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

T-Cell Infiltration and Adaptive Treg Resistance in Response to Androgen Deprivation With or Without Vaccination in Localized Prostate Cancer

Purpose: Previous studies suggest that androgen deprivation therapy (ADT) promotes antitumor immunity in prostate cancer. Whether a vaccine-based approach can augment this effect remains unknown. Experimental Design: Therefore, we conducted a neoadjuvant, randomized study to quantify the immunologic effects of a granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting allogeneic cellular vaccine in combination with low-dose cyclophosphamide (Cy/GVAX) followed by degarelix versus degarelix alone in patients with high-risk localized prostate adenocarcinoma who were planned for radical prostatectomy. Results: Both Cy/GVAX plus degarelix and degarelix alone led to significant increases in intratumoral CD8+ T cell infiltration and PD-L1 expression as compared to a cohort of untreated, matched controls. However, the CD8+ T cell infiltrate was accompanied by a proportional increase in regulatory T cells (Treg), suggesting that adaptive Treg resistance may dampen the immunogenicity of ADT. Although Cy/GVAX followed by degarelix was associated with a modest improvement in time-to-PSA progression and time-to-next treatment as well as an increase in PD-L1, there was no difference in the CD8 T-cell infiltrate as compared to degarelix alone. Gene expression profiling demonstrated that CHIT1, a macrophage marker, was differentially upregulated with Cy/GVAX plus degarelix compared to degarelix alone. Conclusions: Our results highlight that ADT with or without Cy/GVAX induces a complex immune response within the prostate tumor microenvironment. These data have important implications for combining ADT with immunotherapy. In particular, our finding that ADT increases both CD8+ T cells and Tregs, supports the development of regimens combining ADT with Treg-depleting agents in the treatment of prostate cancer

Promoter Hypomethylation of Maspin Inhibits Migration and Invasion of Extravillous Trophoblast Cells during Placentation.

Extravillous trophoblast (EVT) cells invade the endometrium and the maternal spiral arterioles during the first trimester. Mammary Serine Protease Inhibitor (Maspin, SERPINB5) plays a putative role in regulating the invasive activity of cytotrophoblasts. The maspin gene is silenced in various cancers by an epigenetic mechanism that involves aberrant cytosine methylation. We investigated the effect of the methylation status of the maspin promoter on the maspin expression and the aggressiveness of EVT cells.Western blotting was used to detect the maspin protein expression in EVT cells upon hypoxia. The proliferative ability, the apoptosis rate and the migration and invasiveness were measured with Cell Counting Kit-8 assay, Flow Cytometry technology and Transwell methods. Subsequently, we treated cells with recombinant maspin protein. The methylation degree of maspin promoter region upon hypoxia/ decitabine was detected by bisulfite sequencing PCR and methylation-specific PCR. Finally, we explored the effects of decitabine on maspin protein expression and the aggressiveness of EVT cells.Hypoxia effectively increased maspin protein expression in EVT cells and significantly inhibited their aggressiveness. The addition of recombinant maspin protein inhibited this aggressiveness. Decitabine reduced the methylation in the maspin promoter region and effectively increased the maspin protein expression, which significantly weakened the migration and invasiveness of EVT cells.The methylation status of the maspin promoter is an important factor that affects the migration and invasion of EVT cells during early pregnancy. A decrease in the methylation status can inhibit the migration and invasion of EVT cells to affect placentation and can result in the ischemia and hypoxia of placenta

Effect of hypoxia on protein expression of maspin, apoptotic and invasive ability in TEV-1 A: The maspin protein in the normoxic and hypoxic groups expressed as a relative measure compared to GAPDH and normalized to a control sample run on each gel at two different culture time points (24h, 48h).

<p>The protein expression of maspin in the HOX group was significantly higher compared to the NOX group at both 24h and 48h, the protein expression of maspin at 48h was significantly higher compared to 24h; (*P < 0.05). B: Representative Western blot analysis of maspin protein expression in the NOX and HOX groups; C: Apoptosis rate of cells was measured by flow cytometry. The apoptosis rate in the HOX group was significantly higher compared to the NOX group at 12h, 24h and 48h. (*P < 0.05) D: The invasive ability of cells were tested by transwell method. The invaded cells in the HOX group was significantly fewer compared to the NOX group at 48h. (*P < 0.05) E: Photo of invaded cells in the NOX and HOX group (200X).</p

Effect of recombinant maspin protein on proliferative, apoptotic, migrative and invasive ability in TEV-1 A: The number of cells in NOX, HOX, r.M and HOX+r.M groups was measured by ELISA.

