The periplasmic High Temperature Requirement protein A (HtrA) plays important roles in bacterial protein folding and stress responses. However, the role of chlamydial HtrA (cHtrA) in chlamydial pathogenesis is not clear.

Our previous study demonstrated that pretreatment with electroacupuncture (EA) elicited protective effects against transient cerebral ischemia through cannabinoid receptor type 1 receptor (CB1R). In the present study, we investigated whether or not the extracellular signal regulated-kinase 1/2 (ERK1/2) pathway was involved in the ischemic tolerance induced by EA pretreatment through CB1R. At 24h after the end of the last EA pretreatment, focal cerebral ischemia was induced by middle cerebral artery occlusion for 120min in rats. The neurological scores and infarct volumes were evaluated at 24h after reperfusion. The expression of p-ERK1/2 in the brains was also investigated in the presence or absence of CB1R antagonist AM251. EA pretreatment reduced infarct volumes and improved neurological outcome at 24h after reperfusion, and the beneficial effects were abolished by U0126. The blockade of CB1R by AM251 reversed the up-regulation of p-ERK1/2 expression induced by EA pretreatment. Our findings suggest that the ERK1/2 pathway might be involved in EA pretreatment-induced cerebral ischemic tolerance via cannabinoid CB1 receptor in rats.

This study aimed to verify the feasibility and efficacy of ultrasound-targeted microbubble destruction (UTMD)-mediated angiopoietin-1 (Ang-1) gene delivery into the infarcted myocardium. Microbubbles carrying anti-intercellular adhesion molecule-1 (ICAM-1) antibody were prepared and identified. The microbubbles carrying anti-ICAM-1 antibody selectively adhered to the interleukin (IL)-1β-stimulated ECV304 cells and to the ischemic vascular endothelium, and the infarct area was examined to evaluate the targeting ability of ICAM-1 microbubbles in vitro and in vivo. The intravenous administration of the Ang-1 gene was carried out by UTMD in rabbits with acute myocardial infarction (AMI). The rabbits were divided into the control (no treatment), non-targeted microbubble destruction (non-TMB) and the ICAM-1 TMB (TMB) group. Gene delivery by direct intramyocardial injection (IMI) served as a reference. Two weeks later, regional myocardial perfusion and cardiac function were evaluated by echocardiography, and Ang-1 gene-mediated angiogenesis was assessed histologically and biochemically. The results revealed that the ICAM-1-targeted microbubbles selectively adhered to the IL-1β-stimulated ECV304 cells in vitro and to the ischemic vascular endothelium in the infarct area of the rabbits with AMI. Two weeks after the delivery of the Ang-1 gene, compared with the non-TMB group, left ventricular function and myocardial perfusion at the infarct area had improved in the TMB and IMI group (p<0.01). Ang-1 gene expression was detectable in the non-TMB, TMB and IMI group, while its expression was higher in the latter 2 groups (all p<0.01). The microvascular density (MVD) of the infarct area in the non-TMB, TMB and IMI group was 65.6 ± 4.4, 96.7 ± 2.1 and 100.7 ± 3.6, respectively (p<0.01). The findings of our study indicate that UTMD-mediated gene delivery may be used to successfully deliver the Ang-1 gene to the infarcted myocardium, thus improving the efficacy of therapeutic angiogenesis. This may provide a novel strategy for future gene therapy.

