Delta Class Research Articles

Naphthalene and phenanthrene affect differentially two glutathione S-transferases (GSTs) expression, GST activity, and glutathione content in white shrimp P. vannamei

Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants ubiquitous in coastal ecosystems. The white shrimp Penaeus vannamei naturally inhabits in coastal areas and is cultivated in farms located nearby the oceans. PAHs can damage shrimp health, endanger natural populations, and lower shrimp aquaculture productivity. However, crustaceans have enzymes capable of metabolizing organic xenobiotics as PAHs and to neutralize reactive oxygen species (ROS) produced during xenobiotics metabolism. An important superfamily of xenobiotic-metabolizing and antioxidant enzymes are glutathione S-transferases (GSTs). In white shrimp, some GSTs are known, but they have been scarcely studied in response to PAHs. In this study we report the molecular cloning and bioinformatic characterization of two novel nucleotide sequences corresponding to cytosolic GSTs belonging the Delta and Theta classes (GSTD and GSTT). Both proteins genes have tissue-specific patterns of expression under normal conditions, that do not necessarily relate to GST activity and glutathione content. The expression of the GSTD and GSTT, GST activity and glutathione content was analyzed in juvenile P. vannamei exposed to two PAHs, naphthalene (NAP) and phenanthrene (PHE) in sub-lethal concentrations for 96 h. GSTD expression was up-regulated by the two PAHs, while GSTT expression was only induced by NAP. In contrast, GST activity towards CDNB was only up-regulated by PHE, suggesting differential effects of PAHs at gene and protein level. On the other hand, lower reduced glutathione content (GSH) caused by PAHs indicates its utilization for detoxification or antioxidant defenses. However, the GSH/GSSG did not change by PAHs treatment, indicating that shrimp can maintain redox balance during short-term sub-lethal exposure to NAP and PHE. Despite the variations in the responses to NAP and PHE, all these results suggest that the GSTD and GSTT genes could be useful biomarkers for PAH exposure in P. vannamei.

Architecture and potential roles of a delta-class glutathione S-transferase in protecting honey bee from agrochemicals

The European honey bee, Apis mellifera, serves as the principle managed pollinator species globally. In recent decades, honey bee populations have been facing serious health threats from combined biotic and abiotic stressors, including diseases, limited nutrition, and agrochemical exposure. Understanding the molecular mechanisms underlying xenobiotic adaptation of A. mellifera is critical, considering its extensive exposure to phytochemicals and agrochemicals present in the environment. In this study, we conducted a comprehensive structural and functional characterization of AmGSTD1, a delta class glutathione S-transferase (GST), to unravel its roles in agrochemical detoxification and antioxidative stress responses. We determined the 3-dimensional (3D) structure of a honey bee GST using protein crystallography for the first time, providing new insights into its molecular structure. Our investigations revealed that AmGSTD1 metabolizes model substrates, including 1-chloro-2,4-dinitrobenzene (CDNB), p-nitrophenyl acetate (PNA), phenylethyl isothiocyanate (PEITC), propyl isothiocyanate (PITC), and the oxidation byproduct 4-hydroxynonenal (HNE). Moreover, we discovered that AmGSTD1 exhibits binding affinity with the fluorophore 8-Anilinonaphthalene-1-sulfonic acid (ANS), which can be inhibited with various herbicides, fungicides, insecticides, and their metabolites. These findings highlight the potential contribution of AmGSTD1 in safeguarding honey bee health against various agrochemicals, while also mitigating oxidative stress resulting from exposure to these substances.

Structure-activity analysis suggests an olfactory function for the unique antennal delta glutathione transferase of Apis mellifera.

Glutathione transferases (GST) are detoxification enzymes that conjugate glutathione to a wide array of molecules. In the honey bee Apis mellifera, AmGSTD1 is the sole member of the delta class of GSTs, with expression in antennae. Here, we structurally and biochemically characterized AmGSTD1 to elucidate its function. We showed that AmGSTD1 can efficiently catalyse the glutathione conjugation of classical GST substrates. Additionally, AmGSTD1 exhibits binding properties with a range of odorant compounds. AmGSTD1 has a peculiar interface with a structural motif we propose to call 'sulfur sandwich'. This motif consists of a cysteine disulfide bridge sandwiched between the sulfur atoms of two methionine residues and is stabilized by CH…S hydrogen bonds and S…S sigma-hole interactions. Thermal stability studies confirmed that this motif is important for AmGSTD1 stability and, thus, could facilitate its functions in olfaction.

