Structural and functional insights into the flexible ß-hairpin of glycerol dehydrogenase.

During glycerol metabolism, the initial step of glycerol oxidation is catalysed by glycerol dehydrogenase (GDH), which converts glycerol to dihydroxyacetone in a NAD+ -dependent manner via an ordered Bi-Bi kinetic mechanism. Structural studies conducted with GDH from various species have mainly elucidated structural details of the active site and ligand binding. However, the structure of the full GDH complex with both cofactor and substrate bound is not determined, and thus, the structural basis of the kinetic mechanism of GDH remains unclear. Here, we report the crystal structures of Escherichia coli GDH with a substrate analogue bound in the absence or presence of NAD+ . Structural analyses including molecular dynamics simulations revealed that GDH possesses a flexible ß-hairpin, and that during the ordered progression of the kinetic mechanism, the flexibility of the ß-hairpin is reduced after NAD+ binding. It was also observed that this alterable flexibility of the ß-hairpin contributes to the cofactor binding and possibly to the catalytic efficiency of GDH. These findings suggest the importance of the flexible ß-hairpin to GDH enzymatic activity and shed new light on the kinetic mechanism of GDH.

Exploring the substrate scope of glycerol dehydrogenase GldA from E. coli BW25113 towards cis-dihydrocatechol derivatives.

Glycerol dehydrogenase (GldA) from Escherichia coli BW25113, naturally catalyzes the oxidation of glycerol to dihydroxyacetone. It is known that GldA exhibits promiscuity towards short-chain C2-C4 alcohols. However, there are no reports regarding the substrate scope of GldA towards larger substrates. Herein we demonstrate that GldA can accept bulkier C6-C8 alcohols than previously anticipated. Overexpression of the gldA gene in the knockout background, E. coli BW25113 ΔgldA, was strikingly effective converting 2 mM of the compounds: cis-dihydrocatetechol, cis-(1 S,2 R)- 3-methylcyclohexa-3,5-diene-1,2-diol and cis-(1 S,2 R)- 3-ethylcyclohexa-3,5-diene-1,2-diol, into 2.04 ± 0.21 mM of catechol, 0.62 ± 0.11 mM 3-methylcatechol, and 0.16 ± 0.02 mM 3-ethylcatechol, respectively. In-silico studies on the active site of GldA enlightened the decrease in product formation as the steric substrate demand increased. These results are of high interests for E. coli-based cell factories expressing Rieske non-heme iron dioxygenases, producing cis-dihydrocatechols, since such sough-after valuable products can be immediately degraded by GldA, substantially hampering the expected performance of the recombinant platform.

DCXR promotes cell proliferation by promoting the activity of aerobic glycolysis in breast cancer.

The function of human dicarbonyl/L­xylulose reductase (DCXR) in the pathophysiology of breast cancer is yet to be elucidated. The present study aimed to investigate the function of DCXR in glycolysis and the cell cycle of breast cancer cells with respect to cell proliferation. Differential expressed DCXR was identified in The Cancer Genome Atlas (TCGA) database and verified in clinical breast cancer tissue. DCXR silencing and overexpression were induced by RNA interference and lentiviral vectors, respectively. Cell cycle progression, proliferation and glycolytic activity of breast cancer cells were detected by flow cytometry, Cell Counting Kit­8 assay and chemical methods, respectively. Tumorigenicity was detected using nude mice xenograft models. The expression of DCXR was increased in TCGA breast cancer database and the function of DCXR was enriched in 'glycolysis' and 'cell cycle'. Further analysis using clinical breast cancer samples confirmed upregulation of DCXR. The silencing of DCXR suppressed proliferation and cell cycle progression of breast cancer cells and significantly decreased the capacity for glycolysis, thereby demonstrating the effect of DCXR on the function of breast cancer cells. Similar conclusions were obtained in DCXR overexpressing cells; notably, DCXR overexpression promoted proliferation, cell cycle progression at S phase and glycolysis. 2­Deoxy­D­glucose inhibited the effect of DCXR on the proliferation and cell cycle progression of breast cancer cells. The present study revealed that DCXR regulated breast cancer cell cycle progression and proliferation by increasing glycolysis activity and thus may serve as an oncogene for breast cancer.

Detection of Cryptosporidium spp. and Giardia duodenalis in small wild mammals in northeastern Brazil.