Estrogen-related receptor alpha (ERRα) plays an important role in the development of hormone-dependent cancers, but its roles in lung cancer remain elusive. The present study was aimed to investigate the effects of ERRα on the proliferation and metastasis of lung cancer A549 cells. The mRNA and protein levels of ERRα were detected in lung cancer A549 and MCF-7 cells and bronchial epithelial BEAS-2B cells by qRT-PCR and Western blotting, respectively. ERRα plasmid transfection and XCT-790 (an inverse agonist of ERRα) were used to up-regulate or down-regulate ERRα expression in A549 cells, respectively. The viability of A549 cells was measured by cell counting kit-8 (CCK-8) and the motility of A549 cells by wound healing assay and Transwell migration/invasion assay. The epithelial markers E-cadherin (E-Cad) and zona occludin-1 (ZO-1), the mesenchymal markers fibronectin (FN) and vimentin (Vim) and the transcription factors (Snail, Zeb1 Twist and Slug) were further detected at mRNA and protein levels by qRT-PCR and Western blotting, respectively. The results showed that ERRα promoted the growth of lung cancer A549 cells in vitro. XCT-790 significantly inhibited the migration and invasion of A549 cells. Over-expression of ERRα promoted the epithelial-to-mesenchymal transition (EMT) of A549 cells, down-regulated the epithelial makers E-Cad and ZO-1, and up-regulated the mesenchymal makers FN and Vim. Silencing of Slug, but not other transcription factors, significantly abolished the ERRα-induced EMT of A549 cells. It was suggested that ERRα promoted the migration and invasion of A549 cells by inducing EMT, and Slug was involved in the process. Targeting ERRα might be an efficient approach for lung cancer treatment.

The aim of this paper was to explore the long-term effects and pain relief mechanism of acupuncture knife on third lumbar vertebrae (L3) transverse process syndrome. Forty SD rats were randomized into control, model, electroacupuncture (EA), and acupuncture knife (AK) group. Except control rats, other rats were subjected to an operation to emulate L3 transverse process syndrome. Fourteen days after the operation, EA and AK rats were given electroacupuncture and acupuncture knife treatments, respectively. Fifty-six days after the operation, enzyme-linked immunosorbent assay was used to measure substance P (SP), 5-hydroxytryptamine (5-HT), interleukin-1β (IL-1β), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), and transforming growth factor-β (TGF-β) in peripheral blood. The tail flick test was used to observe pain threshold. We found that rats with the simulation operation had significantly higher levels of SP, 5-HT, IL-1, IL-10, TNF-α, and TGF-β, while the AK rats had lower levels. In addition, the pain threshold of AK rats was similar to that of control rats. AK pretreatment could alleviate pain through modulating inflammatory response.

The cerebellum plays an essential role in motor learning. Recently, orexins, the newfound lateral hypothalamic neuropeptides, have been found to excite Purkinje cells in the cerebellar cortex and neurons in the deep cerebellar nuclei (DCN). However, little is known about their roles in cerebellum-dependent motor learning. Therefore, the present study was designed to investigate the functional significance of hypothalamic orexinergic system during trace eyeblink conditioning, a tractable behavioral model system of cerebellum-dependent motor learning. It was revealed that the orexin 1 receptors (OXR1) were specifically localized on the soma of Purkinje cells and large DCN neurons. Furthermore, interfering with the endogenous orexins' effects on the cerebellum via the selective OXR1 antagonist SB-334867 disrupted the timing rather than the acquisition of trace conditioned eyeblink responses. In addition to the behavioral effects, the SB-334867 prevented the increase in peak amplitude of cerebellar theta oscillations with learning. These results suggest that the endogenous orexins may modulate motor learning via the activation of cerebellar OXR1.

The spirochete periplasmic flagellum has many unique attributes. One unusual characteristic is the flagellar hook. This structure serves as a universal joint coupling rotation of the membrane-bound motor to the flagellar filament. The hook is comprised of about 120 FlgE monomers, and in most bacteria these structures readily dissociate to monomers (∼ 50 kDa) when treated with heat and detergent. However, in spirochetes the FlgE monomers form a large mass of over 250 kDa [referred to as a high molecular weight complex (HMWC)] that is stable to these and other denaturing conditions. In this communication, we examined specific aspects with respect to the formation and structure of this complex. We found that the Lyme disease spirochete Borrelia burgdorferi synthesized the HMWC throughout the in vitro growth cycle, and also in vivo when implanted in dialysis membrane chambers in rats. The HMWC was stable to formic acid, which supports the concept that the stability of the HMWC is dependent on covalent cross-linking of individual FlgE subunits. Mass spectrometry analysis of the HMWC from both wild type periplasmic flagella and polyhooks from a newly constructed ΔfliK mutant indicated that other proteins besides FlgE were not covalently joined to the complex, and that FlgE was the sole component of the complex. In addition, mass spectrometry analysis also indicated that the HMWC was composed of a polymer of the FlgE protein with both the N- and C-terminal regions remaining intact. These initial studies set the stage for a detailed characterization of the HMWC. Covalent cross-linking of FlgE with the accompanying formation of the HMWC we propose strengthens the hook structure for optimal spirochete motility.

