Background

Previous work has shown that disruption of the gene for group X secreted phospholipase A2 (sPLA2-X) markedly diminishes airway hyperresponsiveness and remodeling in a mouse asthma model. With the large number of additional sPLA2s in the mammalian genome, the involvement of other sPLA2s in the asthma model is possible – in particular, the group V sPLA2 (sPLA2-V) that like sPLA2-X is highly active at hydrolyzing membranes of mammalian cells.

Methodology and Principal Findings

The allergen-driven asthma phenotype was significantly reduced in sPLA2-V-deficient mice but to a lesser extent than observed previously in sPLA2-X-deficient mice. The most striking difference observed between the sPLA2-V and sPLA2-X knockouts was the significant impairment of the primary immune response to the allergen ovalbumin (OVA) in the sPLA2-V−/− mice. The impairment in eicosanoid generation and dendritic cell activation in sPLA2-V−/− mice diminishes Th2 cytokine responses in the airways.

Conclusions

This paper illustrates the diverse roles of sPLA2s in the immunopathogenesis of the asthma phenotype and directs attention to developing specific inhibitors of sPLA2-V as a potential new therapy to treat asthma and other allergic disorders.

Background. This study aimed to investigate possible associations between FAF1 expression and aspects of gastric cancer, in particular its clinical characteristics and Helicobacter infection. Materials and Methods. RT-PCR and immunohistochemistry were used to analyze expression of FAF1 mRNA and protein in 40 gastric cancer patients. H. pylori infection was detected by three staining protocols. Results. The expression level of FAF1 mRNA was significantly lower in gastric cancer tissue than in normal gastric mucosa from the same patient (P < 0.05). FAF1 mRNA expression was significantly lower in stage IV gastric cancer than in stage I+II or IIIA+IIIB (P = 0.004) and also significantly lower in gastric cancer with distant metastasis. FAF1 mRNA expression was higher in well-differentiated cancer than in poorly-differentiated cancer (0.39 ± 0.06 versus 0.19 ± 0.06, t = 9.966, P < 0.01). FAF1 protein was detected in 15 of 40 (37.5%) cancerous tissue samples and in 29 of 40 (72.5%) corresponding normal tissue samples (P < 0.01). FAF1 mRNA expression was lower in H. pylori-positive cancerous tissue samples than in H. pylori-negative ones (P < 0.05). Conclusions. Downregulation of FAF1 expression may be related to the carcinogenesis and progression of gastric cancer, and H. pylori infection during gastric carcinogenesis may downregulate FAF1 expression.

The nuclear export of the influenza A virus ribonucleoprotein (vRNP) is crucial for virus replication. As a major component of the vRNP, nucleoprotein (NP) alone can also be shuttled out of the nucleus by interacting with chromosome region maintenance 1 (CRM1) and is therefore hypothesized to promote the nuclear export of the vRNP. In the present study, three novel nuclear export signals (NESs) of the NP—NES1, NES2, and NES3—were identified as being responsible for mediating its nuclear export. The nuclear export of NES3 was CRM1 dependent, whereas that of NES1 or NES2 was CRM1 independent. Inactivation of these NESs led to an overall nuclear accumulation of NP. Mutation of all three NP-NESs significantly impaired viral replication. Based on structures of influenza virus NP oligomers, these three hydrophobic NESs are found present on the surface of oligomeric NPs. Functional studies indicated that oligomerization is also required for nuclear export of NP. Together, these results suggest that the nuclear export of NP is important for virus replication and relies on its NESs and oligomerization.

