Form Of Enzyme Research Articles

P-260 A more physiological approach to support oocyte activation in cases with fertilization failure

Abstract Study question Can we use a physiological compound to trigger oocyte activation in couples undergoing ICSI with history of fertilization failure due to sperm activating factor deficiency? Summary answer In a pilot sibling oocyte study, oocyte activation using recombinant human phospholipase C-zeta (rhPLCζ) yielded a comparable fertilization compared to conventional method using ionomycin. What is known already ICSI overcomes most forms of male gamete defects, however partial or total fertilization failure still occurs regardless of adequate semen parameters and optimal oocyte maturity. This has been attributed to the lack of a cytosolic factor identified as PLCζ. To address this, various approaches of assisted oocyte activation have been proposed, including gamete exposure to chemical or electrical stimuli. The most popular approach remains to be ionomycin. Despite its apparent safety, this stimulus is unnatural and may be aggressive on the oocytes. Therefore, we propose a more physiological approach by utilizing a recombinant form of this specific enzyme. Study design, size, duration In the past 2 months, couples with poor fertilization at their initial ICSI cycles were identified and counseled for assisted oocyte activation (AOA) in the subsequent cycle after confirmation of a PLCζ deficiency. Oocytes were separated into 2 equal cohorts activated either by ionomycin or co-injection with recombinant human rhPLCζ during ICSI. Fertilization rates between ionomycin-activated and rhPLCζ-activated cohorts were compared, as well as with the couples’ history cycles. Participants/materials, setting, methods Six couples with suboptimal fertilization outcomes consented for the study (IRB 0712009553) and were offered AOA with their subsequent ICSI cycles. Conventional AOA was carried out by exposing post-ICSI oocytes to 50 mM ionomycin. The rhPLCζ had a confirmed >95% purity. Our physiological rhPLCζ-activation method was performed by co-injecting spermatozoa with 0.4pL of a rhPLCζ (5mg/mL in glycerol) during ICSI. Main results and the role of chance Couples (n = 6, female age: 36.2 ± 6yrs, male age: 38.0 ± 7yrs) had negative infertility workups with normal semen parameters (3.3 ± 3 mL volume, 42.1 ± 35 x106/mL concentration, 30.8 ± 23% motility, 2.0 ± 1% normal morphology). They underwent 7 unsuccessful ICSI cycles with 20.0% (12/60) fertilization rate and yielded a 91.7% cleavage rate, of those, 3 were transferred at cleavage stage but did not yield clinical pregnancy. A total of 2 blastocysts were obtained but were all aneuploid. In their subsequent treatment cycles, overall AGT achieved a significantly improved fertilization rate of 51.6% (32/62) (P<0.001). We then compared the efficiency between the two AGT methods in equally distributed sibling oocyte cohort. Although mathematically insignificant, we observed a trend that rhPLCζ-activation outperforms ionomycin-activation in total activation (67.9% vs 50.0%) as well as fertilization (57.1% vs 47.0%). The new rhPLCζ-activation method yielded a comparable embryo cleavage rate at 87.5% compared to the conventional method at 81.3%. Although fresh embryo transfer at cleavage stage did not yield pregnancy rate yet, rhPLCζ-activated cohort had one euploid blastocyst while the ionomycin-activated cohort did not yield euploid blastocyst. Limitations, reasons for caution While we observed that a more physiological rhPLCζ-activation yields a comparable fertilization compared to the conventional ionomycin method, the study is preliminary, and a confirmatory clinical outcome is not yet available. Wider implications of the findings This is the first attempt to use recombinant sperm-specific protein to mimic physiological oocyte activation for patient treatment. If this data is confirmed in a larger series, this may represent a way to normalize fertilization in cases of sperm activating factor deficiency even in different sources of spermatozoa. Trial registration number N/A

Structural and mechanistic insights into the DNA glycosylase AAG-mediated base excision in nucleosome

