Construction of diverse spirooxindoles via a domino reaction of arylamines, but-2-ynedioates and 3-hydroxy-3-(indol-3-yl)indolin-2-ones.

An iodine-promoted domino reaction of arylamines/benzylamines, dialkyl but-2-ynedioates and 3-hydroxy-3-(indol-3-yl)indolin-2-ones showed very interesting molecular diversity. The reaction in acetonitrile at 65 °C in the presence of 30% mmol I2 resulted in spiro[indoline-3,1'-pyrido[4,3-b]indoles] in satisfactory yields. When anilines without para-substituents were used in the reaction, a direct substitution of the hydroxyl group to 2-(phenylamino)maleate at the para-position of aniline gave chain products in good yields. Additionally, similar reactions with benzylamines not only gave spiro[indoline-3,1'-pyrido[4,3-b]indoles], but also afforded spiro[indoline-3,1'-pyrano[4,3-b]indol]-2-ones in lower yields. A plausible domino annulation mechanism was rationally proposed for the formation of different kinds of polycyclic compounds.

A dynamic and multilocus metabolic regulation strategy using quorum-sensing-controlled bacterial small RNA.

Metabolic regulation strategies have been developed to redirect metabolic fluxes to production pathways. However, it is difficult to screen out target genes that, when repressed, improve yield without affecting cell growth. Here, we report a strategy using a quorum-sensing system to control small RNA transcription, allowing cell-density-dependent repression of target genes. This strategy is shown with convenient operation, dynamic repression, and availability for simultaneous regulation of multiple genes. The parameters Ai, Am, and RA (3-oxohexanoyl-homoserine lactone [AHL] concentrations at which half of the maximum repression and the maximum repression were reached and value of the maximum repression when AHL was added manually, respectively) are defined and introduced to characterize repression curves, and the variant LuxRI58N is identified as the most suitable tuning factor for shake flask culture. Moreover, it is shown that dynamic overexpression of the Hfq chaperone is the key to combinatorial repression without disruptions on cell growth. To show a broad applicability, the production titers of pinene, pentalenene, and psilocybin are improved by 365.3%, 79.5%, and 302.9%, respectively, by applying combinatorial dynamic repression.

Analysis of PTEN expression and promoter methylation in Uyghur patients with mild type 2 diabetes mellitus.

Phosphatase and tension homolog deleted on chromosome 10 (PTEN) was considered as a promising target in type 2 diabetes mellitus (T2DM) because of its negative effects on insulin resistance. Alteration in DNA methylation is thought to play a role in the pathogenesis of T2DM. The aim of the present study was to quantitatively evaluate the promoter methylation of PTEN in Uyghur patients with mild T2DM. We evaluated methylation levels in 21 CpG sites from -2515 bp to -2186 bp relative to the translation initiation site in 55 cases of T2DM and 50 cases of normal glucose tolerance (NGT) using the MassARRAY spectrometry. In addition, PTEN mRNA and protein levels were measured by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blotting to determine whether DNA methylation alterations were responsible for PTEN expression. Compared with NGT groups, the PTEN mRNA expression was significantly higher in Uyghur patients with mild T2DM groups. We also showed that PTEN protein expression was upregulated in Uyghur patients with mild T2DM groups, but the level of protein kinase B (AKT) was downregulated. PTEN methylation in T2DM patients was significantly lower than that in NGT groups. In addition, 2 CpG units demonstrated a significant difference between the NGT and Uyghur patients with mild T2DM groups. Furthermore, there was a negative association between promoter methylation and PTEN expression. Together, these findings suggest that epigenetic inactivation of PTEN plays an important role in Uyghur patients with mild T2DM. The aberrant methylation of CpG sites within the PTEN promoter may serve as a potential candidate biomarker for T2DM in the Uyghur population.

Combined Effects of Temperature and the Microcystin MC-LR on the Feeding Behavior of the Rotifer Brachionus calyciflorus.

