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bioRxiv2018

Biolistic transformation of Haematococcus pluvialis with constructs based on the flanking sequences of its endogenous alpha tubulin gene

The complete sequence information of the alpha tubulin (tub) genes was obtained from both Haematococcus pluvialis NIES144 and SCCAP K0084., Putative transcriptional elements and polyadenylation signals were identified respectively in their 5 and 3 flanking regions. Three selection cassettes of tub/aadA, tub/hyr and tub/ble with 3 different antibiotic-resistant genes fused between the 5 and 3 flanking sequences of the tub gene were constructed and utilized for biolistic transformation of H.pluvialis. Antibiotic resistant transformants were obtained in the bombardments with the tub/aadA cassette in 2 strains. It was found that, the foreign tub/aadA DNA could be completely transferred and inherited in their genome through non-homologous recombination. Moreover, transcripts of the insert and spectinomycin resistance were identified. Transformation efficiencies up to 3x10-5 per g DNA could be obtained in H.pluvialis NIES144 or SCCAP K0084 through utilization of a culture with a high percentage of flagellate cells and by optimizing bombarding protocol. The presented selection marker and optimized transforming procedures in this report should strengthen the platform technology for genetical manipulation and modification of H.pluvialis.

molecular biology↗

bioRxiv2018

IQGAP3 Overexpression Correlates with Poor Prognosis and Radiation Therapy Resistance in Breast Cancer

Background: IQ motif-containing GTPase activating protein 3 (IQGAP3), the latest found protein of IQGAP family, may act as a crucial factor in the process of cancer development and progression; however, its clinical value in breast cancer remains unestablished so far. Our team explored the correlation between IQGAP3 expression profile and the clinicopathological features in breast cancer. Methods: IQGAP3 levels in breast cancer cell lines and tumor tissues were detected by real-time PCR and western blotting and compared to the normal control groups. Protein expression of IQGAP3 was evaluated immunohistochemically in specimens (archived paraffin embedded) of 257 breast cancer patients. We also analyze the association between IQGAP3 expression and the clinical characters and prognosis. The relationship between IQGAP3 expression and sensitivity to radiation therapy was determined by subgroup analysis. Results: There was significant upregulation of IQGAP3 in breast cancer cell lines and human tumor tissues at both the mRNA and protein level compared to the normal ones. In addition, 110/257 (42.8%) of archived paraffin embedded breast cancer specimens had high protein expression of IQGAP3. High expression of IQGAP3 was significantly related to clinical stage (P=0.001), T category (P=0.002), N category (P=0.001), locoregional recurrence(P=0.002), distant metastasis (P=0.001), and vital status (P=0.001). Univariate and multivariate statistical analysis showed that IQGAP3 was an independent prognostic factor of the whole cohort breast cancer patients (P=0.003, P=0.001). Subgroup analysis revealed IQGAP3 expression correlates with radiation therapy resistance and was also an independent predictor for radiation therapy outcome. Conclusions: Our findings suggest that high IQGAP3 expression predicts poor prognosis and radiation therapy resistance in breast cancer. In addition, IQGAP3 may be a reliable novel biomarker to provide personalized prognostication and identify patients who can profit from more aggressive RT regimen for improving the survival of breast cancer patients.

cancer biology↗