Reverse-transcription polymerase chain reaction (RT-PCR) is used to detect CK19 mRNA in sentinel lymph node biopsy (SLNB) tissues from breast cancer patients. We examined whether CK19 mRNA in peripheral blood is predictive of non-sentinel lymph node (nSLN) metastasis. Breast cancer cases diagnosed with clinical stage cT1-3cN0 and registered in our medical biobank were identified retrospectively. This study then included 120 breast cancer cases treated at Zhejiang Cancer Hospital from Aug 2014 to Aug 2015, including 60 SLN-positive and 60 SLN-negative cases. CK19 mRNA levels in peripheral blood samples were assessed using RT-PCR prior to tumor removal. During surgery, if SLNB tissue showed evidence of metastasis, axillary lymph node dissection (ALND) was performed. No ALND was performed if SLNB and nSLN tissues were both negative for metastasis. CK19 expression was higher in nSLN-positive patients than in nSLN-negative patients (p < 0.05). Logistic regression indicated that lymphatic vessel invasion and CK19 levels were predictive of nSLN status (p < 0.05). The area under the ROC curve for CK19 was 0.878 (p < 0.05). We conclude that high CK19 levels in peripheral blood may independently predict nSLN metastasis in breast cancer patients.

The purpose of this study was to determine whether aldosterone has a rapid action in the nucleus tractus solitarius (NTS) that increases sodium intake, and to examine whether this effect of aldosterone, if present, is mediated by G protein-coupled estrogen receptor (GPER). Adult male Sprague-Dawley rats with a stainless-steel cannula in the NTS were used. Aldosterone was injected into the NTS at the doses of 1, 5, 10 and 20 ng 0.1 μl-1. A rapid dose-related increase of 0.3 M NaCl intake was induced within 30 min and this increase was not suppressed by the mineralocorticoid receptor (MR) antagonist spironolactone (10 ng 0.1 μl-1). Water intake was not affected by aldosterone. The GPER agonist G-1 produced a parallel and significant increase in sodium intake, while pre-treatment with GPER antagonist G15 (10 ng 0.1 μl-1) blocked the G-1 or aldosterone-induced rapid sodium intake. In addition, sodium intake induced by sodium depletion or low-sodium diet fell within 30 min after injection into the NTS of the MR antagonist spironolactone, while G15 had no effect. Our results confirm previous reports, and support the hypothesis that aldosterone evokes rapid sodium intake through a non-genomic mechanism involving GPER in NTS.

As a major protein constituent of high density lipoprotein, Apolipoprotein A1 (ApoA-1) might be associated with cancer progression. Our study investigated the serum ApoA-1 level for the prognosis of 443 patients with hepatocellular carcinoma (HCC) and its effects on tumor cells. We found that the serum ApoA-1 level was significantly lower in HCC patients with tumor recurrence, and was an independent indicator of tumor-free survival and overall survival. Low serum ApoA-1 levels were significantly associated with multiple tumors and high Barcelona Clinic Liver Cancer stage. The circulating tumor cell (CTC) levels were significantly higher in patients with low serum ApoA-1 compared with those with high serum ApoA-1 levels (4.03 ± 0.98 vs. 1.48 ± 0.22; p=0.001). In patients with detectable CTCs, those with low ApoA-1 levels had higher recurrence rates and shorter survival times. In vitro experiments showed that ApoA-1 can inhibit tumor cell proliferation through cell cycle arrest and promote apoptosis through down regulating mitogen-activated protein kinase (MAPK) pathway. In addition, ApoA-1 might impair extracellular matrix degradation properties of tumor cells. Taken together, our findings indicate that decreased serum ApoA-1 levels are a novel prognostic factor for HCC, and the role of ApoA-1 in inhibition of proliferation and promotion of apoptosis for tumor cells during their hematogenous dissemination are presumably responsible for the poor prognosis of patients with low ApoA-1 levels. Furthermore, AopA-1 might be a promising therapeutic target to reduce recurrence and metastasis for HCC patients after resection.