Construction of learning environments: A multiple case study in special education settings

AbstractEducation in segregated settings for pupils with intellectual disability (ID) has often been portrayed as a unified form of schooling. There is a risk of providing a simplified picture of these settings and of what pupils with ID need to learn. This is generally stipulated in policy documents, leaving room for staff in school to interpret curricula and form learning environments. The aim of this study is to explore how four different learning environments are constructed in segregated settings where pupils with ID are educated in Sweden. The study was conducted as a multiple case study, using observations and interviews with teachers and pupils in four special classes for pupils with ID (SCIDs). Drawing on curriculum theory, the four learning environments, in the results named as Alpha, Beta, Gamma and Delta class, respectively, appeared to be characterized by different ideas of schooling, labelled as knowledge‐mediating, socializing, functional life skills and caring as well as teacher‐centred classrooms. The study contributes to a deepened understanding of the complexity of education for pupils with ID in segregated settings.

Functional characterization of SlGSTD3 and SlGSTD4 associated with phoxim and chlorpyrifos resistance in Spodoptera litura

Glutathione S-transferases (GSTs) is one of the main detoxification enzyme systems in insects and play important roles in insecticide resistance by direct metabolism, sequestration and antioxidant activity. Several GSTs genes in Spodoptera litura, a polyphagous agricultural pest, have been demonstrated to be overexpressed and involved in organophosphates and pyrethroids resistance. Previous studies have indicated the significant overexpression of two delta class GSTs genes (SlGSTd3 and SlGSTd4) in organophosphates and pyrethroids resistant populations. Here, they were heterologous expressed, and their metabolism activity and antioxidant activity were determined. Results indicated that the recombinant protein SlGSTD3 and SlGSTD4 both showed metabolism activity to phoxim and chlorpyrifos, but not to fenvalerate, cyhalothrin or beta cypermethrin. The metabolism activity of SlGSTD3 to phoxim and chlorpyrifos is higher than that of SlGSTD4. The recombinant vector of SlGSTD3 and SlGSTD4 both showed antioxidant activity after exposure to cumene hydroperoxide. Further modeling and docking analysis indicated that the 3D structure of SlGSTD3 and SlGSTD4 were well shaped for phoxim and chlorpyrifos, and the binding affinity for phoxim was stronger than that of chlorpyrifos. Our work provides evidence that SlGSTd3 and SlGSTd4 both play roles in phoxim and chlorpyrifos resistance in S. litura.

Functional analysis of an overexpressed glutathione S-transferase BdGSTd5 involved in malathion and malaoxon detoxification in Bactrocera dorsalis

Glutathione S-transferases (GSTs) are one of the three detoxification enzyme families. The constitutive and inducible overexpression of GSTs genes plays an important role in insecticide resistance. Previous study showed that malathion resistance was polygenic, and elevated GSTs activity was one of the important factor participating in malathion resistance of Bactrocera dorsalis (Hendel), a serious economic pest worldwide. BdGSTd5 overexpression was inducible upon exposure to malathion. However, the involvement of BdGSTd5 in malathion resistance has not been clarified. In this study, we found that BdGSTd5 sequence harbored the conserved region of delta class GSTs, which were overexpressed in malathion resistant strain of B. dorsalis compared to malathion susceptible strain. The highest mRNA expression level of BdGSTd5 was found in 1-day-old adult, and the levels decreased with aging. The dsBdGSTd5 injection effectively silenced (73.4% reduction) the expression of BdGSTd5 and caused significant increase in susceptibility to malathion with a cumulative mortality increasing of 13.5% at 72 h post malathion treatment (p < 0.05). Cytotoxicity assay demonstrated that BdGSTd5 was capable of malathion detoxification. Molecular docking analysis further indicated the interactive potential of BdGSTd5 with malathion and its toxic oxide malaoxon. The recombinant BdGSTd5 exhibited glutathione-conjugating activity toward 1-chloro-2, 4-dinitrobenzene and malathion and malaoxon metabolic capacity with significant reduction (p < 0.05) of the peak areas by 90.0% and 73.1%, respectively. Taken together, the overexpressed BdGSTd5 contributes to malathion metabolism and resistance, which detoxify the malathion in B. dorsalis via directly depleting malathion and malaoxon.

