miR-186 and 326 Predict the Prognosis of Pancreatic Ductal Adenocarcinoma and Affect the Proliferation and Migration of Cancer Cells

MicroRNAs can function as key tumor suppressors or oncogenes and act as biomarkers for cancer diagnosis or prognosis. Although high-throughput assays have revealed many miRNA biomarkers for pancreatic ductal adenocarcinoma (PDAC), only a few have been validated in independent populations or investigated for functional significance in PDAC pathogenesis. In this study, we correlated the expression of 36 potentially prognostic miRNAs within PDAC tissue with clinico-pathological features and survival in 151 Chinese patients. We then analyzed the functional roles and target genes of two miRNAs in PDAC development. We found that high expression of miR-186 and miR-326 predict poor and improved survival, respectively. miR-186 was over-expressed in PDAC patients compared with controls, especially in patients with large tumors (>2 cm), lymph node metastasis, or short-term survival (< 24 months). In contrast, miR-326 was down-regulated in patients compared with controls and displayed relatively increased expression in the patients with long-term survival or without venous invasion. Functional experiments revealed that PDAC cell proliferation and migration was decreased following inhibition and enhanced following over-expression of miR-186. In contrast, it was enhanced following inhibition and decreased after over-expression of miR-326. A luciferase assay indicated that miR-186 can bind directly to the 3′-UTR of NR5A2 to repress gene expression. These findings suggest that miR-186 over-expression contributes to the invasive potential of PDAC, likely via suppression of NR5A2, thereby leading to a poor prognosis; high miR-326 expression prolongs survival likely via the decreasing invasive potential of PDAC cells. These two miRNAs can be used as markers for clinical diagnosis and prognosis, and they represent therapeutic targets for PDAC.     

Carpal Tunnel Syndrome Assessment with Ultrasonography: Value of Inlet-to-Outlet Median Nerve Area Ratio in Patients versus Healthy Volunteers

Objective

To evaluate the diagnostic value of the Inlet-to-outlet median nerve area ratio (IOR) in patients with clinically and electrophysiologically confirmed carpal tunnel syndrome (CTS).

Methods

Forty-six wrists in 46 consecutive patients with clinical and electrodiagnostic evidence of CTS and forty-four wrists in 44 healthy volunteers were examined with ultrasonography. The cross-sectional area (CSA) of the median nerve was measured at the carpal tunnel inlet (the level of scaphoid-pisiform) and outlet (the level of the hook of the hamate), and the IOR was calculated for each wrist. Ultrasonography and electrodiagnostic tests were performed under blinded conditions. Electrodiagnostic testing combined with clinical symptoms were considered to be the gold standard test. Receiver operating characteristic (ROC) curves were used to evaluate the diagnostic value between the inlet CSA and IOR.

Results

The study population included 16 men and 30 women (mean age, 45.3 years; range, 18–83 years). The control population included 18 men and 26 women (mean age, 50.4 years; range, 18–79 years). The mean inlet CSA was 8.7 mm² in healthy controls and 14.6mm² in CTS group (P<0.001). The mean IOR in healthy volunteers (1.0) was smaller than that in patients (1.6, P<0.001). Receiver operating characteristic analysis revealed a diagnostic advantage to using the IOR rather than the inlet CSA (P<0.01). An IOR cutoff value of ≥ 1.3 would yield 93% specificity and 91% sensitivity in the diagnosis of CTS.

Conclusion

The IOR of median nerve area promises to be an effective means in the diagnosis of CTS. A large-scale, randomized controlled trial is required to determine how and when this parameter will be used.     