The engagement of a DNA glycosylase with a damaged DNA base marks the initiation of base excision repair. Nucleosome-based packaging of eukaryotic genome obstructs DNA accessibility, and how DNA glycosylases locate the substrate site on nucleosomes is currently unclear. Here, we report cryo-electron microscopy structures of nucleosomes bearing a deoxyinosine (DI) in various geometric positions and structures of them in complex with the DNA glycosylase AAG. The apo nucleosome structures show that the presence of a DI alone perturbs nucleosomal DNA globally, leading to a general weakening of the interface between DNA and the histone core and greater flexibility for the exit/entry of the nucleosomal DNA. AAG makes use of this nucleosomal plasticity and imposes further local deformation of the DNA through formation of the stable enzyme–substrate complex. Mechanistically, local distortion augmentation, translation/rotational register shift and partial opening of the nucleosome are employed by AAG to cope with substrate sites in fully exposed, occluded and completely buried positions, respectively. Our findings reveal the molecular basis for the DI-induced modification on the structural dynamics of the nucleosome and elucidate how the DNA glycosylase AAG accesses damaged sites on the nucleosome with different solution accessibility.

PEMANFAATAN SAMPAH ORGANIK MENJADI ECO ENZYME SEBAGAI SOLUSI PUPUK RAMAH LINGKUNGAN DI LINGKUNGAN SD WONOSARI 01, KECAMATAN PUGER, KABUPATEN JEMBER

This study aims to utilize organic waste that has not been considered by the community so far to become a versatile product in the form of eco enzyme in Wonosari Village, Puger District, Jember Regency. This research was conducted in May 2023 using a mixed method of qualitative and quantitative methods using analytic descriptive analysis. Data collection techniques used are interviews, questionnaires, observation, and documentation. The results of this study indicate that the existence of organic waste in the community is still being neglected. There are many ways that can be used to manage organic waste, one of which is by making eco enzymes from organic waste. The training on making eco enzymes in the Elementary School Wonosari 01 area received a positive response. It was found that there was a significant increase in knowledge about organic waste management, from a percentage of 0% to 81.8% in the good category. While the moderate and less categories decreased from 36.4% to 18.2% the moderate category and from 63.6% the less category became 0%.

The prognostic significance of the activities of matrix metalloproteinases-2 and -9 in dogs for mammary gland neoplasia (pilot study)

The aim of this study was to determine the activity of matrix metalloproteinase-2 and -9 in blood and tumour tissue of female dogs with benign and malignant mammary gland neoplasias. The increased activity of matrix metalloproteinase-2 and -9 was registered in 3.8–8.9% of canines with benign neoplasias (adenoma, mixed tumour) and in 32.5–63.5% of those with malignant neoplasias (carcinoma: mixed type, ductal, tubular, tubulopapillary). Neoplastic transformation is accompanied by blood level increase of both latent and active enzyme forms. The dogs with malignant mammary gland neoplasias were diagnosed with credible increase of matrix metalloproteinases-2 activity up to 1.59–1.96 in blood plasma and up to 21.57–24.84 in tumour tissue and the increase of matrix metalloproteinases-9 activity up to 2.16–2.67 and 29.53–35.26 respectively. For benign mammary neoplasms, the proportion of dogs with enhanced expression of matrix metalloproteinase-2 was higher than the number of patients in whom an increase in the level of matrix metalloproteinase-9 or both of these enzymes was registered by 1.7 times, for dogs with malignant tumours – in blood plasma – by 1.4–1.6 times, dogs with neoplastic changed functional tissue – 1.7 and 1.9 times, respectively. Histological type and metastatic foci presence did not correlate with enzymes’ activity. The enzymes’ activity figures in benign neoplasias fluctuated within those in clinically healthy animals. A positive characteristic of determining the plasma and tissue expression level of matrix metalloproteinases in dogs with mammary tumours is the low degree of invasiveness of the method against the background of the high informativeness of the results obtained in the preoperative period. The obtained results prove the possibility of using matrix metalloproteinases-2 and -9 to predict the course and to control the treatment of mammary neoplasia.

MAO inhibiting phytochemicals from the roots of Glycyrrhiza glabra L.