Molecular identification and biochemical characteristics of a delta class glutathione S-transferase gene (FcδGST) from Chinese shrimp Fenneropenaeus chinensis

Molecular identification and biochemical characteristics of a delta class glutathione S-transferase gene (FcδGST) from Chinese shrimp Fenneropenaeus chinensis

Recombinant expression and characterization of GSTd3 from a resistant population of Anopheles arabiensis and comparison of DDTase activity with GSTe2

The development of insecticide resistance in malaria vectors is a challenge for the global effort to control and eradicate malaria. Glutathione S-transferases (GSTs) are multifunctional enzymes involved in the detoxification of many classes of insecticides. For mosquitoes, it is known that overexpression of an epsilon GST, GSTe2, confers resistance towards DDT and pyrethroids. In addition to GSTe2, consistent overexpression of a delta class GST, GSTd3, has been observed in insecticide resistant populations of different malaria vector species. However, the functional role of GSTd3 towards DDT resistance has not yet been investigated. Here, we recombinantly expressed both GSTe2 and GSTd3 from Anopheles arabiensis and compared their metabolic activities against DDT. Both AaGSTd3 and AaGSTe2 exhibited CDNB-conjugating and glutathione peroxidase activity and DDT metabolism was observed for both GSTs. However, the DDT dehydrochlorinase activity exhibited by AaGSTe2 was much higher than for AaGSTd3, and AaGSTe2 was also able to eliminate DDE although the metabolite could not be identified. Molecular modeling revealed subtle differences in the binding pocket of both enzymes and a better fit of DDT within the H-site of AaGSTe2. The overexpression but much lower DDT metabolic activity of AaGSTd3, might suggest that AaGSTd3 sequesters DDT. These findings highlight the complexity of insecticide resistance in the major malaria vectors and the difficulties associated with control of the vectors using DDT, which is still used for indoor residual spraying.

Utilization of English in the Classroom and Instructional Strategies by Delta Class Teachers' in Terms of English Language Teaching Competence

Utilization of English in the Classroom and Instructional Strategies by Delta Class Teachers' in Terms of English Language Teaching Competence

Comparative analysis of detoxification-related gene superfamilies across five hemipteran species

BackgroundHemiptera is one of the most speciose orders of insects, and the most speciose considering Hemimetabola. Through their evolutive history, hemipterans with different feeding habits have adapted to deal with different chemical challenges. Three major gene families are involved in xenobiotic detoxification in insects: the cytochromes P450 (CYPs), carboxyl/cholinesterases (CCEs), and glutathione transferases (GSTs). Here we perform a comparative analysis on the complement of these gene superfamilies across five hemipteran species; four heteropterans (the pentatomid plant feeders Nezara viridula and Halyomorpha halys; the hematophagous Cimex lectularius, Cimicidae, and Rhodnius prolixus, Reduviidae), and one Auchenorrhyncha plant feeder (Nilaparvata lugens).ResultsOur results point to an expansion of several enzyme families associated with xenobiotic detoxification in heteropterans with respect to other species and the existence of a dynamic evolution pattern including CYP3 clan, hormone and pheromone processing class in the CCE superfamily, and sigma class in GST superfamily. Other detoxification-related families are reduced in the hemipteran species analyzed here: reduction or even absence of epsilon class and reduced delta class in GST superfamily; absence of mitochondrial CYP12 family; absence of CYP9 family in CYP3 clan; and reduction or even absence of some dietary/detoxification groups of CCEs. Interestingly, the most polyphagous species analyzed here (H. halys) is also the one that presents the largest repertoire of detoxification enzymes. Gene cluster analysis suggests that this could be due to gene duplication events.ConclusionsThe evolutionary analysis performed here reveals characteristics that are both common and particular for heteropterans. The composition and organization of detoxification-related gene families could shed light on evolutionary forces that shaped their divergence. These families are important for both the detoxification of diet products and for conferring tolerance or resistance to synthetic insecticides. Furthermore, we present the first comprehensive analysis of detoxification gene superfamilies in N. viridula, an understudied species in spite of its economic relevance as a crop pest. The information obtained is of interest for basic insect science as well as for the control of harmful species and the management of insecticide resistance.

In Vivo Glutathione S-Transferases Superfamily Proteome Analysis: An Insight into Aedes albopictus Mosquitoes upon Acute Xenobiotic Challenges.