Development and manufacturability assessment of chemically‐defined medium for the production of protein therapeutics in CHO cells

Advantages of using internally developed chemically‐defined (CD) media for cell culture‐based therapeutic protein production over commercial media include better raw material control and medium vendor options, and most importantly, flexibility for process development and subsequent optimization needed for therapeutic protein production. Through several rounds of design of experiment (DOE) screening, and medium component supplementation and optimization studies, we successfully developed a CD basal medium (CDM) for CHO cell culture. The internally prepared liquid CDM demonstrated comparable cell culture performance to that from a commercially available control medium. However, when the same CDM formulation was transferred to two major commercial medium suppliers for manufacturing, cell culture performance utilizing these newly prepared media was significantly reduced compared with the in‐house prepared counterpart. An investigation was launched to assess whether key medium components were sensitive to large‐scale preparation of the final bulk media by the vendors. Further work necessitated the reformulation of the original CDM formulation into a core medium that was suitable for large‐scale media manufacturing. The modified preparation of the core medium with two separate supplements to generate the final CDM was able to recover the expected cell culture performance and monoclonal antibody (mAb) productivity. Confirmation of cell culture robustness in cell growth and production was corroborated in two additional mAb‐expressing cell lines. This work demonstrates that a robust CD medium is not only one that performs during the development stage, but also one that must be reproducible by commercial media vendors. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1163–1171, 2015     

重组阳离子抗肿瘤肽AIK的原核表达、纯化及活性测定

利用Gateway克隆技术构建重组抗瘤肽AIK的原核表达体系,建立表达及纯化重组AIK的最优条件,为深入研究和利用AIK奠定基础。首先,设计含AttB重组位点的引物,通过重叠PCR技术扩增出Att B-TEV-FLAG-AIK序列,利用BP重组反应将目的序列TEV-FLAG-AIK克隆到供体载体pDONR223中,构建入门载体,再通过LR重组反应,将目的序列转移到目的载体pDEST15中,构建GST-AIK融合蛋白原核表达质粒。随后,在BL21(DE3)工程菌中优化诱导融合蛋白表达的条件。以谷胱甘肽磁珠纯化GST-AIK融合蛋白,再以rTEV酶切除GST,获得FLAG-AIK重组蛋白。最后以MTS法检测FLAG-AIK对白血病细胞HL-60的细胞毒性。菌液PCR验证和测序分析表明成功构建了重组抗瘤肽AIK的入门质粒和原核表达质粒。在BL21(DE3)工程菌中实现了GST-AIK融合蛋白的高效可溶性表达。并测得在37℃下以0.1 mmol/L IPTG诱导工程菌(OD600=1.0)4 h,重组蛋白表达量占菌体总蛋白的30%以上。经GST亲和层析、rTEV酶切除GST标签及二次GST亲和层析获得纯度高于95%的FLAG-AIK蛋白。MTS法测得所制备的FLAG-AIK蛋白抑瘤活性与化学合成的AIK相当。总之,本课题应用Gateway克隆系统成功构建了抗瘤肽AIK的原核表达质粒,实现了GST-AIK融合蛋白的高效可溶性表达,经亲和层析获得了有生物活性的重组AIK多肽,为后续深入研究和大规模制备奠定了基础。     

利用絮凝进行微藻采收的研究进展

微藻可生产不饱和脂肪酸及色素等多种高附加值产品,同时也可用来生产可再生清洁能源如生物柴油等,具有良好的应用前景。但是,目前微藻细胞的采收成本高居不下,已成为限制微藻生物技术大规模应用的重要因素之一。与其他方法相比,絮凝采收成本低、操作简便,是很有应用前景的采收方法。本文综述了国内外利用化学絮凝、物理絮凝及生物絮凝等方法对不同微藻细胞进行采收的研究,重点对生物絮凝方法进行了总结。利用微生物絮凝剂及微藻细胞的自絮凝进行微藻生物量的回收,是微藻采收技术中环境友好、低成本和行之有效的新方法之一。     

