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[EN] beta-Galactosidase (beta-Gal) is a widely used enzyme as a reporter gene in the field of molecular biology which hydrolyzes the beta-galactosides into monosaccharides. beta-Gal is an essential enzyme in humans and its deficiency or its overexpression results in several rare diseases. Cellular senescence is probably one of the most relevant physiological disorders that involve beta-Gal enzyme. In this review, we assess the progress made to date in the design of molecular-based probes for the detection of beta-Gal both in vitro and in vivo. Most of the reported molecular probes for the detection of beta-Gal consist of a galactopyranoside residue attached to a signalling unit through glycosidic bonds. The beta-Gal-induced hydrolysis of the glycosidic bonds released the signalling unit with remarkable changes in color and/or emission. Additional examples based on other approaches are also described. The wide applicability of these probes for the rapid and in situ detection of de-regulation beta-Gal-related diseases has boosted the research in this fertile field, R.M laboratory members received the financial support from the Spanish Government (project RTI2018-100910-B-C41) and the Generalitat Valenciana (project PROMETEO 2018/024). B.L-T. received support from the Spanish Ministry of Economy for their PhD grants (FPU15/02707). J. F.-B received fellowship (CD19/00038)

Alzheimer's disease (AD) is a progressive, chronic and age-related neurodegenerative disorder that affects millions of people across the world. In pursuit of new anti-AD remedies, 2-[Hydroxy-(4-nitrophenyl)methyl]-cyclopentanone (NMC), a β hydroxyl ketone derivative was studied to explore its neuroprotective potentials against AD. The in-vitro AChE and BuChE enzymes inhibition were evaluated by Ellman protocol and antioxidant potentials of NMC by DPPH free radical scavenging assay. In-vivo behavioral studies were performed in the transgenic 5xFAD mice model of AD using shallow water maze (SWM), Paddling Y-Maze (PYM), elevated plus maze (EPM) and balance beam (BB) tests. Also, the ex-vivo cholinesterase inhibitory effects of NMC and histopathological analysis of amyloid-β plaques were determined in the frontal cortex and hippocampal regions of the mice brain. NMC exhibited significant in vitro anti-cholinesterase enzyme potentials with an IC50 value of 67 μg/ml against AChE and 96 μg/ml against BuChE respectively. Interestingly, the activities of AChE and BuChE enzymes were also significantly lower in the cortex and hippocampus of NMC-treated groups. Also, in the DPPH assessment, NMC displayed substantial antioxidant properties with an IC50 value observed as 171 μg/ml. Moreover, histopathological analysis via thioflavin-s staining displayed significantly lower plaques depositions in the cortex and hippocampus region of NMC-treated mice groups. Furthermore, SWM, PYM, EPM, and BB behavioral analysis indicated that NMC enhanced spatial learning, memory consolidation and improved balance performance. Altogether, to the best of our knowledge, we believe that NMC may serve as a potential and promising anti-cholinesterase, antioxidant and neuroprotective agent against AD.

Among the 2-arylbenzofuran derivatives isolated from Morus alba, the farnesylated 2-arylbenzofuran is a rarer constituent. The derivative has been reported to exert anti-obesity effect; however, its inhibitory effect on protein tyrosine phosphatase 1B (PTP1B) has not been investigated. In the previous study, the presence of the farnesyl group in the structure of 2-arylbenzofurans was found to have positive influences on their pancreatic lipase inhibitory activity. In the present study, we have confirmed the authenticity of the notation based on the PTP1B inhibitory activity of farnesylated 2-arylbenzofurans. Specifically, two farnesylated 2-arylbenzofurans [morusalfurans B (2) and C (3)] showed strong inhibitory effects on PTP1B with IC50 values of 8.92 and 7.26 µM, respectively, which was significantly higher than that of the positive controls [sodium orthovanadate (IC50 = 15.10 µM) and ursolic acid (IC50 = 11.34 µM)]. Besides, two 2-arylbenzofurans [morusalfurans A (1) and F (6)], one flavonoid [morusalnol B (9)], and one geranylated stilbene [morusibene A (11)] exhibited PTP1B inhibition with IC50 values ranging from 11.02 to 26.56 µM. Kinetic studies revealed compounds 2, 3, 6, and 11 as mixed type PTP1B inhibitors, while 1 and 9 are known as noncompetitive. Molecular docking simulations demonstrated that these active compounds can bind with the respective catalytic or/and allosteric sites of PTP1B with negative binding energies and the results are in accordance with that of the kinetic studies. To the best of our knowledge, this is the first time, the PTP1B inhibitory activity of eleven compounds (1–11), as well as the mechanism of action underlying the effects on PTP1B enzyme of the active compounds, were investigated. In vitro and in silico results suggest that the farnesylated 2-arylbenzofurans from M. alba may potentially be utilized as an effective treatment therapy for type 2 diabetes mellitus and its associated complications.

Sensitive detection of two biological events in vivo has long been a goal in bioluminescence imaging. Antares, a fusion of the luciferase NanoLuc to the orange fluorescent protein CyOFP, has emerged as a bright bioluminescent reporter with orthogonal substrate specificity to firefly luciferase (FLuc) and its derivatives such as AkaLuc. However, the brightness of Antares in mice is limited by the poor solubility and bioavailability of the NanoLuc substrate furimazine. Here, we report a new substrate, hydrofurimazine, whose enhanced aqueous solubility allows delivery of higher doses to mice. In the liver, Antares with hydrofurimazine exhibited similar brightness to AkaLuc with its substrate AkaLumine. Further chemical exploration generated a second substrate, fluorofurimazine, with even higher brightness in vivo. We used Antares with fluorofurimazine to track tumor size and AkaLuc with AkaLumine to visualize CAR-T cells within the same mice, demonstrating the ability to perform two-population imaging with these two luciferase systems. NanoLuc substrates with improved solubility and bioavailability, hydrofurimazine and fluorofurimazine, strongly enhance bioluminescence signals in vivo and enable bright dual-color bioluminescent imaging with AkaLuc and AkaLumine.

Purification of DPP-IV enzyme from porcine serum, is presented in this study for the first time. The high molecular weight DPP-IV from porcine serum was fractioned using Sephadex G-75 gel filtration followed by DEAE Sephadex anion exchange and Sephadex G-100 gel filtration chromatography columns with a final yield of 11.25%. The SDS-PAGE of the purified sample showed a single band of molecular mass nearing 160 kDa. Distinct single band was observed after PAS staining confirmed it to be a glycoprotein. The purified enzyme showed an optimum pH and temperature of 8 and 37 °C, respectively. The enzyme effectively cleaved fluorogenic substrate Gly-Pro-AMC with Km and Vmax of 4.578 µM and 90.84 nmoles/min, respectively. Purified DPP-IV activity was inhibited by Diprotin A with an IC50 value of 8.473 µM. Among the three plant extracts used to study DPP-IV inhibition, the aqueous hot extract of Terminalia chebula showed the highest inhibition of 87.19%, followed by the aqueous cold extract of Momordica carantia, ( 31.6%) and Azadirachta indica (34.16%) at the concentration of 25 µg.

Present estimates suggest that of the 359 million tons of plastics produced annually worldwide1, 150–200 million tons accumulate in landfill or in the natural environment2. Poly(ethylene terephthalate) (PET) is the most abundant polyester plastic, with almost 70 million tons manufactured annually worldwide for use in textiles and packaging3. The main recycling process for PET, via thermomechanical means, results in a loss of mechanical properties4. Consequently, de novo synthesis is preferred and PET waste continues to accumulate. With a high ratio of aromatic terephthalate units—which reduce chain mobility—PET is a polyester that is extremely difficult to hydrolyse5. Several PET hydrolase enzymes have been reported, but show limited productivity6,7. Here we describe an improved PET hydrolase that ultimately achieves, over 10 hours, a minimum of 90 per cent PET depolymerization into monomers, with a productivity of 16.7 grams of terephthalate per litre per hour (200 grams per kilogram of PET suspension, with an enzyme concentration of 3 milligrams per gram of PET). This highly efficient, optimized enzyme outperforms all PET hydrolases reported so far, including an enzyme8,9 from the bacterium Ideonella sakaiensis strain 201-F6 (even assisted by a secondary enzyme10) and related improved variants11–14 that have attracted recent interest. We also show that biologically recycled PET exhibiting the same properties as petrochemical PET can be produced from enzymatically depolymerized PET waste, before being processed into bottles, thereby contributing towards the concept of a circular PET economy. Computer-aided engineering produces improvements to an enzyme that breaks down poly(ethylene terephthalate) (PET) into its constituent monomers, which are used to synthesize PET of near-petrochemical grade that can be further processed into bottles.