Glycyrrhizin, a natural compound that is substantially present in Glycyrrhiza glabra L. (Gg) root. Monoamine oxidase B (MAOB) inhibitor is used for the treatment of several important neuropsychological diseases like Parkinson’s disease. Gg is known to possess psychoactive properties which relates to its MAO inhibitory potential. This study sought to determine the MAO inhibition property of glycyrrhizin from Gg root extract. The Aqueous extract containing glycyrrhizin was isolated from the root of Gg and characterized using TLC, HPLC, and LC-MS techniques. In silico docking was conducted using Extra precision Glide 2018, Schrödinger docking suite. In addition, the pharmacokinetic properties of the compounds were predicted using SwissADME. The binding energies of the glycyrrhizin correlated well with their in vitro MAO inhibitory potential. Glycyrrhizin exhibited potent inhibitory activity towards MAOB whereas, an aqueous extract of Gg root inhibits both A and B forms of MAO enzyme. Further, molecular docking and molecular dynamics simulation showed that liquiritigenin and methoxyglabridin showed higher stability than other inhibitor compounds from the Gg root extract. These observations suggest that the phytochemicals from the Gg root extract have potent MAO inhibition properties, which can be exploited for the treatment of neurodegenerative disorders. Communicated by Ramaswamy H. Sarma

Mechanism of D-cycloserine inhibition of D-amino acid transaminase from <i>Haliscomenobacter hydrossis</i>

D-cycloserine inhibits pyridoxal-5′-phosphate (PLP)-dependent enzymes. The inhibition efficiency depends on the organization of their active center and the mechanism of the catalyzed reaction. D-cycloserine interacts with the PLP form of enzyme similarly to substrate amino donor, and the interaction is predominantly reversible. Inhibition products include hydroxyisoxazole-pyridoxamine-5′-phosphate, oxime between PLP and β-aminooxy-D-alanine, ketimine between pyridoxamine-5′-phosphate and cyclic or open forms of D-cycloserine, pyridoxamine-5′-phosphate, etc. For some enzymes the formation of a stable aromatic product - hydroxyisoxazole can lead to irreversible D-cycloserine inhibition at certain pH value. The aim of this work was to study the mechanism of D-cycloserine inhibition of PLP-dependent D-amino acid transaminase from the bacterium Haliscomenobacter hydrossis. Spectral methods revealed several products of the interaction of D-cycloserine with PLP in the active site of transaminase: oxime between PLP and β-aminooxy-D-alanine, ketimine between pyridoxamine-5′-phosphate and cyclic or open forms of D-cycloserine, pyridoxamine-5′-phosphate. The formation of hydroxyisoxazole-pyridoxamine-5′-phosphate was not observed. The 3D structure of the complex of transaminase with D-cycloserine was obtained by X-ray diffraction analysis. In the active site of transaminase, a ketimine adduct between pyridoxamine-5′-phosphate and D-cycloserine in the cyclic form was found; the ketimine occupied two positions and was coordinated via hydrogen bonds with different active site residues. Using kinetic and spectral methods we have shown that D-cycloserine inhibition is reversible, and the activity of transaminase from H. hydrossis can be restored by adding an excess of keto substrate as well as by adding an excess of cofactor. The results obtained confirm the reversibility of D-cycloserine inhibition and the conversion of various adducts of D-cycloserine and PLP into each other.

Dehydroepiandrosterone (DHEA) increases undirected singing behavior and alters dopaminergic regulation of undirected song in non-breeding male European starlings (Sturnus vulgaris).