This study investigated the occurrence of Giardia duodenalis and Cryptosporidium spp. in rodents and marsupials from the Atlantic Forest in southern Bahia, northeastern Brazil. Two hundred and four fecal samples were collected from different forest areas in the municipalities of Ilhéus, Una, Belmonte, and Mascote. Identifications were performed using PCR and nested PCR followed by sequencing of the gdh and tpi genes for G. duodenalis, and the gp60 and Hsp-70 genes for Cryptosporidium. The total frequency of positive PCR samples for both G. duodenalis and Cryptosporidium spp. was 5.4% (11/204). Giardia duodenalis occurred in 2.94% (4/136) of rodents and 2.94% (2/68) of marsupials. The prevalence of Cryptosporidium in rodents and marsupials was 1.47% (2/136) and 4.41% (3/68), respectively. In the areas sampled, the frequency of parasitism was 50% (7/14), while the Mascote region alone had no parasitized animals. The G. duodenalis subgenotype AI was identified in the rodent species Hylaeamys laticeps, Oecomys catherinae, Oligoryzomys nigripes and Akodon cursor, and in the marsupials Gracilinanus agilis and Monodelphis americana. In the rodents Rhipidomys mastacalis, H. laticeps and in the marsupial Marmosa murina the protozoa Cryptosporidium fayeri, Cryptosporidium parvum and Cryptosporidium ubiquitum with subtypes IIa and IVg by the gp60 gene were found. In conclusion, this study provides the genetic characterization of Giardia and Cryptosporidium species and genotypes in rodents and marsupials. And, these findings reinforce that the rodent and marsupial species mentioned above play a role as new hosts for Giardia and Cryptosporidium.

Milestones in treatments for inborn errors of metabolism: Reflections on Where chemistry and medicine meet.

From Sir Archibald Garrod's initial description of the tetrad of albinism, alkaptonuria, cystinuria, and pentosuria to today, the field of medicine dedicated to inborn errors of metabolism has evolved from disease identification and mechanistic discovery to the development of therapies designed to subvert biochemical defects. In this review, we highlight major milestones in the treatment and diagnosis of inborn errors of metabolism, starting with dietary therapy for phenylketonuria in the 1950s and 1960s, and ending with current approaches in genetic manipulation.

Mannitol-1-phosphate dehydrogenase, MpdA, is required for mannitol production in vegetative cells and involved in hyphal branching, heat resistance of conidia and sexual development in Aspergillus nidulans.

Aspergillus nidulans produces cleistothecia as sexual reproductive organs in a process affected by genetic and external factors. To gain a deeper insight into A. nidulans sexual development, we performed comparative proteome analyses based on the wild type developmental periods. We identified sexual development-specific proteins with a more than twofold increase in production during hypoxia or the sexual period compared to the asexual period. Among the sexual development-specific proteins analyzed by gene-deletion experiments and functional assays, MpdA, a putative mannitol-1-phosphate 5-dehydrogenase, plays multiple roles in growth and differentiation of A. nidulans. The most distinct mpdA-deletion phenotype was ascosporogenesis failure. Genetic mpdA deletion resulted in small cleistothecia with no functional ascospores. Transcriptional analyses indicated that MpdA modulates the expression of key development- and meiosis-regulatory genes during sexual development. The mpdA deletion increased hyphal branching and decreased conidial heat resistance. Mannitol production in conidia showed no difference, whereas it was decreased in mycelia and sexual cultures. Addition of mannitol during vegetative growth recovered the defects in conidial heat resistance and ascospore genesis. Taken together, these results indicate that MpdA plays an important role in sexual development, hyphal branching, and conidial heat resistance in Aspergillus nidulans.

Opening a Novel Biosynthetic Pathway to Dihydroxyacetone and Glycerol in Escherichia coli Mutants through Expression of a Gene Variant (fsaAA129S) for Fructose 6-Phosphate Aldolase.

