Accumulating evidence suggests that the adult murine hypothalamus, a control site of several fundamental homeostatic processes, has neurogenic capacity. Correspondingly, the adult hypothalamus exhibits considerable cell proliferation that is ongoing even in the absence of external stimuli, and some of the newborn cells have been shown to mature into cells that express neuronal fate markers. However, the identity and characteristics of proliferating cells within the hypothalamic parenchyma have yet to be thoroughly investigated. Here we show that a subset of NG2-glia distributed throughout the mediobasal hypothalamus are proliferative and express the stem cell marker Sox2. We tracked the constitutive differentiation of hypothalamic NG2-glia by employing genetic fate mapping based on inducible Cre recombinase expression under the control of the NG2 promoter, demonstrating that adult hypothalamic NG2-glia give rise to substantial numbers of APC+ oligodendrocytes and a smaller population of HuC/D+ or NeuN+ neurons. Labelling with the cell proliferation marker BrdU confirmed that some NG2-derived neurons have proliferated shortly before differentiation. Furthermore, patch-clamp electrophysiology revealed that some NG2-derived cells display an immature neuronal phenotype and appear to receive synaptic input indicative of their electrical integration in local hypothalamic circuits. Together, our studies show that hypothalamic NG2-glia are able to take on neuronal fates and mature into functional neurons, indicating that NG2-glia contribute to the neurogenic capacity of the adult hypothalamus.
Pig is an important agricultural animal for meat production and provides a valuable model for many human diseases. Functional studies have demonstrated that microRNAs (miRNAs) play critical roles in almost all aspects of skeletal muscle development and disease pathogenesis. To investigate the miRNAs involved in regulating different periods of skeletal muscle development, we herein performed a comprehensive research for porcine microRNAome (miRNAome) during 10 skeletal muscle developmental stages including 35, 49, 63, 77, 91 dpc (days post coitum) and 2, 28, 90, 120, 180 dpn (days postnatal) using Solexa sequencing technology. Our results extend the repertoire of pig miRNAome to 247 known miRNAs processed from 210 pre-miRNAs and 297 candidate novel miRNAs through comparison with known miRNAs in the miRBase. Expression analysis of the 15 most abundant miRNAs in every library indicated that functional miRNAome may be smaller and tend to be highly expressed. A series of muscle-related miRNAs summarized in our study present different patterns between myofibers formation phase and muscle maturation phase, providing valuable reference for investigation of functional miRNAs during skeletal muscle development. Analysis of temporal profiles of miRNA expression identifies 18 novel candidate myogenic miRNAs in pig, which might provide new insight into regulation mechanism mediated by miRNAs underlying muscle development.
In assisted reproductive treatments, embryos remaining after fresh embryo transfer are usually selected for cryopreservation based on traditional morphology assessment. Our previous report has demonstrated that array comparative genomic hybridization (aCGH) screening for IVF patients with good prognosis significantly improves clinical and ongoing pregnancy rates in fresh embryo transfer cycles. The current study further investigates the efficiency of applying aCGH in the selection of euploid embryos for cryopreservation as related to pregnancy and implantation outcomes in subsequent frozen embryo transfer (FET) cycles.
First-time IVF patients with good prognosis undergoing fresh single embryo transfer and having at least one remaining blastocyst for cryopreservation were prospectively randomized into two groups: 1) Group A patients had embryos assessed by morphology first and then by aCGH screening of trophectoderm cells and 2) Group B patients had embryos evaluated by morphology alone. All patients had at least one blastocyst available for cryopreservation after fresh embryo transfer. There were 15 patients in Group A and 23 patients in Group B who failed to conceive after fresh embryo transfer and completed the FET cycles. Blastocyst survival and implantation rates were compared between the two groups.
There were no significant differences in blastocyst survival rates between Group A and Group B (90.9% vs. 91.3%, respectively; p >0.05). However, a significantly higher implantation rate was observed in the morphology assessment plus aCGH screening group compared to the morphology assessment alone group (65.0% vs. 33.3%, respectively; p = 0.038). There was no miscarriage observed in Group A while a 16.7% miscarriage rate was recorded in Group B (0% vs. 16.7%, respectively; p >0.05).
