Background and Purpose

Recently, we invented a computerized endoscopic balloon manometry (CEBM) to measure variceal pressure (VP) in cirrhotic patient. The purpose of this study was to evaluate the reliability and feasibility of this method, and whether this technique provided further information to pharmacological therapy.

Patients and Methods

VP measurements were performed in 83 cirrhotic patients and compared with HVPG as well as endoscopic bleeding risk parameters. Furthermore, VP was assessed before and during propranolol therapy in 30 patients without previous bleeding.

Results

VP measurements were successful in 96% (83/86) of all patients. Of the 83 patients, the VP correlated closely with the HVPG (P<0.001). The presence of red colour signs and the size of varices were strongly associated with VP. Patients with previous bleeding had higher VP than those who had not yet experienced bleeding. In univariate analysis, the level of VP, the size of varices, and red color signs predicted a higher risk of bleeding. The multiple logistic regression model revealed that VP was the major risk factor for bleeding. In 30 patients receiving propranolol, VP significantly decreased from 21.1±3.5 mmHg before therapy to 18.1±3.3 mmHg after 3 months and to 16.3±4.0 mmHg after 6 months. Comparing the mean decrease in VP with that in hepatic venous pressure gradient (HVPG), the decrease in VP was more obvious than HVPG response to propranolol.

Conclusions

This study showed that CEBM is safe and practical to assess VP in cirrhotic patient. It has the potential to be used as a clinical method to assess the risk of variceal bleeding and the effects of pharmacological therapy.

Trial registration

Effect of vasoactive drugs on esophageal variceal hemodynamics in patients with portal hypertension. Chinese Clinical Trial Registry –TRC-08000252.

SUMMARY

Plasticity in growth and reproductive behavior is found in many vertebrate species, but is common in male teleost fish. Typically, “bourgeois” males are considerably larger and defend breeding territories while “parasitic” variants are small and use opportunistic breeding strategies. The P locus mediates this phenotypic variation in Xipophorus and encodes variant alleles of the melanocortin-4 receptor (MC4R). However, deletion of the MC4R has modest effects on somatic growth and reproduction in mammals, suggesting a fundamental difference in the neuroendocrine function of central melanocortin signaling in teleosts. Here we show in a teleost that the hypothalamic proopiomelanocortin and AgRP neurons are hypophysiotropic, projecting to the pituitary to coordinately regulate multiple pituitary hormones. Indeed, AgRP-mediated suppression of MC4R appears essential for early larval growth. This identifies the mechanism by which the central melanocortin system coordinately regulates growth and reproduction in teleosts, and suggests it is an important anatomical substrate for evolutionary adaptation.

Chemotherapy regiments have been widely used in the treatment of a variety of human malignancies including hepatocellular carcinoma (HCC). A major cause of failure in chemotherapy is drug resistance of cancer cells. Resistance to doxorubicin (DOX) is a common and representative obstacle to treat cancer effectively. Individual microRNA (miRNA) has been introduced in the evolution of DOX resistance in HCC in recent studies. However, a global and systematic assessment of the miRNA expression profiles contributing to DOX resistance is still lacking. In the present study, we applied high-throughput Illumina sequencing to comprehensively characterize miRNA expression profiles in both human HCC cell line (HepG2) and its DOX-resistant counterpart (HepG2/DOX). A total of 269 known miRNAs were significantly differentially expressed, of which 23 were up-regulated and 246 were down-regulated in HepG2/DOX cells, indicating that part of them might be involved in the development of DOX resistance. In addition, we have identified 9 and 13 novel miRNAs up- and down-expressed significantly in HepG2/DOX cells, respectively. miRNA profiling was then validated by quantitative real-time PCR for selected miRNAs, including 22 known miRNAs and 6 novel miRNAs. Furthermore, we predicted the putative target genes for the deregulated miRNAs in the samples. Function annotation implied that these selected miRNAs affected many target genes mainly involved in MAPK signaling pathway. This study provides us a general description of miRNA expression profiling, which is helpful to find potential miRNAs for adjunct treatment to overcome DOX resistance in future HCC chemotherapy.