In this study, the induction of glutathione S-transferase (GST) enzymatic activities in Aedes albopictus under 24 h of xenobiotic challenges was investigated. From LCMS analysis, 23 GST isoforms were identified under Delta, Epsilon, Sigma, Zeta, Omega, and Iota classes, together with one GSTX1-1 isoform, in both treated and untreated samples. Using STRING 11.5, the functional enrichment network of Gene Ontology (GO) analysis, the identified peptides were found to be involved in the glutathione metabolic biological process (GO:0006749, p-value: 1.93 × 10-29), and the molecular functions involved are due to glutathione transferase (GO:0016848, p-value: 2.92 × 10-8) aside from carbon-halide lyase activity (GO:004364, p-value: 1.21 × 10-31). The Protein-Protein Interaction (PPI) network (STRING 11.5) showed significant interactions within the GST superfamily and some of the GST classes interacted with other proteins among the input domain of the identified peptides (p-value &lt; 1.0 × 10-16). In TMT labeling for the quantification of peptide abundance, isoforms from Delta (GSTD1-2, GSTD1-3, GSTD1-4) and Epsilon (GSTE3-1, GSTE4-2) were found to be overexpressed (between 1.5-fold and 2-fold changes). In the PPI analysis, 12 common enriched pathways of Kyoto Encyclopedia of Genes and Genomes (KEGG) were found to be intercorrelated with the identified GSTs at PPI enrichment p-value &lt; 1.0 × 10-16. Overall, this study indicates that distinct GST enzymes, which were identified up to their specific protein isoforms, are involved in the metabolic mechanisms underlying xenobiotic stress.

Aerodynamic optimization of a propeller airfoil at low Reynolds numbers

The aerodynamic optimization method was presented to design a propeller airfoil for a stratosphere airship at low Reynolds numbers. The delta class and shape transformation were adopted to describe the airfoil geometry as the design variables. The flow field was calculated by an open-source code XFOIL to obtain the aerodynamic characteristics as the design targets. The separated particle swarm optimization was employed as the optimization algorithm. The optimization case started with a typical low Reynolds number airfoil E387 and obtained a thicker and more cambered airfoil using the proposed method. The flow fields of the optimized and initial airfoils were analysed by CFD. Results suggested that the lift-to-drag ratio of the optimized airfoil changed from 35.7 to 45.9 with an increasing rate by 28.5% at design cruise condition. The overall aerodynamic performance of the optimized airfoil has been significantly improved at both design and off-design conditions within a wide range of attack angle. The optimization platform presented here is an efficient and effective method for the low-Reynolds-number airfoil designs.

Expression Patterns of Drosophila Melanogaster Glutathione Transferases.

Simple SummaryGlutathione transferases are key enzymes found in all living species. In insects, two classes of these enzymes are specific: the Delta and Epsilon classes. These classes of glutathione transferases are generally considered supports during the evolution for the adaptability of the species of an insect. We measured the expression levels of the 25 enzymes forming these two classes in different organs of Drosophila melanogaster. Some glutathione transferases are tissue-specific, others are specifically underexpressed in some tissues, and some appear expressed at the same level in all the tested tissues. All glutathione transferases present a specific pattern that does not depend on their sequence proximity. This study allows us to better understand the potential role of these enzymes by analyzing their expression profile and evolutionary links.Glutathione transferases (GSTs) are ubiquitous enzymes that catalyze the conjugation of glutathione to various molecules. Among the 42 GSTs identified in Drosophila melanogaster, Delta and Epsilon are the largest classes, with 25 members. The Delta and Epsilon classes are involved in different functions, such as insecticide resistance and ecdysone biosynthesis. The insect GST number variability is due mainly to these classes. Thus, they are generally considered supports during the evolution for the adaptability of the insect species. To explore the link between Delta and Epsilon GST and their evolution, we analyzed the sequences using bioinformatic tools. Subgroups appear within the Delta and Epsilon GSTs with different levels of diversification. The diversification also appears in the sequences showing differences in the active site. Additionally, amino acids essential for structural stability or dimerization appear conserved in all GSTs. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the transcripts corresponding to these two classes are heterogeneously expressed within D. melanogaster. Some GSTs, such as GSTD1, are highly expressed in all tissues, suggesting their general function in detoxification. Conversely, some others, such as GSTD11 or GSTE4, are specifically expressed at a high level specifically in antennae, suggesting a potential role in olfaction.

