Staining-Enhanced Peroxidase-Mimicking Gold Nanoparticles in Nano-ELISA for Highly Sensitive Detection of Klebsiella pneumoniae.

Klebsiella pneumoniae, a member of the family Enterobacteriaceae, is a rod-shaped, Gram-negative bacterium, mainly found in the hospital environment and medical tools. It is the leading cause of nosocomial infection, characterized by bloodstream infection, wound site infection, urinary tract infection, and sepsis, mostly in older adults, newborn infants, and immunocompromised patients. This present study demonstrated a novel diagnostic method for K. pneumoniae detection based on the gold nanozyme activity for the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. The nanozyme activity of AuNPs with staining enhancement was statistically three times higher than that of the bare AuNPs in solid absorption at 650 nm. Nano-ELISA with staining enhancement could detect as low as 102 CFUs/mL of K. pneumoniae concentration, as the cutoff value was determined to be 0.158, which boosted the sensitivity of the immunoreactions by up to 100-fold. The detection limit of our assays was 26.023 CFUs/mL, and the limit of quantification was 78.857 CFUs/mL. There was no cross-reaction against other bacteria, which proved the immunoassays' remarkable specificity for recognizing K. pneumoniae. Taken together, we successfully developed and optimized the highly sensitive and decently specific nano-ELISA strategy that might be applicable for detecting various other bacterial pathogens.

P450-driven plastic-degrading synthetic bacteria.

Plastic contamination currently threatens a wide variety of ecosystems and presents damaging repercussions and negative consequences for many wildlife species. Sustainable plastic waste management is an important approach to environmental protection and a necessity in the current life cycle of plastics in nature. Plastic biodegradation by microorganisms is a notable possible solution. This opinion article includes a proposal to use hypothetical P450 enzymes with an engineered active site as potent trigger biocatalysts to biodegrade polyethylene (PE) via in-chain hydroxylation into smaller products of linear aliphatic alcohols and alkanoic acids based on cascade enzymatic reactions. Furthermore, we propose the adoption of P450 into plastic-eating synthetic bacteria for PE biodegradation. This strategy can be applicable to other dense plastics, such as polypropylene (PP) and polystyrene (PS).

Roles of Human Liver Cytochrome P450 Enzymes in Tenatoprazole Metabolism.

Tenatoprazole, a newly developed proton pump inhibitor candidate, was developed as an acid inhibitor for gastric acid hypersecretion disorders such as gastric ulcer and reflux esophagitis. It is known that tenatoprazole is metabolized to three major metabolites of 5'-hydroxy tenatoprazole, tenatoprazole sulfide, and tenatoprazole sulfone in human liver, primarily catalyzed by CYPs 2C19 and 3A4. While CYP2C19 prefers the hydroxylation of tenatoprazole at C-5' position, CYP3A4 is mainly involved in sulfoxidation reaction to make tenatoprazole sulfone. Tenatoprazole sulfide is a major human metabolite of tenatoprazole and is formed spontaneously and non-enzymatically from tenatoprazole. However, its metabolic fate in the human liver is not fully known. Furthermore, no systematic metabolic study has been performed to study tenatoprazole or tenatoprazole sulfide. Here, we studied the functions of human cytochromes P450 in the metabolic pathway of tenatoprazole and tenatoprazole sulfide by using recombinant human P450s and human liver microsomes. Both CYP 2C19 and CYP3A4 showed distinct regioselective and stereospecific monooxygenation activities toward tenatoprazole and tenatoprazole sulfide. Furthermore, a new major metabolite of tenatoprazole sulfide was found, 1'-N-oxy-5'-hydroxytenatoprzole sulfide, which has never been reported. In conclusion, the metabolic fates of tenatoprazole and tenatoprazole sulfide should be considered in the clinical use of tenatoprazole.

Biodegradation of polystyrene by bacteria from the soil in common environments.

Polystyrene (PS), a major plastic waste, is difficult to biodegrade due to its unique chemical structure that comprises phenyl moieties attached to long linear alkanes. In this study, we investigated the biodegradation of PS by mesophilic bacterial cultures obtained from various soils in common environments. Two new strains, Pseudomonas lini JNU01 and Acinetobacter johnsonii JNU01, were specifically enriched in non-carbonaceous nutrient medium, with PS as the only source of carbon. Their growth after culturing in basal media increased more than 3-fold in the presence of PS. Fourier transform infrared spectroscopy analysis, used to confirm the formation of hydroxyl groups and potentially additional chemical bond groups, showed an increase in the amount of oxidized PS samples. Moreover, field emission scanning electron microcopy analysis confirmed PS biodegradation by biofilms of the screened microbes. Water contact angle measurement additionally offered insights into the increased hydrophilic characteristics of PS films. Bioinformatics and transcriptional analysis of A. johnsonii JNU01 revealed alkane-1-monooxygenase (AlkB) to be involved in PS biodegradation, which was confirmed by the hydroxylation of PS using recombinant AlkB. These results provide significant insights into the discovery of novel functions of Pseudomonas sp. and Acinetobacter sp., as well as their potential as PS decomposers.