It has been proposed that in species that defend territories across multiple life history stages, brain metabolism of adrenal dehydroepiandrosterone (DHEA) regulates aggressive behavior at times when gonadal androgen synthesis is low (i.e. the non-breeding season). To date, a role for DHEA in the regulation of other forms of social behavior that are expressed outside of the context of breeding remains unknown. In this experiment, we used the European starling (Sturnus vulgaris) model system to investigate a role for DHEA in the neuroendocrine regulation of singing behavior by males in non-breeding condition. Starling song in a non-breeding context is spontaneous, not directed towards conspecifics, and functions to maintain cohesion of overwintering flocks. Using within-subjects design, we found that DHEA implants significantly increase undirected singing behavior by non-breeding condition male starlings. Given that DHEA is known to modulate multiple neurotransmitter systems including dopamine (DA) and DA regulates undirected song, we subsequently used immunohistochemistry for phosphorylated tyrosine hydroxylase (pTH, the active form of the rate-limiting enzyme in DA synthesis) to investigate the effect of DHEA on dopaminergic regulation of singing behavior in a non-breeding context. Pearson correlation analysis revealed a positive linear association between undirected singing behavior and pTH immunoreactivity in the ventral tegmental area and midbrain central gray of DHEA-implanted, but not control-implanted, males. Taken together, these data suggest that undirected singing behavior by non-breeding starlings is modulated by effects of DHEA on dopaminergic neurotransmission. More broadly, these data expand the social behavior functions of DHEA beyond territorial aggression to include undirected, affiliative social communication.

Development and validation of a proteomic correlation profiling technique to detect and identify enzymes involved in metabolism of drugs of concern.

It is important to be able to detect previously unknown and unsuspected enzymes involved in drug metabolism, in order to predict the variation of pharmacological or toxicological effect caused by pharmacokinetic variance. We investigated the use of proteomic correlation profiling (PCP) as a technique to identify the enzymes involved in metabolism of drugs of concern. By evaluating the metabolic activities of each enzyme (including isoforms of cytochrome P450, uridine 5' diphospho‑glucuronosyltransferase, and hydrolases, plus aldehyde oxidase and carbonyl reductase) on their typical substrates using a panel of human liver samples, we were able to show the validity of PCP for this purpose. Pearson or Spearman's rank correlation coefficient and P values were calculated for the association between the protein abundance profile of each protein and the metabolic rate profile of each typical substrate. For the 18 enzymatic activities examined, 13 of the enzymes reported to be responsible for the reactions had correlation coefficients higher than 0.7, and were ranked first to third. For the remaining five activities, the responsible enzymes had correlation coefficients lower than 0.7 and lower rankings. The reasons for this were diverse, including confounding resulting from low protein abundance ratios, artificially high correlations of other enzymes due to limited sample numbers, the presence of inactive enzyme forms, and genetic polymorphisms. Overall, PCP was able to identify the majority of responsible drug-metabolizing enzymes across several enzyme classes (oxidoreductase, transferase, hydrolase); use of this methodology could allow more timely and accurate identification of unknown drug-metabolizing enzymes. Significance Statement Proteomic correlation profiling using samples from individual human donors was proven to be a useful methodology for the identification of enzymes responsible for drug-metabolism. This methodology could accelerate the identification of unknown drug-metabolizing enzymes in the future.

Contribution of vesicle trafficking dysregulation to the pathomechanism of mucopolysaccharidosis

Although mucopolysaccharidoses (MPS) are monogenic diseases, caused by mutations in genes coding for enzymes involved in degradation of glycosaminoglycans (GAGs), recent studies suggested that changes in expressions of various genes might cause secondary and tertiary cellular dysfunctions modulating the course of these diseases. In this report, we demonstrate that vesicle trafficking regulation is affected in fibroblasts derived from patients suffering from 11 different types of MPS due to changes in levels of crucial proteins (estimated by automated Western-blotting) involved in this process, including caveolin, clathrin, huntingtin (Htt), APPL1, EEA1, GOPC, Rab5, and Rab7. Microscopic studies confirmed these results, while investigations of tissue samples derived from the MPS I mouse model indicated differences between various organs in this matter. Moreover, transcriptomic analyses provided a global picture for changes in expressions of genes related to vesicle trafficking in MPS cells. We conclude that vesicle trafficking is dysregulated in MPS cells and changes in this process might contribute to the molecular mechanisms of this disease. Most probably, primary GAG storage might cause a cellular stress response leading to dysregulation of expression of many genes which, in turn, results in changes in cellular processes like vesicle trafficking. This can significantly modulate the course of the disease due to enhancing accumulation of GAGs and altering crucial cellular processes. This hypothesis has been supported by normalization of levels of clathrin in MPS cells treated with either an active form of the deficient GAG-degrading enzyme or a compound (5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) indirectly reducing the efficiency of GAG synthesis.