The aim of this study was to investigate the responses in filtration and grazing rates of five rotifer strains of the species Brachionus calyciflorus under different temperatures and MC-LR concentrations. The results showed that strain identity, MC-LR concentration, temperature, and the interactions of these factors significantly affected both response variables, with the exception of the interaction of strain and MC-LR on the grazing rates. At low MC-LR concentrations and for the control group, the filtration and grazing rates increased with increasing temperature. The filtering and grazing rates of B. calyciflorus exposed to higher MC-LR concentrations, however, showed no evident enhancement with increasing of temperature. At high temperatures, the filtration and grazing rates of all rotifer strains decreased significantly with increasing concentration of MC-LR, however B. calyciflorus exhibited a refractory stability in the presence of increased MC-LR levels at lower temperatures.

OEPR Cloning: an Efficient and Seamless Cloning Strategy for Large- and Multi-Fragments.

Here, an efficient cloning strategy for large DNA fragments and for simultaneous assembly of multiple DNA fragments assembly is presented. This strategy is named OEPR (based on Overlap Extension PCR and Recombination in vivo). OEPR cloning is a seamless, restriction- and ligation-independent method. The method takes advantage of both homologous recombination enzymes in E. coli and overlap PCR. Using OEPR cloning, a long fragment (1-6 kb) or multiple fragments (2-4 fragments) can be easily constructed and simultaneously assembled into a target vector.

Mega primer-mediated molecular cloning strategy for chimaeragenesis and long DNA fragment insertion.

Molecular cloning methods based on primer and overlap-extension PCR are widely used due to their simplicity, reliability, low cost and high efficiency. In this article, an efficient mega primer-mediated (MP) cloning strategy for chimaeragenesis and long DNA fragment insertion is presented. MP cloning is a seamless, restriction/ligation-independent method that requires only three steps: (i) the first PCR for mega primer generation; (ii) the second PCR for exponential amplification mediated by the mega primers and (iii) DpnI digestion and transformation. Most importantly, for chimaeragenesis, genes can be assembled and constructed into the plasmid vector in a single PCR step. By employing this strategy, we successfully inserted four DNA fragments (approximately 500 bp each) into the same vector simultaneously. In conclusion, the strategy proved to be a simple and efficient tool for seamless cloning.

Heterologous expression and purification of neurotoxic Hainantoxin-III in E. coli.

In the present study, we used Escherichia coli to produce recombinant Hainantoxin-III (rHNTX-III), a 33-amino acid peptic toxin from the tarantula spider Haplopelma hainanum. The toxin has three pairs of disulfide bonds. A pET-HS-HNTX-III vector was constructed and transformed into the E. coli strain SHuffleTM. rHNTX-III was expressed using auto-induction medium. After using a Ni-NTA column, the expressed fusion protein was digested using SUMO protease (ULP1) to remove the HIS-SUMO tag, and then RP-HPLC and ultrafiltration were used for further purification. Then the rHNTX-III was identified by MALDI-TOF/TOF mass spectrometry. The purified rHNTX-III was further analyzed using a whole-cell patch-clamp assay. It was shown that the rHNTX-III was able to block currents generated by human Nav1.7 (hNav1.7) at an IC50 of 225 nM and also have high selectivity for different voltage-gated sodium channels. Therefore, it has very similar activity to the natural one.

Spatial organization of heterologous metabolic system in vivo based on TALE.

For years, prokaryotic hosts have been widely applied in bio-engineering. However, the confined in vivo enzyme clustering of heterologous metabolic pathways in these organisms often results in low local concentrations of enzymes and substrates, leading to a low productive efficacy. We developed a new method to accelerate a heterologous metabolic system by integrating a transcription activator-like effector (TALE)-based scaffold system into an Escherichia coli chassis. The binding abilities of the TALEs to the artificial DNA scaffold were measured through ChIP-PCR. The effect of the system was determined through a split GFP study and validated through the heterologous production of indole-3-acetic acid (IAA) by incorporating TALE-fused IAA biosynthetic enzymes in E. coli. To the best of our knowledge, we are the first to use the TALE system as a scaffold for the spatial organization of bacterial metabolism. This technique might be used to establish multi-enzymatic reaction programs in a prokaryotic chassis for various applications.

Impaired secretion of glucagon-like peptide 1 during oral glucose tolerance test in patients with newly diagnosed type 2 diabetes mellitus.

