NPC (nasopharyngeal carcinoma) is a common malignancy in southern China without defined aetiology. Recent studies have shown that TGFBR3 (transforming growth factor type III receptor, also known as betaglycan), exhibits anticancer activities. This study was to investigate the effects of TGFBR3 on NPC growth and the mechanisms for its actions. Effects of TGFBR3 overexpression on cell viability and apoptosis were measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], AO/EB (acridine orange/ethidium bromide) staining and electron microscopy in human NPC CNE-2Z cells. The expression of apoptosis-related proteins, p-Bad, Bad, XIAP (X-linked inhibitor of apoptosis), AIF (apoptosis-inducing factor), Bax and Bcl-2, was determined by Western blot or immunofluorescence analysis. Caspase 3 activity was measured by caspase 3 activity kit and [Ca2+]i (intracellular Ca2+ concentration) was detected by confocal microscopy. Transfection of TGFBR3 containing plasmid DNA at concentrations of 0.5 and 1 μg/ml reduced viability and induced apoptosis in CNE-2Z in concentration- and time-dependent manners. Forced expression of TGFBR3 up-regulated pro-apoptotic Bad and Bax protein, and down-regulated anti-apoptotic p-Bad, Bcl-2 and XIAP protein. Furthermore, transient overexpression of TGFBR3 also enhanced caspase 3 activity, increased [Ca2+]i and facilitated AIF redistribution from the mitochondria to the nucleus in CNE-2Z cells, which is independent of the caspase 3 pathway. These events were associated with TGFBR3-regulated multiple targets involved in CNE-2Z proliferation. Therefore transient overexpression of TGFBR3 may be a novel strategy for NPC prevention and therapy.

A new family of Hyper-Crosslinked (HC) phases has been recently introduced for use under very aggressive acid conditions including those encountered in ultra-fast, high temperature Two-Dimensional Liquid Chromatography (2DLC). This type of stationary phase showed significantly enhanced acid and thermal stability compared to the most acid stable, commercial RPLC phases. In addition, the use of “orthogonal” chemistry to make surface-confined polymer networks ensures good reproducibility and high efficiency. One of the most interesting features of the HC phases is the ability to derivatize the surface aromatic groups with various functional groups. This led to the development of a family of hyper-crosslinked phases possessing a wide variety of chromatographic selectivities by attaching hydrophobic (e.g. –C8), ionizable (e.g. -COOH, -SO3H), aromatic (e.g. –toluene) or polar (e.g. -OH) species to the aromatic polymer network. HC reversed phases with various degrees of hydrophobicity and mixed-mode HC phases with added strong and weak cation exchange sites have been synthesized, characterized and applied. These silica-based acid-stable HC phases, with their attractive chromatographic properties, should be very useful in the separations of bases or biological analytes in acidic media, especially at elevated temperatures. This work reviews the prior research on HC phases and introduces a novel HC phase made by alternative chemistry.

Ceramic prostheses often fail from fracture and wear. We hypothesize that these failures may be substantially mitigated by an appropriate grading of elastic modulus at the ceramic surface. In this study, we elucidate the effect of elastic modulus profile on the flexural damage resistance of functionally graded materials (FGMs), providing theoretical guidlines for designing FGM with superior load-bearing property. The Young's modulus of the graded structure is assumed to vary in a power-law relation with a scaling exponent n; this is in accordance with experimental observations from our laboratory and elsewhere. Based on the theory for bending of graded beams, we examine the effect of n value and bulk-to-surface modulus ratio (Eb/Es) on stress distribution through the graded layer. Theory predicts that a low exponent (0.15 < n < 0.5), coupled with a relatively small modulus ratio (3 < Eb/Es < 6), is most desirable for reducing the maximum stress and transferring it into the interior, while keeping the surface stress low. Experimentally, we demonstrate that elastically graded materials with various n values and Eb/Es ratios can be fabricated by infiltrating alumina and zirconia with a low-modulus glass. Flexural tests show that graded alumina and zirconia with suitable values of these parameters exhibit superior load-bearing capacity, 20% to 50% higher than their homogeneous counterparts. Improving load-bearing capacity of ceramic materials could have broad impacts on biomedical, civil, structural, and an array of other engineering applications.