Identification of bacterial morphology and catalase coagulation test on propionibacterium acnes bacteria

Infection is a way for microorganisms such as bacteria, viruses and fungi to enter the skin which can cause skin problems such as acne. purpose of this study was to determine the morphology of p. acnes by gram staining method, then the catalase test was used to determine the ability of microorganisms to decompose hydrogen peroxide by producing catalase enzymes and coagulase test is a bacterial examination to detect the formation of coagulase enzymes that bind to the bacterial cell wall. The results obtained were that the morphology of the bacteria was in the form of salt and purple in color, the catalase test was positive and the coagulase test was positive. The conclusion obtained was that P. acnes ATCC 11827 was included in the group of gram-positive bacteria based on the results of gram staining and catalase and coagulase tests.

Aberrant SENP1-SUMO-Sirt3 Signaling Causes the Disturbances of Mitochondrial Deacetylation and Oxidative Phosphorylation in Prion-Infected Animal and Cell Models

Post-translational modifications of proteins, such as acetylation and SUMOylation, play important roles in regulation of protein functions and pathophysiology of different diseases including neurodegenerative diseases. Our previous studies have identified aberrant acetylation profiles and reduced deacetylases Sirt3 and Sirt1 in the brains of prion-infected mouse models. In this study, we have found that the levels of acetylated forms of AceCS2 and LCAD, the key enzymes regulating lipid metabolism, CS and IHD2, the key enzymes regulating complete oxidative metabolism, GDH, the key enzyme regulating the oxidative decomposition of glutamate into the tricarboxylic acid (TCA) cycle, and NDUFA9, the essential component in the complex I of respiratory chain activity, were significantly upregulated in the prion-infected animal and cell models, along with the decrease of Sirt3 activity and mitochondrial cytochrome c oxidase activity. Meanwhile, the increases of SUMO1 modifications and SUMO1-Sirt3 and decrease of SENP1 were identified in the brains and the cultured cells with prion infections. Removal of prion propagation in the cultured cells partially, but significantly, reversed the aberrant situations. Moreover, similar abnormal phenomena were also observed in the cultured 293 T cells transiently expressing cytosolic form PrP (Cyto-PrP), including decreased SENP1, increased SUMO1, decreased Sirt3 activity, increased acetylated forms of the key enzymes, and decreased cytochrome c oxidase activity. Attenuation of the accumulation of Cyto-PrP by co-expression of the p62 protein sufficiently diminished those abnormalities. The data here strongly indicate that deposits of prions in brains or accumulations of Cyto-PrP in cells trigger dysregulation of the SENP1-SUMO1-Sirt pathway and subsequently induce aberrant mitochondrial deacetylation and the mitochondrial respiratory chain.

S-1-propenyl-L-cysteine suppresses lipopolysaccharide-induced expression of matrix metalloproteinase-1 through inhibition of tumor necrosis factor-α converting enzyme-epidermal growth factor receptor axis in human gingival fibroblasts.

Periodontal disease is the most common dental health problem characterized by the destruction of connective tissue and the resorption of alveolar bone resulting from a chronic infection associated with pathogenic bacteria in the gingiva. Aged garlic extract has been reported to improve gingival bleeding index and probing pocket depth score in patients with mild to moderate periodontitis. Although our previous study found that aged garlic extract and its constituents suppressed the tumor necrosis factor-α-induced inflammatory responses in a human gingival epithelial cell line, the mechanism underlying the effect of aged garlic extract on the destruction of the gingiva remains unclear. The present study investigated the effect of S-1-propenyl-L-cysteine, one of the major sulfur bioactive compounds in aged garlic extract, on the lipopolysaccharide-induced expression of matrix metalloproteinases in human gingival fibroblasts HGF-1 cells. Matrix metalloproteinases are well known to be closely related to the destruction of the gingiva. We found that S-1-propenyl-L-cysteine suppressed the lipopolysaccharide-induced expression and secretion of matrix metalloproteinase-1 in HGF-1 cells. In addition, S-1-propenyl-L-cysteine inhibited the lipopolysaccharide-induced phosphorylation of epidermal growth factor receptor and expression of the active form of tumor necrosis factor-α converting enzyme. Furthermore, the inhibitors of epidermal growth factor receptor tyrosine kinase and tumor necrosis factor-α converting enzyme, AG-1478 and TAPI-1, respectively, reduced the lipopolysaccharide-induced protein level of matrix metalloproteinase-1, as did S-1-propenyl-L-cysteine. Taken together, these results suggested that S-1-propenyl-L-cysteine suppresses the lipopolysaccharide-induced expression of matrix metalloproteinase-1 through the blockade of the tumor necrosis factor-α converting enzyme-epidermal growth factor receptor axis in gingival fibroblasts.