In fission yeast, discrete steps in mRNA maturation and synthesis depend on a complex containing the 5′-cap methyltransferase Pcm1 and Cdk9, which phosphorylates the RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) and the processivity factor Spt5 to promote transcript elongation. Here we show that a Cdk9 carboxyl-terminal extension, distinct from the catalytic domain, mediates binding to both Pcm1 and the Pol II CTD. Removal of this segment diminishes Cdk9/Pcm1 chromatin recruitment and Spt5 phosphorylation in vivo and leads to slow growth and hypersensitivity to cold temperature, nutrient limitation, and the IMP dehydrogenase inhibitor mycophenolic acid (MPA). These phenotypes, and the Spt5 phosphorylation defect, are suppressed by Pcm1 overproduction, suggesting that normal transcript elongation and gene expression depend on physical linkage between Cdk9 and Pcm1. The extension is dispensable, however, for recognition of CTD substrates “primed” by Mcs6 (Cdk7). On defined peptide substrates in vitro, Cdk9 prefers CTD repeats phosphorylated at Ser7 over unmodified repeats. In vivo, Ser7 phosphorylation depends on Mcs6 activity, suggesting a conserved mechanism, independent of chromatin recruitment, to order transcriptional CDK functions. Therefore, fission yeast Cdk9 comprises a catalytic domain sufficient for primed substrate recognition and a multivalent recruitment module that couples transcription with capping.

Several operative approaches are available at present for the exposure and fixation of distal fibular fractures combined with posterior malleolus fractures. The present study was designed to study the anatomical characteristics of the distal fibula and to thereby evaluate the advantages and limitations of various operative approaches, as well as their indications for specific conditions. Ten leg specimens from below the knee joint were dissected using posterior, lateral and posterolateral approaches to the fibula. The adjacent vulnerable structures, including nerves, blood vessels, tendons and ligaments, were carefully examined and their distances from the posterior malleolus were recorded. The distance was 7.2±4.1 mm between the sural nerve and the posterior section of the fibula, 79.2±23.5 mm between the lateral malleolus tip and the point where the shape changes in the lower fibula and 66.4±17.4 mm between the lateral malleolus and the jointed tendon of the peroneal and flexor hallux longus muscles. The widest anteroposterior diameter of the distal fibula was 27.3±3.5 mm. Various approaches have certain advantages and limitations when these anatomical factors are taken into account. The choice should be based on the height of the fibular fracture line, the type of posterior malleolus fracture, the effect of the fracture on the stability of the ankle joint and the materials used for internal fixation.

Understanding magnetism and electron correlation in many unconventional superconductors is essential to explore mechanism of superconductivity. In this work, we perform a systematic numerical study of the magnetic and pair binding properties in recently discovered polycyclic aromatic hydrocarbon (PAH) superconductors including alkali-metal-doped picene, coronene, phenanthrene, and dibenzopentacene. The π-electrons on the carbon atoms of a single molecule are modelled by the one-orbital Hubbard model, and the energy difference between carbon atoms with and without hydrogen bonds is taking into account. We demonstrate that the spin polarized ground state is realized for charged molecules in the physical parameter regions, which provides a reasonable explanation of local spins observed in PAHs. In alkali-metal-doped dibenzopentacene, our results show that electron correlation may produce an effective attraction between electrons for the charged molecule with one or three added electrons.

microRNAs (miRNAs) are small noncoding RNAs that regulate genes and contribute to many kinds of human diseases, including cancer. Two miRNAs, miR-511 and miR-1297, were investigated for a possible role in adenocarcinoma based on predicted binding sites for the TRIB2 oncogene by microRNA analysis software, and the pcDNA-GFP-TRIB2–3′UTR vector was constructed to investigate the interaction between TRIB2 and miR-511/1297 in the adenocarcinoma cell line A549. Green fluorescent protein (GFP) expression was estimated by fluorescence microscopy and flow cytometry after A549 cells were co-transfected with miR-511 (or miR-1297) and pcDNA-GFP-TRIB2–3′UTR vector. The expression of GFP in the miR-511- and miR-1297-treated cells was significantly downregulated in contrast with the negative-control (NC) miRNA-treated cells. The decreased expression of TRIB2 was further detected after miR-511 (or miR-1297) treatment by western blotting. The MTT test showed inhibition of A549 cell proliferation and Annexin V-FITC/PI dual staining showed increased apoptosis in the miR-511- and miR-1297-treated cells compared to the NC cultures. A transcription factor downstream of TRIB2, the CCAAT/enhancer-binding protein alpha (C/EBPα), was expression at higher levels after miR-511 (or miR-1297) decreasing TRIB2 expression. Our results illustrate that miR-511 and miR-1297 act as tumor suppressor genes, which could suppress A549 cell proliferation in vitro and in vivo by suppressing TRIB2 and further increasing C/EBPα expression.