While aCGH screening has been recently applied to select euploid blastocysts for fresh transfer in young, low-risk IVF patients, this is the first prospective study on the impact of aCGH specifically on blastocyst survival and implantation outcomes in the subsequent FET cycles of IVF patients with good prognosis. The present study demonstrates that aCGH screening of blastocysts prior to cryopreservation significantly improves implantation rates and may reduce the risk of miscarriage in subsequent FET cycles. Further randomized clinical studies with a larger sample size are needed to validate these preliminary findings.
aCGH; Trophectoderm biopsy; Cryopreservation; Implantation
Productive T cell activation requires efficient reorganization of the actin cytoskeleton. We showed previously that the actin-regulatory protein, hematopoietic lineage cell-specific protein 1 (HS1), is required for the stabilization of F-actin and Vav1 at the immunological synapse and for efficient calcium responses. The Tec family kinase IL-2-inducible T cell kinase (Itk) regulates similar aspects of T cell activation, suggesting that these proteins act in the same pathway. Using video microscopy, we show that T cells lacking Itk or HS1 exhibited similar defects in actin responses, extending unstable lamellipodial protrusions upon TCR stimulation. HS1 and Itk could be coimmunoprecipitated from T cell lysates, and GST-pulldown studies showed that Itk’s Src homology 2 domain binds directly to two phosphotyrosines in HS1. In the absence of Itk, or in T cells overexpressing an Itk Src homology 2 domain mutant, HS1 failed to localize to the immunological synapse, indicating that Itk serves to recruit HS1 to sites of TCR engagement. Because Itk is required for phospholipase C (PLC)γ1 phosphorylation and calcium store release, we examined the calcium signaling pathway in HS1−/− T cells in greater detail. In response to TCR engagement, T cells lacking HS1 exhibited diminished calcium store release, but TCR-dependent PLCγ1 phosphorylation was intact, indicating that HS1’s role in calcium signaling is distinct from that of Itk. HS1-deficient T cells exhibited defective cytoskeletal association of PLCγ1 and altered formation of PLCγ1 microclusters. We conclude that HS1 functions as an effector of Itk in the T cell actin-regulatory pathway, and directs the spatial organization of PLCγ1 signaling complexes.
Many functions of vacuole depend on the activity of vacuolar ATPase which is essential to maintain an acidic lumen and create the driving forces for massive fluxes of ions and metabolites through vacuolar membrane. In filamentous fungus Magnaportheoryzae, subcellular colocalization and quinacrine staining suggested that the V1V0 domains of V-ATPase were fully assembled and the vacuoles were kept acidic during infection-related developments. Targeted gene disruption of MoVMA11 gene, encoding the putative c’ subunit of V-ATPase, impaired vacuolar acidification and mimicked the phenotypes of yeast V-ATPase mutants in the poor colony morphology, abolished asexual and sexual reproductions, selective carbon source utilization, and increased calcium and heavy metals sensitivities, however, not in the typical pH conditional lethality. Strikingly, aerial hyphae of the MoVMA11 null mutant intertwined with each other to form extremely thick filamentous structures. The results also implicated that MoVMA11 was involved in cell wall integrity and appressorium formation. Abundant non-melanized swollen structures and rare, small appressoria without penetration ability were produced at the hyphal tips of the ΔMovma11 mutant on onion epidermal cells. Finally, the MoVMA11 null mutant lost pathogenicity on both intact and wounded host leaves. Overall, our data indicated that MoVMA11, like other fungal VMA genes, is associated with numerous cellular functions and highlighted that V-ATPase is essential for infection-related morphogenesis and pathogenesis in M. oryzae.
Heme-copper oxidase (HCO) performs efficient four-electron reduction of oxygen to water without releasing toxic, reactive oxygen species (ROS). Essential for this function is a post-translationally modified histidine–tyrosine cross-link (Tyr-His) in its heme a3/CuB oxygen reduction center. Through the genetic incorporation of the Tyr-His ligand and CuB site into myoglobin, we recapitulated important features of HCO into this small soluble protein, which exhibits selective O2 reduction activity while generating less than 6% ROS, at more than 1000 turnovers. These results support that Tyr-His crosslink is indeed important for HCO function, and creates the exciting opportunity to rapidly evolve better HCO model proteins to achieve higher activity and selectivity, which may be suitable as alternatives to precious metal catalyst in fuel cells.