Safflower (Carthamus tinctorius L.) has received a significant amount of attention as a medicinal plant and oilseed crop. Gene expression studies provide a theoretical molecular biology foundation for improving new traits and developing new cultivars. Real-time quantitative PCR (RT-qPCR) has become a crucial approach for gene expression analysis. In addition, appropriate reference genes (RGs) are essential for accurate and rapid relative quantification analysis of gene expression. In this study, fifteen candidate RGs involved in multiple metabolic pathways of plants were finally selected and validated under different experimental treatments, at different seed development stages and in different cultivars and tissues for real-time PCR experiments. These genes were ABCS, 60SRPL10, RANBP1, UBCL, MFC, UBCE2, EIF5A, COA, EF1-β, EF1, GAPDH, ATPS, MBF1, GTPB and GST. The suitability evaluation was executed by the geNorm and NormFinder programs. Overall, EF1, UBCE2, EIF5A, ATPS and 60SRPL10 were the most stable genes, and MBF1, as well as MFC, were the most unstable genes by geNorm and NormFinder software in all experimental samples. To verify the validation of RGs selected by the two programs, the expression analysis of 7 CtFAD2 genes in safflower seeds at different developmental stages under cold stress was executed using different RGs in RT-qPCR experiments for normalization. The results showed similar expression patterns when the most stable RGs selected by geNorm or NormFinder software were used. However, the differences were detected using the most unstable reference genes. The most stable combination of genes selected in this study will help to achieve more accurate and reliable results in a wide variety of samples in safflower.

MiR-15a is likely to be associated with autoimmunity. Here, we aimed to examine the expression of miR-15 cluster in PBMCs from myasthenia gravis (MG) patients and investigate the potential roles of miR-15a in MG. We found that the expression of all miR-15 cluster was decreased in MG, furthermore, miR-15a levels in ocular MG (oMG) were much lower, while CXCL10 production was increased in MG. We display that CXCL10 was a functional target gene of miR-15a in MG. Increasing miR-15a expression could reduce CXCL10 expression and alleviate the abnormal T cells activation in immune response, while decreasing miR-15a expression could activate immune response abnormally. Moreover, miR-15a expression was significantly decreased after stimulation, and prednisone treatment could upregulate miR-15a expression in steroid-responsive MG patients. Take together, our data suggest that decreased miR-15a expression facilitates proinflammatory cytokines production and contributes to immune response at least in part via regulating CXCL10 expression in MG.

Calcitriol (1α,25-dihydroxyvitamin D3) has demonstrated anticancer activity against several tumors. However, the underlying mechanism for this activity is not yet fully understood. Our experiment was designed and performed to address one aspect of this issue in cervical cancer. HeLa S3 cells were cultured in media with various concentrations of calcitriol. Cell proliferation and cell cycle were assessed by spectrophotometry and flow cytometry, respectively. The mRNA and protein expression levels of human cervical cancer oncogene (HCCR-1) and p21 were determined by RT-PCR and Western blot, respectively. Results indicated that calcitriol inhibited HeLa S3 cell proliferation and induced cell cycle arrest at the G1 phase. Calcitriol decreased HCCR-1 protein expression in a dose- and time-dependent manner. Furthermore, promoter activity analyses revealed that transcriptional regulation was involved in the inhibition of HCCR-1 expression. Overexpression of HCCR-1 in HeLa S3 cells reversed the inhibition of cell proliferation and G1 phase arrest that resulted from calcitriol treatment. In addition, calcitriol increased p21 expression and promoter activity. HCCR-1 overexpression decreased p21 expression and promoter activity. Thus, our results suggested that calcitriol inhibited HeLa S3 cell proliferation by decreasing HCCR-1 expression and increasing p21 expression.