Plant dehydroascorbate reductase moonlights as membrane integrated ion channel

Plant dehydroascorbate reductases (DHARs) are only known as soluble antioxidant enzymes of the ascorbate-glutathione pathway. They recycle ascorbate from dehydroascorbate, thereby protecting plants from oxidative stress and the resulting cellular damage. DHARs share structural GST fold with human chloride intracellular channels (HsCLICs) which are dimorphic proteins that exists in soluble enzymatic and membrane integrated ion channel forms. While the soluble form of DHAR has been extensively studied, the existence of a membrane integrated form remains unknown. We demonstrate for the first time using biochemistry, immunofluorescence confocal microscopy, and bilayer electrophysiology that Pennisetum glaucum DHAR (PgDHAR) is dimorphic and is localized to the plant plasma membrane. In addition, membrane translocation increases under induced oxidative stress. Similarly, HsCLIC1 translocates more into peripheral blood mononuclear cells (PBMCs) plasma membrane under induced oxidative stress conditions. Moreover, purified soluble PgDHAR spontaneously inserts and conducts ions in reconstituted lipid bilayers, and the addition of detergent facilitates insertion. In addition to the well-known soluble enzymatic form, our data provides conclusive evidence that plant DHAR also exists in a novel membrane-integrated form. Thus, the structure of DHAR ion channel form will help gain deeper insights into its function across various life forms.

Nanotechnology connecting copper metabolism and tumor therapy

AbstractCopper (Cu) is an essential trace element in the human body that is involved in the formation of several natural enzymes, such as superoxide dismutase and cyclooxygenase. Due to the high density of the outer electron cloud of Cu, which allows the transfer of multiple electrons, Cu is often used as the catalytic center in various metabolic enzymes. However, both deficiency and excessive accumulation of Cu can result in irreversible damage to cells. Therefore, strategies to regulate Cu metabolism, such as Cu exhaustion and Cu supplementation, have emerged as attractive approaches in anticancer therapy, due to the potential damages caused by Cu metabolism disorders. Notably, recent advancements in nanotechnology have enabled the development of nanomaterials that can regulate Cu metabolism, making this therapy applicable in vivo. In this review, we provide a systematic discussion of the physical and chemical properties of Cu and summarize the applications of nanotechnology in Cu metabolism‐based antitumor therapy. Finally, we outline the future directions and challenges of nano‐Cu therapy, emphasizing the scientific problems and technical bottlenecks that need to be addressed for successful clinical translation.

COVID-19 related liver injuries in pregnancy.

While severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) quickly spread across the globe, our understanding of its pathogenic mechanisms evolved. Importantly, coronavirus disease 2019 (COVID-19) is now considered a syndromic multisystem inflammatory disease involving not only the respiratory system but also the cardiovascular, excretory, nervous, musculoskeletal, and gastrointestinal systems. Moreover, a membrane-bound form of angiotensin-converting enzyme 2, the entry receptor for SARS-CoV-2, is expressed on the surface of cholangiocytes and hepatocytes, suggesting the potential of COVID-19 to involve the liver. With the widespread distribution of SARS-CoV-2 throughout the population, infection during pregnancy is no longer a rare occurrence; however, little is known about the course of hepatic injuries and related outcomes in pregnant SARS-CoV-2-positive women. Thus, the understudied topic of COVID-related liver disease during pregnancy poses a great challenge for the consulting gynecologist and hepatologist. In this review, we aim to describe and summarize potential liver injuries in pregnant women with COVID-19.