post-translational modification; tyrosine-histidine crosslink; protein design; heme copper oxidase; oxygen reduction
During human immunodeficiency virus type-1 (HIV-1) virion maturation, capsid proteins undergo a major rearrangement to form a conical core that protects the viral nucleoprotein complexes. Mutations in the capsid sequence that alter the stability of the capsid core are deleterious to viral infectivity and replication. Recently, capsid assembly has become an attractive target for the development of a new generation of anti-retroviral agents. Drug screening efforts and subsequent structural and mechanistic studies require gram quantities of active, homogeneous and pure protein. Conventional means of laboratory purification of Escherichia coli expressed recombinant capsid protein rely on column chromatography steps that are not amenable to large-scale production. Here we present a function-based purification of wild-type and quadruple mutant capsid proteins, which relies on the inherent propensity of capsid protein to polymerize and depolymerize. This method does not require the packing of sizable chromatography columns and can generate double-digit gram quantities of functionally and biochemically well-behaved proteins with greater than 98% purity. We have used the purified capsid protein to characterize two known assembly inhibitors in our in-house developed polymerization assay and to measure their binding affinities. Our capsid purification procedure provides a robust method for purifying large quantities of a key protein in the HIV-1 life cycle, facilitating identification of the next generation anti-HIV agents.
Epidemiological evidence has clearly indicated that chronic infection with the hepatitis B virus (HBV) is the major risk factor for developing hepatocellular carcinoma (HCC). Nonetheless, the mechanisms by which HBV contributes to the pathogenesis of HCC have not been fully elucidated.
Our aim was to characterize differential gene expression profiles related to the Wnt signaling pathway between primary tumor and adjacent normal tissues in HCC patients with concomitant HBVinfection .
Materials and Methods
An oligoGEArray® (an oligonucleotide-based gene expression array platform) containing 126 Wnt signaling pathway-related genes was used to compare gene expressions between primary HCC and adjacent non-tumorous liver tissues from 10 patients with HCC. Selected differential genes were identified with real-time RT-PCR and immunohistochemistry (IHC). In particular, the protein of the differential gene DVL3 (disheveled, dsh homolog 3 [Drosophila]) was chosen to investigate whether it is up regulated in primary tumor correlated with the clinic pathological characteristics of HCC patients. For this purpose we examined 56 HCC tissue samples via IHC for the presence of DVL3 protein.
Sixteen genes were identified with significant differential expression between HCC and adjacent non-tumorous liver tissue. These genes have been previously associated with the Frizzled signaling pathway, cell cycle, transcription, or protein degradation. All (100%) of the tumor samples results from 56 HCC patients tested were positive for DVL3 via IHC. Based on the intensity of DVL3 immunoreactivity, 25 (44.6%) and 31 (55.4%) of the patients were classified aslow and high-DVL3, respectively, which correlated with tumor stage (P = 0.029).
This study clarified a number of Wnt pathway-related genes which are dysregulated in HBV-associated HCC. These genes may be contributedto the frequent activation of the Wnt signaling pathway. Our results promote the role of the Wnt signaling pathway in HBV-associated HCC.
Carcinoma, Hepatocellular; Hepatitis B Virus; Oligonucleotide Array Sequence Analysis; Gene Expression Profiling; Disheveled Proteins
As an important factor affecting meat quality, intramuscular fat (IMF) content is a topic of worldwide concern. Emerging evidences indicate that microRNAs play important roles in adipocyte differentiation. However, miRNAome has neither been studied during porcine intramuscular preadipocyte differentiation, nor compared with subcutaneous preadipocytes. The objectives of this study were to identify porcine miRNAs involved in adipogenesis in primary preadipocytes, and to determine whether intramuscular and subcutaneous adipocytes differ in the expression and regulation of miRNAs.
miRNAomes in primary intramuscular and subcutaneous adipocytes during differentiation were first sequenced using the Solexa deep sequencing method. The sequences and relative expression levels of 224 known (98.2% in miRbase 18.0) and 280 potential porcine miRNAs were identified. Fifty-four of them changed in similar pattern between intramuscular vascular stem cells (IVSC) and subcutaneous vascular stem cells (SVSC) differentiation, such as miR-210, miR-10b and miR-99a. Expression levels of 10 miRNAs were reversely up-or down-regulated between IVSC and SVSC differentiation, 19 were up-or down-regulated only during IVSC differentiation and 55 only during SVSC differentiation. Additionally, 30 miRNAs showed fat-depot specific expression pattern (24 in cells of intramuscular origin and 6 in cells of subcutaneous origin). These adipogenesis-related miRNAs mainly functioned by targeting similar pathways in adipogenesis, obesity and syndrome.