Solar-Powered Whole-Cell P450 Catalytic Platform for C-Hydroxylation Reactions.

Invited for this month's cover is the joint research group of Prof. Chan Beum Park at the Korea Advanced Institute of Science and Technology (KAIST) and Prof. Chul-Ho Yun at the Chonnam National University (CNU). The image shows how the use of a natural photosensitizer, flavin mononucleotide, and visible light can lead to a cost-effective, green, and sustainable process for P450-catalyzed reactions in a whole-cell system. The Communication itself is available at 10.1002/cssc.202100944.

Solar-Powered Whole-Cell P450 Catalytic Platform for C-Hydroxylation Reactions.

Photobiocatalysis is a green platform for driving redox enzymatic reactions using solar energy, not needing high-cost cofactors and redox partners. Here, a visible light-driven whole-cell platform for human cytochrome P450 (CYP) photobiocatalysis was developed using natural flavins as a photosensitizer. Photoexcited flavins mediate NADPH/reductase-free, light-driven biocatalysis by human CYP2E1 both in vitro and in the whole-cell systems. In vitro tests demonstrated that the photobiocatalytic activity of CYP2E1 is dependent on the substrate type, the presence of catalase, and the acid type used as a sacificial electron donor. A protective effect of catalase was found against the inactivation of CYP2E1 heme by H2 O2 and the direct transfer of photo-induced electrons to the heme iron not by peroxide shunt. Furthermore, the P450 photobiocatalysis in whole cells containing human CYPs 1A1, 1A2, 1B1, and 3A4 demonstrated the general applicability of the solar-powered, flavin-mediated P450 photobiocatalytic system.

An Unlikely Case of Benztropine Misuse in an Elderly Schizophrenic.

Although data on the prevalence of anticholinergic misuse is scarce, it has been reported among psychiatric patients. Anticholinergic drugs can act as potent indirect dopamine agonists in the limbic system, a mechanism that has been hypothesized to explain their misuse potential among patients. In psychiatric practice settings, the use of typical antipsychotics in conjunction with anticholinergics is common, with the latter mainly used to manage extrapyramidal side effects of the former. Haloperidol is a first-generation (typical) antipsychotic with weak anticholinergic properties that may sometimes be potentiated when it is used in combination with other anticholinergic medications. This combination can induce significant gastrointestinal hypomotility, constipation, and rarely even paralytic ileus. We present the case of a 67-year-old African American male with a history of schizophrenia, benign prostatic hyperplasia, and essential hypertension, who abruptly started misusing benztropine, without any prior history of a substance use disorder. This case highlights the importance of obtaining a detailed history when previously stable psychiatric patients develop acute physical symptoms. It also illustrates the importance of care coordination among care providers and the central role of the psychiatrist in the care of patients with medical comorbidities.

Biosensor-Based Directed Evolution of Methanol Dehydrogenase from Lysinibacillus xylanilyticus.

Methanol dehydrogenase (Mdh), is a crucial enzyme for utilizing methane and methanol as carbon and energy sources in methylotrophy and synthetic methylotrophy. Engineering of Mdh, especially NAD-dependent Mdh, has thus been actively investigated to enhance methanol conversion. However, its poor catalytic activity and low methanol affinity limit its wider application. In this study, we applied a transcriptional factor-based biosensor for the direct evolution of Mdh from Lysinibacillus xylanilyticus (Lxmdh), which has a relatively high turnover rate and low KM value compared to other wild-type NAD-dependent Mdhs. A random mutant library of Lxmdh was constructed in Escherichia coli and was screened using formaldehyde-detectable biosensors by incubation with low methanol concentrations. Positive clones showing higher fluorescence were selected by fluorescence-activated cell sorting (FACS) system, and their catalytic activities toward methanol were evaluated. The successfully isolated mutants E396V, K318N, and K46E showed high activity, particularly at very low methanol concentrations. In kinetic analysis, mutant E396V, K318N, and K46E had superior methanol conversion efficiency, with 79-, 23-, and 3-fold improvements compared to the wild-type, respectively. These mutant enzymes could thus be useful for engineering synthetic methylotrophy and for enhancing methanol conversion to various useful products.