Painful diabetic neuropathy (PDN) is a common complication of diabetes mellitus and adversely affects the patients’ quality of life. Evidence has accumulated that PDN is associated with hyperexcitability of peripheral nociceptive primary sensory neurons. However, the precise cellular mechanism underlying PDN remains elusive. This may result in the lacking of effective therapies for the treatment of PDN. The phenolic glucoside, gastrodin, which is a main constituent of the Chinese herbal medicine Gastrodia elata Blume, has been widely used as an anticonvulsant, sedative, and analgesic since ancient times. However, the cellular mechanisms underlying its analgesic actions are not well understood. By utilizing a combination of behavioral surveys and electrophysiological recordings, the present study investigated the role of gastrodin in an experimental rat model of STZ-induced PDN and to further explore the underlying cellular mechanisms. Intraperitoneal administration of gastrodin effectively attenuated both the mechanical allodynia and thermal hyperalgesia induced by STZ injection. Whole-cell patch clamp recordings were obtained from nociceptive, capsaicin-sensitive small diameter neurons of the intact dorsal root ganglion (DRG). Recordings from diabetic rats revealed that the abnormal hyperexcitability of neurons was greatly abolished by application of GAS. To determine which currents were involved in the antinociceptive action of gastrodin, we examined the effects of gastrodin on transient sodium currents (INaT) and potassium currents in diabetic small DRG neurons. Diabetes caused a prominent enhancement of INaT and a decrease of potassium currents, especially slowly inactivating potassium currents (IAS); these effects were completely reversed by GAS in a dose-dependent manner. Furthermore, changes in activation and inactivation kinetics of INaT and total potassium current as well as IAS currents induced by STZ were normalized by GAS. This study provides a clear cellular basis for the peripheral analgesic action of gastrodin for the treatment of chronic pain, including PDN.

Many virus infections and stresses can induce endoplasmic reticulum (ER) stress response, a host self-defense mechanism against viral invasion and stress. During this event, viral and cellular gene expression is actively regulated and often encounters a switching of the translation initiation from cap-dependent to internal ribosome-entry sites (IRES)-dependent. This switching is largely dependent on the mRNA structure of the 5′ untranslated region (5′ UTR) and on the particular stress stimuli. Picornaviruses and some other viruses contain IRESs within their 5′ UTR of viral genome and employ an IRES-driven mechanism for translation initiation. Recently, a growing number of cellular genes involved in growth control, cell cycle progression and apoptosis were also found to contain one or more IRES within their long highly structured 5′ UTRs. These genes initiate translation usually by a cap-dependent mechanism under normal physiological conditions; however, in certain environments, such as infection, starvation, and heat shock they shift translation initiation to an IRES-dependent modality. Although the molecular mechanism is not entirely understood, a number of studies have revealed that several cellular biochemical processes are responsible for the switching of translation initiation to IRES-dependent. These include the cleavage of translation initiation factors by viral and/or host proteases, phosphorylation (inactivation) of host factors for translation initiation, overproduction of homologous proteins of cap-binding protein eukaryotic initiation factors (eIF)4E, suppression of cap-binding protein eIF4E expression by specific microRNA, activation of enzymes for mRNA decapping, as well as others. Here, we summarize the recent advances in our understanding of the molecular mechanisms for the switching of translation initiation, particularly for the proteins involved in cell survival and apoptosis in the ER stress pathways during viral infections.

The advent of mammalian gene engineering and genetically modified mouse models has led to renewed interest in developing resources for referencing and quantitative analysis of mouse brain anatomy. In this study, we used diffusion tensor imaging (DTI) for quantitative characterization of anatomical phenotypes in the developing mouse brain. As an anatomical reference for neuroscience research using mouse models, this paper presents DTI based atlases of ex vivo C57BL/6 mouse brains at several developmental stages. The atlas complements existing histology and MRI-based atlases by providing users access to three-dimensional, high-resolution images of the developing mouse brain, with distinct tissue contrasts and segmentations of major gray matter and white matter structures. The usefulness of the atlas and database was demonstrated by quantitative measurements of the development of major gray matter and white matter structures. Population average images of the mouse brain at several postnatal stages were created using large deformation diffeomorphic metric mapping and their anatomical variations were quantitatively characterized. The atlas and database enhance our ability to examine the neuroanatomy in normal or genetically engineered mouse strains and mouse models of neurological diseases.

Background

The nucleoprotein (NP) of influenza A virus is a multifunctional protein that plays a critical role in the replication and transcription of the viral genome. Therefore, examining host factors that interact with NP may shed light on the mechanism of host restriction barriers and the tissue tropism of influenza A virus. Here, Cyclophilin E (CypE), a member of the peptidyl-propyl cis-trans isomerase (PPIase) family, was found to bind to NP and inhibit viral replication and transcription.