Comparison of miRNAomes in IVSC and SVSC during differentiation revealed that many different miRNAs are involved in adipogenesis, and they regulate SVSC and IVSC differentiation through similar pathways. These miRNAs may serve as biomarkers or targets for enhancing IMF content, and uncovering their function in IMF development will be of great value in the near future.
Although the dimerization of KIT, a receptor tyrosine kinase, plays a major role in a number of tumors, correlations between the clinicopathological parameters and KIT receptor dimers have not been identified. In the current study, a method for the detection of KIT receptor dimer expression was described and correlations between the clinicopathological parameters and KIT receptor dimers were analyzed. A single center cohort study of 49 patients with gastrointestinal stromal tumors (GISTs) was conducted to analyze the expression of KIT receptor dimers by SDS-PAGE, Native-PAGE and modified Native-PAGE. Immunohistochemistry was used to examine the expression of ki-67, c-kit and stem cell factor (SCF). Mutations of the c-kit gene were examined in 48 GISTs according to the polymerase chain reaction (PCR) and direct sequencing methods. Based on the data, a signal for the KIT receptor monomer was obtained by SDS-PAGE. Faint bands were observed on the nitrocellulose membrane by Native-PAGE, while clear bands were identified for KIT receptor dimers and monomers using modified Native-PAGE (15 out of 49 cases). The tumor size was larger in KIT receptor dimer-positive cases compared with that in KIT receptor dimer-negative cases. Analysis of KIT receptor dimer expression levels and risk stratification demonstrated that KIT receptor dimer-positive cases belonged to the higher risk classification. In addition, there was no significant correlation between the existence of KIT receptor dimers and c-kit gene mutations, including SCF expression. In conclusion, this study established a method for the detection of the existence of KIT receptor dimers in tissues and confirmed that KIT receptor dimers were correlated with risk stratification. Data also indicated that ligand-dependent SCF/KIT dimerization is an independent crucial mechanism in GIST cell proliferation and increases the risk of GIST. Therefore, blocking KIT dimerization may prove to be an effective approach for the treatment of GISTs.
gastrointestinal stromal tumors; dimerization; KIT-dimer
Tegobuvir (TGV) is a novel non-nucleoside inhibitor (NNI) of HCV RNA replication with demonstrated antiviral activity in patients with genotype 1 chronic HCV infection. The mechanism of action of TGV has not been clearly defined despite the identification of resistance mutations mapping to the NS5B polymerase region. TGV does not inhibit NS5B enzymatic activity in biochemical assays in vitro, suggesting a more complex antiviral mechanism with cellular components. Here, we demonstrate that TGV exerts anti-HCV activity utilizing a unique chemical activation and subsequent direct interaction with the NS5B protein. Treatment of HCV subgenomic replicon cells with TGV results in a modified form of NS5B with a distinctly altered mobility on a SDS-PAGE gel. Further analysis reveals that the aberrantly migrating NS5B species contains the inhibitor molecule. Formation of this complex does not require the presence of any other HCV proteins. The intensity of the aberrantly migrating NS5B species is strongly dependent on cellular glutathione levels as well as CYP 1A activity. Furthermore analysis of NS5B protein purified from a heterologous expression system treated with TGV by mass spectrometry suggests that TGV undergoes a CYP- mediated intracellular activation step and the resulting metabolite, after forming a glutathione conjugate, directly and specifically interacts with NS5B. Taken together, these data demonstrate that upon metabolic activation TGV is a specific, covalent inhibitor of the HCV NS5B polymerase and is mechanistically distinct from other classes of the non-nucleoside inhibitors (NNI) of the viral polymerase.
During IVF, non-transferred embryos are usually selected for cryopreservation on the basis of morphological criteria. This investigation evaluated an application for array comparative genomic hybridization (aCGH) in assessment of surplus embryos prior to cryopreservation.
First-time IVF patients undergoing elective single embryo transfer and having at least one extra non-transferred embryo suitable for cryopreservation were offered enrollment in the study. Patients were randomized into two groups: Patients in group A (n=55) had embryos assessed first by morphology and then by aCGH, performed on cells obtained from trophectoderm biopsy on post-fertilization day 5. Only euploid embryos were designated for cryopreservation. Patients in group B (n=48) had embryos assessed by morphology alone, with only good morphology embryos considered suitable for cryopreservation.