The mechanisms that underlie the extensive phenotypic diversity in genetic disorders are poorly understood. Here, we develop a large-scale assay to characterize the functional valence (gain or loss-of-function) of missense variants identified in UBE3A, the gene whose loss-of-function causes the neurodevelopmental disorder Angelman syndrome. We identify numerous gain-of-function variants including a hyperactivating Q588E mutation that strikingly increases UBE3A activity above wild-type UBE3A levels. Mice carrying the Q588E mutation exhibit aberrant early-life motor and communication deficits, and individuals possessing hyperactivating UBE3A variants exhibit affected phenotypes that are distinguishable from Angelman syndrome. Additional structure-function analysis reveals that Q588 forms a regulatory site in UBE3A that is conserved among HECT domain ubiquitin ligases and perturbed in various neurodevelopmental disorders. Together, our study indicates that excessive UBE3A activity increases the risk for neurodevelopmental pathology and suggests that functional variant analysis can help delineate mechanistic subtypes in monogenic disorders., UBE3A gene dysregulation is associated with neurodevelopmental disorders, but predicting the function of UBE3A variants remains difficult. The authors use a high-throughput assay to categorize variants by functional activity, and show that UBE3A hyperactivity increases the risk of neurodevelopmental disease.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening thrombotic microangiopathy caused by severely reduced activity of the von-Willebrand factor-cleaving protease ADAMTS13, mainly caused by anti-ADAMTS-13 antibodies. Although several test systems for ADAMTS13 measurement exist, long turn-around times hamper the usability in daily practice. We performed a method comparison study for two commercially available ADAMTS13 assays and evaluated the agreement between the fully-automated rapid turn-over HemosIL AcuStar ADAMTS13 Activity assay and the manually performed TECHNOZYM ADAMTS-13 Activity assay. Twenty-four paired test samples derived from 10 consecutively recruited patients (n = 8, acquired TTP; n = 1, atypical hemolytic uremic syndrome; n = 1, control), of which nine test samples were collected in case of clinically apparent TTP and 13 samples were collected from TTP patients in clinical remission were included. Overall correlation between the TECHNOZYM and AcuStar assay was good with a Pearson R of 0.93 (p or

Reductions in the activities of mitochondrial electron transport chain (ETC) enzymes have been implicated in the pathogenesis of numerous chronic neurodegenerative disorders. Maintenance of the mitochondrial membrane potential (Δψm) is a primary function of these enzyme complexes, and is essential for ATP production and neuronal survival. We examined the effects of inhibition of mitochondrial ETC complexes I, II/III, III and IV activities by titrations of respective inhibitors on Δψm in synaptosomal mitochondria. Small perturbations in the activity of complex I, brought about by low concentrations of rotenone (1–50 nM), caused depolarisation of Δψm. Small decreases in complex I activity caused an immediate and partial Δψm depolarisation, whereas inhibition of complex II/III activity by more than 70% with antimycin A was required to affect Δψm. A similarly high threshold of inhibition was found when complex III was inhibited with myxothiazol, and inhibition of complex IV by more than 90% with KCN was required. The plasma membrane potential (Δψp) had a complex I inhibition threshold of 40% whereas complex III and IV had to be inhibited by more than 90% before changes in Δψp were registered. These data indicate that in synaptosomes, both Δψm and Δψp are more susceptible to reductions in complex I activity than reductions in the other ETC complexes. These findings may be of relevance to the mechanism of neuronal cell death in Parkinson’s disease in particular, where such reductions in complex I activity are present.

Human carboxylesterase 2 (hCE2), one of the most principal drug-metabolizing enzymes, catalyzes the hydrolysis of a variety of endogenous esters, anticancer agents, and environmental toxicants. The significant roles of hCE2 in both endobiotic and xenobiotic metabolism sparked great interest in the discovery and development of efficacious and selective inhibitors. However, the safe and effective inhibitors of hCE2 are scarce, due to the lack of efficient screening and evaluation systems for complex biological systems. To offer a solution to this problem, a high-content analysis (HCA)-based cell imaging and multiparametric assay method was constructed for evaluating the inhibitory effect and safety of hCE2 inhibitors in living cell system. In this study, we first established a cell imaging-based method for identifying hCE2 inhibitors at the living cell level with hCE2 fluorescent probe NCEN. Meanwhile, two nuclear probes, Hoechst 33342 and PI, were integrated to evaluate the potential cytotoxicity of compounds simultaneously. Then, the accuracy of the HCA-based method was verified by the LC-FD-based method with a positive inhibitor BNPP, and the results showed that the HCA-based method exhibited excellent precision, robustness, and reliability. Finally, the newly established HCA-based multiparametric assay panel was successfully applied to re-evaluate a series of reported hCE2 inhibitors in living cells. In summary, the HCA-based multiparametric method could serve as an efficient tool for the accuracy measurement inhibitory effect and cytotoxicity of compounds against hCE2 in living cell system. Graphical abstract.

A substrate of 4-nitrophenyl phosphate was enzymatically hydrolyzed by alkaline phosphatase (ALP) in a capillary tube, while an injected zone of the substrate was electrophoretically migrating in the separation buffer containing the enzyme by capillary electrophoresis (CE). During CE migration of the substrate from the start time of the electrophoresis to the detection time of the substrate, the substrate was continuously hydrolyzed by ALP to form a product of 4-nitrophenolate, and a plateau signal of 4-nitrophenolate was detected as a result of the zero-order kinetic reaction. The height of the plateau signal was directly related to the reaction rate, and it was used for the determination of a Michaelis-Menten constant through Lineweaver-Burk plots. Since the plateau signal is attributed to the dynamic formation of the product by the enzymatic reaction in CE, this analysis method is named as capillary electrophoresis/dynamic frontal analysis (CE/DFA). In CE/DFA, the CE separation is included on detecting the plateau signal, and the hydrolysis product before the sample injection is resolved from the dynamically and continuously formed product. The inhibition of the enzyme with the product is also eliminated in CE/DFA by the CE separation.

Avian leukosis virus subgroup J (ALV-J) is an important pathogen for various neoplasms and causes significant economic losses in the poultry industry. Serological detection of specific antibodies against ALV-J infection is important for successful clinical diagnosis. Here, a 293F stable cell line was established to stably express gp85 protein. In this cell line, gp85 protein was expressed at approximately 30 mg/L. A subgroup-specific indirect enzyme-linked immunosorbent assay (iELISA) was developed using ALV-J gp85 protein as coated antigen to detect antibodies against ALV-J. The sensitivity of the iELISA (1:51200 diluted in serum) was 16 times more than that of indirect immunofluorescence assay (IFA; 1:3200 diluted in serum). Moreover, there was no crossreactivity with antibodies against other common avian viruses and other avian leukosis virus subgroups, such as subgroups A and B. The practicality of the iELISA was further evaluated by experimental infection and clinical samples. The results from experimental infection indicated that anti-ALV-J antibodies were readily detected by iELISA as early as 4 weeks after ALV-J infection, and positive antibodies were detected until 20 weeks, with an antibody-positive rate of 11.1% to 33.3%. Moreover, analysis of clinical samples showed that 9.49% of samples were positive for anti-ALV-J antibodies, and the concordance rate of iELISA and IFA was 99.24%. Overall, these results suggested that the subgroup-specific iELISA developed in this study had good sensitivity, specificity, and feasibility. This iELISA will be very useful for epidemiological surveillance, diagnosis, and eradication of ALV-J in poultry farms.

