Inhibition of Citrinin Production in Penicillium citrinum Cultures by Neem [Azadirachta indica A. Juss (Meliaceae)]

The efficacy of different concentrations of aqueous neem leaf extract (3.12 to 50 mg/mL) on growth and citrinin production in three isolates of Penicillium citrinum was investigated under laboratory conditions. Mycotoxin production by the isolates was suppressed, depending on the concentration of the plant extract added to culture media at the time of spore inoculation. Citrinin production in fungal mycelia grown for 21 days in culture media containing 3.12 mg/mL of the aqueous extract of neem leaf was inhibited by approximately 80% in three isolates of P. citrinum. High-performance liquid chromatography was performed to confirm the spectrophotometric results. Vegetative growth was assessed, but neem extract failed to inhibit it. Neem leaf extract showed inhibition of toxin production without retardation in fungal mycelia growth.     

Antioxidant Properties of the Edible Basidiomycete Armillaria mellea in Submerged Cultures

Antioxidant components, ascorbic acid, total flavonoids and total phenols are produced effectively by Armillaria mellea submerged cultures. Dried mycelia and mycelia-free broths obtained by A. mellea submerged cultures are extracted with methanol and hot water and investigated for antioxidant properties. Methanolic extracts from dried mycelia (MEM) and mycelia-free broth (MEB) and hot water extracts from dried mycelia (HWEM) by A. mellea submerged cultures show good antioxidant properties as evidenced by low EC(50) values (<10 mg/mL). Total flavonoid is mainly found in hot water extracts; however, total phenol is rich in methanol and hot water extracts from mycelia. Ascorbic acid and total phenol contents are well correlated with the reducing power and the scavenging effect on superoxide anions. Total flavonoid content is dependent on the antioxidant activity and the chelating effect on ferrous ions. Total antioxidant component contents are closely related to the antioxidant activity and the scavenging superoxide anion ability. Results confirm that extracts with good antioxidant properties from fermenting products by A. mellea are potential good substitutes for synthetic antioxidants and can be applied to antioxidant-related functional food and pharmaceutical industries.     

Cloning,Expression and Characterization of a Novel Thermophilic Polygalacturonase from Caldicellulosiruptor bescii DSM 6725

We cloned the gene {"type":"entrez-protein","attrs":{"text":"ACM61449","term_id":"222457187","term_text":"ACM61449"}}ACM61449 from anaerobic, thermophilic Caldicellulosiruptor bescii, and expressed it in Escherichia coli origami (DE3). After purification through thermal treatment and Ni-NTA agarose column extraction, we characterized the properties of the recombinant protein (CbPelA). The optimal temperature and pH of the protein were 72 °C and 5.2, respectively. CbPelA demonstrated high thermal-stability, with a half-life of 14 h at 70 °C. CbPelA also showed very high activity for polygalacturonic acid (PGA), and released monogalacturonic acid as its sole product. The V_max and K_m of CbPelA were 384.6 U·mg⁻¹ and 0.31 mg·mL⁻¹, respectively. CbPelA was also able to hydrolyze methylated pectin (48% and 10% relative activity on 20%–34% and 85% methylated pectin, respectively). The high thermo-activity and methylated pectin hydrolization activity of CbPelA suggest that it has potential applications in the food and textile industry.     

Lower Temperature Cultures Enlarge the Effects of Vitreoscilla Hemoglobin Expression on Recombinant Pichia pastoris

An heterologous expression of Vitreoscilla hemoglobin (VHb) for improving cell growth and recombinant protein production has been successfully demonstrated in various hosts, including Pichia pastoris. Lower temperature cultures can enhance target protein production in some studies of P. pastoris. In this study, the strategy of combining heterologous VHb expression and lower temperature cultures in P. pastoris showed that final cell density and viability of VHb⁺ strain at 23 °C were higher than that at 30 °C. In addition, the effects of VHb expression on recombinant β-galactosidase production and oxygen uptake rate were also higher at 23 °C than at 30 °C. Consequently, lower temperature cultures can enlarge VHb effectiveness on cell performance of P. pastoris. This is because VHb activity obtained at 23 °C cultures was twofold higher than that at 30 °C cultures, due to a different heme production. This strategy makes P. pastoris an excellent expression host particularly suitable for increasing the yields of the low-stability and aggregation-prone recombinant proteins.     