Identification of new sources of aluminum resistance in wheat

Aluminum (Al) toxicity is a major constraint for wheat production in acidic soils. An Al resistance gene on chromosome 4DL that traces to Brazilian wheat has been extensively studied, and can provide partial protection from Al damage. To identify potentially new sources of Al resistance, 590 wheat accessions, including elite wheat breeding lines from the United States and other American and European countries, landraces and commercial cultivars from East Asia, and synthetic wheat lines from CIMMYT, Mexico, were screened for Al resistance by measuring relative root elongation in culture with a nutrient solution containing Al, and by staining Al-stressed root tips with hematoxylin. Eighty-eight wheat accessions demonstrated at least moderate resistance to Al toxicity. Those selected lines were subjected to analysis of microsatellite markers linked to an Al resistance gene on 4DL and a gene marker for the Al-activated malate transporter (ALMT1) locus. Many of the selected Al-resistant accessions from East Asia did not have the Al-resistant marker alleles of ALMT1, although they showed Al resistance similar to the US Al-resistant cultivar, Atlas 66. Most of the cultivars derived from Jagger and Atlas 66 have the Al-resistant marker alleles of ALMT1. Cluster analysis separated the selected Al-resistant germplasm into two major clusters, labeled as Asian and American–European clusters. Potentially new germplasm of Al resistance different from those derived from Brazil were identified. Further investigation of Al resistance in those new germplasms may reveal alternative Al-resistance mechanisms in wheat. Electronic supplementary material The online version of this article (doi:contains supplementary material, which is available to authorized users. Responsible Editor: Thomas B. Kinraide.     

Disrupted-In-Schizophrenia 1 regulates integration of newly generated neurons in the adult brain

Adult neurogenesis occurs throughout life in discrete regions of the adult mammalian brain. Little is known about the mechanism governing the sequential developmental process that leads to integration of new neurons from adult neural stem cells into the existing circuitry. Here, we investigated roles of Disrupted-In-Schizophrenia 1 (DISC1), a schizophrenia susceptibility gene, in adult hippocampal neurogenesis. Unexpectedly, downregulation of DISC1 leads to accelerated neuronal integration, resulting in aberrant morphological development and mispositioning of new dentate granule cells in a cell-autonomous fashion. Functionally, newborn neurons with DISC1 knockdown exhibit enhanced excitability and accelerated dendritic development and synapse formation. Furthermore, DISC1 cooperates with its binding partner NDEL1 in regulating adult neurogenesis. Taken together, our study identifies DISC1 as a key regulator that orchestrates the tempo of functional neuronal integration in the adult brain and demonstrates essential roles of a susceptibility gene for major mental illness in neuronal development, including adult neurogenesis.     

RNAi knockdown of Oryza sativa root meander curling gene led to altered root development and coiling which were mediated by jasmonic acid signalling in rice

Jasmonic acid (JA) is a well-known defence hormone, but its biological function and mechanism in rice root development are less understood. Here, we describe a JA-induced putative receptor-like protein (OsRLK, AAL87185) functioning in root development in rice. RNA in situ hybridization revealed that the gene was expressed largely in roots, and a fusion protein showed its localization on the plasma membrane. The primary roots in RNAi transgenic rice plants meandered and curled more easily than wild-type (WT) roots under JA treatment. Thus, this gene was renamed Oryza sativa root meander curling (OsRMC). The transgenic primary roots were shorter, the number of adventitious roots increased and the number of lateral roots decreased as compared to the WT. As well, the second sheath was reduced in length. Growth of both primary roots and second sheaths was sensitive to JA treatment. No significant change of JA level appeared in the roots between the transgenic rice line and WT. Expression of RSOsPR10, involved in the JA signalling pathway, was induced in transgenic rice. Western blotting revealed OsRMC induced by JA. Our results suggest that OsRMC of the DUF26 subfamily involved in JA signal transduction mediates root development and negatively regulates root curling in rice.     

Enhancement of biodesulfurization in two-liquid systems by heterogeneous expression of vitreoscilla hemoglobin

The vgb gene, encoding Vitreoscilla hemoglobin (VHb), was introduced into a specific desulfurization bacterium, Rhodococcus erythropolis LSSE8-1. The VHb-specific spectrum was observed for the recombinant. Compared to the wild type, the strain bearing vgb showed a higher biomass yield and desulfurizing activity.