OBJECTIVES: To assess glucagon-like peptide 1 (GLP-1) secretion after oral glucose tolerance tests (OGTTs) in subjects with newly diagnosed type 2 diabetes mellitus (T2DM), impaired glucose tolerance (IGT), and normal glucose tolerance (NGT) to clarify changes in GLP-1 secretion during the course of T2DM.  METHODS: In this cross sectional study, 80 subjects were divided into the NGT, IGT, and T2DM groups after undergoing a 75 g OGTT from March to December 2014 at the School of Medicine, First Affiliated Hospital, Shihezi University, Xinjiang, China. Plasma total GLP-1 was measured at 0, 30, 60, 120, and 180 minutes. Homeostasis model assessment of insulin resistance (HOMA-IR), islet ß-cell function (HOMA-ß), Gutt index, Matsuda index, incremental GLP-1 (ΔGLP-1), and areas under the curves of GLP-1 (AUCglp-1), glucose (AUCg), and insulin (AUCins) were calculated. RESULTS: Plasma total GLP-1 at 30-120 minutes and ΔGLP-1 at 30-120 minutes were lower in the T2DM group than in the IGT and NGT groups (p less than 0.05). Peak GLP-1 levels were 35% lower in the T2DM group than in the NGT group. Plasma total GLP-1, ΔGLP-1, and AUCglp-1 correlated negatively with HOMA-IR and AUCg, and positively with HOMA-ß, Gutt index, Matsuda index, and AUCins (p less than 0.05).  CONCLUSION: The GLP-1 secretion after 75 g OGTT was impaired in newly diagnosed T2DM patients, inversely proportional to IR and hyperglycemia, and positively correlated with ß-cell function and insulin sensitivity.

Evolutionary implication of B-1 lineage cells from innate to adaptive immunity.

The paradigm that B cells mainly play a central role in adaptive immunity may have to be reevaluated because B-1 lineage cells have been found to exhibit innate-like functions, such as phagocytic and bactericidal activities. Therefore, the evolutionary connection of B-1 lineage cells between innate and adaptive immunities have received much attention. In this review, we summarized various innate-like characteristics of B-1 lineage cells, such as natural antibody production, antigen-presenting function in primary adaptive immunity, and T cell-independent immune responses. These characteristics seem highly conserved between fish B cells and mammalian B-1 cells during vertebrate evolution. We proposed an evolutionary outline of B cells by comparing biological features, including morphology, phenotype, ontogeny, and functional activity between B-1 lineage cells and macrophages or B-2 cells. The B-1 lineage may be a transitional cell type between phagocytic cells (e.g., macrophages) and B-2 cells that functionally connects innate and adaptive immunities. Our discussion would contribute to the understanding on the origination of B cells specialized in adaptive immunity from innate immunity. The results might provide further insight into the evolution of the immune system as a whole.

Inversion of the Supramolecular Chirality of Nanofibrous Structures through Co-Assembly with Achiral Molecules.

To understand the behavior of chiral nanostructures, it is of critical importance to study how achiral molecules regulate the chirality of such nanostructures and what the main driving forces for the regulation processes are. In this work, the supramolecular chirality of helical nanofibers consisting of phenylalanine-based enantiomers is inverted by achiral bis(pyridinyl) derivatives through co-assembly. This inversion is mainly mediated by intermolecular hydrogen bonding interactions between the achiral additives and the chiral molecules, which may induce stereoselective interactions and different reorientations for the assembled molecules, as confirmed by calculations. This work not only exemplifies a feasible method to invert the helicity of chiral nanostructures by the addition of achiral molecules, but also provides a method to explore their functions in environments where chiral and achiral molecules are in close proximity.

Association of plasma Fetuin-A and clinical characteristics in patients with new-onset type 2 diabetes mellitus.