Background

The hypersensitive response (HR) system of Chenopodium spp. confers broad-spectrum virus resistance. However, little knowledge exists at the genomic level for Chenopodium, thus impeding the advanced molecular research of this attractive feature. Hence, we took advantage of RNA-seq to survey the foliar transcriptome of C. amaranticolor, a Chenopodium species widely used as laboratory indicator for pathogenic viruses, in order to facilitate the characterization of the HR-type of virus resistance.

Methodology and Principal Findings

Using Illumina HiSeq™ 2000 platform, we obtained 39,868,984 reads with 3,588,208,560 bp, which were assembled into 112,452 unigenes (3,847 clusters and 108,605 singletons). BlastX search against the NCBI NR database identified 61,698 sequences with a cut-off E-value above 10−5. Assembled sequences were annotated with gene descriptions, GO, COG and KEGG terms, respectively. A total number of 738 resistance gene analogs (RGAs) and homology sequences of 6 key signaling proteins within the R proteins-directed signaling pathway were identified. Based on this transcriptome data, we investigated the gene expression profiles over the stage of HR induced by Tobacco mosaic virus and Cucumber mosaic virus by using digital gene expression analysis. Numerous candidate genes specifically or commonly regulated by these two distinct viruses at early and late stages of the HR were identified, and the dynamic changes of the differently expressed genes enriched in the pathway of plant-pathogen interaction were particularly emphasized.

Conclusions

To our knowledge, this study is the first description of the genetic makeup of C. amaranticolor, providing deep insight into the comprehensive gene expression information at transcriptional level in this species. The 738 RGAs as well as the differentially regulated genes, particularly the common genes regulated by both TMV and CMV, are suitable candidates which merit further functional characterization to dissect the molecular mechanisms and regulatory pathways of the HR-type of virus resistance in Chenopodium.

This study reveals the basis for how temporal phosphoregulation of Orm protein controls sphingolipid production in response to stress. Orm protein phosphorylation is highly responsive to sphingoid bases, and Ypk1 protein kinase transmits heat stress signals to the sphingolipid biosynthesis pathway via Orm phosphorylation.

Sphingoid intermediates accumulate in response to a variety of stresses, including heat, and trigger cellular responses. However, the mechanism by which stress affects sphingolipid biosynthesis has yet to be identified. Recent studies in yeast suggest that sphingolipid biosynthesis is regulated through phosphorylation of the Orm proteins, which in humans are potential risk factors for childhood asthma. Here we demonstrate that Orm phosphorylation status is highly responsive to sphingoid bases. We also demonstrate, by monitoring temporal changes in Orm phosphorylation and sphingoid base production in cells inhibited for yeast protein kinase 1 (Ypk1) activity, that Ypk1 transmits heat stress signals to the sphingolipid biosynthesis pathway via Orm phosphorylation. Our data indicate that heat-induced sphingolipid biosynthesis in turn triggers Orm protein dephosphorylation, making the induction transient. We identified Cdc55–protein phosphatase 2A (PP2A) as a key phosphatase that counteracts Ypk1 activity in Orm-mediated sphingolipid biosynthesis regulation. In total, our study reveals a mechanism through which the conserved Pkh-Ypk kinase cascade and Cdc55-PP2A facilitate rapid, transient sphingolipid production in response to heat stress through Orm protein phosphoregulation. We propose that this mechanism serves as the basis for how Orm phosphoregulation controls sphingolipid biosynthesis in response to stress in a kinetically coupled manner.

