It is known that chitosan oligosaccharides (COS) suppress LPS-induced vascular endothelial inflammatory response by mechanism involving NF-κB blockade. It remains unknown how COS inhibit NF-κB. We provided evidence both in cultured endothelial cells and mouse model supporting a new mechanism. Regardless of the endothelial cell types, the LPS-induced NF-κB-dependent inflammatory gene expression was suppressed by COS, which was associated with reduced NF-κB nucleus translocation. LPS enhanced O-GlcNAc modification of NF-κB/p65 and activated NF-κB pathway, which could be prevented either by siRNA knockdown of O-GlcNAc transferase (OGT) or pretreatment with COS. Inhibition of either mitogen-activated protein kinase or superoxide generation abolishes LPS-induced NF-κB O-GlcNAcylation. Consistently, aortic tissues from LPS-treated mice presented enhanced NF-κB/p65 O-GlcNAcylation in association with upregulated gene expression of inflammatory cytokines in vascular tissues; however, pre-administration of COS prevented these responses. In conclusion, COS decreased OGT-dependent O-GlcNAcylation of NF-κB and thereby attenuated LPS-induced vascular endothelial inflammatory response.
Chitosan oligosaccharides; LPS (Lipopolysaccharides); Endothelial cells; O-GlcNAcylation; NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells); Inflammatory response
SIRT1 is central to the lifespan and vascular health, but undergoes degradation that contributes to several medical conditions, including diabetes. How SIRT1 turnover is regulated remains unclear. However, emerging evidence suggests that endothelial nitric oxide synthase (eNOS) positively regulates SIRT1 protein expression. We recently identified NO as an endogenous inhibitor of 26S proteasome functionality with a cellular reporter system. Here we extended this finding to a novel pathway that regulates SIRT1 protein breakdown. In cycloheximide (CHX)-treated endothelial cells, NONOate, an NO donor, and A23187, an eNOS activator, significantly stabilized SIRT1 protein. Similarly, NO enhanced SIRT1 protein, but not mRNA expression, in CHX-free cells. NO also stabilized an autophagy-related protein unc-51 like kinase (ULK1), but did not restore SIRT1 protein levels in ULK1-siRNA-treated cells or in mouse embryonic fibroblasts (MEF) from Ulk1−/− mice. This suggests that ULK1 mediated the NO regulation of SIRT1. Furthermore, adenoviral overexpression of ULK1 increased SIRT1 protein expression, while ULK1 siRNA treatment decreased it. Rapamycin-induced autophagy did not mimic these effects, suggesting that the effects of ULK1 were autophagy-independent. Treatment with MG132, a proteasome inhibitor, or siRNA of β-TrCP1, an E3 ligase, prevented SIRT1 reduction induced by ULK1-siRNA. Mechanistically, ULK1 negatively regulated 26S proteasome functionality, which was at least partly mediated by O-linked-GlcNAc transferase (OGT), probably by increased O-GlcNAc modification of proteasomal subunit Rpt2. The NO-ULK1-SIRT1 axis was likely operative in the whole animal: both ULK1 and SIRT1 protein levels were significantly reduced in tissue homogenates in eNOS-knockout mice (lung) and in db/db mice where eNOS is downregulated (lung and heart). Taken together, the results show that NO stabilizes SIRT1 by regulating 26S proteasome functionality through ULK1 and OGT, but not autophagy, in endothelial cells.
Breast cancer is the leading cause of cancer death in women world wide which is closely related to metastasis. Recent studies argue that breast cancer cells that have undergone epithelial-to-mesenchymal transition (EMT) acquire aggressive malignant properties, but the molecular mechanisms underlying this transition are poorly understood. In this study, we found that increased expression of proline-rich protein 11 (PRR11) was associated with the progression of breast cancer and that PRR11 protein levels were significantly elevated in breast cancer. High PRR11 levels also predict shorter overall survival of breast cancer patients. Moreover, we found that the forced expression of PRR11 decreased the expression of the epithelial marker E-cadherin but increased the mesenchymal markers in breast cancer cells. In contrast, silencing PRR11 in metastatic breast tumor cells promoted a shift toward an epithelial morphology concomitant with increased expression of E-cadherin and decreased expression of mesenchymal markers. PRR11 silencing also reduced the expression of EMT-inducing transcription factors (Snail, Slug, ZEB1 and ZEB2).
