus infection). The full-length cDNA of this GST isoenzyme (termed PvGSTU3-3) with complete open reading frame, was isolated using RACE-RT and showed that the deduced amino acid sequence shares high homology with the tau class plant GSTs. PvGSTU3-3 catalyzes several different reactions and exhibits wide substrate specificity. Of particular importance is the finding that the enzyme shows high antioxidant catalytic function and acts as hydroperoxidase, thioltransferase, and dehydroascorbate reductase. In addition, its Km for GSH is about five to ten times lower compared to other plant GSTs, suggesting that PvGSTU3-3 is able to perform efficient catalysis under conditions where the concentration of reduced glutathione is low (e.g., oxidative stress). Its ability to conjugate GSH with isothiocyanates may provide an additional role for this enzyme to act as a regulator of the released isothiocyanates from glucosinolates as a response of biotic stress. Molecular modeling showed that PvGSTU3-3 shares the same overall fold and structural organization with other plant cytosolic GSTs, with major differences at their hydrophobic binding sites (H-sites) and some differences at the level of C-terminal domain and the linker between the C- and N-terminal domains. PvGSTU3-3, in general, exhibits restricted ability to bind xenobiotics in a nonsubstrate manner, suggesting that the biological role of PvGSTU3-3, is restricted mainly to the catalytic function. Our findings highlight the functional and catalytic diversity of plant GSTs and demonstrate their pivotal role for addressing biotic stresses in Phaseolus vulgaris

AdapterSoup: Weight Averaging to Improve Generalization of Pretrained Language Models

Pretrained language models (PLMs) are trained on massive corpora, but often need to specialize to specific domains. A parameter-efficient adaptation method suggests training an adapter for each domain on the task of language modeling. This leads to good in-domain scores but can be impractical for domain- or resource-restricted settings. A solution is to use a related-domain adapter for the novel domain at test time. In this paper, we introduce AdapterSoup, an approach that performs weight-space averaging of adapters trained on different domains. Our approach is embarrassingly parallel: first, we train a set of domain-specific adapters; then, for each novel domain, we determine which adapters should be averaged at test time. We present extensive experiments showing that AdapterSoup consistently improves performance to new domains without extra training. We also explore weight averaging of adapters trained on the same domain with different hyper-parameters, and show that it preserves the performance of a PLM on new domains while obtaining strong in-domain results. We explore various approaches for choosing which adapters to combine, such as text clustering and semantic similarity. We find that using clustering leads to the most competitive results on novel domains.Comment: Accepted at EACL 2023; camera-ready versio

Molecular and catalytic characterization of the herbicide-inducible glutathione transferases from Phaseolus vulgaris

Plant glutathione transferases (GSTs) comprise a large family of inducible enzymes that play important roles in stress tolerance and herbicide detoxification. Treatment of Phaseolus vulgaris leaves with the aryloxyphenoxypropionic herbicide fluazifop-p-butyl resulted in induction of GST activities. Three inducible GST isoenzymes were identified and separated by affinity chromatography. Their full-length cDNAs with complete open reading frame were isolated using RACE-RT and information from N-terminal amino acid sequences. Analysis of the cDNA clones showed that the deduced amino acid sequences share high homology with GSTs that belong to phi and tau classes. The three isoenzymes were expressed in E. coli and their substrate specificity was determined towards 20 different substrates. The results showed that the fluazifop-inducible glutathione transferases from P. vulgaris (PvGSTs) catalyze a broad range of reactions and exhibit quite varied substrate specificity. Molecular modeling and structural analysis was used to identify key structural characteristics and to provide insights into the substrate specificity and the catalytic mechanism of these enzymes. These results provide new insights into catalytic and structural diversity of GSTs and the detoxifying mechanism used by P. vulgaris

Inhibition of human glutathione transferases by pesticides: Development of a simple analytical assay for the quantification of pesticides in water

Glutathione transferases (GSTs; EC 2.5.1.18) form a group of multifunctional enzymes that are involved in phase II cellular detoxification mechanism. Here, screening of the inhibition potency of a wide range of pesticides toward selected human GST isoenzymes (hGSTA1-1, hGSTP1-1, hGSTT2-2 and hGSTO1-1) was carried out. hGSTA1-1 was found more susceptible to inhibition by pesticides than other isoenzymes. The insecticides dieldrin and spiromesifen were identified as potent reversible inhibitors toward hGSTA1- 1 with IC50 values equal to 17.9 ± 1.7 M and 12.1 ± 3.4 M, respectively. Based on in silico docking analysis and kinetic inhibition studies it was concluded that dieldrin and spiromesifen bind specifically to the enzyme presumably at a distinct position that partially overlaps with both the G- and H-site. The ability of dieldrin and spiromesifen to inhibit hGSTA1-1 activity was exploited for the development of analytical quantification assays for these two pesticides. Linear calibration curves were obtained for dieldrin and spiromesifen, with useful concentration in the range of 0–10 M. The reproducibility of the assay response, expressed by relative standard deviation, was in the order of 4.1% (N = 28). The method was successfully applied to the determination of these pesticides in real water samples without sample preparation steps