Phosphofructokinase (PFK) plays a pivotal role in glycolysis. By deletion of the genes pfkA, pfkB (encoding the two PFK isoenzymes), and zwf (glucose 6-phosphate dehydrogenase) in Escherichia coli K-12, a mutant strain (GL3) with a complete block in glucose catabolism was created. Introduction of plasmid-borne copies of the fsaA wild type gene (encoding E. coli fructose 6-phosphate aldolase, FSAA) did not allow a bypass by splitting fructose 6-phosphate (F6P) into dihydroxyacetone (DHA) and glyceraldehyde 3-phosphate (G3P). Although FSAA enzyme activity was detected, growth on glucose was not reestablished. A mutant allele encoding for FSAA with an amino acid exchange (Ala129Ser) which showed increased catalytic efficiency for F6P, allowed growth on glucose with a µ of about 0.12 h-1. A GL3 derivative with a chromosomally integrated copy of fsaAA129S (GL4) grew with 0.05 h-1 on glucose. A mutant strain from GL4 where dhaKLM genes were deleted (GL5) excreted DHA. By deletion of the gene glpK (glycerol kinase) and overexpression of gldA (of glycerol dehydrogenase), a strain (GL7) was created which showed glycerol formation (21.8 mM; yield approximately 70% of the theoretically maximal value) as main end product when grown on glucose. A new-to-nature pathway from glucose to glycerol was created.

Conversion of levoglucosan into glucose by the coordination of four enzymes through oxidation, elimination, hydration, and reduction.

Levoglucosan (LG) is an anhydrosugar produced through glucan pyrolysis and is widely found in nature. We previously isolated an LG-utilizing thermophile, Bacillus smithii S-2701M, and suggested that this bacterium may have a metabolic pathway from LG to glucose, initiated by LG dehydrogenase (LGDH). Here, we completely elucidated the metabolic pathway of LG involving three novel enzymes in addition to LGDH. In the S-2701M genome, three genes expected to be involved in the LG metabolism were found in the vicinity of the LGDH gene locus. These four genes including LGDH gene (lgdA, lgdB1, lgdB2, and lgdC) were expressed in Escherichia coli and purified to obtain functional recombinant proteins. Thin layer chromatography analyses of the reactions with the combination of the four enzymes elucidated the following metabolic pathway: LgdA (LGDH) catalyzes 3-dehydrogenation of LG to produce 3-keto-LG, which undergoes ß-elimination of 3-keto-LG by LgdB1, followed by hydration to produce 3-keto-D-glucose by LgdB2; next, LgdC reduces 3-keto-D-glucose to glucose. This sequential reaction mechanism resembles that proposed for an enzyme belonging to glycoside hydrolase family 4, and results in the observational hydrolysis of LG into glucose with coordination of the four enzymes.

Identification and functional characterization of glycerol dehydrogenase reveal the role in kojic acid synthesis in Aspergillus oryzae.

Glycerol dehydrogenase has been identified and characterized functionally in many species. However, little is known about glycerol dehydrogenase genes and their functions in Aspergillus oryzae. Here, a total of 45 glycerol dehydrogenase genes in Aspergillus oryzae were identified and renamed from AoGld1 to AoGld45 according to their chromosome distribution. They were classified into three groups based on phylogenetic analysis. Synteny analysis revealed that thirteen AoGld genes are conserved among Aspergillus species. Promoter analysis displayed that AoGld3 and AoGld13 harbored multiple binding elements of GATA-type transcription factors and zinc-finger protein msnA that were involved in nitrogen and kojic acid metabolism, respectively. Moreover, the AoGld3 deletion strain Δgld3 was generated by the CRISPR/Cas9 system, which had no visible growth defects compared with the control wild-type strain under the control and osmotic stress treatments. However, disruption of AoGld3 led to the inhibition of kojic acid production, and the expression of kojA, kojR was down-regulated in the Δgld3 strain. Furthermore, when kojA or kojR was overexpressed in the Δgld3 strain, the yield of kojic acid was restored, suggesting that AoGld3 is involved in kojic acid production through affecting the expression of kojR and kojA. Taken together, these findings provide new insights into our understanding of glycerol dehydrogenase and establish foundation for further study of their roles in Aspergillus oryzae.

Purification and characterization of a novel medium-chain ribitol dehydrogenase from a lichen-associated bacterium Sphingomonas sp.