The cyclin-dependent kinases (CDKs) that promote cell-cycle progression are targets for negative regulation by signals from damaged or unreplicated DNA, but also play active roles in response to DNA lesions. The requirement for activity in the face of DNA damage implies that there are mechanisms to insulate certain CDKs from checkpoint inhibition. It remains difficult, however, to assign precise functions to specific CDKs in protecting genomic integrity. In mammals, Cdk2 is active throughout S and G2 phases, but Cdk2 protein is dispensable for survival, owing to compensation by other CDKs. That plasticity obscured a requirement for Cdk2 activity in proliferation of human cells, which we uncovered by replacement of wild-type Cdk2 with a mutant version sensitized to inhibition by bulky adenine analogs. Here we show that transient, selective inhibition of analog-sensitive (AS) Cdk2 after exposure to ionizing radiation (IR) enhances cell-killing. In extracts supplemented with an ATP analog used preferentially by AS kinases, Cdk2as phosphorylated the Nijmegen Breakage Syndrome gene product Nbs1—a component of the conserved Mre11-Rad50-Nbs1 complex required for normal DNA damage repair and checkpoint signaling—dependent on a consensus CDK recognition site at Ser432. In vivo, selective inhibition of Cdk2 delayed and diminished Nbs1-Ser432 phosphorylation during S phase, and mutation of Ser432 to Ala or Asp increased IR–sensitivity. Therefore, by chemical genetics, we uncovered both a non-redundant requirement for Cdk2 activity in response to DNA damage and a specific target of Cdk2 within the DNA repair machinery.

Author Summary

Multiple cyclin-dependent kinases (CDKs) control human cell proliferation, but it remains unclear how functions of different CDKs are coordinated during unperturbed cell division or after dividing cells incur DNA damage. DNA lesions activate checkpoint signaling pathways to inhibit CDK activity, arrest the cell division cycle, and thus prevent loss of genetic information; but an effective response to damage also requires CDK activity to modify components of repair and checkpoint pathways. We took a chemical-genetic approach to ask if a specific CDK, Cdk2, played a specialized, non-redundant role in protecting genomic integrity of human cells. By sensitizing Cdk2 to chemical inhibition, we were able to detect a specific requirement for its catalytic activity in survival of cells after exposure to ionizing radiation (IR). We identified Nbs1, product of the gene mutated in the cancer-predisposing Nijmegen Breakage Syndrome, as a Cdk2 substrate and showed that mutant forms of Nbs1 that cannot be modified by Cdk2 are defective in protecting cells from death due to IR–induced DNA damage. Therefore, our work defines a DNA damage response pathway that depends on catalytic activity of a specific CDK in human cells and suggests a mechanism to promote efficient repair without triggering inappropriate cell division.

Transcript elongation by RNA polymerase II (RNAPII) is accompanied by conserved patterns of histone modification. Whereas histone modifications have established roles in transcription initiation, their functions during elongation are not understood. Mono-ubiquitylation of histone H2B (H2Bub1) plays a key role in coordinating co-transcriptional histone modification by promoting site-specific methylation of histone H3. H2Bub1 also regulates gene expression through an unidentified, methylation-independent mechanism. Here we reveal bidirectional communication between H2Bub1 and Cdk9, the ortholog of metazoan positive transcription elongation factor b (P-TEFb), in the fission yeast Schizosaccharomyces pombe. Chemical and classical genetic analyses indicate that lowering Cdk9 activity or preventing phosphorylation of its substrate, the transcription processivity factor Spt5, reduces H2Bub1 in vivo. Conversely, mutations in the H2Bub1 pathway impair Cdk9 recruitment to chromatin and decrease Spt5 phosphorylation. Moreover, an Spt5 phosphorylation-site mutation, combined with deletion of the histone H3 Lys4 methyltransferase Set1, phenocopies morphologic and growth defects due to H2Bub1 loss, suggesting independent, partially redundant roles for Cdk9 and Set1 downstream of H2Bub1. Surprisingly, mutation of the histone H2B ubiquitin-acceptor residue relaxes the Cdk9 activity requirement in vivo, and cdk9 mutations suppress cell-morphology defects in H2Bub1-deficient strains. Genome-wide analyses by chromatin immunoprecipitation also demonstrate opposing effects of Cdk9 and H2Bub1 on distribution of transcribing RNAPII. Therefore, whereas mutual dependence of H2Bub1 and Spt5 phosphorylation indicates positive feedback, mutual suppression by cdk9 and H2Bub1-pathway mutations suggests antagonistic functions that must be kept in balance to regulate elongation. Loss of H2Bub1 disrupts that balance and leads to deranged gene expression and aberrant cell morphologies, revealing a novel function of a conserved, co-transcriptional histone modification.