Per a 5-derived T-cell peptides modulate NF-kB signalling to ameliorate allergic inflammation systemically in murine model of cockroach allergic hyper-reactivity.

Peptide immunotherapy (PIT) represents a safe and efficacious therapeutic regimen with in-consequential side-effects. The present study aims to identify T-cell epitopes of Per a 5 allergen, a delta class GST from Periplaneta americana and investigate effect of peptide treatment in murine model of cockroach allergen-mediated hyper-reactivity. The epitopes (TC-P1, TC-P2, and TC-P3) were identified as promiscuous MHC-II binders by MHC-Pred, ProPred, and IEDB analysis tool. Murine model of cockroach allergic hyper-reactivity was generated in Balb/c mice. A marked reduction in cellular infiltration in lungs (3-fold compared with Non-IT) was observed in T3-IT group as evidenced by total leucocyte count in BALF and histology. Specific IgE levels were reduced 3-fold in T2-IT and T3-IT compared with Non-IT with increase in IgG2a levels. IL-4 and IL-13 were reduced upto 2.5-fold in treatment groups compared with Non-IT group. Splenocytes revealed significant increase in levels of CD4+FoxP3+ T cells in TC-P1 and TC-P2 mice demonstrating a systemic shift towards Tregs. Peptide treatment downregulated NF-kB signalling in lung and enhanced the levels of immune-regulatory molecules α1-antitrypsin and elafin. Our results indicate that TC-P1 and TC-P3 alter Th2 cytokine milieu and antibody isotype ratio to suppress allergic inflammation. PIT modulates local and systemic mechanisms to resolve inflammation and possess potential for treatment in cockroach allergy.

Amplification and Characterization of DDT Metabolizing Delta Class GST in Sand Fly, Phlebotomus argentipes (Diptera: Psychodidae) From Bihar, India.

Phlebotomus argentipes is an established vector for Visceral leishmaniasis prevalent in the Indian subcontinent. Insect Glutathione S-transferases (GST) enzyme plays a pivotal role in the metabolism of xenobiotics and chemical insecticides. We report herein the identification and characterization of a delta class GST from the sandfly, P. argentipes. The resulting clone (rParg-GSTδ) is successfully sequenced, which revealed 76.43% and 66.32% gene identity with GST from Phlebotomus papatasi (Scopoli; Diptera: Psychodidae) and Lutzomiya longipalpis (Lutz and Neiva; Diptera: Psychodidae), respectively. The identified rParg-GST amino acid Blast results revealed 82.6% homology to delta class GST of Phlebotomus papatasi and more than 50% homology to Lepidoptera which comprises butterflies and moths. The Phylogenetic analysis of Parg-GST with different classes of Insect GSTs further supported its classification as delta class. A functional recombinant Parg-GSTδ protein (rParg-GSTδ) was expressed in Escherichia coli (Migula; Enterobacterales: Enterobacteriaceae) cells in a soluble form, purified to homogeneity and found to be active against a substrate 1-chloro-2,4-dintrobenzene (CDNB) and lipid peroxidation by-product 4-Hydrxynonenal (4-HNE). Interestingly, rParg-GSTδ demonstrates high dehydrochlorination activity against dichlorodiphenyltrichloroethane (DDT) i.e., 16.27 nM/µg in high performance liquid chromatography (HPLC) assay. These results provide evidence of direct DDT metabolism property exhibited by P. argentipes GST and set the foundation to decipher the metabolic resistance mechanism in P. argentipes against insecticides.

Streptozotocin activates inflammation-associated signalling and antioxidant response in the lobster cockroach; Nauphoeta cinerea (Blattodea: Blaberidae)