The phosphotriesterase-like lactonase (PLL) enzymes in the amidohydrolase superfamily hydrolyze various lactones and exhibit latent phosphotriesterase activities. These enzymes serve as attractive templates for in vitro evolution of neurotoxic organophosphates (OPs) with hydrolytic capabilities that can be used as bioremediation tools. Here, a thermostable PLL from Geobacillus kaustophilus HTA426 (GkaP) was targeted for joint laboratory evolution with the aim of enhancing its catalytic efficiency against OP pesticides. By a combination of site saturation mutagenesis and whole-gene error-prone PCR approaches, several improved variants were isolated. The most active variant, 26A8C, accumulated eight amino acid substitutions and demonstrated a 232-fold improvement over the wild-type enzyme in reactivity (kcat/Km) for the OP pesticide ethyl-paraoxon. Concomitantly, this variant showed a 767-fold decrease in lactonase activity with δ-decanolactone, imparting a specificity switch of 1.8 × 105-fold. 26A8C also exhibited high hydrolytic activities (19- to 497-fold) for several OP pesticides, including parathion, diazinon, and chlorpyrifos. Analysis of the mutagenesis sites on the GkaP structure revealed that most mutations are located in loop 8, which determines substrate specificity in the amidohydrolase superfamily. Molecular dynamics simulation shed light on why 26A8C lost its native lactonase activity and improved the promiscuous phosphotriesterase activity. These results permit us to obtain further insights into the divergent evolution of promiscuous enzymes and suggest that laboratory evolution of GkaP may lead to potential biological solutions for the efficient decontamination of neurotoxic OP compounds.

Background

Synovial sarcoma, X breakpoint 2 interacting protein (SSX2IP), which has been identified as an acute myeloid leukemia associated antigen, is a potential target for leukemia immunotherapy. In rodents, its homologous gene, ADIP, plays an important role in the regulation of cell adhesion and migration, underlying its potential role in promoting metastasis of other cancers.

Methods

To investigate the correlation between the expression level of SSX2IP and the clinicopathologic factors of hepatocellular carcinoma (HCC), 53 cases were studied by qPCR and statisted. To directly testing SSX2IP’s contribution to HCC in animal models, 45 nude mice were enrolled in peritoneal spreading and liver metastasis models. For the migration and invasion assays, cell culture experiments were performed using QCMTM 24-Well Colorimetric Migration Assay Kit and Cell Invasion Assay Kit (Millipore). Moreover we examined the influence of SSX2IP overexpression on the chemosensitivity of hepatocellular carcinoma cells to two most common chemotherapy drugs (5-Fu and CDDP) using Cell counting kit-8 (CCK-8). The chemotherapeutic drugs sensitivity was evaluated by IC50 parameter.

Results

Statistical analysis of clinical cases revealed that the SSX2IP high expression group had inclinations towards larger tumor size, more tumor thrombus and shorter survival period, implying a strong correlation between the expression level of SSX2IP and HCC tumorigenesis. Consistently in abdominal cavity metastasis and liver metastasis models of immune-deficient mice, SSX2IP was able to promote the metastasis of hepatoma cells. At the cytological level, SSX2IP stimulates the wound healing, metastasis and invasion of hepatoma cells, and reduces the sensitivity of hepatoma cells to 5-Fu and CDDP.

Conclusions

Our results showed that SSX2IP promotes the development and metastasis of hepatocellular carcinoma and contributes to the drug resistance of hepatoma cells, suggesting that SSX2IP is expected to become a new diagnostic and prognostic marker and a new target of the treatment of hepatocellular carcinoma.

Background

Although brachytherapy is one of the most effective ways to treat metastatic spinal tumor with little damage to surrounding healthy tissue, it may cause radiation myelopathy if an overdose occurs. Establishing a valuable animal model can help to find a method to overcome its complications. In the current study, we set up a banna mini-pig model to mimic percutaneous vertebroplasty with 125I seed implantation.

Methods

Percutaneous vertebroplasty (PVP) combined with interstitial implantation of 125I seeds, 125I seeds were transplanted into the vertebral body at the T13 level of the spine in banna mini-pigs. After raising them for up to eight months, the spinal cord and vertebral body were collected for pathological analysis.

Results

A potential animal model had been successfully established, no case of radiation myelopathy was found in any of the treated banna pigs, and no significant cellular impairment was noted by pathological analysis.

Conclusions

It proves that PVP with 125I brachytherapy is an effective method to treat metastasis spinal tumor, and that the banna mini-pig can be a suitable model to investigate the mechanism of brachytherapy complications.