Author Summary

Modification of histone proteins is an important transcriptional regulatory mechanism in eukaryotic cells. Although various histone modifications are found primarily within the coding regions of transcribed genes, how they influence transcription elongation remains unclear. Among these modifications is mono-ubiquitylation of histone H2B (H2Bub1), which is needed for co-transcriptional methylation of histone H3 at specific sites. Here we show that H2Bub1 and Cdk9, the kinase component of positive transcription elongation factor b (P-TEFb), are jointly regulated by a positive feedback loop: Cdk9 activity is needed for co-transcriptional H2Bub1, and H2Bub1 in turn stimulates Cdk9 activity toward one of its major substrates, the conserved elongation factor Spt5. We provide genetic evidence that the combined action of H2Bub1 on Spt5 phosphorylation and histone methylation accounts for the gene-regulatory effects of this modification. Surprisingly, our genetic and genome-wide studies indicate that P-TEFb and H2Bub1 act in opposition on elongating RNA polymerase. We suggest that the positive feedback linking P-TEFb and H2Bub1 helps to maintain a balance between their opposing actions. These results highlight a novel regulatory role for a conserved histone modification during transcription elongation.

Introduction

Spontaneous osteonecrosis of the navicular bone in adults is a rare entity, known as Müller-Weiss syndrome. We report here on our experience with six patients with Müller-Weiss syndrome accompanied by flatfoot deformity, but on a literature search found no reports on this phenomenon. Because the natural history and treatment are controversial, an understanding of how to manage this deformity may be helpful for surgeons when choosing the most appropriate operative procedure.

Case presentation

Six patients (five women, one man; average age, 54 years) with flatfoot caused by osteonecrosis of the navicular bone were followed up between January 2005 and December 2008 (mean follow-up period, 23.2 months). Conservative treatment, such as physical therapy, and non-steroidal anti-inflammatory drugs were used, but failed. Physical examinations revealed flattening of the medial arch of the involved foot and mild tenderness at the mid-tarsal joint. Weight-bearing X-rays (anterior-posterior and lateral views), computed tomography, and MRI scans were performed for each case. Talonavicular joint arthrodesis was performed in cases of single talonavicular joint arthritis. Triple arthrodesis was performed in cases of triple joint arthritis to reconstruct the medial arch. Clinical outcomes were assessed using the American Orthopaedic Foot and Ankle Society ankle-hindfoot scale; the scores were 63.0 pre-operatively and 89.8 post-operatively. All patients developed bony fusion.

Conclusions

The reason for the development of flatfoot in patients with Müller-Weiss syndrome is unknown. Surgical treatment may achieve favorable outcomes in terms of deformity correction, pain relief, and functional restoration. The choice of operative procedure may differ in patients with both flatfoot and posterior tibial tendon dysfunction.

Human quiescin-sulfhydryl oxidase 1 isoform b (HsQSOX1b) is a highly efficient, multiple-domain enzyme that directly inserts disulfide bonds into client protein. However, previous studies have focused mainly on the catalytic activity of the whole protein rather than its domain structure. In this research, we dissected the structure and function of HsQSOX1b and explored its mechanism as a highly efficient sulfhydryl oxidase by analyzing the truncated variants. The results showed that the first HsQSOX1b thioredoxin domain was essential for thiol oxidase activity. The smallest active fragment (SAQ) was identified to consist of a helix-rich region (HRR) and an essential for respiration and viability/augmenter of liver regeneration (ERV/ALR) domain, which remained highly active to oxidize an artificial non-thiol substrate but not small molecular and protein thiols. Our study clearly demonstrated that SAQ is a highly efficient oxidative engine, which shows high efficiency in the de novo disulfide formation and oxygen reduction and that this more efficient oxidative engine is necessary for the highly efficient catalysis of QSOXs compared to Erv1 and Erv2. This study will help address the roles of different HsQSOX1b domains in de novo disulfide formation and encourage the engineering of more efficient QSOX variants for the in vitro folding of disulfide-containing proteins.