Among biopsied embryos in group A (n=425), euploidy was confirmed in 226 (53.1%). After fresh single embryo transfer, 64 (28.3%) surplus euploid embryos were cryopreserved for 51 patients (92.7%). In group B, 389 good morphology blastocysts were identified and a single top quality blastocyst was selected for fresh transfer. All group B patients (48/48) had at least one blastocyst remaining for cryopreservation. A total of 157 (40.4%) blastocysts were frozen in this group, a significantly larger proportion than was cryopreserved in group A (p=0.017, by chi-squared analysis).
While aCGH and subsequent frozen embryo transfer are currently used to screen embryos, this is the first investigation to quantify the impact of aCGH specifically on embryo cryopreservation. Incorporation of aCGH screening significantly reduced the total number of cryopreserved blastocysts compared to when suitability for freezing was determined by morphology only. IVF patients should be counseled that the benefits of aCGH screening will likely come at the cost of sharply limiting the number of surplus embryos available for cryopreservation.
Fertilization in vitro; Comparative genomic hybridization; Preimplantation genetic diagnosis; Cryopreservation
Single embryo transfer (SET) remains underutilized as a strategy to reduce multiple gestation risk in IVF, and its overall lower pregnancy rate underscores the need for improved techniques to select one embryo for fresh transfer. This study explored use of comprehensive chromosomal screening by array CGH (aCGH) to provide this advantage and improve pregnancy rate from SET.
First-time IVF patients with a good prognosis (age <35, no prior miscarriage) and normal karyotype seeking elective SET were prospectively randomized into two groups: In Group A, embryos were selected on the basis of morphology and comprehensive chromosomal screening via aCGH (from d5 trophectoderm biopsy) while Group B embryos were assessed by morphology only. All patients had a single fresh blastocyst transferred on d6. Laboratory parameters and clinical pregnancy rates were compared between the two groups.
For patients in Group A (n = 55), 425 blastocysts were biopsied and analyzed via aCGH (7.7 blastocysts/patient). Aneuploidy was detected in 191/425 (44.9%) of blastocysts in this group. For patients in Group B (n = 48), 389 blastocysts were microscopically examined (8.1 blastocysts/patient). Clinical pregnancy rate was significantly higher in the morphology + aCGH group compared to the morphology-only group (70.9 and 45.8%, respectively; p = 0.017); ongoing pregnancy rate for Groups A and B were 69.1 vs. 41.7%, respectively (p = 0.009). There were no twin pregnancies.
Although aCGH followed by frozen embryo transfer has been used to screen at risk embryos (e.g., known parental chromosomal translocation or history of recurrent pregnancy loss), this is the first description of aCGH fully integrated with a clinical IVF program to select single blastocysts for fresh SET in good prognosis patients. The observed aneuploidy rate (44.9%) among biopsied blastocysts highlights the inherent imprecision of SET when conventional morphology is used alone. Embryos randomized to the aCGH group implanted with greater efficiency, resulted in clinical pregnancy more often, and yielded a lower miscarriage rate than those selected without aCGH. Additional studies are needed to verify our pilot data and confirm a role for on-site, rapid aCGH for IVF patients contemplating fresh SET.
Evidence suggests that different types of mutation in gastrointestinal stromal tumours (GISTs) correlate with different response rates to imatinib (Glivec, STI571). The purpose of this study was to explore the sensitivity of the PDGFRAL839P mutant, a novel gain-of-function mutation isoform related to GISTs, to imatinib in vitro. The eukaryotic expression vectors pcDNA3.1-PDGFRAWild, pcDNA3.1-PDGFRAD842V and pcDNA3.1-PDGFRAL839P were constructed and transfected into Chinese hamster ovary (CHO) cells by liposome methods. The responses of cells with PDGFRAWild, PDGFRAL839P and PDGFRAD842V mutants to imatinib were determined by methyl thiazolyl tetrazolium (MTT) assay, western blotting and apoptosis assays. Reults of the MTT assay revealed that the growth rate of CHO(PDGFRAL839P) cells decreased to approximately 60% when exposed to 1 μM imatinib and to approximately 50% with 5 μM imatinib. However, the growth rate of CHO(PDGFRAD842V) cells did not significantly change with 5 μM imatinib. Western blot analysis indicated that 1 μM imatinib completely blocked the phosphorylation of PDGFRAL839P, but did not affect PDGFRAD842V phosphorylation. Apoptosis analysis suggested that the percentage of apoptotic CHO(PDGFRAL839P) cells increased approximately 4-fold (from 5.90 to 25.2%) with 1 μM imatinib. Although the treatment of CHO(PDGFRAD842V) and CHO(PDGFRAWild) cells with 5 μM imatinib resulted in a slight increase in the number of apoptotic cells, the percentage of apoptotic cells remained approximately 10% of the total population. Our findings showed that the PDGFRA gene mutation isoform L839P is sensitive to inhibition by imatinib. Screening for PDGFRA mutations in GISTs is essential to identify the response to treatment with imatinib.