A High Molecular-Mass Anoxybacillus sp. SK3-4 Amylopullulanase: Characterization and Its Relationship in Carbohydrate Utilization

An amylopullulanase of the thermophilic Anoxybacillus sp. SK3-4 (ApuASK) was purified to homogeneity and characterized. Though amylopullulanases larger than 200 kDa are rare, the molecular mass of purified ApuASK appears to be approximately 225 kDa, on both SDS-PAGE analyses and native-PAGE analyses. ApuASK was stable between pH 6.0 and pH 8.0 and exhibited optimal activity at pH 7.5. The optimal temperature for ApuASK enzyme activity was 60 °C, and it retained 54% of its total activity for 240 min at 65 °C. ApuASK reacts with pullulan, starch, glycogen, and dextrin, yielding glucose, maltose, and maltotriose. Interestingly, most of the previously described amylopullulanases are unable to produce glucose and maltose from these substrates. Thus, ApuASK is a novel, high molecular-mass amylopullulanase able to produce glucose, maltose, and maltotriose from pullulan and starch. Based on whole genome sequencing data, ApuASK appeared to be the largest protein present in Anoxybacillus sp. SK3-4. The α-amylase catalytic domain present in all of the amylase superfamily members is present in ApuASK, located between the cyclodextrin (CD)-pullulan-degrading N-terminus and the α-amylase catalytic C-terminus (amyC) domains. In addition, the existence of a S-layer homology (SLH) domain indicates that ApuASK might function as a cell-anchoring enzyme and be important for carbohydrate utilization in a streaming hot spring.     

Protease and Hemicellulase Assisted Extraction of Dietary Fiber from Wastes of Cynara cardunculus

The action of protease and hemicellulase for the extraction of fractions enriched in soluble fiber from bracts and stems of Cynara cardunculus was evaluated. Using a two-factor simplex design comprising protease amounts of 0–200 μL and hemicellulase amounts of 0–200 mg for 5 g of material, we explored the effect of a 5 h enzymatic treatment at 40 °C on the chemical composition and yield of the fractions isolated. The fractions contained inulin and pectin. In general, the protein, inulin, and polyphenol contents and also the yields were higher for fractions obtained from stems. The most marked effects were observed when enzymes were used at higher concentrations, especially for hemicellulase. The inclusion of a pre-heating step increased the yield and the inulin content for fractions isolated from bracts and stems and decreased the protein and polyphenol contents, and the galacturonic acid for bracts. These fractions, in general, contained the polyphenolic compounds monocaffeoylquinic acid, apigenin, and pinoresinol.     

Nitric Oxide Functions as a Signal in Ultraviolet-B-Induced Baicalin Accumulation in Scutellaria baicalensis Suspension Cultures

Stress induced by ultraviolet-B (UV-B) irradiation stimulates the accumulation of various secondary metabolites in plants. Nitric oxide (NO) serves as an important secondary messenger in UV-B stress-induced signal transduction pathways. NO can be synthesized in plants by either enzymatic catalysis or an inorganic nitrogen pathway. The effects of UV-B irradiation on the production of baicalin and the associated molecular pathways in plant cells are poorly understood. In this study, nitric oxide synthase (NOS) activity, NO release and the generation of baicalin were investigated in cell suspension cultures of Scutellaria baicalensis exposed to UV-B irradiation. UV-B irradiation significantly increased NOS activity, NO release and baicalin biosynthesis in S. baicalensis cells. Additionally, exogenous NO supplied by the NO donor, sodium nitroprusside (SNP), led to a similar increase in the baicalin content as the UV-B treatment. The NOS inhibitor, N^ω-nitro-l-arginine (LNNA), and NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) partially inhibited UV-B-induced NO release and baicalin accumulation. These results suggest that NO is generated by NOS or NOS-like enzymes and plays an important role in baicalin biosynthesis as part of the defense response of S. baicalensis cells to UV-B irradiation.     