rDNA, the genes encoding ribosomal RNA (rRNA), is highly demanded for ribosome production and protein synthesis in growing cells such as pluripotent stem cells. rDNA transcription activity varies between cell types, metabolism conditions, and specific environmental challenges. Embryonic stem cells (ESCs), partially reprogrammed cells, and somatic cells reveal different epigenetic signatures, including rDNA epigenetic marks. rDNA epigenetic characteristic resetting is not quite clear during induced pluripotent stem cell (iPSC) generation. Little is known that whether the different rDNA epigenetic status in donor cells will result in different rDNA transcription activities, and furthermore affect reprogramming efficiency.

Gene regulatory networks involved in flowering time and photoperiodic responses in legumes remain unknown. Although the major maturity gene E1 has been successfully deciphered in soybean, knowledge on the functional conservation of this gene is limited to a certain extent to E1 homologs in legumes. The ectopic expression of Phvul.009G204600 (PvE1L), an E1 homolog from common bean, delayed the onset of flowering in soybean. By contrast, the ectopic expression of Medtr2g058520 (MtE1L) from Medicago truncatula did not affect the flowering of soybean. Characterization of the late-flowering mte1l mutant indicated that MtE1L promoted flowering in Medicago truncatula. Moreover, all transgenic E1, PvE1L and MtE1L soybean lines exhibited phenotypic changes in terms of plant height. Transgenic E1 or PvE1L plants were taller than the wild-type, whereas transgenic MtE1L plants produced dwarf phenotype with few nodes and short internode. Thus, functional conservation and diversification of E1 family genes from legumes in the regulation of flowering and plant growth may be associated with lineage specification and genomic duplication.

Hepatitis B virus (HBV) infection is a common infectious disease. Here we perform a genome-wide association study (GWAS) among Chinese populations to identify novel genetic loci involved in persistent HBV infection. GWAS scan is performed in 1,251 persistently HBV infected subjects (PIs, cases) and 1,057 spontaneously recovered subjects (SRs, controls), followed by replications in four independent populations totally consisting of 3,905 PIs and 3,356 SRs. We identify a novel locus at 8p21.3 (index rs7000921, odds ratio=0.78, P=3.2 × 10(-12)). Furthermore, we identify significant expression quantitative trait locus associations for INTS10 gene at 8p21.3. We demonstrate that INST10 suppresses HBV replication via IRF3 in liver cells. In clinical plasma samples, we confirm that INST10 levels are significantly decreased in PIs compared with SRs, and negatively correlated with the HBV load. These findings highlight a novel antiviral gene INTS10 at 8p21.3 in the clearance of HBV infection.

Increasing evidence supports that ELL (eleven-nineteen lysine-rich leukaemia) is a key regulator of transcriptional elongation, but the physiological function of Ell in mammals remains elusive. Here we show that ELL functions as an E3 ubiquitin ligase and targets c-Myc for proteasomal degradation. In addition, we identify that UbcH8 serves as a ubiquitin-conjugating enzyme in this pathway. Cysteine 595 of ELL is an active site of the enzyme; its mutation to alanine (C595A) renders the protein unable to promote the ubiquitination and degradation of c-Myc. ELL-mediated c-Myc degradation inhibits c-Myc-dependent transcriptional activity and cell proliferation, and also suppresses c-Myc-dependent xenograft tumour growth. In contrast, the ELL(C595A) mutant not only loses the ability to inhibit cell proliferation and xenograft tumour growth, but also promotes tumour metastasis. Thus, our work reveals a previously unrecognized function for ELL as an E3 ubiquitin ligase for c-Myc and a potential tumour suppressor.