<p>Recombinant maspin protein significantly weakened the proliferative ability of TEV-1 cells in NOX group at 24h, but overall it had no significant effect on the proliferative ability of TEV-1 cells in either NOX or HOX group. B: The apoptosis rate of cells in NOX, HOX, r.M and HOX+r.M groups was detected by FCM. Recombinant maspin protein had no significant effect on the apoptosis rate of TEV-1 cells in either NOX or HOX group. C: The migrated/invaded cells were counted to estimate the effect of recombinant maspin protein on normoxic EVT cells. Recombinant maspin significantly weakened the migrative ability and the invasive ability of TEV-1 cells in the normoxic EVT cells. (*P < 0.05) D: The migrated/invaded cells were counted to estimate the effect of recombinant maspin protein on hypoxic EVT cells. Recombinant maspin significantly weakened the migrative ability and the invasive ability of TEV-1 cells in the hypoxic EVT cells. (*P < 0.05).</p

Effect of DI on maspin expression, aggressive ability of TEV-1 cells.

<p>A: Representative Western blot analysis of maspin protein expression. DI increased the protein expression of maspin in TEV-1 cells in the NOX group at 24h and 48h. DI increased the protein expression of maspin in TEV-1 cells in the HOX group at 24h, but had no significant effect on the protein expression of maspin in TEV-1 cells in the HOX group at 48h. (*P < 0.05). B: We investigated the effect of decitabine on the apoptosis rate of the normoxic/hypoxic EVT cells. DI had no significant effect on the apoptosis rate of TEV-1 cells in the NOX and HOX group. C: Representative Western blot analysis of maspin protein expression in the NOX and HOX groups upon DI at two different culture time points (24h, 48h). (+: with DI;-: without DI). D: The migrated/invaded cells were counted to estimate the effect of decitabine on normoxic EVT cells. Decitabine significantly weakened the migrative ability and the invasive ability of TEV-1 cells in the normoxic EVT cells. (The control was the NOX group. *P < 0.05) E: The migrated/invaded cells were counted to estimate the effect of decitabine on hypoxic EVT cells. Decitabine significantly weakened the migrative ability and the invasive ability of TEV-1 cells in the hypoxic EVT cells. (The control was the NOX group. *P < 0.05) F: Photo of migrated/invaded cells in the NOX, DI, HOX and HOX+DI group (200X).</p

CEPC Conceptual Design Report: Volume 2 - Physics & Detector

The Circular Electron Positron Collider (CEPC) is a large international scientific facility proposed by the Chinese particle physics community to explore the Higgs boson and provide critical tests of the underlying fundamental physics principles of the Standard Model that might reveal new physics. The CEPC, to be hosted in China in a circular underground tunnel of approximately 100 km in circumference, is designed to operate as a Higgs factory producing electron-positron collisions with a center-of-mass energy of 240 GeV. The collider will also operate at around 91.2 GeV, as a Z factory, and at the WW production threshold (around 160 GeV). The CEPC will produce close to one trillion Z bosons, 100 million W bosons and over one million Higgs bosons. The vast amount of bottom quarks, charm quarks and tau-leptons produced in the decays of the Z bosons also makes the CEPC an effective B-factory and tau-charm factory. The CEPC will have two interaction points where two large detectors will be located. This document is the second volume of the CEPC Conceptual Design Report (CDR). It presents the physics case for the CEPC, describes conceptual designs of possible detectors and their technological options, highlights the expected detector and physics performance, and discusses future plans for detector R&D and physics investigations. The final CEPC detectors will be proposed and built by international collaborations but they are likely to be composed of the detector technologies included in the conceptual designs described in this document. A separate volume, Volume I, recently released, describes the design of the CEPC accelerator complex, its associated civil engineering, and strategic alternative scenarios