Summary

Multiple cyclin-dependent kinases (CDKs) control eukaryotic cell division, but assigning specific functions to individual CDKs remains a challenge. During the mammalian cell cycle, Cdk2 forms active complexes before Cdk1, but lack of Cdk2 protein does not block cell-cycle progression. To detect requirements and define functions for Cdk2 activity in human cells when normal expression levels are preserved, and non-physiologic compensation by other CDKs is prevented, we replaced the wild-type kinase with a version sensitized to specific inhibition by bulky adenine analogs. The sensitizing mutation also impaired a non-catalytic function of Cdk2 in restricting assembly of cyclin A with Cdk1, but this defect could be corrected by both inhibitory and non-inhibitory analogs. This allowed either chemical rescue or selective antagonism of Cdk2 activity in vivo, to uncover a requirement in cell proliferation, and non-redundant, rate-limiting roles in restriction point passage and S-phase entry.

Background

As a marker of Helicobacter pylori, Cytotoxin-associated gene A (cagA) has been revealed to be the major virulence factor causing gastroduodenal diseases. However, the molecular mechanisms that underlie the development of different gastroduodenal diseases caused by cagA-positive H. pylori infection remain unknown. Current studies are limited to the evaluation of the correlation between diseases and the number of Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs in the CagA strain. To further understand the relationship between CagA sequence and its virulence to gastric cancer, we proposed a systematic entropy-based approach to identify the cancer-related residues in the intervening regions of CagA and employed a supervised machine learning method for cancer and non-cancer cases classification.

Methodology

An entropy-based calculation was used to detect key residues of CagA intervening sequences as the gastric cancer biomarker. For each residue, both combinatorial entropy and background entropy were calculated, and the entropy difference was used as the criterion for feature residue selection. The feature values were then fed into Support Vector Machines (SVM) with the Radial Basis Function (RBF) kernel, and two parameters were tuned to obtain the optimal F value by using grid search. Two other popular sequence classification methods, the BLAST and HMMER, were also applied to the same data for comparison.

Conclusion

Our method achieved 76% and 71% classification accuracy for Western and East Asian subtypes, respectively, which performed significantly better than BLAST and HMMER. This research indicates that small variations of amino acids in those important residues might lead to the virulence variance of CagA strains resulting in different gastroduodenal diseases. This study provides not only a useful tool to predict the correlation between the novel CagA strain and diseases, but also a general new framework for detecting biological sequence biomarkers in population studies.

Summary: We present a Cytoscape plugin called Mosaic to support interactive network annotation, partitioning, layout and coloring based on gene ontology or other relevant annotations.

Availability: Mosaic is distributed for free under the Apache v2.0 open source license and can be downloaded via the Cytoscape plugin manager. A detailed user manual is available on the Mosaic web site (http://nrnb.org/tools/mosaic).

Contact: apico@gladstone.ucsf.edu

Biomaterials and neurotrophic factors represent promising guidance for neural repair. In this study, we combined poly-(lactic acid-co-glycolic acid) (PLGA) conduits and neurotrophin-3 (NT-3) to generate NT-3-loaded PLGA carriers in vitro. Bioactive NT-3 was released stably and constantly from PLGA conduits for up to 4 weeks. Neural stem cells (NSCs) and Schwann cells (SCs) were coseeded into an NT-releasing scaffold system and cultured for 14 days. Immunoreactivity against Map2 showed that most of the grafted cells (>80%) were differentiated toward neurons. Double-immunostaining for synaptogenesis and myelination revealed the formation of synaptic structures and myelin sheaths in the coculture, which was also observed under electron microscope. Furthermore, under depolarizing conditions, these synapses were excitable and capable of releasing synaptic vesicles labeled with FM1-43 or FM4-64. Taken together, coseeding NSCs and SCs into NT-3-loaded PLGA carriers increased the differentiation of NSCs into neurons, developed synaptic connections, exhibited synaptic activities, and myelination of neurites by the accompanying SCs. These results provide an experimental basis that supports transplantation of functional neural construction in spinal cord injury.

Background

Although metastasis of clear cell renal cell carcinoma (ccRCC) is basically observed in late stage tumors, T1 stage metastasis of ccRCC can also be found with no definite molecular cause resulting inappropriate selection of surgery method and poor prognosis. Notch signaling is a conserved, widely expressed signal pathway that mediates various cellular processes in normal development and tumorigenesis. This study aims to explore the potential role and mechanism of Notch signaling in the metastasis of T1 stage ccRCC.