Associative learning is thought to require coordinated activities among distributed brain regions. For example, to direct behavior appropriately, the medial prefrontal cortex (mPFC) must encode and maintain sensory information and then interact with the cerebellum during trace eyeblink conditioning (TEBC), a commonly-used associative learning model. However, the mechanisms by which these two distant areas interact remain elusive. By simultaneously recording local field potential (LFP) signals from the mPFC and the cerebellum in guinea pigs undergoing TEBC, we found that theta-frequency (5.0-12.0 Hz) oscillations in the mPFC and the cerebellum became strongly synchronized following presentation of auditory conditioned stimulus. Intriguingly, the conditioned eyeblink response (CR) with adaptive timing occurred preferentially in the trials where mPFC-cerebellum theta coherence was stronger. Moreover, both the mPFC-cerebellum theta coherence and the adaptive CR performance were impaired after the disruption of endogenous orexins in the cerebellum. Finally, association of the mPFC -cerebellum theta coherence with adaptive CR performance was time-limited occurring in the early stage of associative learning. These findings suggest that the mPFC and the cerebellum may act together to contribute to the adaptive performance of associative learning behavior by means of theta synchronization.

The neovascularization network of pannus formation plays a crucial role in the development of rheumatoid arthritis (RA). Annexin a2 (Axna2) is an important mediating agent that induces angiogenesis in vascular diseases. The correlation between Axna2 and pannus formation has not been studied. Here, we provided evidence that compared to osteoarthritis (OA) patients and healthy people, the expression of Axna2 and Axna2 receptor (Axna2R) were up-regulated in patients with RA. Joint swelling, inflammation and neovascularization were increased significantly in mice with collagen-induced arthritis (CIA) that were exogenously added Axna2. Cell experiments showed that Axna2 promoted HUVEC proliferation by binding Axna2R, and could activate Hedgehog (HH) signaling and up-regulate the expression of Ihh and Gli. Besides, expression of Ihh, Patched (Ptc), Smoothened (Smo) and Gli and matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2), angiogenic growth factor of HH signaling downstream, were down-regulated after inhibition of expression Axna2R on HUVEC. Together, our research definitely observed that over-expression of Axna2 could promote the development of CIA, especially during the process of pannus formation for the first time. Meanwhile, Axna2 depended on combining Axna2R to activate and enlarge HH signaling and the expression of its downstream VEGF, Ang-2 and MMP-2 to promote HUVEC proliferation, and eventually caused to angiogenesis. Therefore, the role of Axna2 is instructive for understanding the development of RA, suppress the effect of Axna2 might provide a new potential measure for treatment of RA.

Intervertebral disc degeneration (IDD) is characterized by disordered extracellular matrix (ECM) metabolism, implicating subdued anabolism and enhanced catabolic activities in the nucleus pulposus (NP) of discs. Pro-inflammatory cytokines such as interleukin-1β (IL-1β) are considered to be potent mediators of ECM breakdown. Hemeoxygenase-1 (HO-1) has been reported to participate in cellular anti-inflammatory processes. The purpose of this study was to investigate HO-1 modulation of ECM metabolism in human NP cells under IL-1β stimulation. Our results revealed that expression of HO-1 decreased considerably during IDD progression. Induction of HO-1 by cobalt protoporphyrin IX attenuated the inhibition of sulfate glycosaminoglycan and collagen type II (COL-II) synthesis and ameliorated the reduced expressions of aggrecan, COL-II, SOX-6 and SOX-9 mediated by IL-1β. Induction of HO-1 also reversed the effect of IL-1β on expression of the catabolic markers matrix metalloproteinases-1, 3, 9 and 13. This was combined with inhibition of the activation of mitogen-activated protein kinase signaling. These findings suggest that HO-1 might play a pivotal role in IDD, and that manipulating HO-1 expression might mitigate the impairment of ECM metabolism in NP, thus potentially offering a novel therapeutic approach to the treatment of IDD.