Methodology/Principal Findings

In the present study, CypE was found to interact with NP but not with the other components of the viral ribonucleoprotein complex (vRNP): PB1, PB2, and PA. Mutagenesis data revealed that the CypE domain comprised of residues 137–186 is responsible for its binding to NP. Functional analysis results indicated that CypE is a negative regulator in the influenza virus life cycle. Furthermore, knock-down of CypE resulted in increased levels of three types of viral RNA, suggesting that CypE negatively affects viral replication and transcription. Moreover, up-regulation of CypE inhibited the activity of influenza viral polymerase. We determined that the molecular mechanism by which CypE negatively regulates influenza virus replication and transcription is by interfering with NP self-association and the NP-PB1 and NP-PB2 interactions.

Conclusions/Significance

CypE is a host restriction factor that inhibits the functions of NP, as well as viral replication and transcription, by impairing the formation of the vRNP. The data presented here will help us to better understand the molecular mechanisms of host restriction barriers, host adaptation, and tissue tropism of influenza A virus.

The Norrie disease gene (Ndp) codes for a secreted protein, Norrin, that activates canonical Wnt signaling by binding to its receptor, Frizzled-4. This signaling system is required for normal vascular development in the retina and for vascular survival in the cochlea. In mammals, the pattern of Ndp expression beyond the retina is poorly defined due to the low abundance of Norrin mRNA and protein. Here we characterize Ndp expression during mouse development by studying a knock-in mouse that carries the coding sequence of human placental alkaline phosphatase (AP) inserted at the Ndp locus (NdpAP). In the CNS, NdpAP expression is apparent by E10.5 and is dynamic and complex. The anatomically delimited regions of NdpAP expression observed prenatally in the CNS are replaced postnatally by widespread expression in astrocytes in the forebrain and midbrain, Bergman glia in the cerebellum, and Müller glia in the retina. In the developing and adult cochlea, NdpAP expression is closely associated with two densely vascularized regions, the stria vascularis and a capillary plexus between the organ of Corti and the spiral ganglion. These observations suggest the possibility that Norrin may have developmental and/or homeostatic functions beyond the retina and cochlea.

Background

Myocarditis is the major heart disease in infants and young adults. It is very commonly caused by coxsackievirus B3 (CVB3) infection; however, no specific treatment or vaccine is available at present. RNA interference (RNAi)-based anti-viral therapy has shown potential to inhibit viral replication, but this strategy faces two major challenges; viral mutational escape from drug suppression and targeted delivery of the reagents to specific cell populations.

Methodology/Principal Findings

In this study, we designed artificial microRNAs (AmiRs) targeting the 3′untranslated region (3′UTR) of CVB3 genome with mismatches to the central region of their targeting sites. Antiviral evaluation showed that AmiR-1 and AmiR-2 reduced CVB3 (Kandolf and CG strains) replication approximately 100-fold in both HeLa cells and HL-1 cardiomyoctes. To achieve specific delivery, we linked AmiRs to the folate-conjugated bacterial phage packaging RNA (pRNA) and delivered the complexes into HeLa cells, a folate receptor positive cancer cells widely used as an in vitro model for CVB3 infection, via folate-mediated specific internalization. We found that our designed pRNA-AmiRs conjugates were tolerable to target mutations and have great potential to suppress viral mutational escape with little effect on triggering interferon induction.

Conclusion/Significance

This study provides important clues for designing AmiRs targeting the 3′UTR of viral genome. It also proves the feasibility of specific deliver of AmiRs using conjugated pRNA vehicles. These small AmiRs combined with pRNA-folate conjugates could form a promising system for antiviral drug development.