Methanol Dehydrogenases as a Key Biocatalysts for Synthetic Methylotrophy.

One-carbon (C1) chemicals are potential building blocks for cheap and sustainable re-sources such as methane, methanol, formaldehyde, formate, carbon monoxide, and more. These resources have the potential to be made into raw materials for various products used in our daily life or precursors for pharmaceuticals through biological and chemical processes. Among the soluble C1 substrates, methanol is regarded as a biorenewable platform feedstock because nearly all bioresources can be converted into methanol through syngas. Synthetic methylotrophy can be exploited to produce fuels and chemicals using methanol as a feedstock that integrates natural or artificial methanol assimilation pathways in platform microorganisms. In the methanol utilization in methylotrophy, methanol dehydrogenase (Mdh) is a primary enzyme that converts methanol to formaldehyde. The discovery of new Mdhs and engineering of present Mdhs have been attempted to develop synthetic methylotrophic bacteria. In this review, we describe Mdhs, including in terms of their enzyme properties and engineering for desired activity. In addition, we specifically focus on the application of various Mdhs for synthetic methylotrophy.

Regioselective Hydroxylation of Phloretin, a Bioactive Compound from Apples, by Human Cytochrome P450 Enzymes.

Phloretin, the major polyphenol compound in apples and apple products, is interesting because it shows beneficial effects on human health. It is mainly found as a form of glucoside, phlorizin. However, the metabolic pathway of phloretin in humans has not been reported. Therefore, identifying phloretin metabolites made in human liver microsomes and the human cytochrome P450 (P450) enzymes to make them is interesting. In this study, the roles of human liver P450s for phloretin oxidation were examined using human liver microsomes and recombinant human liver P450s. One major metabolite of phloretin in human liver microsomes was 3-OH phloretin, which is the same product of a bacterial CYP102A1-catalyzed reaction of phloretin. CYP3A4 and CYP2C19 showed kcat values of 3.1 and 5.8 min-1, respectively. However, CYP3A4 has a 3.3-fold lower Km value than CYP2C19. The catalytic efficiency of a CYP3A4-catalyzed reaction is 1.8-fold higher than a reaction catalyzed by CYP2C19. Whole-cell biotransformation with CYP3A4 was achieved 0.16 mM h-1 productivity for 3-OH phlorein from 8 mM phloretin at optimal condition. Phloretin was a potent inhibitor of CYP3A4-catalyzed testosterone 6ß-hydroxylation activity. Antibodies against CYP3A4 inhibited up to 90% of the microsomal activity of phloretin 3-hydroxylation. The immunoinhibition effect of anti-2C19 is much lower than that of anti-CYP3A4. Thus, CYP3A4 majorly contributes to the human liver microsomal phloretin 3-hydroxylation, and CYP2C19 has a minor role.

Biocatalytic Production of a Potent Inhibitor of Adipocyte Differentiation from Phloretin Using Engineered CYP102A1.

In this study, we investigated an efficient enzymatic strategy for producing potentially valuable phloretin metabolites from phlorizin, a glucoside of phloretin that is rich in apple pomace. Almond ß-glucosidase efficiently removed phlorizin's glucose moiety to produce phloretin. CYP102A1 engineered by site-directed mutagenesis, domain swapping, and random mutagenesis catalyzed the highly regioselective C-hydroxylation of phloretin into 3-OH phloretin with high conversion yields. Under the optimal hydroxylation conditions of 15 g cells L-1 and a 20 mM substrate for whole-cell biocatalysis, phloretin was regioselectively hydroxylated into 3.1 mM 3-OH phloretin each hour. Furthermore, differentiation of 3T3-L1 preadipocytes into adipocytes and lipid accumulation were dramatically inhibited by 3-OH phloretin but promoted by phloretin. Consistent with these inhibitory effects, the expression of adipogenic regulator genes was downregulated by 3-OH phloretin. We propose a platform for the sustainable production and value creation of phloretin metabolites from apple pomace capable of inhibiting adipogenesis.

The Impact of Removal of Ovarian Hormones on Cholinergic Muscarinic Receptors: Examining Prepulse Inhibition and Receptor Binding.