We previously showed that electroacupuncture (EA) activates medulla-spinal serotonin-containing neurons. The present study investigated the effects of intrathecal 5,7-dihydroxytryptamine creatinine sulfate (5,7-DHT), a selective neurotoxin for serotonergic terminals, the 5-hydroxytryptamine 1A receptor (5-HT1AR) antagonist NAN-190 hydrobromide and the 5-HT2C receptor (5-HT2CR) antagonist SB-242,084 on EA anti-hyperalgesia. EA was given twice at acupoint GB30 after complete Freund’s adjuvant (CFA) injection into hind paw. CFA-induced hyperalgesia was measured by assessing hind paw withdrawal latency (PWL) to a noxious thermal stimulus 30 min post-EA. Serotonin depletion and the 5-HT1AR antagonist blocked EA anti-hyperalgesia; the 5-HT2CR antagonist did not. Immunohistochemical staining showed that spinal 5-HT1AR was expressed and that 5-HT2CR was absent in naive and CFA-injected animals 2.5 hr post-CFA. These results show a correlation between EA anti-hyperalgesia and receptor expression. Collectively, the data show that EA activates supraspinal serotonin neurons to release 5-HT, which acts on spinal 5-HT1AR to inhibit hyperalgesia.

Although studies demonstrate that electroacupuncture (EA) alleviates the sensory dimension of pain, they have not addressed EA’s effect on the affective dimension. An inflammatory pain rat model, produced by a complete Freund adjuvant (CFA) injection into the hind paw, was combined with a conditioned place avoidance (CPA) test to determine EA’s effects and its underpinning mechanism on the affective dimension of pain. CFA-injected rats showed place aversion, i.e. the affective dimension of pain, by spending less time in a pain-paired compartment after conditioning than before, while saline-injected rats did not. CFA rats given EA treatment at GB30 before a postconditioning test showed no aversion to the pain-paired compartment, indicating that EA inhibited the affective response. Intra-rostral anterior cingulate cortex (rACC) administration of a κ-, but not μ-opioid receptor antagonist, blocked EA action. These data demonstrate that EA activates opioid receptors in the rACC to inhibit the affective dimension of pain.

The association between promoter methylation status and survival was investigated in a large cohort of women with breast cancer, participants in the Long Island Breast Cancer Study Project. Archived tumor tissues (n=839) were collected from women diagnosed with a first primary invasive or in situ breast cancer in 1996-1997. Vital status was followed through the end of 2005 with a mean follow up time of 8 years. Promoter methylation of 8 breast cancer-related genes was assessed by MethyLight. The frequencies of methylation for HIN1, RASSF1A, DAPK1, GSTP1, CyclinD2, TWIST, CDH1 and RARβ were 62.9%, 85.2%, 14.1%, 27.8%, 19.6%, 15.3%, 5.8% and 27.6%, respectively. Since survival rates of in situ and invasive breast cancers are substantially different, survival analyses were conducted within 670 invasive cases with complete data on all genes. Age-adjusted Cox-proportional hazards models revealed that GSTP1, TWIST and RARβ methylation was significantly associated with higher breast cancer-specific mortality. Methylation of GSTP1 and RARβ were significantly associated with higher all-cause mortality. To investigate the relationship between the number of methylated genes and breast cancer-specific mortality, we included previously published MethyLight data on p16 and APC methylation status. Breast cancer-specific mortality increased in a dose-dependent manner with increasing number of methylated genes (Ptrend = 0.002), although confidence intervals were wide. Our results suggest that promoter methylation, particularly for a panel of genes, has the potential to be used as a biomarker for predicting prognosis in breast cancer.

Since the KCNB1 encoding Kv2.1 channel accounts for the majority of Kv currents modulating insulin secretion by pancreatic islet beta-cells, we postulated that KCNB1 is a plausible candidate gene for genetic variation contributing to the variable compensatory secretory function of beta-cells in type-2 diabetes (T2D). We conducted two studies, a case-control study and a cross-section study, to investigate the association of common single-nucleotide polymorphisms (SNPs) in KCNB1 with T2D and its linking traits. In the case-control study, we first examined the association of 20 tag SNPs of KCNB1 with T2D in a population with 226 T2D patients and non-diabetic subjects (screening study). We then identified the association in an enlarged population of 412 T2D patients and non-diabetic subjects (replication study). In the cross-sectional study, we investigated the linkage between the candidate SNP rs1051295 and T2D by comparing beta-cell function and insulin sensitivity among rs1051295 genotypes in a general population of 1051 subjects at fasting and after glucose loading (oral glucose tolerance tests, OGTT) in 84 fasting glucose impaired subjects, and several T2D-related traits. We found that among the 19 available tag SNPs, only the KCNB1 rs1051295 was associated with T2D (P = 0.027), with the rs1051295 TT genotype associated with an increased risk of T2D compared with genotypes CC (P = 0.009). At fasting, rs1051295 genotype TT was associated with a 9.8% reduction in insulin sensitivity compared to CC (P = 0.008); along with increased plasma triglycerides (TG) levels (TT/CC: P = 0.046) and increased waist/hip (W/H) ratio (TT/CC: P = 0.013; TT/TC: P = 0.002). OGTT confirmed that genotype TT exhibited reduced insulin sensitivity by 16.3% (P = 0.030) compared with genotype TC+CC in a fasting glucose impaired population. The KCNB1 rs1051295 genotype TT in the Chinese Han population is associated with decreased insulin sensitivity and increased plasma TG and W/H ratio, which together contribute to an increased risk for T2D.