3-hydroxy-3-methyl glutaryl-CoA reductase, also known as HMGR, plays a crucial role in regulating cholesterol biosynthesis and represents the main pharmacological target of statins. In mammals, this enzyme localizes to the endoplasmic reticulum membrane. HMGR includes different regions, an integral N-terminal domain connected by a linker-region to a cytosolic C-terminal domain, the latter being responsible for enzymatic activity. The aim of this work was to design a simple strategy for cloning, expression, and purification of the catalytic C-terminal domain of the human HMGR (cf-HMGR), in order to spectrophotometrically test its enzymatic activity. The recombinant cf-HMGR protein was heterologously expressed in Escherichia coli, purified by Ni+-agarose affinity chromatography and reconstituted in its active form. MALDI mass spectrometry was adopted to monitor purification procedure as a technique orthogonal to the classical Western blot analysis. Protein identity was validated by MS and MS/MS analysis, confirming about 82% of the recombinant sequence. The specific activity of the purified and dialyzed cf-HMGR preparation was enriched about 85-fold with respect to the supernatant obtained from cell lysate. The effective, cheap, and easy method here described could be useful for screening statin-like molecules, so simplifying the search for new drugs with hypocholesterolemic effects.

Naturally occurring fructosamines are of high clinical significance due to their potential use in diabetes mellitus monitoring (quantification of fructosylated hemoglobin, HbA1c) or for the investigation of their reactivity in consecutive reactions and harmfulness towards the organism. Here we report the specific synthesis of the fructosylated dipeptide l-valyl-l-histidine (Fru-Val-His) and fructosylated l-valine (Fru-Val). Both are basic tools for the development and validation of enzymatic HbA1c assays. The two fructosamine derivatives were synthesized via a protected glucosone intermediate which was coupled to the primary amine of Val or Val-His, performing a reductive amination reaction. Overall yields starting from fructose were 36% and 34% for Fru-Val and Fru-Val-His, respectively. Both compounds were achieved in purities > 90%. A HILIC-ESI-MS/MS method was developed for routine analysis of the synthesized fructosamines, including starting materials and intermediates. The presented method provides a well-defined and efficient synthesis protocol with purification steps and characterization of the desired products. The functionality of the fructosylated dipeptide has been thoroughly tested in an enzymatic HbA1c assay, showing its concentration-dependent oxidative degradation by fructosyl-peptide oxidases (FPOX).

Estrogens are key factors in the development of the estrogen receptor–positive (ER+) breast cancer. Estrogens, estrone (E1), and estradiol (E2) production is achieved by aromatase, a cytochrome P450 enzyme that has androgens, androstenedione (AD), and testosterone (T) as substrates. Nowadays, third-generation aromatase inhibitors (AIs) are considered the gold-standard treatment for ER+ breast cancer in postmenopausal women as well as in premenopausal women with ovary ablation. Aromatase activity assessment still relies on radiometric assays that are expensive, hazardous, and non-environmentally friendly. Thus, in order to overcome these disadvantages, a new methodology was developed to evaluate aromatase activity, based on dispersive liquid-liquid microextraction (DLLME) followed by gas chromatography-mass spectrometry (GC-MS). The enzymatic reaction was carried out in human placental microsomes, using AD as substrate, and the anti-aromatase activity was measured by determining the conversion percentage of AD into E1 (ratio E1/AD) using isotopic analogues as internal standards. The method showed good linearity (r2 = 0.9908 for AD and 0.9944 for E1), high accuracy (more than 74% for AD and more than 66% for E1), high extraction efficiency, and good intra-day and inter-day precision (below 14%, 4 levels). In this work, the IC50 values of the third-generation AIs, anastrozole, letrozole, and exemestane, obtained from the radiometric assay are also compared, and similar IC50 values are described. This method is a good alternative to the current radiometric assay, being fast and sensitive with a good extraction efficiency, accuracy, and recovery. In addition, it may be applied for the evaluation of the anti-aromatase activity of new potential AIs.

The synthetic pathways of life’s building blocks are envisaged to be through a series of complex prebiotic reactions and processes. However, the strategy to compartmentalize and concentrate biopolymers under prebiotic conditions remains elusive. Liquid-liquid phase separation is a mechanism by which membraneless organelles form inside cells, and has been hypothesized as a potential mechanism for prebiotic compartmentalization. Associative phase separation of oppositely charged species has been shown to partition RNA, but the strongly negative charge exhibited by RNA suggests that RNA-polycation interactions could inhibit RNA folding and its functioning inside the coacervates. Here, we present a prebiotically plausible pathway for non-associative phase separation within an evaporating all-aqueous sessile droplet. We quantitatively investigate the kinetic pathway of phase separation triggered by the non-uniform evaporation rate, together with the Marangoni flow-driven hydrodynamics inside the sessile droplet. With the ability to undergo liquid-liquid phase separation, the drying droplets provide a robust mechanism for formation of prebiotic membraneless compartments, as demonstrated by localization and storage of nucleic acids, in vitro transcription, as well as a three-fold enhancement of ribozyme activity. The compartmentalization mechanism illustrated in this model system is feasible on wet organophilic silica-rich surfaces during early molecular evolution., Prebiotic compartmentalization could prove essential for the evolution of life. Guo et al. show that liquid-liquid separation in an aqueous two-phase system driven by evaporation may already suffice to facilitate chemical processes required for the RNA world hypothesis.

In our present paper, the influence of a pyramidal structure on physicochemical properties of a protein in buffer solution has been studied. The pyramidal structure employed herein was similar to those produced industrially for anechoic chambers. Pyramidal structures are also used as elements of biosensors. Herein, horseradish peroxidase (HRP) enzyme was used as a model protein. HRP macromolecules were adsorbed from their solution onto an atomically smooth mica substrate, and then visualized by atomic force microscopy (AFM). In parallel, the enzymatic activity of HRP was estimated by conventional spectrophotometry. Additionally, attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) has been employed in order to find out whether or not the protein secondary structure changes after the incubation of its solution either near the apex of a pyramid or in the center of its base. Using AFM, we have demonstrated that the incubation of the protein solution either in the vicinity of the pyramid’s apex or in the center of its base influences the physicochemical properties of the protein macromolecules. Namely, the incubation of the HRP solution in the vicinity of the top of the pyramidal structure has been shown to lead to an increase in the efficiency of the HRP adsorption onto mica. Moreover, after the incubation of the HRP solution either near the top of the pyramid or in the center of its base, the HRP macromolecules adsorb onto the mica surface predominantly in monomeric form. At that, the enzymatic activity of HRP does not change. The results of our present study are useful to be taken into account in the development of novel biosensor devices (including those for the diagnosis of cancer in humans), in which pyramidal structures are employed as sensor, noise suppression or construction elements.

Nicotinamide adenine dinucleotide (NAD) and its reduced form are indispensable cofactors in life. Diverse NAD mimics have been developed for applications in chemical and biological sciences. Nicotinamide cytosine dinucleotide (NCD) has emerged as a non-natural cofactor to mediate redox transformations, while cells are fed with chemically synthesized NCD. Here, we create NCD synthetase (NcdS) by reprograming the substrate binding pockets of nicotinic acid mononucleotide (NaMN) adenylyltransferase to favor cytidine triphosphate and nicotinamide mononucleotide over their regular substrates ATP and NaMN, respectively. Overexpression of NcdS alone in the model host Escherichia coli facilitated intracellular production of NCD, and higher NCD levels up to 5.0 mM were achieved upon further pathway regulation. Finally, the non-natural cofactor self-sufficiency was confirmed by mediating an NCD-linked metabolic circuit to convert L-malate into D-lactate. NcdS together with NCD-linked enzymes offer unique tools and opportunities for intriguing studies in chemical biology and synthetic biology., Nicotinamide cytosine dinucleotide is an NAD mimic that can be used to mediate redox reactions. Here the authors design an NCD synthetase for the intracellular production of NCD.