Ovarian hormones, such as estrogens and progesterone, are known to exert beneficial effects on cognition and some psychiatric disorders. The basis of these effects is not fully understood, but may involve altered cholinergic neurotransmission. This study aimed to investigate how a lack of ovarian hormones would impact muscarinic receptor-induced deficits in prepulse inhibition (PPI) and muscarinic receptor density in several brain regions. Adult female rats were either ovariectomized, to remove the source of ovarian hormones, or left intact (sham-operated). PPI is a measure of sensorimotor gating that is typically impaired in schizophrenia patients, and similar deficits can be induced in rats by administering scopolamine, a muscarinic receptor antagonist. Our results revealed no significant effects of ovariectomy on PPI after saline or scopolamine treatment. Autoradiography was performed to measure cholinergic muscarinic receptor binding density using [3H]-pirenzepine, [3H]-AF-DX, and [3H]-4-DAMP, to label M1, M2/M4, and M3 receptors, respectively. We examined the amygdala, caudate putamen, dorsal hippocampus, motor cortex, retrosplenial cortex, and ventromedial hypothalamus. There were no significant group differences in any region for any muscarinic receptor type. These results suggest that removing peripheral ovarian hormones does not influence the cholinergic muscarinic receptor system in the context of PPI or receptor binding density.

Phenolic compounds from the stems of Fissistigma polyanthoides and their anti-oxidant activities.

The stems of Fissistigma polyanthoides (A.DC.) Merr. are traditionally used for the treatment of rheumatism and for recuperating women after childbirth. In our continuous phytochemical investigation of this plant, four new (1, 2, 5, and 19) and fifteen known (3, 4, and 6-18) phenolic compounds were isolated. The structures of all compounds were elucidated based on extensive spectroscopic analyses (1D-, 2D-NMR, and MS), and in comparison with reported literature data. The new natural products showed to be two poly-methoxylated chalcones (1 and 2) and two flavonoid glycosides, with 19 containing an uncommon sugar moiety (quinovose). Compounds with sufficient amount were tested for their anti-oxidant activity in a cell-based assay using the human bronchial epithelial cell line BEAS-2B. The compounds' capacity to inhibit the peroxyl radical triggered formation of dichlorofluorescein (DCF) was investigated in a dose-dependent manner. Both, anti-oxidant (3, 4, 6, 8-12, and 14) and pro-oxidative (5 and 16) properties were found for the investigated substances. The half maximal concentrations (IC50) for the inhibition of ROS formation ranged between 18.8 µM and 63.5 µM. Compounds, which acted protectively in the cellular antioxidant activity (CAA) assay and did not negatively affect cell viability, could be interesting targets for further investigations.

Medical Care for Undocumented Immigrants: National and International Issues.

The number of undocumented immigrants (UIs) varies worldwide, and most reside in the United States. With more than 12 million UIs in the United States, addressing the health care needs of this population presents unique challenges and opportunities. Most UIs are uninsured and rely on the safety-net health system for their care. Because of young age, this population is often considered to be healthier than the overall US population, but they have specific health conditions and risks. Adequate coverage is lacking; however, there are examples of how to better address the health care needs of UIs.

Peroxide-dependent oxidation reactions catalyzed by CYP191A1 from Mycobacterium smegmatis.

OBJECTIVES: To find the catalytic activities of CYP191A1 from Mycobacterium smegmatis, in which functions of most P450s are unknown, by using a set of reductase systems, peroxides, and various substrates including fatty acids and human drugs. RESULTS: CYP191A1 was functionally expressed in Escherichia coli and purified. Its catalytic activities were examined with fatty acids, chromogenic and fluorogenic substrates, and several human P450 substrates, in the presence of six different types of electron transfer systems, such as rat NADPH-P450 reductase, Candida NADPH-P450 reductase, ferredoxin/ferredoxin reductase, putidaredoxin/putidaredoxin reductase, and peroxides (H2O2 and t-butyl hydroperoxide). The reactions catalyzed by CYP191A1 included the hydroxylation and O-dealkylation of several substrates. CONCLUSIONS: CYP191A1 preferentially catalyzes the peroxide-dependent oxidation of various substrates over the reductase-dependent reaction. Its peroxygenase activity may be used an effective biocatalytic tool to synthesize the metabolites of drugs.

Medical Care for Undocumented Immigrants: National and International Issues.

The number of undocumented immigrants (UIs) varies worldwide, and most reside in the United States. With more than 12 million UIs in the United States, addressing the health care needs of this population presents unique challenges and opportunities. Most UIs are uninsured and rely on the safety-net health system for their care. Because of young age, this population is often considered to be healthier than the overall US population, but they have specific health conditions and risks. Adequate coverage is lacking; however, there are examples of how to better address the health care needs of UIs.