Methodology/Principal Findings

The expression of Notch1 and Jagged1 were analyzed in tumor tissues and matched normal adjacent tissues obtained from 51 ccRCC patients. Compared to non-tumor tissues, Notch1 and Jagged1 expression was significantly elevated both in mRNA and protein levels in tumors. Tissue samples of localized and metastatic tumors were divided into three groups based on their tumor stages and the relative mRNA expression of Notch1 and Jagged1 were analyzed. Compared to localized tumors, Notch1 expression was significantly elevated in metastatic tumors in T1 stage while Jagged1 expression was not statistically different between localized and metastatic tumors of all stages. The average size of metastatic tumors was significantly larger than localized tumors in T1 stage ccRCC and the elevated expression of Notch1 was significantly positive correlated with the tumor diameter. The functional significance of Notch signaling was studied by transfection of 786-O, Caki-1 and HKC cell lines with full-length expression plasmids of Notch1 and Jagged1. Compared to the corresponding controls, all cell lines demonstrated significant promotion in cell proliferation and migration while cell cycle remained unaffected.

Conclusions/Significance

High-level expression of Notch signaling increased the risk of metastasis in T1 stage ccRCC by stimulating the proliferation and migration of tumor cells, which may be helpful for the selection of suitable operation method and prognosis of ccRCC.

Albuvirtide (ABT) is a 3-maleimimidopropionic acid (MPA)-modified peptide HIV fusion inhibitor that can irreversibly conjugate to serum albumin. Previous studies demonstrated its in vivo long half-life and potent anti-HIV activity. Here, we focused to characterize its biophysical properties and evaluate its antiviral spectrum. In contrast to T20 (Enfuvirtide, Fuzeon), ABT was able to form a stable α-helical conformation with the target sequence and block the fusion-active six-helix bundle (6-HB) formation in a dominant-negative manner. It efficiently inhibited HIV-1 Env-mediated cell membrane fusion and virus entry. A large panel of 42 HIV-1 pseudoviruses with different genotypes were constructed and used for the antiviral evaluation. The results showed that ABT had potent inhibitory activity against the subtypes A, B and C that predominate the worldwide AIDS epidemics, and subtype B′, CRF07_BC and CRF01_AE recombinants that are currently circulating in China. Furthermore, ABT was also highly effective against HIV-1 variants resistant to T20. Taken together, our data indicate that the chemically modified peptide ABT can serve as an ideal HIV-1 fusion inhibitor.

The melanocortin signaling system is integral in regulating energy homeostasis. The melanocortin-4 receptor (MC4R) activates several signaling pathways in performance of this function. The effect of MC4R on insulin-stimulated mammalian target of rapamycin (mTOR), a cellular energy sensor, signaling was investigated. The GT1-1 cell line which expresses MC4R expression was utilized. mTOR signaling was measured by Western blotting analysis using Phospho-mTOR (Ser2448) antibody. NDP-MSH dose-dependently enhanced insulin-stimulated mTOR phosphorylation. The MC4R antagonist SHU9119 blocked this effect, demonstrating specificity. The protein kinase A - cyclic AMP pathway and the MAP kinase pathway were not involved in NDP-MSH actions on insulin-stimulated mTOR phosphorylation. In contrast, the AMP-activated protein kinase agonist, AICAR, attenuated this effect. MC4R activation potentiates insulin-stimulated mTOR signaling via the AMPK pathway.

Background

Histamine is an established growth factor for gastrointestinal malignancies. The effect of histamine is largely determined locally by the histamine receptor expression pattern. Histamine receptor H4 (HRH4), the newest member of the histamine receptor family, is positively expressed on the epithelium of the gastrointestinal tract, and its function remains to be elucidated. Previously, we reported the decreased expression of HRH4 in colorectal cancers and revealed its correlation with tumor proliferation. In the current study, we aimed to investigate the abnormalities of HRH4 gene in gastric carcinomas (GCs).