A novel method is described for the immobilization of alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae onto carboxymethyl dextran (CMD) coated magnetic nanoparticles (CMD-MNPs) activated with epoxy groups, using epichlorohydrin (EClH). EClH was used as an activating agent to bind ADH molecules on the surface of CMD-MNPs. Optimal immobilization conditions (activating agent concentration, temperature, rotation speed, medium pH, immobilization time and enzyme concentration) were set to obtain the highest expressed activity of the immobilized enzyme. ADH that was immobilized onto epoxy-activated CMD-MNPs (ADH-CMD-MNPs) maintained 90% of the expressed activity. Thermal stability of ADH-CMD-MNPS after 24 h at 20 °C and 40 °C yielded 79% and 80% of initial activity, respectively, while soluble enzyme activity was only 19% at 20 °C and the enzyme was non-active at 40 °C. Expressed activity of ADH-CMD-MNPs after 21 days of storage at 4 °C was 75%. Kinetic parameters (KM, vmax) of soluble and immobilized ADH were determined, resulting in 125 mM and 1.2 µmol/min for soluble ADH, and in 73 mM and 4.7 µmol/min for immobilized ADH.

By using graphene quantum dots (GQDs) and o-phenylenediamine (OPD), a ratiometric fluorescence probe was designed for the highly sensitive and selective detection of AChE. GQDs with strong fluorescence were synthesized by the one-step hydrothermal method. The optimal emission wavelength of GQDs was 450 nm at the excitation wavelength of 375 nm. MnO2 nanosheets with a wide absorption band of 300–600 nm were prepared at room temperature. Because of the extensive overlap between the absorption spectrum of MnO2 nanosheets and the excitation and emission spectra of GQDs, the fluorescence of GQDs at 450 nm was efficiently quenched by the inner-filter effect. Meanwhile, due to the peroxidase-like activity of MnO2 nanosheets, OPD was catalytically oxidized to 2,3-diaminophenazine (oxOPD), a yellow fluorescent substance with a new emission peak at 572 nm. When AChE was present, the substrate acetylthiocholine (ATCh) was hydrolyzed to thiocholine (TCh) that is capable of decomposing MnO2 nanosheets. Therefore, the quench of GQDs and the oxidation of OPD by MnO2 nanosheets were suppressed, resulting in the fluorescence recovery of GQDs at 450 nm, while the fluorescence decrease of oxOPD at 572 nm. Utilizing the fluorescence intensity ratio F450/F572 as the signal readout, the ratiometric fluorescence method was established to detect AChE activity. The ratio F450/F572 against the AChE concentration demonstrated two linear relationships in the range 0.1–2.0 and 2.0–4.5 mU mL−1 with a detection limit of 0.09 mU mL−1. The method was applied to the detection of positive human serum samples and the analysis of the inhibitor neostigmine. Due to the advantages of high sensitivity, favorable selectivity, and strong anti-interference, the method possesses an application prospect in clinical diagnosis of AChE and the screening of inhibitors.

A turn-on method for determining α-glucosidase activity is described using a chemical redox strategy in which the fluorescence of red fluorescent carbon dots (CDs) is modulated. The red fluorescent CDs were prepared using a solvothermal method with p-phenylenediamine and sodium citrate. The excitation and emission maxima of the CDs were 490 and 618 nm, respectively. Ce4+ ions catalyze the oxidation of the colorless substrate 3,3′,5,5′-tetramethylbenzidine (TMB) to give a blue oxidized TMB product (oxTMB). Absorption by oxTMB overlaps with the red light emitted by the CDs because of the fluorescence inner filter effect; therefore the presence of oxTMB decreases the intensity of fluorescence emission by the CDs. However, hydrolysis of L-ascorbic acid-2-O-α-D-glucopyranosyl by the enzyme α-glucosidase causes formation of ascorbic acid . Ascorbic acid reduces oxTMB to TMB, so that the inner filter effect disappeared and the fluorescence recovered. The strategy allows α-glucosidase activity to be successfully determined down to 0.02 U mL−1 and gives a dynamic linear range of 0–5.5 U mL−1. The strategy is very selective for α-glucosidase activity in the presence of potentially interfering substances. The method has been successfully applied to the determination of α-glucosidase activity in spiked human serum samples and gave satisfactory results.

Human infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) and there is no cure currently. The 3CL protease (3CLpro) is a highly conserved protease which is indispensable for CoVs replication, and is a promising target for development of broad-spectrum antiviral drugs. In this study we investigated the anti-SARS-CoV-2 potential of Shuanghuanglian preparation, a Chinese traditional patent medicine with a long history for treating respiratory tract infection in China. We showed that either the oral liquid of Shuanghuanglian, the lyophilized powder of Shuanghuanglian for injection or their bioactive components dose-dependently inhibited SARS-CoV-2 3CLpro as well as the replication of SARS-CoV-2 in Vero E6 cells. Baicalin and baicalein, two ingredients of Shuanghuanglian, were characterized as the first noncovalent, nonpeptidomimetic inhibitors of SARS-CoV-2 3CLpro and exhibited potent antiviral activities in a cell-based system. Remarkably, the binding mode of baicalein with SARS-CoV-2 3CLpro determined by X-ray protein crystallography was distinctly different from those of known 3CLpro inhibitors. Baicalein was productively ensconced in the core of the substrate-binding pocket by interacting with two catalytic residues, the crucial S1/S2 subsites and the oxyanion loop, acting as a "shield" in front of the catalytic dyad to effectively prevent substrate access to the catalytic dyad within the active site. Overall, this study provides an example for exploring the in vitro potency of Chinese traditional patent medicines and effectively identifying bioactive ingredients toward a specific target, and gains evidence supporting the in vivo studies of Shuanghuanglian oral liquid as well as two natural products for COVID-19 treatment.

High-throughput function-based screening techniques remain the major bottleneck in the novel biocatalysts development pipeline. In the present study, we customized protocols for amylolytic activity determination (Somogyi-Nelson and starch-iodine tests) to micro-volume thermalcycler-based assays (linearity range 60–600 μM of reducing sugar, R2 = 0.9855; 0–2 mg/mL of starch, R2 = 0.9921, respectively). Exploitation of a thermalcycler enabled rapid and accurate temperature control, further reduction of reagents and samples volumes, and limited evaporation of the reaction mixtures, meeting several crucial requirements of an adequate enzymatic assay. In the optimized micro-volume Somogyi-Nelson protocol, we were able to reduce the time required for high-temperature heating sixfold (down to 5 min) and further increase sensitivity of the assay (tenfold), when compared to the previous MTP-based protocol. The optimized microassays have complementary scope of specificities: micro-starch-iodine test for endoglucanases, micro-Somogyi-Nelson test for exoglucanases. Due to rapid, micro-volume and high-throughput character, the methods can complement toolbox assisting development of novel biocatalysts and analysis of saccharides-containing samples. Electronic supplementary material The online version of this article (10.1007/s00253-018-09610-0) contains supplementary material, which is available to authorized users.

© The Author(s) 2020., Matrix metalloproteinases (MMPs) occur in 23 human paralogues with key functions in physiology, and their activity is controlled by protein inhibitors. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), which is essential for embryogenesis and tumour suppression, has been reported to inhibit MMPs. Here, we developed eukaryotic and bacterial expression systems for different RECK variants and analysed their inhibitory capacity against representative MMPs in vitro. We could not detect any significant inhibition. Instead, we found that partially purified RECK from the conditioned medium of transfected Expi293F cells but not that of ExpiCHO-S or Drosophila Schneider cells contained a contaminant with proteolytic activity. The contaminant was removed through treatment with a small-molecule serine peptidase inhibitor and additional chromatographic purification. A tantamount contaminant was further detected in an equivalent expression system of the N-terminal fragment of the proteoglycan testican 3, but not in those of two other proteins. These results indicate that previous reports of inhibitory activity of recombinant RECK on MMPs, which were performed with partially purified samples, were probably masked by a coeluting contaminant present in the supernatant of HEK293-derived cells. Thus, RECK is probably not a direct inhibitor of MMP catalytic activity but may still regulate MMPs through other mechanisms., This study was supported in part by grants from Spanish and Catalan public and private bodies (grant/ fellowship references BFU2015–64487R, MDM-2014–0435, BES-2015–074583, BES-2016–076877, 2017SGR3 and Fundació “La Marató de TV3” 201815).