A Novel Cold-Active Lipase from Candida albicans: Cloning, Expression and Characterization of the Recombinant Enzyme

A novel lipase gene lip5 from the yeast Candida albicans was cloned and sequenced. Alignment of amino acid sequences revealed that 86-34% identity exists with lipases from other Candida species. The lipase and its mutants were expressed in the yeast Pichia pastoris, where alternative codon usage caused the mistranslation of 154-Ser and 293-Ser as leucine. 154-Ser to leucine resulted in loss of expression of Lip5, and 293-Ser to leucine caused a marked reduction in the lipase activity. Lip5-DM, which has double mutations that revert 154 and 293 to serine residues, showed good lipase activity, and was overexpressed and purified by (NH(4))(2)SO(4) precipitation and ion-exchange chromatography. The pure Lip5-DM was stable at low temperatures ranging from 15-35 °C and pH 5-9, with the optimal conditions being 15-25 °C and pH 5-6. The activation energy of recombinant lipase was 8.5 Kcal/mol between 5 and 25 °C, suggesting that Lip5-DM was a cold-active lipase. Its activity was found to increase in the presence of Zn(2+), but it was strongly inhibited by Fe(2+), Fe(3+), Hg(2+) and some surfactants. In addition, the Lip5-DM could not tolerate water-miscible organic solvents. Lip5-DM exhibited a preference for the short-and medium-chain length p-nitrophenyl (C4 and C8 acyl group) esters rather than the long chain length p-nitrophenyl esters (C12, C16 and C18 acyl group) with highest activity observed with the C8 derivatives. The recombinant enzyme displayed activity toward triacylglycerols, such as olive oil and safflower oil.     

Biology,Pest Status,Microbiome and Control of Kudzu Bug (Hemiptera: Heteroptera: Plataspidae): A New Invasive Pest in the U.S.

Soybean is an important food crop, and insect integrated pest management (IPM) is critical to the sustainability of this production system. In recent years, the introduction into the United States of the kudzu bug currently identified as Megacopta cribraria (F.), poses a threat to soybean production. The kudzu bug was first discovered in the state of Georgia, U.S. in 2009 and since then has spread to most of the southeastern states. Because it was not found in the North American subcontinent before this time, much of our knowledge of this insect comes from research done in its native habitat. However, since the U.S. introduction, studies have been undertaken to improve our understanding of the kudzu bug basic biology, microbiome, migration patterns, host selection and management in its expanding new range. Researchers are not only looking at developing IPM strategies for the kudzu bug in soybean, but also at its unique relationship with symbiotic bacteria. Adult females deposit bacterial packets with their eggs, and the neonates feed on these packets to acquire the bacteria, Candidatus Ishikawaella capsulata. The kudzu bug should be an informative model to study the co-evolution of insect function and behavior with that of a single bacteria species. We review kudzu bug trapping and survey methods, the development of bioassays for insecticide susceptibility, insecticide efficacy, host preferences, impact of the pest on urban environments, population expansion, and the occurrence of natural enemies. The identity of the kudzu bug in the U.S. is not clear. We propose that the kudzu bug currently accepted as M. cribraria in the U.S. is actually Megacoptapunctatissima, with more work needed to confirm this hypothesis.     