Metabarcoding successfully tracks temporal changes in eukaryotic communities in coastal sediments

Metabarcoding is a method that combines high-throughput DNA sequencing and DNA-based identification. Previously, this method has been successfully used to target spatial variation of eukaryote communities in marine sediments, however, the temporal changes in these communities remain understudied. Here, we follow the temporal changes of the eukaryote communities in Baltic Sea surface sediments collected from two coastal localities during three seasons of two consecutive years. Our study reveals that the structure of the sediment eukaryotic ecosystem was primarily driven by annual and seasonal changes in prevailing environmental conditions, whereas spatial variation was a less significant factor in explaining the variance in eukaryotic communities over time. Therefore, our data suggests that shifts in regional climate regime or large-scale changes in the environment are the overdriving factors in shaping the coastal eukaryotic sediment ecosystems rather than small-scale changes in local environmental conditions or heterogeneity in ecosystem structure. More studies targeting temporal changes are needed to further understand the long-term trends in ecosystem stability and response to climate change. Furthermore, this work contributes to the recent efforts in developing metabarcoding applications for environmental biomonitoring, proving a comprehensive option for traditional monitoring approaches.Peer reviewe

Sol-gel immobilization of glutathione transferase: efficient tool for bioremediation

Glutathione transferases are multi-functional enzymes with an important role in xenobiotic detoxification. They catalyse the nucleophilic addition of the sulfur atom of glutathione (γ-L-Glu-L-Cys-Gly, GSH) to the electrophilic groups of a large variety of hydrophobic molecules including organic halides, epoxides, arene oxides, α- and β-unsaturated carbonyls, organic nitrate esters, and organic thiocyanates. The conjugation of GSH to such molecules increases their solubility and reduces their toxicity. GSTs represent a versatile tool with a variety of biotechnological applications, in the field of bioremediation to clean up environmentally contaminated sites. The purpose of this project was the study of GST immobilization for the biodegradation of toxic compounds

Structure and catalytic properties of human glutathione transferase p1-1

Glutathione transferases (EC 2.5.1.18, GSTs) catalyze the nucleophilic attack of glutathione (GSH) on the electrophilic centre of a number of electrophilic compounds helping to detoxify a diverse array of toxic xenobiotics including carcinogenic, and pharmacologically active compounds. In this review, detailed descriptions are given on the structure and catalytic properties of human glutathione transferase P1-1 (hGSTP1-1) an enzyme that ubiquitously expressed in human tissues and exhibits many biological functions and multiple roles. The detoxification properties of hGSTP1-1 have been a primary research focus for the last years. However, now it has become apparent that the noncatalytic functions of GSTP1-1 have expanded the biological roles of this enzyme in cell survival, cell death and stress signalling mechanism

Preconceptual care for couples seeking fertility treatment, an evidence-based approach

There is accumulating evidence demonstrating that positive lifestyle modification and the optimization of the preconceptual period can influence the reproductive potential for both men and women. However, a large percentage of couples attending fertility clinics with potential to improve preconception habits may not always receive appropriate preconceptual advice. In addition, supplements and adjuncts that promise to increase fertility treatment success rates are marketed to infertile patients despite lack of convincing evidence supporting their benefit. This review aims to identify possible associations between lifestyle factors for couples seeking fertility treatment and fertility treatment outcomes and to offer possible explanations of the biological basis of these associations. An electronic search was conducted from 1978 through July 2019 linking preconceptual behaviors for women and men with the outcome of fertility treatment. The literature search explored the importance of numerous factors, including smoking, caffeine, alcohol, obesity, physical exercise, recreational drugs, stress, diet, supplements, alternative medicine, environmental factors, and pollutants. Some associations were found to be more significant than others. The preconceptual period is undeniably a delicate and important window which should not be overlooked during fertility counseling. Simple lifestyle modifications could positively influence fertility treatment outcomes. Fertility teams, consisting of clinicians, fertility nurses, dieticians, psychologists, exercise advisors and others, should dedicate time to offer evidence-based preconceptual advice and targeted interventions to couples seeking fertility treatment