The rapid detection of blood stream infections (BSI) by carbapenemase-producing Enterobacterales (CPE) is indispensable to early optimize antibiotic treatment and to improve survival. While phenotypic tests are time-consuming and PCR is expensive and not available in many routine laboratories, colorimetric tests (e.g., Carba NP test) can provide rapid results at moderate cost. However, up to now, the detection of CPE-BSI requires a further 3-h incubation in broth supplemented with zinc sulfate and imipenem after a blood culture has become positive, thereby causing delay and additional hands-on time. The purpose of this study was to develop and evaluate a new method for the detection of CPE directly from positive blood culture without the need for incubation in broth, based on the commercially available colorimetric β-CARBA test. For the evaluation, blood cultures spiked with 140 different Enterobacterales isolates producing diverse beta-lactamases were tested with the new method. Of these, 70 were CPE (OXA-48-like, NDM, KPC, VIM, and GIM). After blood cultures turned positive, blood culture fluid was drawn, and erythrocytes were hemolyzed with SDS, washed, and equilibrated before the β-CARBA was performed on the bacterial pellet. All carbapenemases were reliably detected, including weak carbapenemases of the OXA-48 group. The sensitivity was 100% (95% CI 94.9-100) and the specificity 94.3% (95% CI 89.2-99.4). The time to result was 20 to 45 min. Carbapenemases can rapidly and reliably be detected directly from blood cultures using the new method, which could help to improve the outcome of these difficult-to-treat infections.

The project SAFEPEDRUG aims to provide guidelines for drug research in children, based on bottom-up and top-down approaches. Propofol, one of the studied model compounds, was selected because it is extensively metabolized in liver and kidney, with an important role for the glucuronidation pathway. Besides, being a lipophilic molecule, it is distributed into fat tissues, from where it redistributes into the systemic circulation. In the past, both bottom-up (Physiologically based pharmacokinetic, PBPK) and top-down approaches (population pharmacokinetic, popPK) were applied to describe its pharmacokinetics (PK). In this work, a combination of the two was used to check their performance to describe PK in children and neonates (both term and preterm) using propofol as a case compound. First, in vitro data was generated in human liver microsomes and recombinant enzymes and used to develop an adult PBPK model in Simcyp®. Activity adjustment factors (AAFs) were calculated to account for differences between in vitro and in vivo enzyme activity. Clinical data were analyzed using a 3-compartment model in NONMEM. These data were used to construct a retrograde PBPK model and for qualification of the PBPK models. Once an accurate in vivo clearance was obtained accounting for the contribution of the different metabolic pathways, the resulting PBPK models were challenged with new data for qualification. After that, the constructed adult PPBK model for propofol was extrapolated to the pediatric population. Both the default built-in and in vivo derived ontogeny functions were used to do so. The models were qualified by comparing their predicted PK parameters to published values, and by comparison of predicted concentration-time profiles to available clinical data. Clearance values were predicted well, especially when compared with values obtained from trials where long-term sampling was applied, whereas volume of distribution was lower compared to the most common popPK model predictions. Concentration-time profiles were predicted well up until and including the preterm neonatal population. In this work, it was thus shown that PBPK can be used to predict the PK up to and including the preterm neonatal population without the use of pediatric in vivo data. This work adds weight to the need for further development of PBPK models, especially regarding distribution modeling and the use of in vivo derived ontogeny functions. ispartof: JOURNAL OF PHARMACOKINETICS AND PHARMACODYNAMICS vol:45 issue:6 pages:765-785 ispartof: location:United States status: published

Made available in DSpace on 2018-12-11T17:22:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-01-01 L-asparaginase or ASNase (L-asparagine aminohydrolase, E.C.3.5.1.1) is an enzyme clinically accepted as an antitumor agent to treat acute lymphoblastic leukemia (ALL) and lymphosarcoma through the depletion of L-asparagine (L-Asn) resulting in cytotoxicity to leukemic cells. ASNase is also important in the food industry, preventing acrylamide formation in processed foods. Several quantification techniques have been developed and used for the measurement of the ASNase activity, but standard pharmaceutical quality control methods were hardly reported, and in general, no official quality control guidelines were defined. To overcome this lack of information and to demonstrate the advantages and limitations, this work properly compares the traditional colorimetric methods (Nessler; L-aspartic acid β-hydroxamate (AHA); and indooxine) and the high-performance liquid chromatography (HPLC) method. A comparison of the methods using pure ASNase shows that the colorimetric methods both overestimate (Nessler) and underestimate (AHA and indooxine) the ASNase activity when compared to the values obtained with HPLC, considered the most precise method as this method monitors both substrate consumption and product formation, allowing for overall mass-balance. Correlation and critical analysis of each method relative to the HPLC method were carried out, resulting in a demonstration that it is crucial to select a proper method for the quantification of ASNase activity, allowing bioequivalence studies and individualized monitoring of different ASNase preparations. [Figure not available: see fulltext.] Department of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP), Rodovia Araraquara-Jaú/Km 01, Campos Ville Biochemistry and Technology Chemistry Department Chemistry Institute São Paulo State University (UNESP) Department of Chemistry Earlham College, 801 National Road West Department of Biochemical-Pharmaceutical Technology Pharmaceutical Biotechnology Laboratory University of Sao Paulo (USP) Department of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP), Rodovia Araraquara-Jaú/Km 01, Campos Ville Biochemistry and Technology Chemistry Department Chemistry Institute São Paulo State University (UNESP)

The serum and glucocorticoid-regulated kinase (SGK) family has been implicated in the regulation of many cellular processes downstream of the PI3K pathway. It plays a crucial role in PI3K-mediated tumorigenesis, making it a potential therapeutic target for cancer. SGK family consists of three isoforms (SGK1, SGK2, and SGK3), which have high sequence homology in the kinase domain and similar substrate specificity with the AKT family. In order to identify novel compounds capable of inhibiting SGK3 activity, a high-throughput screening campaign against 50,400 small molecules was conducted using a fluorescence-based kinase assay that has a Z' factor above 0.5. It identified 15 hits (including nitrogen-containing aromatic, flavone, hydrazone, and naphthalene derivatives) with IC(50) values in the low micromolar to sub-micromolar range. Four compounds with a similar scaffold (i.e., a hydrazone core) were selected for structural modification and 18 derivatives were synthesized. Molecular modeling was then used to investigate the structure-activity relationship (SAR) and potential protein–ligand interactions. As a result, a series of SGK inhibitors that are active against both SGK1 and SGK3 were developed and important functional groups that control their inhibitory activity identified.

A nitrogen-fixing, endospore-forming bacterium, designated strain L201T was isolated from the leaves of Bryophyllum pinnatum growing in South China Agricultural University. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain L201T is affiliated with the genus Paenibacillus, and closely related to Paenibacillus albidus Q4-3T (97.4%), Paenibacillus odorifer DSM 15391T (97.3%) and Paenibacillus borealis DSM 13188T (97.2%). The main fatty acids components was anteiso-C15:0 (48.1%). The predominant isoprenoid quinone was MK-7. The major polar lipids were found to be diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The G+C content of strain L201T was 43.9%. DNA–DNA relatedness between L201T and the reference strain was 29.8%. Biological and biochemical tests, protein patterns, genomic DNA fingerprinting and comparison of cellular fatty acids distinguished strain L201T from the closely related Paenibacillus species. Based on these data, the novel species Paenibacillus bryophyllum sp. nov. is proposed, with the type strain L201T(= KCTC 33951 T = GDMCC 1.1251 T).