AIM

To compare the retinal nerve fiber layer (RNFL) thickness and macular thickness in the amblyopic eye with that in the sound eye of children with hyperopic anisometropic amblyopia using optical coherence tomography (OCT).

METHODS

A prospective, nonrandom, intraindividual comparative cohort study includes 72 children with hyperopic anisometropic amblyopia in a single center. Macular thickness, macular foveola thickness, and peripapillary RNFL thickness were compared between the amblyopia eyes and the contralateral sound eyes.

RESULTS

There were 38 male and 34 female patients, with a mean age as 9.7±1.9 years (range, 5–16 years). Hyperopic was +3.62±1.16D (range +2.00D to +6.50D) in the amblyopic eyes, which was significantly higher in the control eyes with +0.76±0.90D (range 0D to +2.00D) (P < 0.01). The mean peripapillary RNFL thickness was 113.9±7.2µm and 109.2±6.9µm in the amblyopic eye and the normal eye, respectively, reaching statistical significance (P = 0.02). The mean macular foveola thickness was significantly thicker in the amblyopic eyes than the contralateral sound eyes (181.4±14.2µm vs 175.2±13.3µm, P < 0.01), but the 1mm, 3mm or 6mm macular thickness central macular thickness was not significantly different. Degree of anisometropia in the contralateral eyes was not significantly correlated with differences of peripapillary RNFL, macular foveola thickness or central macular thickness.

CONCLUSION

Eyes with hyperopic anisometropic amblyopia are found thicker macular foveola and peripapillary RNFL than the contralateral eyes in children.

Background

Xylanase is an important component of hemicellulase enzyme system. Since it plays an important role in the hydrolysis of hemicellulose into xylooligosaccharides (XOs), high thermostable xylanase has been the focus of much recent attention as powerful enzyme as well as in the field of biomass utilization.

Results

A xylanase gene (xyn10A) with 3,474 bp was cloned from the extremely thermophilic bacterium Thermotoga thermarum that encodes a protein containing 1,158 amino acid residues. Based on amino acid sequence homology, hydrophobic cluster and three dimensional structure analyses, it was attested that the xylanase belongs to the glycoside hydrolase (GH) families 10 with five carbohydrate binding domains. When the xylanase gene was cloned and expressed in Escherichia coli BL21 (DE3), the specific enzyme activity of xylanase produced by the recombinant strain was up to 145.8 U mg-1. The xylanase was optimally active at 95°C, pH 7.0. In addition, it exhibited high thermostability over broad range of pH 4.0-8.5 and temperature 55-90°C upon the addition of 5 mM Ca2+. Confirmed by Ion Chromatography System (ICS) analysis, the end products of the hydrolysis of beechwood xylan were xylose, xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose.

Conclusions

The xylanase from T. thermarum is one of the hyperthermophilic xylanases that exhibits high thermostability, and thus, is a suitable candidate for generating XOs from cellulosic materials such as agricultural and forestry residues for the uses as prebiotics and precursors for further preparation of furfural and other chemicals.

This study is aimed to investigate the effect of human resistin on myocyte differentiation and insulin resistance. The human resistin eukaryotic expression vector was stable transfected into C2C12 myocyte cells and was transiently transfected into COS7 cells. The effects of human resistin on cell proliferation, cell cycle, and myogenic differentiation of C2C12 cells were examined. Glucose uptake assays was performed on C2C12 myotubes by using [3H] 2-deoxy-D-glucose. The mRNA levels of insulin receptor (IR) and glucose transporter 4 (GLUT4) were evaluated by semiquantitative RT-PCR. Results showed by the C2C12 cells transfected with human resistin gene compared with that without transfecting gene are as follows: (1) cell proliferation was significantly promoted, (2) after inducing differentiation, the myotube's diameters and expression of desmin and myoglobin decreased, and (3) glucose uptake ratio was lowered and expression of IR and GLUT4 decreased. However, there was no significant difference in the glucose uptake ratio between C2C12 myotubes treated with a human resistin conditioned medium of COS7 cells and treated with control medium. These results suggest that maybe human resistin has not a direct role on insulin sensitivity of myocytes. However, maybe it impaired the insulin sensitivity of myocytes through suppressing myogenesis and stimulating proliferation of myoblasts.

