Intergeneric protoplast fusion between Kluyveromyces and Saccharomyces cerevisiae – to produce sorbitol from Jerusalem artichokes

The construction of inulin-assimilating and sorbitol-producing fusants was achieved by intergeneric protoplast fusion between Kluyveromyces sp. Y-85 and Saccharomyces cerevisiae E-15. The cells of parental strains were pretreated with 0.1% EDTA (w/v) and 2-mercaptoethanol (0.1%, v/v) and then exposed to 2.0% (w/v) Zymolase at 30 °C for 30–40 min. The optimized fusion condition demonstrated that with the presence of 30% (w/v) polyethylene glycol 6000 (PEG-6000) and 10 mM CaCl₂ for 30 min, the fusion frequency reached 2.64 fusants/10⁶ parental cells. The fusants were screened by different characters between two parental strains and further identified by DNA contents, inulinase activity and sorbitol productivity. One of the genetically stable fusants, Strain F27, reached a maximal sorbitol production of 4.87 g/100 ml under optimal fermentation condition.     

Symbiosis-promoting and deleterious effects of NopT, a novel type 3 effector of Rhizobium sp. strain NGR234

Establishment of symbiosis between certain host plants and nitrogen-fixing bacteria ("rhizobia") depends on type 3 effector proteins secreted via the bacterial type 3 secretion system (T3SS). Here, we report that the open reading frame y4zC of strain NGR234 encodes a novel rhizobial type 3 effector, termed NopT (for nodulation outer protein T). Analysis of secreted proteins from NGR234 and T3SS mutants revealed that NopT is secreted via the T3SS. NopT possessed autoproteolytic activity when expressed in Escherichia coli or human HEK 293T cells. The processed NopT exposed a glycine (G50) to the N terminus, which is predicted to be myristoylated in eukaryotic cells. NopT with a point mutation at position C93, H205, or D220 (catalytic triad) showed strongly reduced autoproteolytic activity, indicating that NopT is a functional protease of the YopT-AvrPphB effector family. When transiently expressed in tobacco plants, proteolytically active NopT elicited a rapid hypersensitive reaction. Arabidopsis plants transformed with nopT showed chlorotic and necrotic symptoms, indicating a cytotoxic effect. Inoculation experiments with mutant derivatives of NGR234 indicated that NopT affected nodulation either positively (Phaseolus vulgaris cv. Yudou No. 1; Tephrosia vogelii) or negatively (Crotalaria juncea). We suggest that NopT-related polymorphism may be involved in evolutionary adaptation of NGR234 to particular host legumes.     

Characterization of a novel thermo-stable lipase from endophyte <Emphasis Type="Italic">Pseudomonas putida</Emphasis> in <Emphasis Type="Italic">Pistacia chinensis</Emphasis> Bunge

A novel lipolytic enzyme-producing endophytic strain PC2 was successfully isolated from the seeds of an ideal bioenergy plant Pistacia chinensis Bunge. Based on the analysis of morphology and 16S rRNA sequence, bacterial strain PC2 was identified as a subspecies of Pseudomonas putida, therefore named as P. putida PC2. Whole-genome sequencing showed PC2 contained a 1224-nucleotide lipase gene (named lip-PC2) predicted to encode a 407-amino-acid protein. Purified lipases from both the original PC2 strain and heterologously expressed Escherichia coli were nearly 50 kD with specific activity of 9.48 U/mL. LIP-PC2 displayed the maximal activity at 50°C or pH 8.0, and maintained above 80% relative activity in the range of from 40 to 60°C or pH in the range of from 6.0 to 8.0, indicating thermostable and alkaline properties. Enzyme activity was enhanced by Mg²⁺, Na⁺ and Mn²⁺, but strongly inhibited by Cu²⁺, Zn²⁺ Co²⁺, EDTA as well as organic solvents and surfactants. Additionally, the analysis of amino acid sequence and structure indicated that LIP-PC2 was a novel member belonging to family I.3 of bacterial lipolytic enzymes and its catalytic triad was consisted of Ser-200, Asp-342 and His-374.     

Analysis of Mid-Infrared Surface Plasmon Modes in a Graphene-Based Cylindrical Hybrid Waveguide

A graphene-based cylindrical hybrid surface plasmon polariton waveguide, composed of a silicon nanowire core surrounded by a silica layer and then a graphene layer, is investigated using the finite-difference time-domain method. The analytical solutions and the numerical simulation show that an ultra-small mode area and a large propagation length can be achieved with this waveguide. Utilizing the perturbation theory of coupled mode, we demonstrate that the six lowest-order coupling modes originate from the coupling of the three lowest-order single-waveguide modes, and the m?=?1 order yy-coupling mode possesses the maximum coupling length and the minimum crosstalk. This waveguide can be used for photonic integrated circuits in the mid-infrared range.     