DNA methyltransferase (MTase) is related to transcriptional repressor activity in biological functions. It is an essential for cancer diagnosis and therapeutics to detect DNA MTase activity sensitively. Here, a fluorescent system based on polymerase amplification has been developed to detect DNA adenine MTase (Dam) activity sensitively. The amplification is triggered by the probe DNA regions a, which are the primes of a polymerase-induced replicated reaction. They come from methylation and a digestion reaction of DNA S1-S1, including a 5'-GATC-3' sequence recognized by Dam MTase and methylation sensitive restriction endonuclease Dpn I. The intensities of fluorescence are dependent on the Dam MTase activity. The method shows fine sensitivity with a detection limit of 3.2 × 10-4 U mL-1 and specificity for Dam MTase. In human serum samples, the method has been successfully applied, and it has also been used to screen the inhibitors, which means that the developed method can be a powerful and potential tool for drug development and clinical diagnosis in the future.

A graphene quantum dots (GQDs) and horse radish peroxidase (HRP) hybrid system was designed for the sensing of alkaline phosphatase (ALP) activity and inhibitor screening. We found that the photoluminescence (PL) intensity of GQDs could be quenched efficiently in the presence of phenol, H2O2 and HRP. Moreover, ALP could hydrolyze disodium phenyl phosphate (DPP) to produce phenol, and also could result in the photoluminescence quenching of GQDs. The decrease in the PL intensity was linear to the activity of ALP in the concentration range of 0.02 - 0.8 U/L, with a detection limit of 0.008 U/L. The proposed GQDs/HRP hybrid system was successfully applied to ALP determination in human serum samples. The inhibition study was further analyzed, and Na3VO4 (as an ALP inhibitor) showed a clear inhibition effect. The results suggest that the GQDs/HRP hybrid system has good potential applications for the assay of ALP activity and inhibitors screening in related biochemical fields.

Self-assembly on target membranes is one of the important properties of all dynamin family proteins. Drp6, a dynaminrelated protein in Tetrahymena, controls nuclear remodelling and undergoes cycles of assembly/disassembly on the nuclear envelope. To elucidate the mechanism of Drp6 function, we have characterized its biochemical and biophysical properties using size exclusion chromatography, chemical cross-linking and electron microscopy. The results demonstrate that Drp6 readily forms high-molecular-weight self-assembled structures as determined by size exclusion chromatography and chemical cross-linking. Negative stain electron microscopy revealed that Drp6 assembles into rings and spirals at physiological ionic strength. We have also shown that the recombinant Drp6 expressed in bacteria is catalytically active and its GTPase activity is not enhanced by low salt. These results suggest that, in contrast to dynamins but similar to MxA, Drp6 self-assembles in the absence of membrane templates, and its GTPase activity is not affected by ionic strength of the buffer. We discuss the self-assembly structure of Drp6 and explain the basis for lack of membrane-stimulated GTPase activity.

Made available in DSpace on 2018-12-11T17:35:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-08-01 Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) It has been suggested that food storage inside the nest may offer termites with a nutritional provision during low resource availability. Additionally, feces employed as construction material provide an excellent environment for colonization by microorganisms and, together with the storage of plant material inside the nest, could thus provide some advantage to the termites in terms of lignocellulose decomposition. Here, we conducted for the first time a comprehensive study of the microbial communities associated to a termite exhibiting food storage behavior using Illumina sequencing of the 16S and (ITS2) regions of rRNA genes, together with enzymatic assays and data collected in the field. Cornitermes cumulans (Syntermitinae) stored grass litter in nodules made from feces and saliva located in the nest core. The amount of nodules increased with nest size and isolation, and interestingly, the soluble fraction of extracts from nodules showed a higher activity against hemicellulosic substrates compared to termite guts. Actinobacteria and Sordariales dominated microbial communities of food nodules and nest walls, whereas Spirochetes and Pleosporales dominated gut samples of C. cumulans. Within Syntermitinae, however, gut bacterial assemblages were dissimilar. On the other hand, there is a remarkable convergence of the bacterial community structure of Termitidae nests. Our results suggest that the role of nodules could be related to food storage; however, the higher xylanolytic activity in the nodules and their associated microbiota could also provide C. cumulans with an external source of predigested polysaccharides, which might be advantageous in comparison with litter-feeding termites that do not display food storage behavior. Departamento de Biologia Instituto de Biociências Universidade Estadual Paulista (UNESP) Universidade Positivo Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE) Centro Nacional de Pesquisa em Energia e Materiais Programa em Processos Tecnológicos e Ambientais Universidade de Sorocaba (UNISO) CCNH—Center for Natural Sciences and Humanities Federal University of ABC (UFABC) Instituto de Ciências da Natureza Universidade Federal de Alfenas Department of Food Technology School of Food Engineering University of Campinas (UNICAMP) Departamento de Biologia Instituto de Biociências Universidade Estadual Paulista (UNESP) FAPEMIG: 00878-12 FAPESP: 2015/21497-6

International audience; Enthalpy–entropy compensation supposes that differences in activation enthalpy ∆H ‡ for different reactions (or, typically in biochemistry, the same reaction catalysed by enzymes obtained from different species) may be compensated for by differences in activation entropy ∆S ‡. At the isokinetic temperature the compensation is exact, so that all samples have the same activity. These ideas have been controversial for several decades, but examples are still frequently reported as evidence of a real phenomenon, nearly all of the reports ignoring or discounting the possibility of a statistical artefact. Even for measurements in pure chemistry artefacts occur often, and they are almost inescapable in enzyme kinetics and other fields that involve biological macromolecules, on account of limited stability and the fact that kinetic equations are normally valid only over a restricted range of temperature. Here I review the current status and correct an error in a recent book chapter.

Deletion or loss-of-function mutation of LKB1, frequently occurring in non-small cell lung cancers (NSCLCs), is a predominant caution of NSCLC initiation and progression. However, the upstream signaling pathways governing LKB1 activation are largely unknown. Here, we report that LKB1 undergoes Aurora kinase A (AURKA)-mediated phosphorylation, which largely compromises the LKB1/AMPK signaling axis, in turn leading to the elevation of NSCLC cell proliferation, invasion and migration. Mechanically, AURKA-mediated phosphorylation of LKB1 impairs LKB1 interaction with and phosphorylation of its downstream target AMPKα, which has critical roles in governing cancer cell energy metabolic homeostasis and tumorigenesis. Clinically, AURKA displays high levels in NSCLC patients, and correlates with poor outcome of patients with lung adenocarcinoma. Pathologically, the amplification or activation of AURKA-induced impairment of the LKB1/AMPK signaling pathway contributes to NSCLC initiation and progression, highlighting AURKA as a potential therapeutic target for combatting hyperactive AURKA-driven NSCLCs.

The β-secretase (BACE1) initiates the generation of toxic amyloid-β peptide (Aβ) from amyloid-β precursor protein (APP), which was widely considered to play a key role in the pathogenesis of Alzheimer’s disease (AD). Here, a novel microfluidics-based mobility shift assay (MMSA) was developed, validated, and applied for the screening of BACE1 inhibitors for AD. First, the BACE1 activity assay was established with a new fluorescent peptide substrate (FAM-EVNLDAEF) derived from the Swedish mutant APP, and high-quality ratiometric data were generated in both endpoint and kinetic modes by electrophoretic separation of peptide substrate from the BACE1 cleaved product (FAM-EVNL) before fluorescence quantification. To validate the assay, the inhibition and kinetic parameter values of two known inhibitors (AZD3839 and AZD3293) were evaluated, and the results were in good agreement with those reported by other methods. Finally, the assay was applied to screen for new inhibitors from a 900-compound library in a 384-well format, and one novel hit (IC50 = 26.5 ± 1.5 μM) was identified. Compared with the common fluorescence-based assays, the primary advantage of the direct MMSA was to discover novel BACE1 inhibitors with lower auto-fluorescence interference, and its superb capability for kinetic study.