Shifts in functional traits and interactions patterns of soil methane-cycling communities following forest-to-pasture conversion in the Amazon Basin.

Deforestation threatens the integrity of the Amazon biome and the ecosystem services it provides, including greenhouse gas mitigation. Forest-to-pasture conversion has been shown to alter the flux of methane gas (CH4 ) in Amazonian soils, driving a switch from acting as a sink to a source of atmospheric CH4 . This study aimed to better understand this phenomenon by investigating soil microbial metagenomes, focusing on the taxonomic and functional structure of methane-cycling communities. Metagenomic data from forest and pasture soils were combined with in situ CH4 fluxes and soil edaphic factors measurements and analysed using multivariate statistical approaches. We found a significantly higher abundance and diversity of methanogens in pasture soils. As inferred by co-occurrence networks, these microorganisms seem to be less interconnected within the soil microbiota in pasture soils. Metabolic traits were also different between land uses, with increased hydrogenotrophic and methylotrophic pathways of methanogenesis in pasture soils. Land-use change also induced shifts in taxonomic and functional traits of methanotrophs, with bacteria harboring genes encoding the soluble form of methane monooxygenase enzyme (sMMO) depleted in pasture soils. Redundancy analysis and multimodel inference revealed that the shift in methane-cycling communities was associated with high pH, organic matter, soil porosity, and micronutrients in pasture soils. These results comprehensively characterize the effect of forest-to-pasture conversion on the microbial communities driving the methane-cycling microorganisms in the Amazon rainforest, which will contribute to the efforts to preserve this important biome.

Activation of TrkB in Parvalbumin interneurons is required for the promotion of reversal learning in spatial and fear memory by antidepressants

Critical period-like plasticity (iPlasticity) can be reinstated in the adult brain by several interventions, including drugs and optogenetic modifications. We have demonstrated that a combination of iPlasticity with optimal training improves behaviors related to neuropsychiatric disorders. In this context, the activation of TrkB, a receptor for BDNF, in Parvalbumin-positive (PV+) interneurons has a pivotal role in cortical network changes. However, it is unknown if the activation of TrkB in PV+ interneurons is important for other plasticity-related behaviors, especially for learning and memory. Here, using mice with heterozygous conditional TrkB deletion in PV+ interneurons (PV-TrkB hCKO) in IntelliCage and fear erasure paradigms, we show that chronic treatment with fluoxetine, a widely prescribed antidepressant drug that is known to promote the activation of TrkB, enhances behavioral flexibility in spatial and fear memory, largely depending on the expression of the TrkB receptor in PV+ interneurons. In addition, hippocampal long-term potentiation was enhanced by chronic treatment with fluoxetine in wild-type mice, but not in PV-TrkB hCKO mice. Transcriptomic analysis of PV+ interneurons after fluoxetine treatment indicated intrinsic changes in synaptic formation and downregulation of enzymes involved in perineuronal net formation. Consistently, immunohistochemistry has shown that the fluoxetine treatment alters PV expression and reduces PNNs in PV+ interneurons, and here we show that TrkB expression in PV+ interneurons is required for these effects. Together, our results provide molecular and network mechanisms for the induction of critical period-like plasticity in adulthood.

The Isolation, Immobilization, and Characterization of Urease from The Seeds of Winged Bean (Psophocarpus tetragonolobus (L.) DC.