PDGFRA mutant; imatinib; drug sensitivity; gastrointestinal stromal tumour
The evidence is increasing that cancer stem cells (CSCs) expressing embryonic and neuronal stem cell markers are present in human retinoblastoma (Rb). This study was conducted to determine whether stem-like cancer cells (SLCCs) in Rb express retinal stem cell–related genes and whether SLCCs can directly differentiate into retinal neurons.
The cancer stem cell characteristics in WERI-Rb1 cells were determined with Hoechst 33,342 staining, clone formation assay, and CD133 flow cytometry. The expression of embryonic stem cell and retinal stem cell–related genes was analyzed with real-time PCR and immunofluorescence. The SLCCs were induced to differentiate into retinal neurons by the addition of Dickkopf-related protein 1 and Lefty-A.
A small but persistent population of cells excluding Hoechst dye in a verapamil-sensitive manner exhibited a cancer stem cell–like phenotype. The SLCCs displayed highly clonogenic abilities and increased CD133 expression with isolation and expansion in culture in serum-free medium. By comparing the expression of stem cell markers, we found Oct3/4 was more highly expressed in the SLCCs than in human embryonic stem cells. Together with the properties of intrinsic retinal stem cell–related gene expression, we found SLCCs can be induced into neuron-like cells that express glial fibrillary acidic protein and rhodopsin (a photoreceptor cell marker).
These findings provide new insight into cancer stem cells and used a strategy of an artificial change of cancer stem cell fate with transcription factors.
HIV-1 RNase H breaks down the intermediate RNA-DNA hybrids during reverse transcription, requiring two divalent metal ions for activity. Pyrimidinol carboxylic acid and N-hydroxy quinazolinedione inhibitors were designed to coordinate the two metal ions in the active site of RNase H. High-resolution (1.4 Å to 2.1 Å) crystal structures were determined with the isolated RNase H domain and reverse transcriptase (RT), which permit accurate assessment of the metal and water environment at the active site. The geometry of the metal coordination suggests that the inhibitors mimic a substrate state prior to phosphodiester catalysis. Surface plasmon resonance studies confirm metal-dependent binding to RNase H and demonstrate that the inhibitors do not bind at the polymerase active site of RT. Additional evaluation of the RNase H site reveals an open protein surface with few additional interactions to optimize active-site inhibitors.
Highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) has caused large economic losses in swine industry in recent years. However, current antiviral strategy could not effectively prevent and control this disease. In this research, five artificial microRNAs (amiRNAs) respectively targeted towards ORF5 (amirGP5-243, -370) and ORF6 (amirM-82, -217,-263) were designed and incorporated into a miRNA-based vector that mimics the backbone of murine miR-155 and permits high expression of amiRNAs in a GFP fused form mediated by RNA Pol II promoter CMV.
It was found that amirGP5-370 could effectively inhibit H-PRRSV replication. The amirM-263-M-263, which was a dual pre-amiRNA expression cassette where two amirM-263s were chained, showed stronger virus inhibitory effects than single amirM-263. H-PRRSV replication was inhibited up to 120 hours in the MARC-145 cells which were stably transduced by recombinant lentiviruses (Lenti-amirGP5-370, -amirM-263-M-263). Additionally, efficacious dose of amirGP5-370 and amirM-263 expression did not trigger the innate interferon response.
Our study is the first attempt to suppress H-PRRSV replication in MARC-145 cells through vector-based and lentiviral mediated amiRNAs targeting GP5 or M proteins coding sequences of PRRSV, which indicated that artificial microRNAs and recombinant lentiviruses might be applied to be a new potent anti-PRRSV strategy.