Influenza virus RNA-dependent RNA polymerase scavenges the 5′ cap from host pre-mRNA to prime viral transcription initiation. It is also well established that viral RNA-dependent RNA polymerase (vRNP) associates with cellular RNA polymerase II (Pol II), on which viral replication depends. Here we report that cyclin T1/CDK9 can interact with influenza virus polymerase and facilitate its association with cellular Pol II. The immunodepletion of cyclin T1/CDK9 totally abolished the association of vRNP with the C-terminal domain (CTD) Ser-2-phosphorylated form of RNA polymerase II. Further studies showed that overexpression of cyclin T1/CDK9 increased the transcription activity of vRNP, while knockdown of cyclin T1/CDK9 impaired viral replication. Our results suggest that cyclin T1/CDK9 serves as an adapter to mediate the interaction of vRNP and RNA Pol II and promote viral transcription.

Background

Inadequate surgical hemostasis may lead to transfusion and/or other bleeding-related complications. This study examines the incidence and costs of bleeding-related complications and/or blood product transfusions occurring as a consequence of surgery in various inpatient surgical cohorts.

Methods

A retrospective analysis was conducted using Premier's Perspective™ hospital database. Patients who had an inpatient procedure within a specialty of interest (cardiac, vascular, non-cardiac thoracic, solid organ, general, reproductive organ, knee/hip replacement, or spinal surgery) during 2006-2007 were identified. For each specialty, the rate of bleeding-related complications (including bleeding event, intervention to control for bleeding, and blood product transfusions) was examined, and hospital costs and length of stay (LOS) were compared between surgeries with and without bleeding-related complications. Incremental costs and ratios of average total hospital costs for patients with bleeding-related complications vs. those without complications were estimated using ordinary least squares (OLS) regression, adjusting for demographics, hospital characteristics, and other baseline characteristics. Models using generalized estimating equations (GEE) were also used to measure the impact of bleeding-related complications on costs while accounting for the effects related to the clustering of patients receiving care from the same hospitals.

Results

A total of 103,829 cardiac, 216,199 vascular, 142,562 non-cardiac thoracic, 45,687 solid organ, 362,512 general, 384,132 reproductive organ, 246,815 knee/hip replacement, and 107,187 spinal surgeries were identified. Overall, the rate of bleeding-related complications was 29.9% and ranged from 7.5% to 47.4% for reproductive organ and cardiac, respectively. Overall, incremental LOS associated with bleeding-related complications or transfusions (unadjusted for covariates) was 6.0 days and ranged from 1.3 to 9.6 days for knee/hip replacement and non-cardiac thoracic, respectively. The incremental cost per hospitalization associated with bleeding-related complications and adjusted for covariates was highest for spinal surgery ($17,279) followed by vascular ($15,123), solid organ ($13,210), non-cardiac thoracic ($13,473), cardiac ($10,279), general ($4,354), knee/hip replacement ($3,005), and reproductive organ ($2,805).

Conclusions

This study characterizes the increased hospital LOS and cost associated with bleeding-related complications and/or transfusions occurring as a consequence of surgery, and supports implementation of blood-conservation strategies.

Cardiomyocyte apoptosis is a hallmark of coxsackievirus B3 (CVB3)-induced myocarditis. We used cardiomyocytes and HeLa cells to explore the cellular response to CVB3 infection, with a focus on pathways leading to apoptosis. CVB3 infection triggered endoplasmic reticulum (ER) stress and differentially regulated the three arms of the unfolded protein response (UPR) initiated by the proximal ER stress sensors ATF6a (activating transcription factor 6a), IRE1-XBP1 (X box binding protein 1), and PERK (PKR-like ER protein kinase). Upon CVB3 infection, glucose-regulated protein 78 expression was upregulated, and in turn ATF6a and XBP1 were activated via protein cleavage and mRNA splicing, respectively. UPR activity was further confirmed by the enhanced expression of UPR target genes ERdj4 and EDEM1. Surprisingly, another UPR-associated gene, p58IPK, which often is upregulated during infections with other types of viruses, was downregulated at both mRNA and protein levels after CVB3 infection. These findings were observed similarly for uninfected Tet-On HeLa cells induced to overexpress ATF6a or XBP1. In exploring potential connections between the three UPR pathways, we found that the ATF6a-induced downregulation of p58IPK was associated with the activation of PKR (PERK) and the phosphorylation of eIF2α, suggesting that p58IPK, a negative regulator of PERK and PKR, mediates cross-talk between the ATF6a/IRE1-XBP1 and PERK arms. Finally, we found that CVB3 infection eventually produced the induction of the proapoptoic transcription factor CHOP and the activation of SREBP1 and caspase-12. Taken together, these data suggest that CVB3 infection activates UPR pathways and induces ER stress-mediated apoptosis through the suppression of P58IPK and induction/activation of CHOP, SREBP1, and caspase-12.