Methodology/Principal Findings

We analyzed H4R expression in collected GC samples by quantitative PCR, Western blot analysis, and immunostaining. Our results showed that the protein and mRNA levels of HRH4 were reduced in some GC samples, especially in advanced GC samples. Copy number decrease of HRH4 gene was observed (17.6%, 23 out of 131), which was closely correlated with the attenuated expression of H4R. In vitro studies, using gastric cancer cell lines, showed that the alteration of HRH4 expression on gastric cancer cells influences tumor growth upon exposure to histamine.

Conclusions/Significance

We show for the first time that deletion of HRH4 gene is present in GC cases and is closely correlated with attenuated gene expression. Down-regulation of HRH4 in gastric carcinomas plays a role in histamine-mediated growth control of GC cells.

Post-synaptic density 95 (PSD-95), the major scaffold at excitatory synapses, is critical for synapse maturation and learning. In rodents, eye opening, the onset of pattern vision, triggers a rapid movement of PSD-95 from visual neuron somata to synapses. We previously showed that the PI3 kinase-Akt pathway downstream of BDNF/TrkB signaling stimulates synaptic delivery of PSD-95 via vesicular transport. However, vesicular transport requires PSD-95 palmitoylation to attach it to a lipid membrane. Also PSD-95 insertion at synapses is known to require this lipid modification. Here, we show that BDNF/TrkB signaling is also necessary for PSD-95 palmitoylation and its transport to synapses in mouse visual cortical layer 2/3 neurons. However, palmitoylation of PSD-95 requires the activation of another pathway downstream of BDNF/TrkB, namely signaling through PLCγ and the brain-specific PKC variant PKMζ. We find that PKMζ selectively regulates phosphorylation of the palmitoylation enzyme ZDHHC8. Inhibition of PKMζ results in a reduction of synaptic PSD-95 accumulation in vivo, which can be rescued by over-expression ZDHHC8. Therefore, TrkB and PKMζ, two critical regulators of synaptic plasticity, facilitate PSD-95 targeting to synapses. These results also indicate that palmitoylation can be regulated by a trophic factor. Our findings have implications for neurodevelopmental disorders as well as ageing brains.

We investigated the effects of polysaccharides from the brown seaweed Sargassum graminifolium (Turn.) (SGP) on calcium oxalate crystallization, and determined its antioxidant activities. To examine the effects of SGP on calcium oxalate crystallization, we monitored nucleation and aggregation of calcium oxalate monohydrate crystals, using trisodium citrate as a positive control. We assessed antioxidant activities of SGP by determining its reducing power, its ability to scavenge superoxide radicals, and its activity in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The nucleation inhibition ratio of trisodium citrate and SGP was 58.5 and 69.2%, respectively, and crystal aggregation was inhibited by 71.4 and 76.8%, respectively. Increasing concentrations of SGP resulted in increased scavenging of superoxide anions and DPPH radicals (IC50 = 1.9 and 0.6 mg/mL, respectively). These results suggest that SGP could be a candidate for treating urinary stones because of its ability to inhibit calcium oxalate crystallization and its antioxidant properties.

A specific oligodeoxynucleotide (ODN), ODN MT01, was found to have positive effects on the proliferation and activation of the osteoblast-like cell line MG 63. In this study, the detailed signaling pathways in which ODN MT01 promoted the differentiation of osteoblasts were systematically examined. ODN MT01 enhanced the expression of osteogenic marker genes, such as osteocalcin and type I collagen. Furthermore, ODN MT01 activated Runx2 phosphorylation via ERK1/2 mitogen-activated protein kinase (MAPK) and p38 MAPK. Consistently, ODN MT01 induced up-regulation of osteocalcin, alkaline phosphatase (ALP) and type I collagen, which was inhibited by pre-treatment with the ERK1/2 inhibitor U0126 and the p38 inhibitor SB203580. These results suggest that the ERK1/2 and p38 MAPK pathways, as well as Runx2 activation, are involved in ODN MT01-induced up-regulation of osteocalcin, type I collagen and the activity of ALP in MG 63 cells.

Class IIa bacteriocins are heat-stable, unmodified peptides with a conserved amino acids sequence YGNGV on their N-terminal domains, and have received much attention due to their generally recognized as safe (GRAS) status, their high biological activity, and their excellent heat stability. They are promising and attractive agents that could function as biopreservatives in the food industry. This review summarizes the new developments in the area of class IIa bacteriocins and aims to provide uptodate information that can be used in designing future research.