CONTEXT: Fetuin-A is an abundant plasma protein known to inhibit insulin signaling and pathologic calcification, has emerged as a promising candidate biomarker for diabetes risk. OBJECTIVE: The objective of this study was to investigate the relationships between plasma Fetuin-A level with clinical characteristics in patients with new-onset type 2 diabetes mellitus (nT2DM). SUBJECTS AND METHODS: Plasma Fetuin-A levels, and clinical characteristics were assessed in 100 patients with nT2DM and 100 normal glucose tolerance (NGT). RESULTS: nT2DM subjects had significantly higher Fetuin-A levels than NGT subjects (368.5 ± 15.6 vs 152.7 ± 7.1 mg/ml, P < 0.01). In the Pearson's correlation coefficients, Fetuin-A levels and clinical parameters. Fetuin-A was positively correlated with HOMA-insulin resistance index (HOMA-IR), carotid intima media thickness(CIMT), HbA1c, triglyceride (TG), Low-density lipoprotein cholesterol (LDL-C), body mass index (BMI), systolic blood pressure (SBP), fasting plasma glucose (FBG) and 2 h post-glucose load blood glucose (2 h OGTT) (P < 0.05 and P < 0.01), but negatively with fasting plasma insulin (FINS), 2 h plasma insulin after glucose overload (PINS), High-density lipoprotein cholesterol (HDL-C) and HOMA-beta-cell insulin secretion index (HOMA-IS) (P < 0.05 and P < 0.01). However, no significant relationships were observed between plasma Fetuin-A levels and estimated glomerular filtration rate (eGFR), age and gender in nT2DM subjects. In a multiple linear regression analysis, Fetuin-A levels were independently associated with FBG, 2 h OGTT, HOMA-IS, TG, and CIMT (R(2) = 0.6760). CIMT were negatively associated with FINS and HDL-C (r = -0.33, P = 0.008; r = -0.31, P = 0.01, respectively) in the Pearson's analyses. Moreover, they were positively associated with HOMA-IR (r = 0.28, P = 0.03). It showed significant correlations of plasma CIMT with FINS, PINS and HOMA-IR (R(2) = 0.6760). CONCLUSIONS: Our study suggests that the plasma Fetuin-A levels may be associated with macroangiopathies in nT2DM patients. Therefore, detecting early plasma Fetuin-A levels nT2DM provides an opportunity to intervene of carotid artery disease in diabetic patients and giving timely treatment for the prevention of diabetic vascular complications.

An efficient strategy for heterologous expression and purification of active peptide hainantoxin-IV.

Hainantoxin-IV (HNTX-IV) from the venom of the spider Selenocosmia hainana is a potent antagonist that specifically inhibits the tetrodotoxin-sensitive (TTX-S) sodium channels. The toxin peptide consists of 35 amino acids and adopts a typical inhibitory cystine knot (ICK) motif. To obtain adequate HNTX-IV peptides for further insight into the structure-activity relationships of the toxin, a novel strategy including cloning, expression and purification was developed in an E. coli expression system. For this purpose, a seamless restriction-free (RF) cloning method was employed for the construction of an expression vector to avoid introducing unwanted sequences into the target gene. Furthermore, the solubility of recombinant HNTX-IV could be promoted efficiently by the combination of a glutathione S-transferase (GST) tag and a small ubiquitin-related modifier (SUMO) tag. Finally, an affinity-chromatography-free purification strategy was developed by cut-off dialysis tubing combined with trichloroacetic acid (TCA) extraction. Further HPLC purification yielded recombinant, tag-free HNTX-IV with high yield and purity. The molecular weight of recombinant HNTX-IV (rHNTX-IV) is identical to its theoretical value according to Matrix-Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) analysis. The recombinant toxin has similar activity (IC50 value of 120 nM) on the tetrodotoxin-sensitive (TTX-S) sodium channels in adult rat dorsal root ganglion (DRG) neurons to native toxins. In the report, an efficient and cost-effective strategy for producing rHNTX-IV was developed, which paved the way for the further study of structure-activity relationships of rHNTX-IV and its pharmaceutical applications.

Association between fetuin-A levels with insulin resistance and carotid intima-media thickness in patients with new-onset type 2 diabetes mellitus.