Disorders of retinal vascular growth and function are responsible for vision loss in a variety of diseases, including diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, and retinal artery or vein occlusion. Over the past decade, a new signaling pathway that controls retinal vascular development has emerged from the study of inherited disorders - in both humans and mice - that are characterized by retinal hypovascularization. This pathway utilizes a glial-derived extracellular ligand, Norrin, that acts on a transmembrane receptor, Frizzled4, a coreceptor, Lrp5, and an auxiliary membrane protein, Tspan12, on the surface of developing endothelial cells. The resulting signal controls a transcriptional program that regulates endothelial growth and maturation. It will be of great interest to determine whether modulating this pathway could represent a therapeutic approach to human retinal vascular disease.

Background

Heat shock protein 70 (Hsp70) was identified as a cellular interaction partner of the influenza virus ribonucleoprotein (RNP) complex. The biological significance of the interaction between Hsp70 and RNP has not been fully investigated.

Principal Findings

Here we demonstrated that Hsp70 was involved in the regulation of influenza A viral transcription and replication. It was found that Hsp70 was associated with viral RNP by directly interacting with the PB1 and PB2 subunits, and the ATPase domain of Hsp70 was required for the association. Immunofluorescence analysis showed that Hsp70 was translocated from the cytoplasm into the nucleus in infected cells. Then we found that Hsp70 negatively regulated the expression of viral proteins in infected cells. Real-time PCR analysis revealed that the transcription and replication of all eight viral segments were significantly reduced in Hsp70 overexpressed cells and greatly increased as Hsp70 was knocked down by RNA interference. Luciferase assay showed that overexpression of Hsp70 could inhibit the viral RNP activity on both vRNA and cRNA promoters. Biochemical analysis demonstrated that Hsp70 interfered with the integrity of RNP. Furthermore, delivered Hsp70 could inhibit the replication of influenza A virus in mice.

Significance

Our study indicated that Hsp70 interacted with PB1 and PB2 of RNP and could interfere with the integrity of RNP and block the virus replication in vitro and in vivo possibly through disrupting the binding of viral polymerase with viral RNA.

Cell cycle progression into S phase requires the induction of histone gene expression to package newly synthesized DNA as chromatin. Cyclin E stimulation of CDK2 at the Restriction point late in G1 controls both histone gene expression by the p220NPAT/HiNF-P pathway and initiation of DNA replication through the pRB/E2F pathway. The three CDK inhibitors (CKIs) p21CIP1/WAF1, p27KIP1 and p57KIP2 attenuate CDK2 activity. Here we find that γ-irradiation induces p21CIP1/WAF1 but not the other two CKIs, while reducing histone H4 mRNA levels but not histone H4 gene promoter activation by the p220NPAT/HiNF-P complex. We also show that p21CIP1/WAF1 is less effective than p27KIP1 and p57KIP2 in inhibiting the CDK2 dependent phosphorylation of p220NPAT at subnuclear foci and transcriptional activation of histone H4 genes. The greater effectiveness of p57KIP2 in blocking the p220NPAT/HiNF-P pathway is attributable in part to its ability to form a specific complex with p220NPAT that may suppress CDK2/cyclin E phosphorylation through direct substrate inhibition. We conclude that CKIs selectively control stimulation of the histone H4 gene promoter by the p220NPAT/HiNF-P complex.