Hydrophobic chromatography of the HL-60 cellular fraction co-binding with hexamethylene bisacetamide

Methods of separating N-acetyl-1,6-diaminohexane (NADAH) and its immobilization to diol-silica have been developed. Hexamethylene bisacetamide (HMBA) and its metabolite NADAH are used as inducers of leukemia cell differentiation. The inducing mechanism of HMBA is still not clear. Experiments show that HMBA and NADAH undergo relatively strong hydrophobic reactions and do not readily undergo ion-exchange with the proteins of the cytosolic fraction of HL-60 cells during immobilization of NADAH; the retention time of the proteins was longer than that of the phosphatides. These results show that the adsorption of HMBA and NADAH to proteins was higher than that to phosphatides. The expected biospecific receptor binding with HMBA has not been found.     

Plasma metabonomics as a novel diagnostic approach for major depressive disorder

Major depressive disorder (MDD) is a socially detrimental psychiatric disorder, contributing to increased healthcare expenditures and suicide rates. However, no empirical laboratory-based tests are available to support the diagnosis of MDD. In this study, a NMR-based plasma metabonomic method for the diagnosis of MDD was tested. Proton nuclear magnetic resonance ((1)H NMR) spectra of plasma sampled from first-episode drug-na??ve depressed patients (n = 58) and healthy controls (n = 42) were recorded and analyzed by orthogonal partial least-squares discriminant analysis (OPLS-DA). The OPLS-DA score plots of the spectra demonstrated that the depressed patient group was significantly distinguishable from the healthy control group. Moreover, the method accurately diagnosed blinded samples (n = 26) in an independent replication cohort with a sensitivity and specificity of 92.8% and 83.3%, respectively. Taken together, NMR-based plasma metabonomics may offer an accurate empirical laboratory-based method applicable to the diagnosis of MDD.     

A meta-analysis of tumor necrosis factor-alpha, interleukin-6, and interleukin-10 in preeclampsia

Inflammation may play a major role in the pathogenesis of preeclampsia (PE). In this meta-analysis, we determined whether maternal polymorphisms and serum concentrations of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) were associated with PE. All studies investigating the associations between PE and maternal polymorphisms of TNF-α-308G/A, IL-6-174G/C, and IL-10-1082A/G or serum concentrations of TNF-α, IL-6, and IL-10 were reviewed. We found that neither maternal TNF-α-308G/A (p=0.86, odds ratio [OR]=0.98, 95% confidence interval [CI], 0.76-1.25), IL-6 174G/C (p=0.14, OR=1.23, 95% CI, 0.93-1.61), nor IL-10-1082A/G (p=0.72, OR=1.07, 95% CI, 0.75-1.52) were associated with PE. On the other hand, maternal TNF-α (p<0.00001, weighted mean difference [WMD]=19.63 pg/ml, 95% CI, 18.54-20.72 pg/ml), IL-6 (p<0.00001, WMD=6.58 pg/ml, 95% CI, 5.49-7.67 pg/ml), and IL-10 (p=0.0005, WMD=19.30 pg/ml, 95% CI, 8.42-30.17 pg/ml) concentrations were significantly higher in PE patients versus controls. Our findings strengthen the clinical evidence that PE is accompanied by exaggerated inflammatory responses, but do not support TNF-α-308G/A, IL-6-174G/C, and IL-10-1082A/G as candidate susceptibility loci in PE.     

Uncovering principles that control septin-septin interactions

Septins comprise a conserved family of GTPases important in cytokinesis. These proteins polymerize into filaments from rod-shaped heteromeric septin complexes. Septins interact with one another at two interfaces (NC and G) that alternate within the complex. Here, we show that small mutations at the N terminus greatly enhance the formation of SEPT2 homopolymers. Taking advantage of this mutation to examine polymer formation using SEPT2 alone, we show that both NC and G interfaces are required for filament formation. However, co-expression of wild type SEPT2 with SEPT2 containing mutations at either NC or G interfaces revealed that only the NC mutant suppressed filament formation. NC mutants are able to interact with one another at putative G interfaces, whereas G mutants fail to interact at NC interfaces. In addition, all promiscuous septin pairwise interactions occur at the G interface. These findings suggest that G interface interactions must occur before NC interactions during polymer formation.     

X-ray Phase Contrast Imaging of Cell Isolation with Super-Paramagnetic Microbeads

Super-paramagnetic microbeads are widely used for cell isolation. Evaluation of the binding affinity of microbeads to cells using optical microscopy has been limited by its small scope. Here, magnetic property of microbeads was first investigated by using synchrotron radiation (SR) in-line x-ray phase contrast imaging (PCI). The cell line mouse LLC (Lewis lung carcinoma) was selected for cell adhesion studies. Targeted microbeads were prepared by attaching anti-VEGFR2 (vascular endothelial growth factor receptor-2) antibody to the shell of the microbeads. The bound microbeads were found to better adhere to LLC cells than unbound ones. PCI dynamically and clearly showed the magnetization and demagnetization of microbeads in PE-50 tube. The cells incubated with different types of microbeads were imaged by PCI, which provided clear and real-time visualization of the cell isolation. Therefore, PCI might be considered as a novel and efficient tool for further cell isolation studies.