PRR11; EMT; breast cancer; migration; invasion
Osteosarcoma is the most common histological form of primary bone cancer, which arises from osteoid tissue. It occurs predominantly in infants and adolescents, with an incidence of 4–5 cases/100,000,000. The 5-year survival rate of patients with osteosarcoma has significantly improved over time; however, there remains a significant proportion of patients that respond poorly to chemotherapy. An improved understanding of the pathology of osteosarcoma is required to provide more effective treatment strategies, identify biomarkers and develop novel chemotherapeutic agents. Disturbance in microRNA (miRNA) expression has been identified in osteosarcoma tissues and cell lines; however, the roles of miRNA during osteosarcoma pathogenesis remain to be elucidated. In the present study, the expression levels of eight selected miRNAs were investigated in osteosarcoma tissues and the results revealed that the expression levels of miR-542-3p and miR-542-5p were significantly upregulated and the expression of miR-199-3p was significantly downregulated. Using a dual luciferase assay and western blot analysis, the present study confirmed that Van Gogh-like 2, which is a non-canonical Wnt pathway suppressor, was a target gene of miR-542-3p. Subsequently, the biological function of miR-542-3p in U2OS cells was examined, which revealed that overexpression of miR-542-3p can enhance the cell proliferation and migration ability of U2OS cells. This indicated that miR-542-3p may act as an oncogene in osteosarcoma pathogenesis. The findings of the present study may provide assistance in understanding the development of osteosarcoma and aid in the development of strategies for the diagnosis and treatment of osteosarcoma.
osteosarcoma; microRNA; Van Gogh-like 2; 3′ untranslated region; proliferation; migration
Preparations utilizing monoclonal antibodies against S100A4 provide useful tools for functional studies to investigate the clinical applications of the human S100A4 protein. In the present study, human S100A4 protein was expressed in Escherichia coli (E. coli) BL21 (DE3), successfully purified by diethylaminoethyl cellulose anion-exchange chromatography and identified by western blot analysis. Soluble S100A4 bioactivity was confirmed by Transwell migration and invasion assays in the human HeLa cell line. Monoclonal antibodies (mAbs) were generated utilizing the standard hybridoma method and were validated by enzyme-linked immunosorbent assay and western blot analysis. The antibody was then used to examine human gastric carcinoma specimens by immunohistochemistry. Recombinant S100A4 was functionally expressed in E. coli and promoted the migration and invasion of HeLa cells. Four hybridoma cell lines, which secreted mAbs specifically against human S100A4 protein, were obtained. One of the four mAbs, namely 2A12D10B2, recognized human S100A4 as indicated by immunohistochemical staining of human gastric carcinoma specimens and recombinant S100A4 was functionally expressed in E. coli. The mAbs of recombinant S100A4 were suitable for detecting S100A4 expression in human tissues and for investigating the subsequent clinical applications of the protein.
human S100A4 protein; monoclonal antibody; preparation; application
Osteosarcoma (OS) is the most prevalent primary malignant bone tumor in children and young adults, its complex etiology involving a combination of environmental and genetic factors. MicroRNA (miRNA) is a short, non-coding regulatory RNA molecule that represses gene expression by imperfectly base-pairing to the 3′ untranslated region of target mRNAs. Evidence has shown that alterations in the expression of miRNA are involved in the initiation, progression, and metastasis of human cancers. It is believed that miRNAs function both as tumor suppressors and oncogenes during cancer development. In the present study, three tumor-associated miRNAs (miR-21, miR-34a and miR-146a) coding regions were screened in Chinese-Han OS patients. A G>A variation in the pre-miR-34a coding region was found to be associated with higher OS morbidity. By detecting the mature miR-34a expression in cells transfected with pre-miR-34a expression vectors of different genotypes using quantitative polymerase chain reaction, it was demonstrated that the G>A variation reduced miR-34a expression in vitro. This was in accordance with the data collected from tumor tissue and patient serum samples. Subsequently, a dual-luciferase reporter assay and western blot analysis were used to detect the site variation effect on the expression of c-Met, a target gene of miR-34a. The G>A variation downregulated the suppression of c-Met in two OS cell lines. Furthermore, it was found that reduced miR-34a expression decreased the suppression of OS cell proliferation in vitro. In conclusion, the present study established the association between miR-34a and the risk of suffering OS in a Chinese Han population by identifying one functional single nucleotide polymorphism site in pre-miR-34a. These findings may give insight into the mechanism of OS development and create an opportunity to approach the diagnosis and treatment of OS.
osteosarcoma; microRNA; pre-miRNA; polymorphism; cell proliferation
The objective of this study was to perform a meta-analysis of published studies for evaluating the impact of neoadjuvant chemotherapy (NAC) on immediate breast reconstruction.