Streptozotocin exhibits tropism to insulin-producing beta-cells in mammals and has been used to model diabetes-like phenotypes in insects. We have previously shown increased brain glucose levels and oxidative stress in STZ-treated nymphs of Nauphoeta cinerea. Here, we validate Nauphoeta cinerea as an experimental organism for studying STZ-induced metabolic disruptions by investigating the potential changes in the expression of inflammation and antioxidant related genes. Cockroaches were injected with 0.8% NaCl, 74 and 740 nmol of STZ. mRNA extracted from the head of cockroaches was used to estimate the RT-qPCR expression of inflammation and antioxidant genes. STZ-treatment upregulated the target genes of the JNK pathway (early growth factor response factor and reaper) but had no effect on PDGF-and VEGF-related factor 1. TOLL 1, the target gene of TOLL/NF-kB pathway was up regulated, while both the activator and target gene of the UPD3/JAK/STAT pathway [unpaired 3 and Suppressor of cytokine signalling at 36E] were upregulated. mRNA levels of primary antioxidants (superoxide dismutase and catalase) were increased in STZ treated nymphs but there was no effect on thioredoxins and Peroxiredoxin 4. Likewise, STZ treatment did not affect the expression of the delta class of the glutathione S-transferase gene family, but the sigma and theta classes of the GST family were upregulated. The STZ-induced N. cinerea gene expression modification demonstrates the involvement of primary antioxidants and the GST detoxification system in the cockroach oxidative stress response and buttresses the proposed crosstalk between inflammatory and redox pathways.

Delta class glutathione S-transferase (TuGSTd01) from the two-spotted spider mite Tetranychus urticae is inhibited by abamectin

GSTs (Glutathione S-transferases) are known to catalyze the nucleophilic attack of the sulfhydryl group of reduced glutathione (GSH) on electrophilic centers of xenobiotic compounds, including insecticides and acaricides. Genome analyses of the polyphagous spider mite herbivore Tetranychus urticae (two-spotted spider mite) revealed the presence of a set of 32 genes that code for secreted proteins belonging to the GST family of enzymes. To better understand the role of these proteins in T. urticae, we have functionally characterized TuGSTd01. Moreover, we have modeled the structure of the enzyme in apo form, as well as in the form with bound inhibitor. We demonstrated that this protein is a glutathione S-transferase that can conjugate glutathione to 1-chloro-2,4-dinitrobenzene (CDNB). We have tested TuGSTd01 activity with a range of potential substrates such as cinnamic acid, cumene hydroperoxide, and allyl isothiocyanate; however, the enzyme was unable to process these compounds. Using mutagenesis, we showed that putative active site variants S11A, E66A, S67A, and R68A mutants, which were residues predicted to interact directly with GSH, have no measurable activity, and these residues are required for the enzymatic activity of TuGSTd01. There are several reports that associate some T. urticae acaricide resistance with increased activity of GSTs . However, we found that TuGSTd01 is not able to detoxify abamectin; in fact, the acaricide inhibits the enzyme with Ki = 101 μM. Therefore, we suggest that the increased GST activity observed in abamectin resistant T. urticae field populations is a part of the compensatory feedback loop. In this case, the increased production of GSTs and relatively high concentration of GSH in cells allow GSTs to maintain physiological functions despite the presence of the acaricide.

Temporal recovery of soil biological activity and diversity after 915-MHz microwave treatments

Two 915-MHz microwave treatments (2 kW-8 min and 4 kW-4 min) were applied to soil and their effects were monitored just after treatments (T0) and 26 days (T26) in soil microcosms. Densities of culturable bacteria, fluorescent pseudomonads and nematodes, hydrolysis activity and soil DNA content declined by over 50% immediately after both microwave treatments (T0), excluding the total fungal 18S rRNA (−13 or −17%) and bacterial 16S rRNA copies (non-significant). A rapid shift in bacterial community composition occurred from T0 towards a large increase in the relative abundance of Firmicutes (+1650%) and a concomitant decrease in various phyla (e.g. Acidobacteria, Actinobacteria, Bacteroidetes, Proteobacteria) from −85 to −61%. At T26 and for both treatments, fluorescein diacetate hydrolysis, density of culturable bacteria, 18S rRNA gene numbers, Simpson diversity, relative abundances of Bacteroidetes and Proteobacteria regained levels similar to controls. Alpha, Delta and Gamma classes Proteobacteria also end up reaching a similar level. In contrast, fluorescent pseudomonad density, nematode diversity and abundance, soil DNA content and relative abundances of some phylum (Acidobacteria, Actinobacteria, Chloroflexi) did not increase in such proportions. Most of the soil biological properties have not been permanently impacted. Nevertheless, the recovery kinetics highly differed according properties, with resilience indices at T26 varying from −87 to +99%.