After a tightly regulated developmental program in the thymus, “mature” single positive (SP) thymocytes leave the thymus and enter the periphery. These newly arrived recent thymic emigrants (RTEs) are phenotypically and functionally immature, and will complete a dynamic maturation in the peripheral lymphoid organs before being licensed to be resident naïve T cells. To study the early events occurring in the RTE maturation process, we identified the phenotype of CD4+ pre-RTEs, a population of CD4+ SP thymocytes that have acquired the thymus egress capability. Compared to peripheral naïve T cells, CD4+ pre-RTEs displayed superior survival capability in lymphoreplete mice and faster proliferation under lymphopenic condition. The differences in Bcl2/Bim expression and/or heightened IL-7 signaling pathway may account for the pre-RTEs’ better responsiveness to homeostatic signals. Qa2, the expression of which indicates the phenotypic maturation of SPs and RTEs, was found to be upregulated in CD4+ pre-RTEs in thymic perivascular space. Migratory dendritic cells that surround this region contribute to Qa2 expression in pre-RTEs. The dendritic cell-driven Qa2 induction of CD4+ pre-RTEs is independent of MHC class II and Aire molecules.

Objective

We conducted a prospective, randomized, open-label, multicenter study to compare busulfan plus fludarabine (BuFlu) with busulfan plus cyclophosphamide (BuCy) as the conditioning regimen in allogeneic hematopoietic stem cell transplantation (allo-HSCT) for acute myeloid leukemia (AML) in first complete remission (CR1).

Methods

Totally 108 AML-CR1 patients undergoing allo-HSCT were randomized into BuCy (busulfan 1.6 mg/kg, q12 hours, -7 ~ -4d; cyclophosphamide 60 mg/kg.d, -3 ~ -2d) or BuFlu (busulfan 1.6 mg/kg, q12 hours, -5 ~ -2d; fludarabine 30 mg/m2.d, -6 ~ -2d) group. Hematopoietic engraftment, regimen-related toxicity (RRT), graft-versus-host disease (GVHD), transplant related mortality (TRM), and overall survival were compared between the two groups.

Results

All patients achieved hematopoietic reconstitution except for two patients who died of RRT during conditioning. All patients obtained complete donor chimerism by day +30 post-transplantation. The incidence of total and III-IV RRT were 94.4% and 81.5% (P = 0.038), and 16.7% and 0.0% (P = 0.002), respectively, in BuCy and BuFlu group. With a median follow up of 609 (range, 3–2130) days after transplantation, the 5-year cumulative incidence of TRM were 18.8 ± 6.9% and 9.9 ± 6.3% (P = 0.104); the 5-year cumulative incidence of leukemia relapse were 16.5 ± 5.8% and 16.2 ± 5.3% (P = 0.943); the 5-year disease-free survival and overall survival were 67.4 ± 7.6% and 75.3 ± 7.2% (P = 0.315), and 72.3 ± 7.5% and 81.9 ± 7.0% (P = 0.177), respectively in BuCy and BuFlu group.

Conclusion

Compared with BuCy, BuFlu as a myeloablative condition regimen was associated with lower toxicities and comparable anti-leukemic activity in AML-CR1 patients undergoing allo-HSCT.

Background

This study aimed to evaluate the feasibility of intraarterial (IA) delivery and in vivo MR imaging of superparamagnetic iron oxide (SPIO)-labeled mesenchymal stem cells (MSCs) in a canine stroke model.

Methodology

MSCs harvested from beagles’ bone marrow were labeled with home-synthesized SPIO. Adult beagle dogs (n = 12) were subjected to left proximal middle cerebral artery (MCA) occlusion by autologous thrombus, followed by two-hour left internal carotid artery (ICA) occlusion with 5 French vertebral catheter. One week later, dogs were classified as three groups before transplantation: group A: complete MCA recanalization, group B: incomplete MCA recanalization, group C: no MCA recanalization. 3×106 labeled-MSCs were delivered through left ICA. Series in vivo MRI images were obtained before cell grafting, one and 24 hours after transplantation and weekly thereafter until four weeks. MRI findings were compared with histological studies at the time point of 24 hours and four weeks.

Principal Findings

Home-synthesized SPIO was useful to label MSCs without cell viability compromise. MSCs scattered widely in the left cerebral hemisphere in group A, while fewer grafted cells were observed in group B and no cell was detected in group C at one hour after transplantation. A larger infarction on the day of cell transplantation was associated with more grafted cells in the brain. Grafted MSCs could be tracked effectively by MRI within four weeks and were found in peri-infarction area by Prussian blue staining.

Conclusion

It is feasible of IA MSCs transplantation in a canine stroke model. Both the ipsilateral MCA condition and infarction volume before transplantation may affect the amount of grafted cells in target brain. In vivo MR imaging is useful for tracking IA delivered MSCs after SPIO labeling.