SUMMARY

Disorders of vascular structure and function play a central role in a wide variety of CNS diseases. Mutations in the Frizzled4 (Fz4) receptor, Lrp5 co-receptor, or Norrin ligand cause retinal hypovascularization, but the role of Norrin/Fz4/Lrp signaling in vascular development has not been defined. Using mouse genetic and cell culture models, we show that loss of Fz4 signaling in endothelial cells causes defective vascular growth, which leads to chronic but reversible silencing of retinal neurons. Loss of Fz4 in all endothelial cells disrupts the blood brain barrier in the cerebellum, while excessive Fz4 signaling disrupts embryonic angiogenesis. Sox17, a transcription factor that is up-regulated by Norrin/Fz4/Lrp signaling, plays a central role in inducing the angiogenic program controlled by Norrin/Fz4/Lrp. These experiments establish a cellular basis for retinal hypovascularization diseases due to insufficient Frizzled signaling, and they suggest a broader role for Frizzled signaling in vascular growth, remodeling, maintenance, and disease.

Background

Pharmacologic control of Cre-mediated recombination using tamoxifen-dependent activation of a Cre-estrogen receptor ligand binding domain fusion protein [CreER(T)] is widely used to modify and/or visualize cells in the mouse.

Methods and Findings

We describe here two new mouse lines, constructed by gene targeting to the Rosa26 locus to facilitate Cre-mediated cell modification. These lines should prove particularly useful in the context of sparse labeling experiments. The R26rtTACreER line provides ubiquitous expression of CreER under transcriptional control by the tetracycline reverse transactivator (rtTA); dual control by doxycycline and tamoxifen provides an extended dynamic range of Cre-mediated recombination activity. The R26IAP line provides high efficiency Cre-mediated activation of human placental alkaline phosphatase (hPLAP), complementing the widely used, but low efficiency, Z/AP line. By crossing with mouse lines that direct cell-type specific CreER expression, the R26IAP line has been used to produce atlases of labeled cholinergic and catecholaminergic neurons in the mouse brain. The R26IAP line has also been used to visualize the full morphologies of retinal dopaminergic amacrine cells, among the largest neurons in the mammalian retina.

Conclusions

The two new mouse lines described here expand the repertoire of genetically engineered mice available for controlled in vivo recombination and cell labeling using the Cre-lox system.

Diets that are high in dietary fiber are reported to have substantial health benefits. We sought to compare the metabolic effects for three types of dietary fibers, i.e. sugar cane fiber (SCF), psyllium (PSY) and cellulose (CEL) on body weight, carbohydrate metabolism and stomach ghrelin gene expression in a high-fat diet fed mouse model. Thirty-six male mice (C57BL/6) were randomly divided into four groups that consumed high fat-diets or high fat diet containing 10% SCF, PSY, and CEL respectively. After baseline measurements were assessed for body weight, plasma insulin, glucose, leptin and glucagon-like peptide-1 (GLP-1), animals were treated for 12 weeks. Parameters were re-evaluated at end of study. Whereas there was no difference at the baseline, body weight gains in the PSY and SCF groups were significantly lower than in CEL group at end of study, No difference in body weight was observed between the PSY and SCF animals. Body composition analysis demonstrated that fat mass in the SCF group was considerably lower than in the CEL and HFD groups. In addition, fasting plasma glucose and insulin and areas under curve of IPGTT were also significantly lower in the SCF and PSY groups than in the CEL and HFD groups. Moreover, fasting plasma concentrations of leptin were significantly lower and GLP-1 level was two-fold higher in the SCF and PSY mice than in the HFD and CEL mice. Ghrelin mRNA levels of stomach in SCF groups were significantly lower than in CEL and HFD groups as well. These results suggest differences in response to dietary fiber intake in this animal model as high fat diets incorporating dietary fibers such as SCF and PSY appeared to attenuate weight gain, enhance insulin sensitivity, and modulate leptin and GLP-1 secretion and gastric ghrelin gene expression.

Objective

UGT2B7 plays a central role in the liver-mediated biotransformation of endogenous and exogenous compounds. The genetic basis of interindividual variability in UGT2B7 function is unknown. This study aimed to discover novel gene variants of functional significance.