Deoxyhypusine synthase (DHS) catalyzes the post-translational modification of eukaryotic translation factor 5A (eIF5A) by the polyamine, spermidine, that converts one specific lysine residue to deoxyhypusine [N e -4-aminobutyl(lysine)], which is subsequently hydroxylated to hypusine [N e -4-amino-2-hydroxybutyl(lysine)]. Hypusine synthesis represents the most critical function of polyamine. As eIF5A has been implicated in various human diseases, identification of specific inhibitors of hypusine modification is of vital importance. DHS catalyzes a complex reaction that occurs in two stages, first, the NAD-dependent cleavage of spermidine to form an enzyme-butylimine intermediate and enzyme-bound NADH, and second, the transfer of the butylimine moiety from the enzyme intermediate to the eIF5A precursor and subsequent reduction of the eIF5A-butylimine intermediate by enzyme-bound NADH to form deoxyhypusine [N e -4-aminobutyl(lysine)]. Our data demonstrate that there is a measurable release of enzyme-bound NADH in the absence of eIF5A precursor and that the DHS activity can be determined by coupling the first phase reaction with the NADH-Glo assay in which the generation of luminescence is dependent on NADH derived from the DHS partial reaction. The conventional DHS assay that measures the incorporation of radioactivity from [1,8-3H]spermidine into the eIF5A precursor in the complete reaction cannot be readily adapted for high throughput screening (HTS). In contrast, the non-radioactive DHS/NADH-Glo coupled assay is highly specific, sensitive and reproducible and could be configured for HTS of small molecule libraries for the identification of new inhibitors of DHS. Furthermore, the coupled assay provides new insights into the dynamics of the DHS reaction especially regarding the fate of NADH.

The twin-arginine translocase (Tat) pathway is involved in the transport of folded proteins in bacteria, and has been implicated in virulence and pathogenesis. A simple but efficient assay based on the quantification of the exopolysaccharide colanic acid was developed as a new means to study Tat function. Colanic acid contains a methylpentose (L-fucose) component, and its production is directly linked to the Tat pathway through the transport of enzymes involved in polysaccharide biosynthesis. Monitoring of L-fucose levels can be applied for identification of new Tat substrates and high-throughput screening of Tat inhibitors for therapeutic applications.

Polyunsaturated fatty acids have been reported to play a protective role in a wide range of diseases characterized by an increased metalloproteinases (MMPs) activity. The recent finding that omega-3 and omega-6 fatty acids exert an anti-inflammatory effect in periodontal diseases has stimulated the present study, designed to determine whether such properties derive from a direct inhibitory action of these compounds on the activity of MMPs. To this issue, we investigated the effect exerted by omega-3 and omega-6 fatty acids on the activity of MMP-2 and MMP-9, two enzymes that actively participate to the destruction of the organic matrix of dentin following demineralization operated by bacteria acids. Data obtained (both in vitro and on ex-vivo teeth) reveal that omega-3 and omega-6 fatty acids inhibit the proteolytic activity of MMP-2 and MMP-9, two enzymes present in dentin. This observation is of interest since it assigns to these compounds a key role as MMPs inhibitors, and stimulates further study to better define their therapeutic potentialities in carious decay.

A chlorosome is a large and efficient light-harvesting antenna system found in some photosynthetic bacteria. This system comprises self-aggregates of bacteriochlorophyll (BChl) c, d, or e possessing a chiral 1-hydroxyethyl group at the 3-position, which plays a key role in the formation of the supramolecule. Biosynthesis of chlorosomal pigments involves stereoselective conversion of 3-vinyl group to 3-(1-hydroxyethyl) group facilitated by a 3-vinyl hydratase. This 3-vinyl hydration also occurs in BChl a biosynthesis, followed by oxidation that introduces an acetyl group at the 3-position. Herein, we present in vitro enzymatic assays of paralogous 3-vinyl hydratases derived from green sulfur bacteria, Chlorobaculum tepidum and Chlorobaculum limnaeum, the filamentous anoxygenic phototroph Chloroflexus aurantiacus, and the chloracidobacterium Chloracidobacterium thermophilum. All the hydratases showed hydration activities. The biosynthetic pathway of BChl a and other chlorosomal pigments is discussed considering the substrate specificity and stereoselectivity of the present hydratases.

The significance and frequency of marine microorganisms as producers of bioactive metabolites-a natural source of drug discovery had varied significantly during the last decades, making marine ecosystem a huge treasure trove of novel isolates and novel compounds. Among the twelve actinomycetes isolated from marine sediment sample (Lat. 17°41′962″N, Long. 83°19′633″E), amylase, protease, lipase and cellulase activities were exhibited by 8,7,4,3 isolates respectively. Five isolates exhibited l -asparaginase activity, while 5, 6, 2 isolates exhibited antibacterial, antifungal and antimicrobial activities respectively. One isolate VMS-A10 efficiently producing alpha-amylase (25.53 ± 0.50 U/mL), protease (19.26 ± 0.25 U/mL), lipase (36.25 ± 0.10 U/mL), cellulase (14.43 ± 0.513 U/mL), l -asparaginase (0.125 ± 0.004 U/mL), antimicrobial metabolites against B. subtilis (503.33 ± 5.77 U/mL) , S. aureus (536.66 ± 5.77 U/mL) , E. coli (533.33 ± 5.77 U/mL) , P. aeruginosa (500.00 ± 10.0 U/mL) , MRSA (538.33 ± 5.77 U/mL), C. albicans (353.33 ± 11.54 U/mL) and A. niger (443.33 ± 15.27 U/mL) was selected, identified on the basis of morphological, cultural, physiological, and biochemical properties together with 16S rDNA sequence, designated as Streptomyces parvulus strain sankarensis-A10 and sequencing product (1490 bp) was deposited in the GenBank database under accession number KT906299, Culture Deposit No: NCIM-5601 . Isolation and characterization of each potential actinobacteria having immense industrial and therapeutic value on an unprecedented scale from marine sediments of Visakhapatnam coast will have a burgeoning effect.

We studied the effects of some aniline and dioxaborininopyridine derivatives on the rate of oxidative deamination of putrescine and polyamines in a tissue with high mitotic index. These effects were evaluated quantitatively by measuring diamine oxidase and polyamine oxidase activities in a model cell-free test system of regenerating rat liver tissue. Aniline derivatives exhibited mainly antiproliferative effects and promoted oxidative degradation of putrescine, spermidine, and spermine. Dioxaborininopyridine derivatives inhibited this process, thus exhibiting carcinogenic properties.

The biological significance of the DHTKD1-encoded 2-oxoadipate dehydrogenase (OADH) remains obscure due to its catalytic redundancy with the ubiquitous OGDH-encoded 2-oxoglutarate dehydrogenase (OGDH). In this work, metabolic contributions of OADH and OGDH are discriminated by exposure of cells/tissues with different DHTKD1 expression to the synthesized phosphonate analogues of homologous 2-oxodicarboxylates. The saccharopine pathway intermediates and phosphorylated sugars are abundant when cellular expressions of DHTKD1 and OGDH are comparable, while nicotinate and non-phosphorylated sugars are when DHTKD1 expression is order(s) of magnitude lower than that of OGDH. Using succinyl, glutaryl and adipoyl phosphonates on the enzyme preparations from tissues with varied DHTKD1 expression reveals the contributions of OADH and OGDH to oxidation of 2-oxoadipate and 2-oxoglutarate in vitro. In the phosphonates-treated cells with the high and low DHTKD1 expression, adipate or glutarate, correspondingly, are the most affected metabolites. The marker of fatty acid β-oxidation, adipate, is mostly decreased by the shorter, OGDH-preferring, phosphonate, in agreement with the known OGDH dependence of β-oxidation. The longest, OADH-preferring, phosphonate mostly affects the glutarate level. Coupled decreases in sugars and nicotinate upon the OADH inhibition link the perturbation in glucose homeostasis, known in OADH mutants, to the nicotinate-dependent NAD metabolism.