We searched medical databases to identify appropriate studies that assessed the impact of NAC on immediate breast reconstruction from the inception of this technique through April 2013. We then performed a meta-analysis of these studies.
Our searches identified 11 studies among 1,840 citations. In the meta-analysis, NAC did not increase the overall rate of complications after immediate breast reconstruction (odds ratio [OR] = 0.59; 95% confidence interval[CI] = 0.38–0.91). The complication rate was also unaffected by NAC when we considered infections (OR = 0.82; 95% CI = 0.46–1.45), hematomas (OR = 1.35; 95% CI = 0.57–3.21), and seromas (OR = 0.77; 95% CI = 0.23–2.55). Additionally, expander or implant loss did not significantly increase in patients after NAC (OR = 1.59; 95% CI = 0.91–2.79). Only 2 studies (202 procedures) had reported total autologous flap loss, and they were included in our analysis; both studies found no association between NAC and total flap loss.
Our analysis suggests that NAC does not increase the complication rate after immediate breast reconstruction. For appropriately selected patients, immediate breast reconstruction following NAC is a safe procedure. The best way to study this issue in the future is to conduct a multicenter prospective study with a longer follow-up period and more clearly defined parameters.
The 26S proteasome plays a fundamental role in almost all eukaryotic cells, including vascular endothelial cells. However, it remains largely unknown how proteasome functionality is regulated in the vasculature. Endothelial nitric oxide (NO) synthase (eNOS)-derived NO is known to be essential to maintain endothelial homeostasis. The aim of the present study was to establish the connection between endothelial NO and 26S proteasome functionality in vascular endothelial cells. The 26S proteasome reporter protein levels, 26S proteasome activity, and the O-GlcNAcylation of Rpt2, a key subunit of the proteasome regulatory complex, were assayed in 26S proteasome reporter cells, human umbilical vein endothelial cells (HUVEC), and mouse aortic tissues isolated from 26S proteasome reporter and eNOS knockout mice. Like the other selective NO donors, NO derived from activated eNOS (by pharmacological and genetic approach) increased O-GlcNAc modification of Rpt2, reduced proteasome chymotrypsin-like activity, and caused 26S proteasome reporter protein accumulation. Conversely, inactivation of eNOS reversed all the effects. SiRNA knockdown of O-GlcNAc transferase (OGT), the key enzyme that catalyzes protein O-GlcNAcylation, abolished NO-induced effects. Consistently, adenoviral overexpression of O-GlcNAcase (OGA), the enzyme catalyzing the removal of the O-GlcNAc group, mimicked the effects of OGT knockdown. Finally, compared to eNOS wild type aortic tissues, 26S proteasome reporter mice lacking eNOS exhibited elevated 26S proteasome functionality in parallel with decreased Rpt2 O-GlcNAcylation, without changing the levels of Rpt2 protein. In conclusion, the eNOS-derived NO functions as a physiological suppressor of the 26S proteasome in vascular endothelial cells.
Low incidence of GVHD provides the major rational for pursuing UCB stem cell transplant (UCB SCT). Considerable evidence also suggests a lower rate of recurrence after UCB SCT than after transplantation from adult donors. Recent advances in understanding of the human fetal immune development provide a rational underpinning for these clinical outcomes. The fetal immune system is geared toward maintaining tolerance to foreign antigens, particularly to the maternal antigens to which it is exposed throughout gestation. To this purpose it is dominated by a unique population of peripheral T regulatory cells which actively maintain tolerance. This and other features of the UCB lymphoid system explains the low incidence of GVHD and superior outcomes of UCB SCT with NIMA (non-inherited maternal antigens)-matched grafts. At the same time, highly sensitized maternal microchimeric cells are frequently detected in UCB and likely contribute to superior GVL effects and low rates of disease recurrence in IPA (inherited paternal antigen) matched UCB recipients.