Highly pathogenic PRRSV; RNAi; artificial miRNA; Lentivirus
GS-8374 is a novel bis-tetrahydrofuran HIV-1 protease (PR) inhibitor (PI) with a unique diethylphosphonate moiety. It was selected from a series of analogs containing various di(alkyl)phosphonate substitutions connected via a linker to the para position of a P-1 phenyl ring. GS-8374 inhibits HIV-1 PR with high potency (Ki = 8.1 pM) and with no known effect on host proteases. Kinetic and thermodynamic analysis of GS-8374 binding to PR demonstrated an extremely slow off rate for the inhibitor and favorable contributions of both the enthalpic and entropic components to the total free binding energy. GS-8374 showed potent antiretroviral activity in T-cell lines, primary CD4+ T cells (50% effective concentration [EC50] = 3.4 to 11.5 nM), and macrophages (EC50 = 25.5 nM) and exhibited low cytotoxicity in multiple human cell types. The antiviral potency of GS-8374 was only moderately affected by human serum protein binding, and its combination with multiple approved antiretrovirals showed synergistic effects. When it was tested in a PhenoSense assay against a panel of 24 patient-derived viruses with high-level PI resistance, GS-8374 showed lower mean EC50s and lower fold resistance than any of the clinically approved PIs. Similar to other PIs, in vitro hepatic microsomal metabolism of GS-8374 was efficiently blocked by ritonavir, suggesting a potential for effective pharmacokinetic boosting in vivo. In summary, results from this broad in vitro pharmacological profiling indicate that GS-8374 is a promising candidate to be further assessed as a new antiretroviral agent with potential for clinical efficacy in both treatment-naïve and -experienced patients.
The structural mechanism by which non-structural protein 3 (NS3) from the hepatitis C virus (HCV) translocates along RNA is currently unknown. HCV NS3 is an ATP-dependent motor protein essential for viral replication and a member of the superfamily 2 (SF2) helicases. Crystallographic analysis using a labeled RNA oligonucleotide allowed us to unambiguously track the positional changes of RNA bound to full-length HCV NS3 during two discrete steps of the ATP hydrolytic cycle. The crystal structures of HCV NS3, NS3 bound to bromine-labeled RNA, and a tertiary complex of NS3 bound to labeled RNA and a non-hydrolyzable ATP analog provide a direct view of how large domain movements resulting from ATP binding and hydrolysis allow the enzyme to translocate along the phosphodiester backbone. While directional translocation of HCV NS3 by a single base pair per ATP hydrolyzed is observed, the 3’-end of the RNA does not shift register with respect to a conserved tryptophan residue, supporting a “spring-loading” mechanism that leads to larger steps by the enzyme as it moves along a nucleic acid substrate.
Parvovirus B19 (B19V) is pathogenic for humans and has an extreme tropism for human erythroid progenitors. We report cell type-specific expression of the B19V capsid genes (VP1 and VP2) and greatly increased B19V capsid protein production in nonpermissive cells by codon optimization. Codon usage limitation, rather than promoter type and the 3′ untranslated region of the capsid genes, appears to be a key factor in capsid protein production in nonpermissive cells. Moreover, B19 virus-like particles were successfully generated in nonpermissive cells by transient transfection of a plasmid carrying both codon-optimized VP1 and VP2 genes.
Understanding the dynamics of muscle transcriptome during development and between breeds differing in muscle growth is necessary to uncover the complex mechanism underlying muscle development. Herein, we present the first transcriptome-wide longissimus dorsi muscle development research concerning Lantang (LT, obese) and Landrace (LR, lean) pig breeds during 10 time-points from 35 days-post-coitus (dpc) to 180 days-post-natum (dpn) using Solexa/Illumina's Genome Analyzer. The data demonstrated that myogenesis was almost completed before 77 dpc, but the muscle phenotypes were still changed from 77 dpc to 28 dpn. Comparative analysis of the two breeds suggested that myogenesis started earlier but progressed more slowly in LT than in LR, the stages ranging from 49 dpc to 77 dpc are critical for formation of different muscle phenotypes. 595 differentially expressed myogenesis genes were identified, and their roles in myogenesis were discussed. Furthermore, GSK3B, IKBKB, ACVR1, ITGA and STMN1 might contribute to later myogenesis and more muscle fibers in LR than LT. Some myogenesis inhibitors (ID1, ID2, CABIN1, MSTN, SMAD4, CTNNA1, NOTCH2, GPC3 and HMOX1) were higher expressed in LT than in LR, which might contribute to more slow muscle differentiation in LT than in LR. We also identified several genes which might contribute to intramuscular adipose differentiation. Most important, we further proposed a novel model in which MyoD and MEF2A controls the balance between intramuscular adipogenesis and myogenesis by regulating CEBP family; Myf5 and MEF2C are essential during the whole myogenesis process while MEF2D affects muscle growth and maturation. The MRFs and MEF2 families are also critical for the phenotypic differences between the two pig breeds. Overall, this study contributes to elucidating the mechanism underlying muscle development, which could provide valuable information for pig meat quality improvement.