Urease has been utilized in the field of health and industry. Urease is commonly used in the form of free enzyme, so that the utilization is limited. Urease efficiency can be improved using immobilization enzyme. This research aimed to do the urease isolation, immobilization, and characterization from the winged bean seeds. This research was started by determining the amino-acid content of winged bean seeds using the Liquid chromatography-mass spectrometry (LCMS). The winged bean seeds were germinated and extracted. The obtained crude extract’s activity was determined using Nessler reagent and measured using UV-Vis spectrophotometer with the wavelength of 500 nm. The urease of winged bean seeds was immobilized using the alginate matrix. The optimization of urease-immobilized beads could be made through the variations of natrium alginate concentration and beads formation periods in solution CaCl2. Characterization free and immobilized urease were made using the variations of urea substrate concentration, pH, temperature, and also the repeated utilization of immobilized urease. Winged bean seeds are rich with essential amino acid, such as leucine, isoleucine, histidine, phenylalanine, and valine. The urease obtained from the winged bean seeds had the optimum activity in the germination period of 8 days. The urease immobilization showed the optimum condition in the natrium alginate concentration of 5% (w/v) and beads formation period in solution CaCl2 for 60 minutes. The characterization results of free urease and immobilization had the optimum condition at the urea substrate of 0.2 M, and pH 7. Free urease had the optimum temperature of 35 oC, while the immobilized urease had the optimum temperature of 40 oC. The immobilized urease had the utilization stability up to 5 times with the relative activity of 48%. The EDX analysis results showed that the alginate did not contain N, while alginate urease beads contained N as much as 12%.

Electrochemical characterization of a dual cytochrome-containing lactate dehydrogenase

Flavin-dependent L-lactate dehydrogenase (LDH) from baker’s yeast (Saccharomyces cerevisiae) reversibly catalyzes the oxidation of L-lactate to L-pyruvate. In this study, four different enzymatic constructs were generated, and their catalytic and electrochemical properties were compared. Specifically, a truncated form of the native enzyme that includes only the catalytic domain, the native enzyme that includes an intrinsic electron-transferring cytochrome b2, a novel artificial enzyme containing a minimal cytochrome c and a version of the enzyme containing a fusion between two cytochromes were designed. All four variants were successfully expressed in Escherichia coli and presented properly matured heme domains. Assessing in vitro biocatalytic performance as reflected by lactate oxidation revealed the fusion-containing enzyme to be ∼ 12 times more active than the native enzyme. Electrochemical studies of electrode drop-casted enzyme variants also showed the superior performance of the dual-cytochrome construct, which displayed a lower average redox-potential for lactate oxidation, oxygen insensitivity in the lactate oxidation potential range and a wider dynamic range for lactate sensing, relative to the native enzyme. Moreover, product inhibition of this variant occurred at much higher lactate concentrations than with the native enzyme. In addition, when lower potentials were scanned using cyclic voltammetry, lactate-dependent oxygen reduction was measured for the dual-cytochrome fusion enzyme.

The binding of nitrogen-donor ligands to the ferric and ferrous forms of cytochrome P450 enzymes

The cytochrome P450 superfamily of heme-thiolate monooxygenase enzymes can catalyse various oxidation reactions. The addition of a substrate or an inhibitor ligand induces changes in the absorption spectrum of these enzymes and UV–visible (UV–vis) absorbance spectroscopy is the most common and readily available technique used to interrogate their heme and active site environment. Nitrogen-containing ligands can inhibit the catalytic cycle of heme enzymes by interacting with the heme. Here we evaluate the binding of imidazole and pyridine-based ligands to the ferric and ferrous forms of a selection of bacterial cytochrome P450 enzymes using UV–visible absorbance spectroscopy. The majority of these ligands interact with the heme as one would expect for type II nitrogen directly coordinated to a ferric heme-thiolate species. However, the spectroscopic changes observed in the ligand-bound ferrous forms indicated differences in the heme environment across these P450 enzyme/ligand combinations. Multiple species were observed in the UV–vis spectra of the ferrous ligand-bound P450s. None of the enzymes gave rise to the isolation of a single species with a Soret band at ∼442–447 nm, indicative of a 6-coordinate ferrous thiolate species with a nitrogen-donor ligand. A ferrous species with Soret band at ∼427 nm coupled with an α-band of increased intensity was observed with the imidazole ligands. With some enzyme-ligand combinations reduction resulted in breaking of the iron‑nitrogen bond yielding a 5-coordinate high-spin ferrous species. In other instances, the ferrous form was readily oxidised back to the ferric form on addition of the ligand.