Methods

Caucasian human livers (n=54) were used. UGT2B7 was resequenced in 12 samples (6 highest and 6 lowest for the formation of morphine-3-glucuronide, M3G). Haplotype-tagging single nucleotide polymorphisms (tSNPs) were genotyped in the entire sample set. Samples were phenotyped for mRNA expression.

Results

10 tSNPs were identified and their haplotypes were inferred. Haplotype 4 (-45597G;-6682_-6683A;372A;IVS1+9_IVS1+10A;IVS1+829T;IVS1+985G;IVS1+999C;IVS1+1250G;80 1T;IVS4+185C) (frequency of 0.12) was associated with an increase in enzyme activity and gene expression. The 1/4 and 4/6 diplotypes had higher M3G formation compared to 1/1 (p<0.05) and 2/3 (p<0.01) diplotypes. Diplotypes containing haplotype 4 resulted in a significant 45% average increase in the formation of M3G compared to diplotypes without haplotype 4 (p=0.002). There was also an association between haplotype 4 and increased mRNA expression. IVS1+985A>G, 735A>G and 1062C>T are the putative functional variants of haplotype 4. We also identified two mRNA splicing variants (UGT2B7_v2 and UGT2B7_v3) splicing out exon 1, 4, 5 and 6 but sharing exons 2 and 3 with the involvement of additional 5' exons. UGT2B7_v2 was detected in all livers tested, but UGT2B7_v3 was present at much lower levels compared to UGT2B7_v2. The UGT2B7 reference sequence mRNA is now named UGT2B7_v1.

Conclusions

UGT2B7 haplotype 4 is functional and its effects on the biotransformation of UGT2B7 substrates should be tested in controlled clinical trials. Biochemical studies should investigate the functional role of the newly discovered mRNA splicing variants.

Biomarkers of Hepatitis B Virus (HBV) infection, aflatoxin B1 (AFB1) exposure and oxidative stress were detected in 71 hepatocellular carcinoma (HCC) patients and 694 controls from southern China. Plasma level of AFB1-Albumin-Adducts (AAA) and protein carbonyl content (PCC) were significantly higher in the 71 HCC cases than in any age/gender matched HBV sero-status groups (P<0.001). HCC patients positive for the p53-249 G-T mutation had a marginally higher level of PCC than those negative for the mutation (p=0.077). HBV infection had a prominent influence on the association between AFB1 exposure and oxidative stress biomarkers in the controls. Our study indicates a significant contribution from HBV infection to oxidative stress in a population with AFB1 exposure which might substantially increase risk for HCC in this region.

We recently demonstrated the pivotal role of the transcription factor (TF) activating TF 3 (ATF3) in dampening inflammation. We demonstrate that ATF3 also ameliorates allergen-induced airway inflammation and hyperresponsiveness in a mouse model of human asthma. ATF3 expression was increased in the lungs of mice challenged with ovalbumin allergen, and this was associated with its recruitment to the promoters of genes encoding Th2-associated cytokines. ATF3-deficient mice developed significantly increased airway hyperresponsiveness, pulmonary eosinophilia, and enhanced chemokine and Th2 cytokine responses in lung tissue and in lung-derived CD4+ lymphocytes. Although several TFs have been associated with enhanced inflammatory responses in the lung, ATF3 attenuates the inflammatory responses associated with allergic airway disease.

Background

Primordial germ cell (PGC) specification is the first crucial step in germ line development. However, owing to significant challenges regarding the in vivo system, such as the complex cellular environment and potential problems with embryo manipulation, it is desirable to generate embryonic stem (ES) cells that are capable of overcoming these aforementioned limitations in order to provide a potential in vitro model to recapitulate the developmental processes in vivo.