Pain has both sensory-discriminative and emotional-affective dimensions. Previous studies demonstrate that electroacupuncture (EA) alleviates the sensory dimension but do not address the affective. An inflammatory pain rat model, produced by a complete Freund adjuvant (CFA) injection into the hind paw, was combined with a conditioned place avoidance (CPA) test to determine whether EA inhibits spontaneous pain-induced affective response and, if so, to study the possibility that rostral anterior cingulate cortex (rACC) opioids underlie this effect. Male Sprague-Dawley rats (250–275g, Harlan) were used. The rats showed place aversion (i.e. affective pain) by spending less time in a pain-paired compartment after conditioning than during a preconditioning test. Systemic non-analgesic morphine (0.5 and 1.0 mg/ kg, i.p.) inhibited the affective reaction, suggesting that the affective dimension is underpinned by mechanisms different from those of the sensory dimension of pain. Morphine at 0.5 and at 1 mg/kg did not induce reward. Rats given EA treatment before pain-paired conditioning at GB 30 showed no aversion to the pain-paired compartment, indicating that EA inhibited the affective dimension. EA treatment did not produce reward or aversive effect. Intra-rACC administration of D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP), a selective mu opioid receptor antagonist, but not norbinaltorphimine (nor-BNI), a selective kappa opioid receptor antagonist, blocked EA inhibition of the affective dimension. These data demonstrate that EA activates opioid receptors in the rACC to inhibit pain-induced affective responses and that EA may be an effective therapy for both the sensory-discriminative and the affective dimensions of pain.

OBJECTIVE:

Ischemia reperfusion injury is partly responsible for the high mortality associated with induced myocardial injury and the reduction in the full benefit of myocardial reperfusion. Remote ischemic preconditioning, perconditioning, and postconditioning have all been shown to be cardioprotective. However, it is still unknown which one is the most beneficial. To examine this issue, we used adult male Wistar rat ischemia reperfusion models to compare the cardioprotective effect of these three approaches applied on double-sided hind limbs.

METHODS:

The rats were randomly distributed to the following five groups: sham, ischemia reperfusion, remote preconditioning, remote perconditioning, and remote post-conditioning. The ischemia/reperfusion model was established by sternotomy followed by a 30-min ligation of the left coronary artery and a subsequent 3-h reperfusion. Remote conditioning was induced with three 5-min ischemia/5-min reperfusion cycles of the double-sided hind limbs using a tourniquet.

RESULTS:

A lower early reperfusion arrhythmia score (1.50±0.97) was found in the rats treated with remote perconditioning compared to those in the ischemia reperfusion group (2.33±0.71). Meanwhile, reduced infarct size was also observed (15.27±5.19% in remote perconditioning, 14.53±3.45% in remote preconditioning, and 19.84±5.85% in remote post-conditioning vs. 34.47±7.13% in ischemia reperfusion, p<0.05), as well as higher expression levels of the apoptosis-relevant protein Bcl-2/Bax following global (ischemia/reperfusion) injury in in vivo rat heart models (1.255±0.053 in remote perconditioning, 1.463±0.290 in remote preconditioning, and 1.461±0.541 in remote post-conditioning vs. 1.003±0.159 in ischemia reperfusion, p<0.05).

CONCLUSION:

Three remote conditioning strategies implemented with episodes of double-sided hind limb ischemia/reperfusion have similar therapeutic potential for cardiac ischemia/reperfusion injury, and remote perconditioning has a greater ability to prevent reperfusion arrhythmia.

Dark-grown seedlings exhibit skotomorphogenic development. Genetic and molecular evidence indicates that a quartet of Arabidopsis Phytochrome (phy)-Interacting bHLH Factors (PIF1, 3, 4, and 5) are critically necessary to maintaining this developmental state and that light activation of phy induces a switch to photomorphogenic development by inducing rapid degradation of the PIFs. Here, using integrated ChIP–seq and RNA–seq analyses, we have identified genes that are direct targets of PIF3 transcriptional regulation, exerted by sequence-specific binding to G-box (CACGTG) or PBE-box (CACATG) motifs in the target promoters genome-wide. In addition, expression analysis of selected genes in this set, in all triple pif-mutant combinations, provides evidence that the PIF quartet members collaborate to generate an expression pattern that is the product of a mosaic of differential transcriptional responsiveness of individual genes to the different PIFs and of differential regulatory activity of individual PIFs toward the different genes. Together with prior evidence that all four PIFs can bind to G-boxes, the data suggest that this collective activity may be exerted via shared occupancy of binding sites in target promoters.