Endogenous Protease Nexin-1 Protects against Cerebral Ischemia

The serine protease thrombin plays a role in signalling ischemic neuronal death in the brain. Paradoxically, endogenous neuroprotective mechanisms can be triggered by preconditioning with thrombin (thrombin preconditioning, TPC), leading to tolerance to cerebral ischemia. Here we studied the role of thrombin’s endogenous potent inhibitor, protease nexin-1 (PN-1), in ischemia and in tolerance to cerebral ischemia induced by TPC. Cerebral ischemia was modelled in vitro in organotypic hippocampal slice cultures from rats or genetically engineered mice lacking PN-1 or with the reporter gene lacZ knocked into the PN-1 locus PN-1HAPN-1-lacZ/HAPN-1-lacZ (PN-1 KI) exposed to oxygen and glucose deprivation (OGD). We observed increased thrombin enzyme activity in culture homogenates 24 h after OGD. Lack of PN-1 increased neuronal death in the CA1, suggesting that endogenous PN-1 inhibits thrombin-induced neuronal damage after ischemia. OGD enhanced β-galactosidase activity, reflecting PN-1 expression, at one and 24 h, most strikingly in the stratum radiatum, a glial cell layer adjacent to the CA1 layer of ischemia sensitive neurons. TPC, 24 h before OGD, additionally increased PN-1 expression 1 h after OGD, compared to OGD alone. TPC failed to induce tolerance in cultures from PN-1^−/− mice confirming PN-1 as an important TPC target. PN-1 upregulation after TPC was blocked by the c-Jun N-terminal kinase (JNK) inhibitor, L-JNKI1, known to block TPC. This work suggests that PN-1 is an endogenous neuroprotectant in cerebral ischemia and a potential target for neuroprotection.     

A New Strategy for Identification of Highly Conserved microRNAs in Non-Model Insect, Spodoptera litura

The indigenous small non-coding RNAs, known as microRNAs (miRNAs), are important regulators of gene expression and many of them are evolutionarily conserved. Whether stem-loop RT-PCR, as a sensitive method, could be utilized to clone conserved miRNAs from non-model insects lacks information. Here, three miRNAs, sli-miR-14, sli-miR-2a and sli-bantam, were cloned from Spodoptera litura by stem-loop RT-PCR. Two groups of primers were designed, and one of them performed especially well and proved stable. The sequences of two highly conserved miRNAs, sli-miR-14 and sli-miR-2a were identical to those in Drosophila melanogaster. To validate the reliability of this strategy, pre-miR-14 and pre-miR-2a in S. litura as representatives were given as well; this shared high homology with those in D. melanogaster and Bombyx mori, and both mature sequences of sli-miR-14 and sli-miR-2a in their precursors shared 100% identity to the results shown by stem-loop RT-PCR. Moreover, expression patterns of these miRNAs were investigated by real-time quantitative PCR. Sli-miR-14 and sli-miR-2a could be detected successfully and their expression patterns showed similar characteristics with those in model insects, further suggesting stem-loop RT-PCR technology can be used for identification of highly conserved miRNAs in non-model insects. These results provide a simplified and efficient strategy for studying the structure and function of highly conserved miRNAs, especially some critical miRNAs in non-model insects.     

Phenotypic and Molecular Convergence of 2q23.1 Deletion Syndrome with Other Neurodevelopmental Syndromes Associated with Autism Spectrum Disorder

Roughly 20% of autism spectrum disorders (ASD) are syndromic with a well-established genetic cause. Studying the genes involved can provide insight into the molecular and cellular mechanisms of ASD. 2q23.1 deletion syndrome (causative gene, MBD5) is a recently identified genetic neurodevelopmental disorder associated with ASD. Mutations in MBD5 have been found in ASD cohorts. In this study, we provide a phenotypic update on the prevalent features of 2q23.1 deletion syndrome, which include severe intellectual disability, seizures, significant speech impairment, sleep disturbance, and autistic-like behavioral problems. Next, we examined the phenotypic, molecular, and network/pathway relationships between nine neurodevelopmental disorders associated with ASD: 2q23.1 deletion Rett, Angelman, Pitt-Hopkins, 2q23.1 duplication, 5q14.3 deletion, Kleefstra, Kabuki make-up, and Smith-Magenis syndromes. We show phenotypic overlaps consisting of intellectual disability, speech delay, seizures, sleep disturbance, hypotonia, and autistic-like behaviors. Molecularly, MBD5 possibly regulates the expression of UBE3A, TCF4, MEF2C, EHMT1 and RAI1. Network analysis reveals that there could be indirect protein interactions, further implicating function for these genes in common pathways. Further, we show that when MBD5 and RAI1 are haploinsufficient, they perturb several common pathways that are linked to neuronal and behavioral development. These findings support further investigations into the molecular and pathway relationships among genes linked to neurodevelopmental disorders and ASD, which will hopefully lead to common points of regulation that may be targeted toward therapeutic intervention.     