Bioinformatic and immunological investigation of Per a 5 (delta class GST) allergen from Periplaneta americana

IntroductionGSTs are multifunctional enzymes involved in cellular detoxification and present as potent allergens in several sources. Present study investigates allergenic relevance of GST from P. americana and determine its cross reactive potential with other indoor allergen sources. MethodsComputational analysis with FASTA and ConSurf webserver was performed to determine potentially cross reactive allergens. Further, Per a 5 gene was cloned in pET 22b+ vector and expressed in E.coli BL21 cells and the rPer a 5 protein was purified using Ni-NTA affinity chromatography. Enzymatic activity of rPer a 5 was assessed using CDNB and cumene hydroperoxide. ELISA and immunoblot were performed using cockroach hypersensitive patient’s sera. Functional activity of rPer a 5 was evaluated by basophil activation test. Inhibition studies were carried out with D. pteronyssinus, A. alternata and C. lunata extracts. ResultsPer a 5 demonstrates highest sequence similarity with delta class GST of Blattella germanica (94.9%). It also exhibits significant sequence similarity (50–58%) with mite, fungal and helminth allergenic GSTs. ConSurf analysis reveals high degree of evolutionary similarity in N terminal region of Per a 5, especially at GST dimerization interface. The purified rPer a 5 protein resolved at 27 kDa on SDS-PAGE. The rPer a 5 protein exhibits GST activity and possess upto 65% immunoreactivity with cockroach hypersensitive patient’s sera in ELISA and immunoblot. It upregulates expression of CD203c on basophils signifying its biological ability to activate effector cells. rPer a 5 significantly inhibits corresponding GSTs in P. americana, D. pteronyssinus, A. alternata and C. lunata with EC50 values of 15.5 ng. 38.38 ng, 41.4 ng and 61.66 ng, respectively. ConclusionRecombinant delta class GST of P. americana is a clinically relevant allergen showing upto 65% immunoreactivity with hypersensitive patient’s sera. Per a 5 GST allergen showed phylogenetic similarity with dust mite, fungal and birch allergens thereby demonstrating allergen cross reactivity.

Target-Site Mutations and Glutathione S-Transferases Are Associated with Acequinocyl and Pyridaben Resistance in the Two-Spotted Spider Mite Tetranychus urticae (Acari: Tetranychidae).

Simple SummaryThe two-spotted spider mite Tetranychus urticae is a difficult-to-control pest due to its short life cycle and rapid resistance development. In this study, we characterized field strains collected in 2001 and 2003 that have been selected for acequinocyl resistance and pyridaben resistance, respectively. These strains displayed resistance ratios of 1798.6 and 5555.6, respectively, and were screened for cross-resistance against several currently used acaricides. The acequinocyl resistant strain exhibited pyridaben cross-resistance, but the pyridaben resistant strain showed no cross-resistance. The acequinocyl resistant strain exhibited point mutations in cytb (I256V and N321S) and PSST (H92R). In contrast, the pyridaben resistant strain exhibited the H92R but not the I256V and N321S point mutations. In addition, the increased GST metabolism and GST delta expression might be related to acequinocyl resistance in Tetranychus urticae. We hope that the data and patterns described here can now be exploited in the continued quest for rational resistance management strategies.The two-spotted spider mite Tetranychus urticae is a difficult-to-control pest due to its short life cycle and rapid resistance development. In this study, we characterized field strains collected in 2001 and 2003 that were selected for acequinocyl resistance (AR) and pyridaben resistance (PR), respectively. These strains displayed resistance ratios of 1798.6 (susceptible vs. AR) and 5555.6 (susceptible vs. PR), respectively, and were screened for cross-resistance against several currently used acaricides. The AR strain exhibited pyridaben cross-resistance, but the PR strain showed no cross-resistance. The AR strain exhibited point mutations in cytb (I256V, N321S) and PSST (H92R). In contrast, the PR strain exhibited the H92R but not the I256V and N321S point mutations. In some cases increased glutathione S-transferase (GST) activity has previously been linked to enhanced detoxification. The AR strain exhibited approximately 2.3-, 1.8-, and 2.2-fold increased GST activity against 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), and 4-nitrobenzyl chloride (NBC), respectively. Among the five GST subclass genes (delta, omega, mu, zeta, and kappa), the relative expression of delta class GSTs in the AR strain were significantly higher than the PR and susceptible strain. These results suggest that the I256V and N321S mutations and the increased GST metabolism and GST delta overexpression might be related to acequinocyl resistance in T. urticae.