Author Summary

An important issue in understanding mechanisms of eukaryotic transcriptional regulation is how members of large transcription-factor families, with conserved DNA–binding domains (such as the 162-member Arabidopsis bHLH family), discriminate between target genes. However, the specific question of whether, and to what extent, closely related sub-family members, with potential overlapping functional redundancy (like the quartet of Phytochrome (phy)-Interacting bHLH transcription Factors (PIF1, 3, 4, and 5) studied here), share regulation of target genes through shared binding to promoter-localized consensus motifs does not appear to have been widely investigated. Here, using ChIP–seq analysis, we have identified genes that bind PIF3 to conserved, sequence-specific sites in their promoters; and, using RNA–seq, we have identified those genes displaying altered expression in various pif mutants. Integration of these data identifies those genes that are likely direct targets of transcriptional regulation by PIF3. Our data suggest that the PIF quartet members share directly in transcriptional activation of numerous target genes, potentially via redundant promoter occupancy, in a manner that varies quantitatively from gene to gene. This finding suggests that these PIFs function collectively as a signaling hub, selectively partitioning common upstream signals from light-activated phys at the transcriptional-network interface.

Prostate cancer is the most frequently diagnosed cancer and the second leading cause of cancer deaths in men today. Although virus-based gene therapy is a promising strategy to combat advanced prostate cancer, its current effectiveness is limited partially due to inefficient cellular transduction in vivo. To overcome this obstacle, conditional oncolytic viruses (such as conditional replication adenovirus (CRAD)) are developed to specifically target prostate without (or with minimal) systemic toxicity due to viral self-replication. In this study, we have analyzed and compared three prostate-specific promoters (PSA, probasin, and MMTV LTR) for their specificity and activity both in vitro and in vivo. Both mice model with xenograft prostate tumor model and canine model were used. The best PSP was selected to construct a prostate-specific oncolytic adenovirus (CRAD) by controlling the adenoviral E1 region. The efficacy and specificity of CRAD on prostate cancer cells were examined in cell culture and animal models.

The paper is aimed to investigate the toxicity of nano-TiO2 and its potential harmful impact on human health using meta-analysis of in vitro and short-time animal studies. Data were retrieved according to included and excluded criteria from 1994 to 2011. The combined toxic effects of nano-TiO2 were calculated by the different endpoints by cell and animal models. From analysis of the experimental studies, more than 50% showed positive statistical significance except the apoptosis group, and the cytotoxicity was in a dose-dependent but was not clear in size-dependent manner. Nano-TiO2 was detained in several important organs including the liver, spleen, kidney, and brain after entering the blood through different exposure routes, but the coefficient of the target organs was altered slightly from animal models. It is possible that nano-TiO2 can induce cell damage related to exposure size and dose. Further studies will be needed to demonstrate that nanoparticles have toxic effects on human body, especially in epidemiological studies.

Alpha-hemolysin (α-HL) is a self-assembling, channel-forming toxin produced by most Staphylococcus aureus strains as a 33.2-kDa soluble monomer. Upon binding to a susceptible cell membrane, the monomer self-assembles to form a 232.4-kDa heptamer that ultimately causes host cell lysis and death. Consequently, α-HL plays a significant role in the pathogenesis of S. aureus infections, such as pneumonia, mastitis, keratitis and arthritis. In this paper, experimental studies show that oroxylin A (ORO), a natural compound without anti-S. aureus activity, can inhibit the hemolytic activity of α-HL. Molecular dynamics simulations, free energy calculations, and mutagenesis assays were performed to understand the formation of the α-HL-ORO complex. This combined approach revealed that the catalytic mechanism of inhibition involves the direct binding of ORO to α-HL, which blocks the conformational transition of the critical “Loop” region of the α-HL protein thereby inhibiting its hemolytic activity. This mechanism was confirmed by experimental data obtained from a deoxycholate-induced oligomerization assay. It was also found that, in a co-culture system with S. aureus and human alveolar epithelial (A549) cells, ORO could protect against α-HL-mediated injury. These findings indicate that ORO hinders the lytic activity of α-HL through a novel mechanism, which should facilitate the design of new and more effective antibacterial agents against S. aureus.

Author Summary

The mechanism controlling protein-ligand interactions is one of the most important processes in rational drug design. X-ray crystallography is a traditional tool used to investigate the interaction of ligands and proteins in a complex. However, protein crystallography is inefficient, and the development of crystal technology and research remains unequally distributed. Thus, it seems impractical to explore the structure of the α-hemolysin-ORO monomer complex by crystallography. Therefore, we used molecular dynamics simulations to investigate the receptor-ligand interaction in the α-HL-ORO monomer complex. In this study, we found that oroxylin A (ORO), a natural compound with little anti-S. aureus activity, can inhibit the hemolytic activity of α-HL at low concentrations. Through molecular docking and molecular dynamics simulations, we determined the potential binding mode of the protein-ligand interaction. The data revealed that ORO directly binds to α-HL, an interaction that blacks the conformational transition of the critical “Loop” region in α-HL and thus prevents the formation of the α-HL heptameric transmembrane pore, which ultimately inhibits the hemolytic activity of α-HL. This mechanism was confirmed by experimental data. Furthermore, we demonstrated that ORO could protect against α-HL-mediated injury in human alveolar epithelial (A549) cells.