Sugar alcohols (polyols) are abundant carbohydrates in lichen-forming algae and transported to other lichen symbionts, fungi, and bacteria. Particularly, ribitol is an abundant polyol in the lichen Cetraria sp. Polyols have important physiological roles in lichen symbiosis, but polyol utilization in lichen-associated bacteria has been largely unreported. Herein, we purified and characterized a novel ribitol dehydrogenase (RDH) from a Cetraria sp.-associated bacterium Sphingomonas sp. PAMC 26621 grown on a minimal medium containing D-ribitol (the RDH hereafter referred to as SpRDH). SpRDH is present as a trimer in its native form, and the molecular weight of SpRDH was estimated to be 39 kDa by SDS-PAGE and 117 kDa by gel filtration chromatography. SpRDH converted D-ribitol to D-ribulose using NAD+ as a cofactor. As far as we know, SpRDH is the first RDH belonging to the medium-chain dehydrogenase/reductase family. Multiple sequence alignments indicated that the catalytic amino acid residues of SpRDH consist of Cys37, His65, Glu66, and Glu157, whereas those of short-chain RDHs consist of Ser, Tyr, and Lys. Furthermore, unlike other short-chain RDHs, SpRDH did not require divalent metal ions for its catalytic activity. Despite SpRDH originating from a psychrophilic Arctic bacterium, Sphingomonas sp., it had maximum activity at 60°C and exhibited high thermal stability within the 4-50°C range. Further studies on the structure/function relationship and catalytic mechanism of SpRDH will expand our understanding of its role in lichen symbiosis.

Giardia and Cryptosporidium infections in Danish cats: risk factors and zoonotic potential.

Giardia and Cryptosporidium infections are common in cats, but knowledge is limited about their clinical importance, risk factors, and the role of cats as a reservoir for human infections. Here, we collected faeces and questionnaire data from 284 cats from shelters and veterinary clinics in the Copenhagen Metropolitan Region (= study population). Additionally, 33 samples were analysed separately from catteries with gastrointestinal clinical signs (= cases). (Oo-)cysts were quantified by immunofluorescence microscopy. All Giardia (n = 34) and Cryptosporidium (n = 29) positive samples were analysed by sequencing of the 18S rRNA, gdh and hsp70 loci, and co-infections were detected by McMaster/inverted microscopy. In the study population, 7.0% and 6.7% were positive for Giardia and Cryptosporidium respectively; 48.5% and 36.4% of the breeder cats (cases) were infected. Increased odds of diarrhoea were demonstrated in Giardia (p = 0.0008) and Cryptosporidium (p = 0.034) positive cats. For Giardia, the odds were positively correlated with infection intensity. Co-infection with Cryptosporidium (OR 12.79; p < 0.001), parasitic co-infections other than Cryptosporidium (OR 5.22; p = 0.009), no deworming (OR 4.67; p = 0.035), and male sex (OR 3.63; p = 0.025) were risk factors for Giardia. For Cryptosporidium, co-infection with Giardia was the only risk factor (OR 11.93; p < 0.0001). Genotyping revealed G. duodenalis assemblages A and F, and C. felis, all of them previously detected in humans. In conclusion, excretion of Giardia and Cryptosporidium was associated with clinical disease. Although a public health risk is likely, studies including larger sample sizes, more discriminatory markers and samples from other animals and humans are needed to reveal the full zoonotic potential.

Permeabilized Escherichia coli Whole Cells Containing Co-Expressed Two Thermophilic Enzymes Facilitate the Synthesis of scyllo-Inositol from myo-Inositol.

Scyllo-inositol (SI), a stereoisomer of inositol, is regarded as a promising therapeutic agent for Alzheimer's disease. Here, an in vitro cofactor-balance biotransformation for the production of SI from myo-inositol (MI) by thermophilic myo-inositol 2-dehydrogenase (IDH) and scyllo-inositol 2-dehydrogenase (SIDH) is presented. These two enzymes (i.e., IDH and SIDH from Geobacillus kaustophilus) are co-expressed in Escherichia coli BL21(DE3), and E. coli cells containing the two enzymes are permeabilized by heat treatment as whole-cell catalysts to convert MI to SI. After condition optimizations about permeabilized temperature, reaction temperature, and initial MI concentration, about 82 g L-1 of SI is produced from 250 g L-1 of MI within 24 h without any cofactor supplementation. This final titer of SI produced is the highest to the authors' limited knowledge. This study provides a promising method for the large-scale industrial production of SI.