Associations of MTHFR C677T and MTRR A66G Gene Polymorphisms with Metabolic Syndrome: A Case-Control Study in Northern China

Prior evidence indicates that homocysteine plays a role in the development of metabolic syndrome (MetS). Methylenetetrahydrofolate reductase (MTHFR) C677T and methionine synthase reductase (MTRR) A66G polymorphisms are common genetic determinants of homocysteine levels. To investigate the associations of the MTHFR C677T and MTRR A66G polymorphisms with MetS, 692 Chinese Han subjects with MetS and 878 controls were recruited. The component traits of MetS and the MTHFR C677T and MTRR A66G genotypes were determined. A significant association was observed between the MTHFR 677T allele and increased risk of MetS, high fasting blood glucose, high waist circumference, and increasing number of MetS components. The MTRR A66G polymorphism was associated with an increased risk of MetS when combined with the MTHFR 677TT genotype, although there was no association found between MetS and MTRR A66G alone. Furthermore, the MTRR 66GG genotype was associated with high fasting blood glucose and triglycerides. Our data suggest that the MTHFR 677T allele may contribute to an increased risk of MetS in the northern Chinese Han population. The MTRR A66G polymorphism is not associated with MetS. However, it may exacerbate the effect of the MTHFR C677T variant alone. Further large prospective population-based studies are required to confirm our findings.     

Two New Quinochalcone C-Glycosides from the Florets of Carthamus tinctorius

Two new quinochalcone C-glycosides, named hydroxysafflor yellow B (1) and hydroxysafflor yellow C (2), along with two known quinochalcone C-glycosides, safflomin C (3) and saffloquinoside C (4), and one known flavanone, (2R)-4'',5-dihydroxyl-6,7-di-O-β-d-glucopyranosyl flavanone (5), were isolated from the florets of Carthamus tinctorius. Their structures were determined by extensive spectroscopic (UV, IR, HR-ESI-MS, 1D and 2D NMR) analyses. In addition, these quinochalcone C-glycosides together with hydroxysafflor yellow A and anhydrosafflor yellow B were evaluated for their anti-oxidative effects against H₂O₂-induced cytotoxicity in cultured H9c2 cells. Among them, compound 2 exhibited significant anti-oxidative effects.     

Expression Patterns and Functional Novelty of Ribonuclease 1 in Herbivorous Megalobrama amblycephala

Ribonuclease 1 (RNase1) is an important digestive enzyme that has been used to study the molecular evolutionary and plant-feeding adaptation of mammals. However, the expression patterns and potential biological function of RNase1 in herbivorous fish is not known. Here, we identified RNase1 from five fish species and illuminated the functional diversification and expression of RNase1 in herbivorous Megalobrama amblycephala. The five identified fish RNase1 genes all have the signature motifs of the RNase A superfamily. No expression of Ma-RNase1 was detected in early developmental stages but a weak expression was detected at 120 and 144 hours post-fertilization (hpf). Ma-RNase1 was only expressed in the liver and heart of one-year-old fish but strongly expressed in the liver, spleen, gut, kidney and testis of two-year-old fish. Moreover, the immunostaining localized RNase1 production to multiple tissues of two-year-old fish. A biological functional analysis of the recombinant protein demonstrated that M. amblycephala RNase1 had a relatively strong ribonuclease activity at its optimal pH 6.1, which is consistent with the pH of its intestinal microenvironment. Collectively, these results clearly show that Ma-RNase1 protein has ribonuclease activity and the expression patterns of Ma-RNase1 are dramatically different in one year and two-year-old fish, suggesting the functional differentiation during fish growing.     

