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BackgroundDespite recent evidence demonstrating a potent protective effect of adoptively transferred regulatory T cells (Tregs) in ischemic stroke, the mechanism for Treg mobilization and activation in the ischemic brain is, remarkably, unknown. This study determines the role of C‐C chemokine receptor type 5 (CCR5) in mediating the docking and activation of transferred Tregs in their protection of early blood‐brain barrier disruption after stroke. Methods and ResultsAdoptive transfer of CCR5−/− Tregs failed to reduce brain infarct or neurological deficits, indicating an indispensable role of CCR5 in Treg‐afforded protection against cerebral ischemia. Two‐photon live imaging demonstrated that CCR5 was critical for Treg docking at the injured vessel wall, where they interact with blood‐borne neutrophils/macrophages after cerebral ischemic injury. CCR5 deficiency on donor Tregs deprived of their early protection against blood‐brain barrier damage. Using flow cytometry, real‐time polymerase chain reaction, and immunostaining, we confirmed that the expression of CCL5, a CCR5 ligand, was significantly elevated on the injured endothelium after cerebral ischemia, accompanied by CCR5 upregulation on circulating Tregs. In a Treg‐endothelial cell coculture, CCR5 expression was induced on Tregs on their exposure to ischemia‐injured endothelial cells. Furthermore, CCR5 induction on Tregs enhanced expression of the inhibitory molecule programmed death ligand 1, which in turn inhibited neutrophil‐derived matrix metallopeptidase 9. ConclusionsThese results suggest that CCR5 is a critical molecule for Treg‐mediated blood‐brain barrier protection and a potential target to optimize Treg therapy for stroke.

Chilling stress is considered the major abiotic stress affecting the growth, development, and yield of rice. To understand the transcriptomic responses and methylation regulation of rice in response to chilling stress, we analyzed a cold-tolerant variety of rice (Oryza sativa L. cv. P427). The physiological properties, transcriptome, and methylation of cold-tolerant P427 seedlings under low-temperature stress (2−3 °C) were investigated. We found that P427 exhibited enhanced tolerance to low temperature, likely via increasing antioxidant enzyme activity and promoting the accumulation of abscisic acid (ABA). The Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq) data showed that the number of methylation-altered genes was highest in P427 (5496) and slightly lower in Nipponbare (Nip) and 9311 (4528 and 3341, respectively), and only 2.7% (292) of methylation genes were detected as common differentially methylated genes (DMGs) related to cold tolerance in the three varieties. Transcriptome analyses revealed that 1654 genes had specifically altered expression in P427 under cold stress. These genes mainly belonged to transcription factor families, such as Myeloblastosis (MYB), APETALA2/ethylene-responsive element binding proteins (AP2-EREBP), NAM-ATAF-CUC (NAC) and WRKY. Fifty-one genes showed simultaneous methylation and expression level changes. Quantitative RT-PCR (qRT-PCR) results showed that genes involved in the ICE (inducer of CBF expression)-CBF (C-repeat binding factor)—COR (cold-regulated) pathway were highly expressed under cold stress, including the WRKY genes. The homologous gene Os03g0610900 of the open stomatal 1 (OST1) in rice was obtained by evolutionary tree analysis. Methylation in Os03g0610900 gene promoter region decreased, and the expression level of Os03g0610900 increased, suggesting that cold stress may lead to demethylation and increased gene expression of Os03g0610900. The ICE-CBF-COR pathway plays a vital role in the cold tolerance of the rice cultivar P427. Overall, this study demonstrates the differences in methylation and gene expression levels of P427 in response to low-temperature stress, providing a foundation for further investigations of the relationship between environmental stress, DNA methylation, and gene expression in rice.

The structural integrity of the cervix in pregnancy is necessary for carrying a pregnancy until term, and the organization of human cervical tissue collagen likely plays an important role in the tissue's structural function. Collagen fibers in the cervical extracellular matrix exhibit preferential directionality, and this collagen network ultrastructure is hypothesized to reorient and remodel during cervical softening and dilation at time of parturition. Within the cervix, the upper half is substantially loaded during pregnancy and is where the premature funneling starts to happen. To characterize the cervical collagen ultrastructure for the upper half of the human cervix, we imaged whole axial tissue slices from non-pregnant and pregnant women undergoing hysterectomy or cesarean hysterectomy respectively using optical coherence tomography (OCT) and implemented a pixel-wise fiber orientation tracking method to measure the distribution of fiber orientation. The collagen fiber orientation maps show that there are two radial zones and the preferential fiber direction is circumferential in a dominant outer radial zone. The OCT data also reveal that there are two anatomic regions with distinct fiber orientation and dispersion properties. These regions are labeled: Region 1-the posterior and anterior quadrants in the outer radial zone and Region 2-the left and right quadrants in the outer radial zone and all quadrants in the inner radial zone. When comparing samples from nulliparous vs multiparous women, no differences in these fiber properties were noted. Pregnant tissue samples exhibit an overall higher fiber dispersion and more heterogeneous fiber properties within the sample than non-pregnant tissue. Collectively, these OCT data suggest that collagen fiber dispersion and directionality may play a role in cervical remodeling during pregnancy, where distinct remodeling properties exist according to anatomical quadrant.