First report on Giardia duodenalis assemblage F in Slovakian children living in poor environmental conditions.

BACKGROUND: Giardiasis is one of the most common gastrointestinal infections of humans and animals attributable to complex of eight morphologically identical genetic assemblages, further divided into sub-assemblages. Disease is common for a wide range of hosts and genetic characterization is needed for better understanding of multifaceted epidemiology for this protozoan parasite. The aim of this study was to identify genetic heterogeneity in assemblages and sub-assemblages of Giardiaduodenalis circulating among the children population living in deprived socioeconomic conditions. METHODS: A total of 333 stool samples from children in eastern Slovakia were collected during the period of 2015-2016 and analysed by molecular methods. Molecular characterization of G. duodenalis was performed by sequence analysis of triose phosphate isomerase gene (tpi) and glutamate dehydrogenase gene (gdh). RESULTS: G. duodenalis DNA was detected in 21 samples (6.3%), out of which 14 isolates (66.7%) belonged to assemblage B, 4 isolates (19.0%) to sub-assemblage AII and 3 isolates (14.3%) corresponded to assemblage F. As regards the determination of sub-assemblages of assemblage B, 4 isolates were characterized as sub-assemblage BIII and 6 isolates as sub-assemblage BIV. CONCLUSION: This study is the first finding of cat specific assemblage F in man not only in Slovakia, but also in Europe. The absence of molecular data about G. duodenalis in companion animals in Slovakia establishes a strong need for further investigation for potential sources of giardiasis and understanding the epidemiology will help to improve the preventive strategies in eradication of infection in this population.

Biochemical and Molecular Characterization of Glycerol Dehydrogenas from Klebsiella pneumoniae.

Glycerol dehydrogenase (GlyDH) catalyzes the oxidation of glycerol to dihydroxyacetone (DHA), which is the first step in the glycerol metabolism pathway. GlyDH has attracted great interest for its potential industrial applications, since DHA is a precursor for the synthesis of many commercially valuable chemicals and various drugs. In this study, GlyDH from Klebsiella pneumoniae (KpGlyDH) was overexpressed in E. coli and purified to homogeneity for biochemical and molecular characterization. KpGlyDH exhibits an exclusive preference for NAD+ over NADP+. The enzymatic activity of KpGlyDH is maximal at pH 8.6 and pH 10.0. Of the three common polyol substrates, KpGlyDH showed the highest kcat/Km value for glycerol, which is three times higher than for racemic 2,3-butanediol and 32 times higher than for ethylene glycol. The kcat value for glycerol oxidation is notably high at 87.1 ± 11.3 sec-1. KpGlyDH was shown to exist in an equilibrium between two different oligomeric states, octamer and hexadecamer, by size-exclusion chromatography analysis. KpGlyDH is structurally thermostable, with a Tm of 83.4°C, in thermal denaturation experiment using circular dichroism spectroscopy. The biochemical and biophysical characteristics of KpGlyDH revealed in this study should provide the basis for future research on its glycerol metabolism and possible use in industrial applications.

Engineering Bacillus velezensis with high production of acetoin primes strong induced systemic resistance in Arabidopsis thaliana.

Many plant growth promoting rhizobacteria such as Bacillus velezensis GJ11 can produce acetoin to trigger induced systemic resistance (ISR) in plants. For improving acetoin production, the mutant strains were respectively constructed by knockout of the gene of bdh (2,3-butanediol dehydrogenase) and gdh (glycerol dehydrogenase) in GJ11, but only GJ11Δbdh produced a high level of acetoin triggering strong ISR against Pseudomonas syringae infection in plants. GJ11Δbdh could induce H2O2 accumulation in plants by producing a high level of acetoin. H2O2 was necessary for triggering ISR against the pathogen infection because after scavenging H2O2 with ascorbic acid or catalase, the inhibition role to pathogen infection induced by acetoin almost disappeared in plants. Further investigation found the plants treated with GJ11Δbdh in an obvious "priming" state, in which the mild immune response was observed such as a slight increase of H2O2 production, callose deposition, and enzymes activity related with defence response (e.g. POD, PAL and PPO). The plants in "priming" could rapidly respond to the pathogen infection accompanying with a significant increase of H2O2 production, callose deposition, and enzymes activity. Collectively, this study provides new insight into the role of acetoin as a strong elicitor of defense response, and ascribes a new approach to construct the mutant strains with high production of acetoin for triggering stronger ISR against pathogens infection in plants.