Background While many risk factors for preeclampsia, such as increased body mass index, advanced maternal age, chronic hypertension, diabetes, are now established in clinical practice, maternal lipid profile has not been included in the risk assessment for preeclampsia. We aim to characterize the serum levels of Total Cholesterol (TC), High density lipoprotein (HDL), Low density lipoprotein (LDL), Triglycerides (TG), Apolipoprotein A1, Apolipoprotein B and their ratios TC/HDL and ApoB/ApoA1 in the maternal and fetal circulations of normal pregnancy, preeclampsia (PE), fetal growth restriction (FGR) and PE + FGR. Methods A prospective cross-sectional case control study was conducted measuring maternal and fetal lipid levels by enzymatic analysis and immune-turbidimetric enzymatic assays. FGR was defined by elevated umbilical artery Doppler resistance in association with estimated fetal weight Results (1) TG levels were elevated in maternal PE and cord blood PE + FGR groups compared to normal pregnancies. (2) A statistically significant elevation of fetal ApoB levels was observed in PE, FGR and PE + FGR compared to normal pregnancies. Apolipoprotein levels A1 and B were not different between maternal groups. (3) TC, HDL, LDL and TC/HDL levels did not show any significant gestational variation or between clinical groups in the maternal or fetal circulation. Conclusions Elevation in maternal TG levels may have a role in the pathogenesis of PE. The implications of elevated maternal and fetal TG levels and elevated fetal Apolipoprotein B levels deserves further exploration of their role in long term cardiovascular risk in the mother as well as the offspring.

Protein kinases are enzymes whose abundance, protein-protein interactions, and posttranslational modifications together determine net signaling activity in cells. Large-scale data on cellular kinase activity are limited, because existing assays are cumbersome, poorly sensitive, low throughput, and restricted to measuring one kinase at a time. Here, we surmount the conventional hurdles of activity measurement with a multiplexing approach that leverages the selectivity of individual kinase-substrate pairs. We demonstrate proof of concept by designing an assay that jointly measures activity of five pleiotropic signaling kinases: Akt, IκB kinase (IKK), c-jun N-terminal kinase (JNK), mitogen-activated protein kinase (MAPK)-extracellular regulated kinase kinase (MEK), and MAPK-activated protein kinase-2 (MK2). The assay operates in a 96-well format and specifically measures endogenous kinase activation with coefficients of variation less than 20%. Multiplex tracking of kinase-substrate pairs reduces input requirements by 25-fold, with ~75 μg of cellular extract sufficient for fiveplex activity profiling. We applied the assay to monitor kinase signaling during coxsackievirus B3 infection of two different host-cell types and identified multiple differences in pathway dynamics and coordination that warrant future study. Because the Akt–IKK–JNK–MEK–MK2 pathways regulate many important cellular functions, the fiveplex assay should find applications in inflammation, environmental-stress, and cancer research.

Aldehyde-alcohol dehydrogenase (AdhE) is a key enzyme in bacterial fermentation, converting acetyl-CoA to ethanol, via two consecutive catalytic reactions. Here, we present a 3.5 Å resolution cryo-EM structure of full-length AdhE revealing a high-order spirosome architecture. The structure shows that the aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) active sites reside at the outer surface and the inner surface of the spirosome respectively, thus topologically separating these two activities. Furthermore, mutations disrupting the helical structure abrogate enzymatic activity, implying that formation of the spirosome structure is critical for AdhE activity. In addition, we show that this spirosome structure undergoes conformational change in the presence of cofactors. This work presents the atomic resolution structure of AdhE and suggests that the high-order helical structure regulates its enzymatic activity., Aldehyde-alcohol dehydrogenase (AdhE) converts acetyl-CoA to ethanol and plays an important role in bacterial fermentation. Here the authors present the 3.5 Å cryo-EM structure of full-length E. coli AdhE, which reveals a right-handed helical spirosome structure and they show that the helical structure is required for AdhE activity.

A method is described for the determination of ascorbic acid (AA) in complex biological fluids. It based on maganese(II)-doped zinc/germanium oxide nanoparticles (Mn@ZnGe NPs) with appealing time-resolved phosphorescence (TRP). TRP can provide a background-free reporter signal in analytical methods. The absorption of AA overlaps the excitation band of Mn@ZnGe NPs at 254 nm. This reduces the intensity of fluorescence via an inner filter effect (IFE) with increasing concentration of AA. Typical experimental conditions include an emission peak at 536 nm, a delay time of 50 μs and a counting time of 2 ms. This method can detect AA in a range of 5–500 μM with a 0.13 μM limit of detection. If AA is oxidized by the enzyme AA oxidase (AAOx), dehydroascorbic acid will be formed which doesn’t absorb at 254 nm. Hence, the IFE cannot occur and fluorescence is not reduced. The strategy can be used to quantify AAOx in the activity range of 1–4 U·mL−1. By using a handheld UV lamp and a smart phone with a color-scanning feature, the feasibility for visual detection and real-time/onsite quantitative scanometric monitoring of AA and AAOx is demonstrated.

Catechol O-methyltransferase (COMT) is widely distributed in nature and installs a methyl group onto one of the vicinal hydroxyl groups of a catechol derivative. Enzymes belonging to this family require two cofactors for methyl transfer: S-adenosyl-l-methionine as a methyl donor and a divalent metal cation for regiospecific binding and activation of a substrate. We have determined two high-resolution crystal structures of Rv0187, one of three COMT paralogs from Mycobacterium tuberculosis, in the presence and absence of cofactors. The cofactor-bound structure clearly locates strontium ions and S-adenosyl-l-homocysteine in the active site, and together with the complementary structure of the ligand-free form, it suggests conformational dynamics induced by the binding of cofactors. Examination of in vitro activities revealed promiscuous substrate specificity and relaxed regioselectivity against various catechol-like compounds. Unexpectedly, mutation of the proposed catalytic lysine residue did not abolish activity but altered the overall landscape of regiospecific methylation.

Resistance to 157 different herbicides and 88% of known sites of action has been observed, with many weeds resistant to two or more modes. Coupled with tighter environmental regulation, this demonstrates the need to identify new modes of action and novel herbicides. The plant sphingolipid biosynthetic enzyme, inositol phosphorylceramide synthase (IPCS), has been identified as a novel, putative herbicide target. The non-mammalian nature of this enzyme offers the potential of discovering plant specific inhibitory compounds with minimal impact on animals and humans, perhaps leading to the development of new non-toxic herbicides. The best characterised and most highly expressed isoform of the enzyme in the model-dicot Arabidopsis, AtIPCS2, was formatted into a yeast-based assay which was then utilized to screen a proprietary library of over 11,000 compounds provided by Bayer AG. Hits from this screen were validated in a secondary in vitro enzyme assay. These studies led to the identification of a potent inhibitor that showed selectivity for AtIPCS2 over the yeast orthologue, and activity against Arabidopsis seedlings. This work highlighted the use of a yeast-based screening assay to discover herbicidal compounds and the status of the plant IPCS as a novel herbicidal target.

An ultrasensitive fluorometric and colorimetric dual-mode assay is described for the determination of the activity of alkaline phosphatase (ALP). ALP catalyzes the decomposition of 2-phospho-L-ascorbic acid, and the ascorbic acid thus generated reduces silver ions. In the presence of gold nanoparticles, gold-silver nanoparticles (Au@Ag NPs) are formed. This is accompanied by a color change form pink to deep yellow. The Au@Ag NPs reduce the fluorescence of blue fluorescent graphene quantum dots due to spectral overlap. The changes of absorbance (measured at 410 and 520 nm) and fluorescence (measured at excitation/emission wavelengths of 346/415 nm) correlate well with the ALP activity in the 0.01–6 mU·mL−1 (absorption) and 0.01–2 mU·mL−1 (fluorescence) ranges, and the detection limits are 9 and 5 μU·mL−1 individually.