Hepatitis B Virus (HBV) mutations play a role in the development of hepatocellular carcinoma (HCC). However, the association between HBV polymerase gene mutations and HCC has not been reported. In this study, we conducted a multi-stage study to identify HCC-related mutations in the reverse transcriptase (RT) domain of the HBV polymerase gene.A total of 231 HCCs and 237 non-HCC controls from Qidong, China, were included in this study. The entire sequence of HBV RT was first compared between 29 HCC and 35 non-HCC cases, and candidate mutations were then evaluated in two independent validation sets.There were 15 candidate mutations identified from the discovery set, with A799G and T1055A being consistently associated with HCC across all studies. A pooled analysis of samples revealed that A799G, A987G, and T1055A were independent risk factors for HCC, with adjusted odds ratios of 5.53 [95% confidence interval (CI), 1.69-18.10], 4.20 (95%CI, 1.15-15.35), and 3.78 (95%CI, 1.45-9.86), respectively. A longitudinal study showed that these mutations were detectable 4-5 years prior to HCC diagnosis.Our study provides evidence the first that HBV RT contains naturally occurring mutations that can be used as predictive markers for HCC.

We performed an updated meta-analysis, using a comprehensive strategy of a logistic regression and a model-free approach, to evaluate more precisely the role of the rs4444235 variant near the Bone morphogenetic protein-4 (BMP4) gene in susceptibility to colorectal cancer (CRC).A total of 19 studies with 28770 cases and 28234 controls were included. Metagen system with logistic regression was applied to choose the most plausible genetic model for rs4444235. Generalized odds ratio (ORG) metric was used to provide a global test of relationship between rs4444235 and CRC risk.Metagen analysis suggested the rs4444235 fitted best to an additive model. In assessment of the additive model, heterogeneity was observed (P = 0.059, I2 = 36.1), and pooled per-allele OR was 1.08 (95% CI = 1.05-1.11). Based on the model-free approach, pooled ORG was 1.09 (95% CI = 1.05-1.14) under a random-effect model. Stratified analyses suggested heterogeneity could be in part explained by population ethnicity, study design, sources of controls, and sample size. Sensitivity analysis further supported the robust stability of the current results, by showing similar pooled estimates before and after sequential removal of each study.This meta-analysis provides a robust estimate of the positive association between the rs4444235 and CRC risk and further emphasizes the importance of the rs4444235 in CRC risk prediction.

Purpose: Deregulation or mutation of the EZH2 gene causes various tumors, including clear cell renal cell carcinoma (ccRCC). Although several splice variants of EZH2 have been identified, little is known about how EZH2 splicing is regulated or the contribution of alternative splicing to its protumorigenic functions.Experimental Design: We conducted RT-PCR, Western blot analysis, and IHC techniques to examine EZH2 and its alternative splicing transcript expression in renal cancer tissue and renal cancer cell lines. Proliferation, migration, clonogenicity, and tumorigenicity of renal cancer cells either exhibiting knockdown of EZH2 or its splicing factor SF3B3 were assessed by CCK8, Transwell assay, and murine xenograft experiments.Results: We found that the inclusion of alternative EZH2 exon 14 was significantly increased in ccRCC samples and renal cancer cell lines. In ccRCC lines, enforced expression of EZH2Δ14 inhibited, and EZH2 promoted, cell growth, migration, proliferation, and tumorigenicity in a xenograft model. Mechanistic studies demonstrated that EZH2Δ14 isoform functions as a dominant-negative inhibitor of full-length EZH2. Coexpression of EZH2Δ14 variant with full-length EZH2 not only abrogated DAB2IP and HOXA9 suppression but also inhibited EZH2-driven tumorigenesis. Strikingly, the splicing factor SF3B3 stimulates inclusion of exon14 and has pro-proliferative activity. Importantly, the upregulation of SF3B3 expression observed in clinical ccRCC samples parallels the increased inclusion of EZH2 exon14, and the SF3B3 level is associated with higher tumor stage and poor overall survival.Conclusions: These results suggest SF3B3 as a key regulator of EZH2 pre-mRNA splicing and SF3B3 may represent a novel prognostic factor and potential therapeutic target in ccRCC. Clin Cancer Res; 23(13); 3428-41. ©2016 AACR.