Targeting Mannitol Metabolism as an Alternative Antimicrobial Strategy Based on the Structure-Function Study of Mannitol-1-Phosphate Dehydrogenase in Staphylococcus aureus.

Mannitol-1-phosphate dehydrogenase (M1PDH) is a key enzyme in Staphylococcus aureus mannitol metabolism, but its roles in pathophysiological settings have not been established. We performed comprehensive structure-function analysis of M1PDH from S. aureus USA300, a strain of community-associated methicillin-resistant S. aureus, to evaluate its roles in cell viability and virulence under pathophysiological conditions. On the basis of our results, we propose M1PDH as a potential antibacterial target. In vitro cell viability assessment of ΔmtlD knockout and complemented strains confirmed that M1PDH is essential to endure pH, high-salt, and oxidative stress and thus that M1PDH is required for preventing osmotic burst by regulating pressure potential imposed by mannitol. The mouse infection model also verified that M1PDH is essential for bacterial survival during infection. To further support the use of M1PDH as an antibacterial target, we identified dihydrocelastrol (DHCL) as a competitive inhibitor of S. aureus M1PDH (SaM1PDH) and confirmed that DHCL effectively reduces bacterial cell viability during host infection. To explain physiological functions of SaM1PDH at the atomic level, the crystal structure of SaM1PDH was determined at 1.7-Å resolution. Structure-based mutation analyses and DHCL molecular docking to the SaM1PDH active site followed by functional assay identified key residues in the active site and provided the action mechanism of DHCL. Collectively, we propose SaM1PDH as a target for antibiotic development based on its physiological roles with the goals of expanding the repertory of antibiotic targets to fight antimicrobial resistance and providing essential knowledge for developing potent inhibitors of SaM1PDH based on structure-function studies.IMPORTANCE Due to the shortage of effective antibiotics against drug-resistant Staphylococcus aureus, new targets are urgently required to develop next-generation antibiotics. We investigated mannitol-1-phosphate dehydrogenase of S. aureus USA300 (SaM1PDH), a key enzyme regulating intracellular mannitol levels, and explored the possibility of using SaM1PDH as a target for developing antibiotic. Since mannitol is necessary for maintaining the cellular redox and osmotic potential, the homeostatic imbalance caused by treatment with a SaM1PDH inhibitor or knockout of the gene encoding SaM1PDH results in bacterial cell death through oxidative and/or mannitol-dependent cytolysis. We elucidated the molecular mechanism of SaM1PDH and the structural basis of substrate and inhibitor recognition by enzymatic and structural analyses of SaM1PDH. Our results strongly support the concept that targeting of SaM1PDH represents an alternative strategy for developing a new class of antibiotics that cause bacterial cell death not by blocking key cellular machinery but by inducing cytolysis and reducing stress tolerance through inhibition of the mannitol pathway.

Homologous overexpression of hydrogenase and glycerol dehydrogenase in Clostridium pasteurianum to enhance hydrogen production from crude glycerol.

This study reports engineering of a hypertransformable variant of C. pasteurianum for bioconversion of glycerol into hydrogen (H2). A functional glycerol-triggered hydrogen pathway was engineered based on two approaches: (1) increasing product yield by overexpression of immediate enzyme catalyzing H2 production, (2) increasing substrate uptake by overexpression of enzymes involved in glycerol utilization. The first strategy aimed at overexpression of hydA gene encoding hydrogenase, and the second one, through combination of overexpression of dhaD1 and dhaK genes encoding glycerol dehydrogenase and dihydroxyacetone kinase. These genetic manipulations resulted in two recombinant strains (hydA++/dhaD1K++) capable of producing 97% H2 (v/v), with yields of 1.1 mol H2/mol glycerol in hydA overexpressed strain, and 0.93 mol H2/mol glycerol in dhaD1K overexpressed strain, which was 1.5 fold higher than wild type. Among two strains, dhaD1K++ consumed more glycerol than hydA++ which proves that overexpression of glycerol enzymes has enhanced glycerol intake rate.

Transcriptional regulation of microalgae for concurrent lipid overproduction and secretion.