Methodology and Principal Findings

Here, we studied the detailed process of PGC specification from stella-GFP ES cells. We first observed the heterogeneous expression of stella in ES cells. However, neither Stella-positive ES cells nor Stella-negative ES cells shared a similar gene expression pattern with either PGCs or PGC precursors. Second, we derived PGCs from ES cells using two differentiation methods, namely the attachment culture technique and the embryoid body (EB) method. Compared with PGCs derived via the attachment culture technique, PGCs derived via the EB method that had undergone the sequential erasure of Peg3 followed by Igf2r resulted in a cell line in which the expression dynamics of T, Fgf8 and Sox17, in addition to the expression of the epiblast markers, were more similar to the in vivo expression, thus demonstrating that the process of PGC derivation was more faithfully recapitulated using the EB method. Furthermore, we developed an in vitro model of PGC specification in a completely chemically defined medium (CDM) that indicated that BMP4 and Wnt3a promoted PGC derivation, whereas BMP8b and activinA had no observable effect on PGC derivation.

Conclusions and Significance

The in vitro model we have established can recapitulate the developmental processes in vivo and provides new insights into the mechanism of PGC specification.

Two asymmetrically branched oligosaccharides, LewisX and dimeric LewisX were assembled in one-pot with high yields and exclusive regio- and stereo-selectivities. p-Tolyl thioglycosides were utilized as the sole type of building blocks, thus simplifying the overall synthetic design. The reactivity independent nature of the pre-activation based method allows modular assembly of the dimeric LewisX octasaccharide without the need for tedious protective group manipulation to achieve exact anomeric reactivities.

Two efficient routes for rapid assembly of the tumor-associated carbohydrate antigen Globo-H hexasaccharide 2 by the pre-activation based iterative one pot strategy are reported. The first method involves the sequential coupling of four glycosyl building blocks, leading to the desired hexasaccharide in 47% overall yield in one-pot. Although model study on constructing the challenging Gal-α-1,4-Gal linkage in Gb3 trisaccharide yielded the desired α linkage almost exclusively, similar approach to assemble the hexasaccharide led to formation of significant amount of β anomer. As an alternative, the second synthesis utilizes three components in one pot with the Gal-α-1,4-Gal linkage pre-formed, producing the desired hexasaccharide in a similar overall yield as the four component approach. Both methods demonstrate that oligosaccharides containing α and β linkages within the same molecule can be constructed in one pot via the pre-activation based approach with higher glyco-assembly efficiencies than the automated solid phase synthesis strategy. Furthermore, because glycosylations can be carried out independent of anomeric reactivities of donors, it is not necessary to differentiate anomeric reactivities of building blocks through extensive protective group adjustment for chemoselective glycosylation. This confers great flexibilities in building block design allowing matching of the donor with the acceptor leading to improved overall yield.

Decreases in the expression of connexin 43 and the integrity of gap junctions in cardiac muscle, induced by the constitutive activation of the c-Jun N-terminal kinase (JNK) signaling pathway, have been linked to conduction defects and sudden cardiac failure in mice [16,17]. We examined the membrane cytoskeletal protein, αII-spectrin, which associates with connexin 43, to learn if changes in its association with connexin 43 are linked to the instability of gap junctions. Several forms of αII-spectrin are expressed in heart, including one, termed αII-SH3i, which contains a 20-amino acid sequence next to the SH3 domain of repeat 10. In adult mouse heart, antibodies to all forms of αII-spectrin labeled the sarcolemma, transverse (“t-”) tubules and intercalated disks of cardiomyocytes. In contrast, antibodies specific for αII-SH3i labeled only gap junctions and transverse tubules. In transgenic hearts, in which the JNK pathway was constitutively activated, αII-SH3i was lost specifically from gap junctions but not from t-tubules while other isoforms of αII-spectrin were retained at intercalated disks. Immunoprecipitations confirmed the decreased association of αII-SH3i with connexin 43 in transgenic hearts compared to controls. Furthermore, activation of JNK in neonatal myocytes blocked the formation of gap junctions by exogenously expressed Cx43-GFP fusion protein. Similarly, over-expression of the SH3i fragment in the context of repeats 9-11 of αII–spectrin specifically caused the accumulation of Cx43-GFP in the perinuclear region and inhibited its accumulation at gap junctions. These results support a critical role for the αII-SH3i isoform of spectrin in intracellular targeting of Cx43 to gap junctions and implicates αII-SH3i as a potential target for stress signaling pathways that modulate intercellular communication.