Fetuin-A, which is known to inhibit insulin signaling and pathological calcification, has emerged as a diabetes risk biomarker. In the present study, the association between the fetuin-A levels with insulin resistance (IR) and carotid intima-media thickness (CIMT) was investigated in patients with new-onset type 2 diabetes mellitus (nT2DM). A total of 100 patients with nT2DM (nT2DM group) and 100 normal glucose tolerance (NGT group) controls were evaluated. The serum fetuin-A level was measured by a commercial solid-phase ELISA kit. The estimate of IR was calculated by homeostasis model assessment (HOMA-IR). CIMT was measured by B-mode ultrasound. The association between the serum fetuin-A levels and the metabolic parameters was also analyzed. The serum fetuin-A levels were increased significantly in the nT2DM group compared to the NGT group (368.5±15.6 mg/ml vs. 152.7±7.1 mg/ml, P<0.01). Fetuin-A was positively correlated with HOMA-IR, CIMT, glycated hemoglobin, triglyceride, low-density lipoprotein cholesterol, body mass index, systolic blood pressure, fasting blood glucose and 2 h post-glucose load blood glucose (P<0.05 and P<0.01), but negatively correlated with fasting plasma insulin, 2 h plasma insulin after glucose overload, high-density lipoprotein cholesterol and HOMA-ß-cell insulin secretion index (P<0.05 and P<0.01). To the best of our knowledge, the study demonstrated for the first time that there is a significant association between the serum fetuin-A levels with IR and CIMT in nT2DM. These results indicate that serum fetuin-A levels can be used as independent markers in the diagnosis of macroangiopathies in nT2DM.

Screening for voltage-gated sodium channel interacting peptides.

The voltage-gated sodium channel (VGSC) interacting peptide is of special interest for both basic research and pharmaceutical purposes. In this study, we established a yeast-two-hybrid based strategy to detect the interaction(s) between neurotoxic peptide and the extracellular region of VGSC. Using a previously reported neurotoxin JZTX-III as a model molecule, we demonstrated that the interactions between JZTX-III and the extracellular regions of its target hNav1.5 are detectable and the detected interactions are directly related to its activity. We further applied this strategy to the screening of VGSC interacting peptides. Using the extracellular region of hNav1.5 as the bait, we identified a novel sodium channel inhibitor SSCM-1 from a random peptide library. This peptide selectively inhibits hNav1.5 currents in the whole-cell patch clamp assays. This strategy might be used for the large scale screening for target-specific interacting peptides of VGSCs or other ion channels.

[Quantified diagnositic standard for large intestinal cancer of spleen qi deficiency syndrome.].

OBJECTIVE: To set a quantified diagnostic standard for large intestinal cancer of spleen qi deficiency syndrome. METHODS: The spleen qi deficiency syndrome was identified by experts on the basis of clinical epidemiological investigation of 311 patients suffering from large intestinal cancer. Corresponding points were assigned to the correlative factors (traditional Chinese medicine symptoms) on the basis of symptom differences between spleen qi deficiency syndrome and non-spleen-qi-deficiency syndrome. The best threshold was determined by receiver operating characteristic curve (ROC) according to syndrome differentiation from expert team, and the quantified diagnostic standard was established. The syndrome identification from the expert team which was regarded as golden standard was tested retrospectively. RESULTS: All the traditional Chinese medicine symptoms possibly related to spleen qi deficiency syndrome were analyzed based on the opinions of experts, and 28 symptoms were confirmed as candidate correlative factors. The occurrence of 11 symptoms between spleen qi deficiency syndrome and non-spleen-qi-deficiency syndrome showed statistical differences by means of crosstabs analysis (P<0.05). The 11 symptoms were filtered by logistic regression analysis, and tiredness, fatigue, loose stool, and poor appetite were finally determined as the symptoms relative to large intestinal cancer. These four symptoms were analyzed with conditional probability conversion and endowed with 16, 11, 4 and 8 points respectively. The diagnostic standard of spleen qi deficiency syndrome of large intestinal cancer was over 13 points. The sensitivity, specificity and accuracy of retrospective examination were all above 80%, and its positive likelihood ratio was 9.89. CONCLUSION: The quantified diagnostic standard for spleen qi deficiency syndrome of large intestinal cancer is in accordance with clinical characteristics of large intestine cancer and the characteristics of TCM syndrome diagnosis.

[Research progress in telemonitoring systems and automatic analysis methods of ambulatory ECG signals].

This paper reviews the research progress in telemonitoring systems and automatic analysis of ECG signals. The key problems, urgently to be solved, about the automatic analysis in ECG telemonitoring systems are discussed.