Commercialization of algal lipids and biofuels is still impractical owing to the unavailability of lipogenic strains and lack of economically viable oil extraction strategies. Because lipogenesis is governed by multiple factors, success in generating industrial-suitable algal strains using conventional strategies has been limited. We report the discovery of a novel bZIP1 transcription factor, NobZIP1, whose overexpression results in a remarkable elevation of lipid accumulation and lipid secretion in a model microalga Nannochloropsis oceanica, without impairing other physiological properties. Chromatin immunoprecipitation-quantitative PCR analysis revealed that the key genes up- and down-regulated by NobZIP1 are involved in lipogenesis and cell wall polymer synthesis, respectively, which, in turn, induce lipid overproduction and secretion. Among these regulated genes, UDP-glucose dehydrogenase was shown to alter cell wall composition, thus also boosting lipid secretion. In summary, these results offer a comprehensive strategy for concurrent lipid overproduction and secretion, strongly increasing the commercial potential of microalgae.

GDH and toxin immunoassay for the diagnosis of Clostridioides (Clostridium) difficile infection is not a 'one size fit all' screening test.

Diagnosing Clostridioides (Clostridium) difficile infection is challenged by lack of a clear gold standard. We sought to determine if the two-step algorithm (screening GDH and toxin lateral flow assay followed by tcdB PCR) would have adequate clinical performance at a tertiary care center. Of 486 patients, 310 (63.8%) were immunocompromised. Of 150 PCR-positive specimens, 52 (34.7%) were toxin-positive and 126 (84.0%) were GDH positive. Positive GDH or toxin results corresponded to lower PCR cycle threshold values (P < 0.01). PCR-positive patients had more frequently documented antibiotic usage (78.4% vs 66.9%, P = 0.05) and diarrhea (91.0% vs. 79.4%, P < 0.01) and less frequent alternate etiologies of diarrhea (27.3% vs. 41.1%, P = 0.004) or laxative use (24.6% vs 36.1%, P = 0.02). Toxin positivity was associated with antibiotic use (P < 0.01), but not with neutropenia, diarrhea, malignancy, or chemotherapy (P > 0.05). The application of the 2-step algorithm should be thoroughly evaluated in immunocompromised patient populations before implementation.

Molecular and biochemical characterization of key enzymes in the cysteine and serine metabolic pathways of Acanthamoeba castellanii.

BACKGROUND: Acanthamoeba spp. can cause serious human infections, including Acanthamoeba keratitis, granulomatous amoebic encephalitis and cutaneous acanthamoebiasis. Cysteine biosynthesis and the L-serine metabolic pathway play important roles in the energy metabolism of Acanthamoeba spp. However, no study has confirmed the functions of cysteine synthase (AcCS) in the cysteine pathway and phosphoglycerate dehydrogenase (AcGDH) or phosphoserine aminotransferase (AcSPAT) in the non-phosphorylation serine metabolic pathway of Acanthamoeba. METHODS: The AcCS, AcGDH and AcSPAT genes were amplified by PCR, and their recombinant proteins were expressed in Escherichia coli. Polyclonal antibodies against the recombinant proteins were prepared in mice and used to determine the subcellular localisation of each native protein by confocal laser scanning microscopy. The enzymatic activity of each recombinant protein was also analysed. Furthermore, each gene expression level was analysed by quantitative PCR after treatment with different concentrations of cysteine or L-serine. RESULTS: The AcCS gene encodes a 382-amino acid protein with a predicted molecular mass of 43.1 kDa and an isoelectric point (pI) of 8.11. The AcGDH gene encodes a 350-amino acid protein with a predicted molecular mass of 39.1 kDa and a pI of 5.51. The AcSPAT gene encodes a 354-amino acid protein with a predicted molecular mass of 38.3 kDa and a pI of 6.26. Recombinant AcCS exhibited a high cysteine synthesis activity using O-acetylserine and Na2S as substrates. Both GDH and SPAT catalysed degradation, rather than synthesis, of serine. Exogenous L-serine or cysteine inhibited the expression of all three enzymes in a time- and dose-dependent manner. CONCLUSIONS: This study demonstrated that AcCS participates in cysteine biosynthesis and serine degradation via the non-phosphorylation serine metabolic pathway, providing a molecular basis for the discovery of novel anti-Acanthamoeba drugs.