Highly regioselective hydroxylation of polydatin, a resveratrol glucoside, for one-step synthesis of astringin, a piceatannol glucoside, by P450 BM3.

Enzymatic conversion of natural glycosides to their corresponding hydroxylated products using cytochromes P450 has significant advantages over synthetic chemistry and even enzyme-catalyzed glycosylation of chemicals. At present, the basic strategy for making glycosides of stilbenoid compounds is to use the glycosylation activity of enzymes, such as glycosyltransferases. Here, an efficient synthesis of a valuable (E)-astringin, a piceatannol glucoside, was developed using CYP102A1 via the highly regioselective C-3' hydroxylation of polydatin, a resveratrol glucoside. (E)-astringin is a high added value compound found in plants and wine. Benzylic hydroxylation of polydatin provides an attractive route to (E)-astringin, a catechol product. Thus far, chemical and enzymatic methods of producing (E)-astringin have not been developed. In the present study, a set of CYP102A1 mutants from Bacillus megaterium was found to catalyze regioselective hydroxylation of polydatin at the C-3' position to generate an (E)-astringin, a piceatannol glucoside.

Characterization of a Biflaviolin Synthase CYP158A3 from Streptomyces avermitilis and Its Role in the Biosynthesis of Secondary Metabolites.

Streptomyces avermitilis produces clinically useful drugs such as avermectins and oligomycins. Its genome contains approximately 33 cytochrome P450 genes and they seem to play important roles in the biosynthesis of many secondary metabolites. The SAV_7130 gene from S. avermitilis encodes CYP158A3. The amino acid sequence of this enzyme has high similarity with that of CYP158A2, a biflaviolin synthase from S. coelicolor A3(2). Recombinant S. avermitilis CYP158A3 was heterologously expressed and purified. It exhibited the typical P450 Soret peak at 447 nm in the reduced CO-bound form. Type I binding spectral changes were observed when CYP158A3 was titrated with myristic acid; however, no oxidative product was formed. An analog of flaviolin, 2-hydroxynaphthoquinone (2-OH NQ) displayed similar type I binding upon titration with purified CYP158A3. It underwent an enzymatic reaction forming dimerized product. A homology model of CYP158A3 was superimposed with the structure of CYP158A2, and the majority of structural elements aligned. These results suggest that CYP158A3 might be an orthologue of biflaviolin synthase, catalyzing C-C coupling reactions during pigment biosynthesis in S. avermitilis.

Regioselective C-H hydroxylation of omeprazole sulfide by Bacillus megaterium CYP102A1 to produce a human metabolite.

OBJECTIVES: To find a simple enzymatic strategy for the efficient synthesis of the expensive 5'-hydroxyomeprazole sulfide, a recently identified minor human metabolite, from omeprazole sulfide, which is an inexpensive substrate. RESULTS: The practical synthetic strategy for the 5'-OH omeprazole sulfide was accomplished with a set of highly active CYP102A1 mutants, which were obtained by blue colony screening from CYP102A1 libraries with a high conversion yield. The mutant and even the wild-type enzyme of CYP102A1 catalyzed the high regioselective (98 %) C-H hydroxylation of omeprazole sulfide to 5'-OH omeprazole sulfide with a high conversion yield (85-90 %). CONCLUSIONS: A highly efficient synthesis of 5'-OH omeprazole sulfide was developed using CYP102A1 from Bacillus megaterium as a biocatalyst.

Sox21 regulates the progression of neuronal differentiation in a dose-dependent manner.

Members of the SoxB transcription factor family play critical roles in the regulation of neurogenesis. The SoxB1 proteins are required for the induction and maintenance of a proliferating neural progenitor population in numerous vertebrates, however the role of the SoxB2 protein, Sox21, is less clear due to conflicting results. To clarify the role of Sox21 in neurogenesis, we examined its function in the Xenopus neural plate. Here we report that misexpression of Sox21 expands the neural progenitor domain, and represses neuron formation by binding to Neurogenin (Ngn2) and blocking its function. Conversely, we found that Sox21 is also required for neuron formation, as cells lacking Sox21 undergo cell death and thus are unable to differentiate. Together our data indicate that Sox21 plays more than one role in neurogenesis, where a threshold level is required for cell viability and normal differentiation of neurons, but a higher concentration of Sox21 inhibits neuron formation and instead promotes progenitor maintenance.