But historically erratic and slow hematopoietic recovery after UCB SCT leads to increased early morbidity and mortality, excessive hospitalization and costs. This has held up the widespread utilization of UCB SCT in adults. Here we summarize recent data on UCB SCT with an emphasis on studies of co-infusion of adult CD34 selected hematopoietic stem cells with UCB SCT. This procedure, through transient engraftment of adult hematopoietic stem cells largely overcomes the problem of delayed engraftment. We also briefly discuss unresolved issues and possible future applications of this technology.
S100A4 protein is associated with Ca2+-dependent regulation of intracellular activities and is significant in the invasion, growth and metastasis of cancer. In order to express rat S100A4 functionally and identify its biological activity following purification, an S100A4 gene fragment was optimized and fully synthesized via overlapping polymerase chain reaction. The gene was inserted into the prokaryotic expression vector, pBV220, with phage λ PRPL promoters following confirmation by DNA sequencing. The pBV220-S100A4 plasmid was constructed and transformed into Escherichia coli DH5α. Following temperature induction, rat S100A4 was overexpressed and the protein was observed to be located in the supernatant of the lysates, which was ~30–40% of the total protein within the host. The protein was isolated and purified by metal-chelate affinity chromatography. High purity protein (>98% purity) was obtained and in vitro western blot analysis identified that the recombinant S100A4 was able to bind to the antibody against wild-type S100A4. The bioactivity of the recombinant protein was detected via Transwell migration and invasion assays. The polyclonal antibody of rat S100A4 protein was prepared for rabbit immunization and exhibited similar efficacies when compared with commercial S100A4. Therefore, rat S100A4 was functionally expressed in E. coli; thus, the production of active recombinant S100A4 protein in E. coli may further aid with the investigation and application of S100A4.
rat S100A4; functional expression; gene recombination
The squat exercise was usually performed with varying feet and hip angles by different populations. The objective of this study was to compare and contrast the three-dimensional knee angles, moments, and forces during dynamic squat exercises with varying feet and hip angles. Lower extremity motions and ground reaction forces for fifteen healthy subjects (9 females and 6 males) were recorded while performing the squat with feet pointing straight ahead (neutral squat), 30º feet adduction (squeeze squat) and 30º feet abduction (outward squat). Nonparametric procedures were used to detect differences in the interested measures between the conditions. No significant difference in three-dimensional peak knee angles was observed for three squat exercises (p>0.05), however, the overall tendency of knee rotations was affected by varying feet and hip positions. During the whole cycle, the outward squat mainly displayed adduction moments, while the neutral and squeeze squat demonstrated abduction moments. Peak abduction moments were significantly affected by feet positions (p<0.05). Moreover, the tibiofemoral and patellofemoral joint forces progressively increased as knee flexed and decreased as knee extended, yet peak forces were not affected by varying feet positions (p>0.05). In conclusion, a neutral position is recommended to perform the squat exercise, while the squeeze squat and outward squat might contribute to the occurrence of joint pathologies.
squat performance; foot angle; knee alignment; rehabilitation; osteoarthritis
Aortic dissection is a lift-threatening medical emergency associated with high rates of morbidity and mortality. The incidence rate of aortic dissection is estimated at 5 to 30 per 1 million people per year. The prompt and correct diagnosis of aortic dissection is critical. This study was to compare the ascending aortic image quality and the whole aortic radiation dose of high-pitch dual-source CT angiography and conventional dual-source CT angiography.
A total of 110 consecutive patients with suspected aortic dissection and other aortic disorders were randomly divided into two groups. Group A underwent traditional scan mode and Group B underwent high-pitch dual-source CT scan mode. The image quality and radiation dose of two groups were compared.
Close interobserver agreement was found for image quality scores (κ = 0.87). The image quality of ascending aorta was significantly better in the high-pitch group than in the conventional group (2.78 ± 0.46 vs 1.57 ± 0.43, P < 0.001). There was no significant difference of the CT attenuation values, the aortic image noise and SNR between two groups. The mean radiation dose of high-pitch group was also significantly lower than that of conventional group (2.7 ± 0.6 mSv vs. 3.9 ± 0.9 mSv, P < 0.001).