The raw data have been submitted to Gene Expression Omnibus (GEO) under series GSE25406.
There was a large scale outbreak of the highly pathogenic porcine reproductive and respiratory syndrome (PRRS) in China and Vietnam during 2006 and 2007 that resulted in unusually high morbidity and mortality among pigs of all ages. The mechanisms underlying the molecular pathogenesis of the highly virulent PRRS virus (H-PRRSV) remains unknown. Therefore, the relationship between pulmonary gene expression profiles after H-PRRSV infection and infection pathology were analyzed in this study using high-throughput deep sequencing and histopathology.
H-PRRSV infection resulted in severe lung pathology. The results indicate that aberrant host innate immune responses to H-PRRSV and induction of an anti-apoptotic state could be responsible for the aggressive replication and dissemination of H-PRRSV. Prolific rapid replication of H-PRRSV could have triggered aberrant sustained expression of pro-inflammatory cytokines and chemokines leading to a markedly robust inflammatory response compounded by significant cell death and increased oxidative damage. The end result was severe tissue damage and high pathogenicity.
The systems analysis utilized in this study provides a comprehensive basis for better understanding the pathogenesis of H-PRRSV. Furthermore, it allows the genetic components involved in H-PRRSV resistance/susceptibility in swine populations to be identified.
Two new cytotoxic xanthones were isolated from extracts of the Madagascar rain forest plant Psorospermum cf. molluscum using bioassay guided fractionation with the Escherichia coli SOS chromotest. The structures of the new dihydrofuranoxanthones, designated 3′,4′-deoxy-4′-chloropsoroxanthin-(3′,5′-diol) (1) and psoroxanthin (4), were determined on the basis of 2D-NMR, MS and UV spectroscopic data, and are structurally related to the psorospermins, a known class of plant antitumor agents. A new hydroxyprenylated xanthone (5) is also described. Xanthones (1) and (4) showed selective in vitro cytotoxicity against ABAE cells (bovine endothelial cell line).
In order to study the daily Pb absorption in fetus and to monitor the main Pb sources in prenatal fetus, we have investigated several cases of Pb distribution along the longitudinal axis of fetal hair. The changes of Pb levels in the pregnancy period, even the daily changes of Pb levels can be detected in the hair. Therefore, by analyzing the Pb distribution curves in the fetal hair and the living habits of their mothers, the main sources of Pb in the prenatal fetus can be evaluated. In our study, the main sources of Pb in the two cases of prenatal fetus studied here should be from the polluted aquatics.
Synchrotron radiation micro XRF analysis; Fetal hair; Lead
Porcine reproductive and respiratory syndrome (PRRS) has been one of the most economically important diseases affecting swine industry worldwide and causes great economic losses each year. PRRS virus (PRRSV) replicates mainly in porcine alveolar macrophages (PAMs) and dendritic cells (DCs) and develops persistent infections, antibody-dependent enhancement (ADE), interstitial pneumonia and immunosuppression. But the molecular mechanisms of PRRSV infection still are poorly understood. Here we report on the first genome-wide host transcriptional responses to classical North American type PRRSV (N-PRRSV) strain CH 1a infection using Solexa/Illumina's digital gene expression (DGE) system, a tag-based high-throughput transcriptome sequencing method, and analyse systematically the relationship between pulmonary gene expression profiles after N-PRRSV infection and infection pathology. Our results suggest that N-PRRSV appeared to utilize multiple strategies for its replication and spread in infected pigs, including subverting host innate immune response, inducing an anti-apoptotic and anti-inflammatory state as well as developing ADE. Upregulation expression of virus-induced pro-inflammatory cytokines, chemokines, adhesion molecules and inflammatory enzymes and inflammatory cells, antibodies, complement activation were likely to result in the development of inflammatory responses during N-PRRSV infection processes. N-PRRSV-induced immunosuppression might be mediated by apoptosis of infected cells, which caused depletion of immune cells and induced an anti-inflammatory cytokine response in which they were unable to eradicate the primary infection. Our systems analysis will benefit for better understanding the molecular pathogenesis of N-PRRSV infection, developing novel antiviral therapies and identifying genetic components for swine resistance/susceptibility to PRRS.