The aim of this study was to investigate whether abnormal expression of matrix metalloproteinase (MMP)-9/tissue inhibitors of MMPs (TIMP)-1 and B cell lymphoma 2 (BCL-2)/BCL-2-associated X protein (BAX) are correlated with the characteristic accelerated fibrosis and apoptosis during ageing and in atrial fibrillation (AF). Four groups of dogs were studied: adult dogs in sinus rhythm (SR), aged dogs in SR, adult dogs with AF induced by rapid atrial pacing and aged dogs with AF induced by rapid atrial pacing. The mRNA and protein expression levels of the target gene in the left atrium were measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. Pathohistological and ultrastructural changes were assessed by light and electron microscopy. The apoptotic indices of myocytes were detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL). The mRNA and protein expression levels of MMP-9 and BAX and those of TIMP-1 and BCL-2 were significantly upregulated and down-regulated, respectively, in the aged groups compared with the adult groups. Compared with the control groups, the adult and aged groups with AF exhibited significantly increased mRNA and protein expression levels of MMP-9 and BAX and decreased expression levels of TIMP-1 and BCL-2. Samples of atrial tissue demonstrated abnormal pathohistological and ultrastructural changes, accelerated fibrosis and apoptosis. MMP-9/TIMP-1 and BCL-2/BAX hold potential for use as substrates conducive to AF and their abnormal expression plays a major role in structural remodeling of the atrium.

Background

In the differentiation of mouse embryonic stem (ES) cells into neurons using the 5-stage method, cells in stage 4 are in general used as neural progenitors (NPs) because of their ability to give rise to neurons. The choice of stage 4 raises several questions about neural progenitors such as the type of cell types that are specifically considered to be neural progenitors, the exact time when these progenitors become capable of neurogenesis and whether neurogenesis is an independent and autonomous process or the result of an interaction between NP cells and the surrounding cells.

Methodology/Principal Findings

In this study, we found that the confluent monolayer cells and neural sphere like cell clusters both appeared in the culture of the first 14 days and the subsequent 6 weeks. However, only the sphere cells are neural progenitors that give rise to neurons and astrocytes. The NP cells require 14 days to mature into neural lineages fully capable of differentiation. We also found that although the confluent monolayer cells do not undergo neurogenesis, they play a crucial role in the growth, differentiation, and apoptosis of the sphere cells, during the first 14 days and long term culture, by secreted factors and direct cell to cell contact.

Conclusions/Significance

The sphere cells in stage 4 are more committed to developing into neural progenitors than monolayer cells. Interaction between the monolayer cells and sphere cells is important in the development of stage 4 cell characteristics.

Background

Outcrossing is known to carry genetic advantages in comparison with inbreeding. In many cases, flowering plants develop a self-incompatibility mechanism, along with a floral component adaptation mechanism, to avoid self-pollination and to promote outbreeding. Orchids commonly have a lip in their flower that functions as the a visiting plate for insect pollinators. Aside from the lip, however, many species (including Coelogyne rigida) have sheaths around the axis of inflorescence. The function of these sheaths remains unknown, and has long been a puzzle to researchers.

Methodology/Principal Findings

We investigated the function of these sheaths in relation to the lip and the pollinators, as well as their role in the modes of pollination and reproduction of Coelogyne rigida in 30 flowering populations of orchids in the limestone area of Southeast Yunnan, China. We found that self-incompatible C. rigida developed specialized bird perches around the basal axis of inflorescence to attract sunbirds and to complement their behavioral tendency to change foraging locations frequently. This self-incompatibility mechanism operates separately from the floral component adaptation mechanism. This mechanism thus prevents bees from repeatedly visiting the floral lip of the same plant which, in turn, results in autogamy. In this way, instead of preventing autogamy, C. rigida responds to these negative effects through a highly efficient cross-pollination method that successfully transfers pollen to different plants.

Conclusions

The proposed method ensures reproductive success, while offsetting the infertile self-pollination by insects, thereby reducing mating costs and addressing the lack of cross-pollination. The adaptation provides a novel and striking example of structural adaptation that promotes cross-pollination in angiosperms.