High-pitch dual-source CT angiography of the whole aorta can provide motion-artifact-free imaging of the ascending aorta at a low radiation dose compared to conventional protocol.
Aorta; Radiation dose; Image quality; Dual-source CT
Arabidopsis thaliana cryptochrome 2 (CRY2) mediates light control of flowering time. CIB1 (CRY2-interacting bHLH 1) specifically interacts with CRY2 in response to blue light to activate the transcription of FT (Flowering Locus T). In vitro, CIB1 binds to the canonical E-box (CACGTG, also referred to as G-box) with much higher affinity than its interaction with non-canonical E-box (CANNTG) DNA sequences. However, in vivo, CIB1 binds to the chromatin region of the FT promoter, which only contains the non-canonical E-box sequences. Here, we show that CRY2 also interacts with at least CIB5, in response to blue light, but not in darkness or in response to other wavelengths of light. Our genetic analysis demonstrates that CIB1, CIB2, CIB4, and CIB5 act redundantly to activate the transcription of FT and that they are positive regulators of CRY2 mediated flowering. More importantly, CIB1 and other CIBs proteins form heterodimers, and some of the heterodimers have a higher binding affinity than the CIB homodimers to the non-canonical E-box in the in vitro DNA-binding assays. This result explains why in vitro CIB1 and other CIBs bind to the canonical E-box (G-box) with a higher affinity, whereas they are all associated with the non-canonical E-boxes at the FT promoter in vivo. Consistent with the hypothesis that different CIB proteins play similar roles in the CRY2-midiated blue light signaling, the expression of CIB proteins is regulated specifically by blue light. Our study demonstrates that CIBs function redundantly in regulating CRY2-dependent flowering, and that different CIBs form heterodimers to interact with the non-canonical E-box DNA in vivo.
Arabidopsis thaliana blue light receptor cryptochromes (CRYs) mediate light control of flowering time by interacting with CIB1 (CRY2-interacting bHLH1) in response to blue light. However, it remains unclear how the blue light-dependent CRY2-CIB1 interaction affects the FT transcription. We report here that in addition to CIB1, CRY2 also interact with CIB1 related bHLH proteins, CIBs. These CIBs act redundantly with CIB1 to activate the transcription of FT and flowering. More importantly, CIB1 and the CIBs can form heterodimers and some of those heterodimers have a higher binding affinity to the non-canonical E-box, although their homodimers all prefer canonical E-box (G-box), so they can bind to the non-canonical E-Box sequences of the FT promoter. This is the first example in plants that heterodimerization of bHLH can change the DNA binding affinity or specificity. CIB proteins are involved in blue light signaling and they are specifically stabilized by blue light.
Although the connection of oxidative stress and inflammation has been long recognized in diabetes, the underlying mechanisms are not fully elucidated. This study defined the role of 26S proteasomes in promoting vascular inflammatory response in early diabetes.
Methods and Results
The 26S proteasome functionality, markers of autophagy, and unfolded protein response (UPR) were assessed in: (a) cultured 26S proteasome reporter cells and endothelial cells challenged with high glucose, (b) transgenic reporter (UbG76V-GFP) and wild type (C57BL/6J) mice rendered diabetic, and (c) genetically diabetic (Akita and OVE26) mice. In glucose-challenged cells, and also in aortic, renal, and retinal tissues from diabetic mice, enhanced 26S proteasome functionality was observed, evidenced by augmentation of proteasome (chymotrypsin-like) activities and reduction in 26S proteasome reporter proteins, accompanied by increased nitrotyrosine-containing proteins. Also, while IκBα proteins were decreased, an increase was found in NF-κB nucleus translocation, which enhanced the NF-κB-mediated pro-inflammatory response, without affecting markers of autophagy or UPR. Importantly, the alterations were abolished by MG132 administration, siRNA knockdown of PA700 (proteasome activator protein complex), or superoxide scavenging in vivo.