Identification of two glutamic acid residues essential for catalysis in the {beta}-glycosidase from the thermoacidophilic archaeon Sulfolobus solfataricus

The Sulfolobus solfataricus, strain MT4, ß-glycosidase(Ssßgly) is a thermophilic member of glycohydrolasefamily 1. To identify active-site residues, glutamic acids 206and 387 have been changed to isosteric glutamine by site-directedmutagenesis. Mutant proteins have been purified to homogeneityusing the Schistosoma japonicum glutathione S-transferase (GST)fusion system. The proteolytic cleavage of the chimeric proteinwith thrombin was only obtainable after the introduction ofa molecular spacer between the GST and the Ssß-glydomains. The Glu387 Gin mutant showed no detectable activity,as expected for the residue acting as the nucleophile of thereaction. The Glu206 Gin mutant showed 10- and 60-fold reducedactivities on aryl-galacto and aryl-glucosides, respectively,when compared with the wild type. Moreover, a significant K_mdecrease with plo-nitrophenyl-ß-D-glucoside was observed.The residual activity of the Glu206 Gln mutant lost the typicalpH dependence shown by the wild type. These data suggest thatGlu206 acts as the general acid/base catalyst in the hydrolysisreaction.     

Synthesis and Characterization of Some New Bis-Pyrazolyl-Thiazoles Incorporating the Thiophene Moiety as Potent Anti-Tumor Agents

A new series of 1,4-bis(1-(5-(aryldiazenyl)thiazol-2-yl)-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-3-yl)benzenes 3a–i were synthesized via reaction of 5,5′-(1,4-phenylene)bis(3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide) (1) with hydrazonoyl halides 2a–i. In addition, reaction of 1 with ethyl chloroacetate afforded bis-thiazolone derivative 8 as the end product. Reaction of compound 8 with methyl glyoxalate gave bis-thiazolone derivative 10. The structures of the newly synthesized compounds were established on the basis of spectroscopic evidences and their alternative syntheses. All the synthesized compounds were evaluated for their anti-tumor activities against hepatocellular carcinoma (HepG2) cell lines, and the results revealed promising activities of compounds 3g, 5e, 3e, 10, 5f, 3i, and 3f with IC₅₀ equal 1.37 ± 0.15, 1.41 ± 0.17, 1.62 ± 0.20, 1.86 ± 0.20, 1.93 ± 0.08, 2.03 ± 0.25, and 2.09 ± 0.19 μM, respectively.     

New PCR Assays for the Identification of Fusarium verticillioides, Fusarium subglutinans, and Other Species of the Gibberella fujikuroi Complex

Fusarium verticillioides and Fusarium subglutinans are important fungal pathogens of maize and other cereals worldwide. In this study, we developed PCR-based protocols for the identification of these pathogens targeting the gaoB gene, which codes for galactose oxidase. The designed primers recognized isolates of F. verticillioides and F. subglutinans that were obtained from maize seeds from several producing regions of Brazil but did not recognize other Fusarium spp. or other fungal genera that were either obtained from fungal collections or isolated from maize seeds. A multiplex PCR protocol was established to simultaneously detect the genomic DNA from F. verticillioides and F. subglutinans. This protocol could detect the DNA from these fungi growing in artificially or naturally infected maize seeds. Another multiplex reaction with a pair of primers developed in this work combined with a pre-existing pair of primers has allowed identifying F. subglutinans, F. konzum, and F. thapsinum. In addition, the identification of F. nygamai was also possible using a combination of two PCR reactions described in this work, and another described in the literature.     

Antibacterial Activity of New Oxazolidin-2-One Analogues in Methicillin-Resistant Staphylococcus aureus Strains

Staphylococcus aureus is one of the most common causes of nosocomial infections. The purpose of this study was the synthesis and in vitro evaluation of antimicrobial activity of 10 new 3-oxazolidin-2-one analogues on 12 methicillin resistant S. aureus (MRSA) clinical isolates. S. aureus confirmation was achieved via catalase and coagulase test. Molecular characterization of MRSA was performed by amplification of the mecA gene. Antimicrobial susceptibility was evaluated via the Kirby-Bauer disc diffusion susceptibility test protocol, using commonly applied antibiotics and the oxazolidinone analogues. Only (R)-5-((S)-1-dibenzylaminoethyl)-1,3-oxazolidin-2-one (7a) exhibited antibacterial activity at 6.6 μg. These results, allow us to infer that molecules such as 7a can be potentially used to treat infections caused by MRSA strains.