Early hyperglycemia enhances 26S proteasome functionality, not autophagy or UPR, through peroxynitrite/superoxide-mediated PA700-dependent proteasomal activation, which elevates NF-κB-mediated endothelial inflammatory response in early diabetes.
diabetes; 26S proteasomes; oxidative stress; inflammatory response; NF-κB
The aim of this study was to determine the correlation of S100A4 expression with the progression, prognosis and clinical pathology of gastric cancer (GC) in young pateints. A total of 85 tumor tissues with corresponding adjacent normal tissues and 62 non-metastatic lymph nodes (LNs) with corresponding metastatic LNs were obtained from young GC patients (<40 years old) who underwent surgery between January 2001 and December 2006. The expression of S100A4 was detected by RT-PCR and immunohistochemistry. Differences in the expression of S100A4 mRNA or protein were observed among the GC tissues, matched normal gastric mucosa, non-metastatic LNs and metastatic LNs. The expression of S100A4 mRNA and protein in GC tissues and metastatic LNs was significantly higher compared with that in the matched normal gastric mucosa and non-metastatic LNs, respectively (P<0.05). The overexpression of S100A4 was significantly associated with parameters involved in tumor progression and poor prognosis, including tumor size (P=0.017), Lauren classification (P=0.002), histological classification (P= 0.010), histological differentiation (P= 0.000), Borrmann classification (P=0.020), tumor-node-metastasis (TNM) stage (P=0.000), LN metastasis (P=0.000) and distant metastasis (P=0.024). Multivariate analysis suggested that patient age (P=0.035), tumor size (P=0.002), TNM stage (P=0.001) and S100A4 upregulation (P=0.000) were independent prognostic indicators for the disease. The overexpression of S100A4 in young GC patients is significantly associated with the clinicopathological characteristics. S100A4 may be used as a biomarker to predict the progression and poor prognosis of GC in young patients.
S100A4; gastric cancer; young patients; progression; prognosis; immunohistochemistry
Infrastructure has become an important topic in a variety of areas of the policy debate, including energy saving and climate change. In this paper, we use an energy input-output model to evaluate the amounts of China's embodied energy use in infrastructure investment from 1992 to 2007. We also use the structure decomposition model to analyze the factors impacting the embodied energy use in infrastructure investment for the same time period. The results show that embodied energy use in infrastructure investment accounted for a significant proportion of China's total energy use with an increasing trend and reflect that improper infrastructure investment represents inefficient use of energy and other resources. Some quantitative information is provided for further determining the low carbon development potentials of China's economy.
The blue light receptors cryptochromes mediate various light responses in plants. The photoexcited cryptochrome molecules undergo a number of biophysical and biochemical changes, including electron transfer, phosphorylation, and ubiquitination, resulting in conformational changes to propagate light signals. Two modes of cryptochrome signal transduction have been recently discovered, the CIB (cryptochrome-interacting basic-helix-loop-helix 1)-dependent CRY2 regulation of transcription and the SPA1/COP1 (SUPPRESSOR OF PHYA /CONSTITUTIVELY PHOTOMORPHOGENIC1)-dependent cryptochrome regulation of proteolysis. Both cryptochrome signaling pathways rely on blue light-dependent interactions between the cryptochrome photoreceptor and its signaling proteins to modulate gene expression changes in response to blue light, leading to altered developmental programs of plants.
Light inducible protein-protein interactions are powerful tools to manipulate biological processes. Genetically encoded light-gated proteins for controlling precise cellular behavior are a new and promising technology, called optogenetics. Here we exploited the blue light-induced transcription system in yeast and zebrafish, based on the blue light dependent interaction between two plant proteins, blue light photoreceptor Cryptochrome 2 (CRY2) and the bHLH transcription factor CIB1 (CRY-interacting bHLH 1). We demonstrate the utility of this system by inducing rapid transcription suppression and activation in zebrafish.
MicroRNAs (miRNAs) are small, non-coding RNAs capable of postranscriptionally regulating gene expression. Accurate expression profiling is crucial for understanding the biological roles of miRNAs, and exploring them as biomarkers of diseases.
A novel, highly sensitive, and reliable miRNA quantification approach,termed S-Poly(T) miRNA assay, is designed. In this assay, miRNAs are subjected to polyadenylation and reverse transcription with a S-Poly(T) primer that contains a universal reverse primer, a universal Taqman probe, an oligo(dT)11 sequence and six miRNA-specific bases. Individual miRNAs are then amplified by a specific forward primer and a universal reverse primer, and the PCR products are detected by a universal Taqman probe. The S-Poly(T) assay showed a minimum of 4-fold increase in sensitivity as compared with the stem-loop or poly(A)-based methods. A remarkable specificity in discriminating among miRNAs with high sequence similarity was also obtained with this approach. Using this method, we profiled miRNAs in human pulmonary arterial smooth muscle cells (HPASMC) and identified 9 differentially expressed miRNAs associated with hypoxia treatment. Due to its outstanding sensitivity, the number of circulating miRNAs from normal human serum was significantly expanded from 368 to 518.
With excellent sensitivity, specificity, and high-throughput, the S-Poly(T) method provides a powerful tool for miRNAs quantification and identification of tissue- or disease-specific miRNA biomarkers.
Recently, apoptosis has been considered to be an important regulator for allograft survival. The serine/threonine kinase Pim2 has been implicated in many apoptotic pathways. In a previous study, we found that pim2 was highly expressed in CD4+ T cells in an allograft model. Here, we further investigated the effects of Pim2 on allograft survival and the underlying mechanisms associated with apoptosis. The results showed that pim2 was overexpressed in grafts and spleens, particularly in spleen CD4+ T cells when acute allorejection occurred, and correlated positively with the extent of rejection. In T cells from the spleens of naive BALB/c mice treated with 5 µM 4a (a specific inhibitor of Pim2) for 24 h, the apoptosis rate increased and the phosphorylation of BAD was decreased. Furthermore, adoptive transfer of CD4+ T cells treated with 4a in vitro to allografted severe combined immunodeficiency (SCID) mice effectively prolonged allograft survival from 19.5±1.7 days to 31±2.3 days. Moreover, the results demonstrated that the CD4+CD25− effector T-cell subset was the predominate expresser of the pim2 gene as compared with the CD4+CD25+ regulatory T (Treg) cell subset. Alloantigen-induced CD4+CD25+ T cells displayed less Foxp3 expression and a low suppression of apoptosis compared with effector CD4+CD25− T cells treated with 4a. Collectively, these data revealed that Pim2 facilitated allograft rejection primarily by modulating the apoptosis of effector T cells and the function of Treg cells. These data suggested that Pim2 may be an important target for in vivo anti-rejection therapies and for the ex vivo expansion of CD4+CD25+ T cells.
apoptosis; CD4+ T cell; Pim2; regulatory; transplantation
Diabetes impairs physiological angiogenesis by molecular mechanisms that are not fully understood. Methylglyoxal (MGO), a metabolite of glycolysis, is increased in patients with diabetes. This study defined the role of MGO in angiogenesis impairment and tested the mechanism in diabetic animals. Endothelial cells and mouse aortas were subjected to Western blot analysis of vascular endothelial growth factor receptor 2 (VEGFR2) protein levels and angiogenesis evaluation by endothelial cell tube formation/migration and aortic ring assays. Incubation with MGO reduced VEGFR2 protein, but not mRNA, levels in a time and dose dependent manner. Genetic knockdown of the receptor for advanced glycation endproducts (RAGE) attenuated the reduction of VEGFR2. Overexpression of Glyoxalase 1, the enzyme that detoxifies MGO, reduced the MGO-protein adducts and prevented VEGFR2 reduction. The VEGFR2 reduction was associated with impaired angiogenesis. Suppression of autophagy either by inhibitors or siRNA, but not of the proteasome and caspase, normalized both the VEGFR2 protein levels and angiogenesis. Conversely, induction of autophagy either by rapamycin or overexpression of LC3 and Beclin-1 reduced VEGFR2 and angiogenesis. MGO increased endothelial LC3B and Beclin-1, markers of autophagy, which were accompanied by an increase of both autophagic flux (LC3 punctae) and co-immunoprecipitation of VEGFR2 with LC3. Pharmacological or genetic suppression of peroxynitrite (ONOO−) generation not only blocked the autophagy but also reversed the reduction of VEGFR2 and angiogenesis. Like MGO-treated aortas from normglycemic C57BL/6J mice, aortas from diabetic db/db and Akita mice presented reductions of angiogenesis or VEGFR2. Administration of either autophagy inhibitor ex vivo or superoxide scavenger in vivo abolished the reductions. Taken together, MGO reduces endothelial angiogenesis through RAGE-mediated, ONOO–dependent and autophagy-induced VEGFR2 degradation, which may represent a new mechanism for diabetic angiogenesis impairment.
The role of p53 in tissue protection is not well understood. Loss of p53 blocks apoptosis in the intestinal crypts following irradiation, but paradoxically accelerates gastrointestinal (GI) damage and death. PUMA and p21 are the major mediators of p53-dependent apoptosis and cell cycle checkpoints, respectively. To better understand these two arms of p53 response in radiation-induced GI damage, we compared animal survival, as well as apoptosis, proliferation, cell cycle progression, DNA damage, and regeneration in the crypts of WT, p53 KO, PUMA KO, p21 KO, and p21/PUMA double KO (DKO) mice in a whole body irradiation model. Deficiency in p53 or p21 led to shortened survival but accelerated crypt regeneration associated with massive non-apoptotic cell death. Non-apoptotic cell death is characterized by aberrant cell cycle progression, persistent DNA damage, rampant replication stress and genome instability. PUMA deficiency alone enhanced survival and crypt regeneration by blocking apoptosis, but failed to rescue delayed non-apoptotic crypt death or shortened survival in p21 KO mice. These studies help better understand p53 functions in tissue injury and regeneration, and potentially improve strategies to protect or mitigate intestinal damage induced by radiation.
p53; p21; PUMA; cell cycle arrest; apoptosis; irradiation; small intestine; stem cells
The initial photochemistry of plant cryptochromes has been extensively investigated in recent years. It is hypothesized that cryptochrome photoexcitation involves a Trp-triad-dependent photoreduction. According to this hypothesis, cryptochromes in the resting state contain oxidized FAD; light triggers a sequential electron transfer from three tryptophan residues to reduce FAD to a neutral semiquinone (FADH•); FADH• is the presumed signaling state and it is re-oxidized to complete the photocycle. However, this photoreduction hypothesis is currently under debate. An alternative model argues that the initial photochemistry of cryptochromes involves a photolyase-like cyclic electron shuttle without a bona fide redox reaction mediated by the Trp-triad residues, leading to conformational changes, signal propagation, and physiological responses.
Cryptochromes are photolyase-like blue light receptors originally discovered in Arabidopsis but later found in other plants, microbes, and animals. Arabidopsis has two cryptochromes, CRY1 and CRY2, which mediate primarily blue light inhibition of hypocotyl elongation and photoperiodic control of floral initiation, respectively. In addition, cryptochromes also regulate over a dozen other light responses, including circadian rhythms, tropic growth, stomata opening, guard cell development, root development, bacterial and viral pathogen responses, abiotic stress responses, cell cycles, programmed cell death, apical dominance, fruit and ovule development, seed dormancy, and magnetoreception. Cryptochromes have two domains, the N-terminal PHR (Photolyase-Homologous Region) domain that bind the chromophore FAD (flavin adenine dinucleotide), and the CCE (CRY C-terminal Extension) domain that appears intrinsically unstructured but critical to the function and regulation of cryptochromes. Most cryptochromes accumulate in the nucleus, and they undergo blue light-dependent phosphorylation or ubiquitination. It is hypothesized that photons excite electrons of the flavin molecule, resulting in redox reaction or circular electron shuttle and conformational changes of the photoreceptors. The photoexcited cryptochrome are phosphorylated to adopt an open conformation, which interacts with signaling partner proteins to alter gene expression at both transcriptional and posttranslational levels and consequently the metabolic and developmental programs of plants.
Cobicistat (3, GS-9350) is a newly discovered, potent, and selective inhibitor of human cytochrome P450 3A (CYP3A) enzymes. In contrast to ritonavir, 3 is devoid of anti-HIV activity and is thus more suitable for use in boosting anti-HIV drugs without risking selection of potential drug-resistant HIV variants. Compound 3 shows reduced liability for drug interactions and may have potential improvements in tolerability over ritonavir. In addition, 3 has high aqueous solubility and can be readily coformulated with other agents.
GS-9350; cobicistat; pharmacoenhancer; PK enhancer; CYP3A inhibitor; ritonavir