miR-27b Represses Migration of Mouse MSCs to Burned Margins and Prolongs Wound Repair through Silencing SDF-1a

Background

Interactions between stromal cell-derived factor-1α (SDF-1α) and its cognate receptor CXCR4 are crucial for the recruitment of mesenchymal stem cells (MSCs) from bone marrow (BM) reservoirs to damaged tissues for repair during alarm situations. MicroRNAs are differentially expressed in stem cell niches, suggesting a specialized role in stem cell regulation. Here, we gain insight into the molecular mechanisms involved in regulating SDF-1α.

Methods

MSCs from green fluorescent protein transgenic male mice were transfused to irradiated recipient female C57BL/6 mice, and skin burn model of bone marrow-chimeric mice were constructed. Six miRNAs with differential expression in burned murine skin tissue compared to normal skin tissue were identified using microarrays and bioinformatics. The expression of miR-27b and SDF-1α was examined in burned murine skin tissue using quantitative real-time PCR (qPCR) and immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA). The Correlation of miR-27b and SDF-1α expression was analyzed by Pearson analysis Correlation. miRNAs suppressed SDF-1α protein expression by binding directly to its 3′UTR using western blot and luciferase reporter assay. The importance of miRNAs in MSCs chemotaxis was further estimated by decreasing SDF-1α in vivo and in vitro.

Results

miR-23a, miR-27a and miR-27b expression was significantly lower in the burned skin than in the normal skin (p<0.05). We also found that several miRNAs suppressed SDF-1α protein expression, while just miR-27a and miR-27b directly bound to the SDF-1α 3′UTR. Moreover, the forced over-expression of miR-27a and miR-27b significantly reduced the directional migration of mMSCs in vitro. However, only miR-27b in burn wound margins significantly inhibited the mobilization of MSCs to the epidermis.

Conclusion

miR-27b may be a unique signature of the stem cell niche in burned mouse skin and can suppress the directional migration of mMSCs by targeting SDF-1α by binding directly to its 3′UTR.     

αA-Crystallin Peptide 66 SDRDKFVIFLDVKHF 80 Accumulating in Aging Lens Impairs the Function of α-Crystallin and Induces Lens Protein Aggregation

Background

The eye lens is composed of fiber cells that are filled with α-, β- and γ-crystallins. The primary function of crystallins is to maintain the clarity of the lens through ordered interactions as well as through the chaperone-like function of α-crystallin. With aging, the chaperone function of α-crystallin decreases, with the concomitant accumulation of water-insoluble, light-scattering oligomers and crystallin-derived peptides. The role of crystallin-derived peptides in age-related lens protein aggregation and insolubilization is not understood.

Methodology/Principal Findings

We found that αA-crystallin-derived peptide, ⁶⁶ SDRDKFVIFLDVKHF ⁸⁰, which accumulates in the aging lens, can inhibit the chaperone activity of α-crystallin and cause aggregation and precipitation of lens crystallins. Age-related change in the concentration of αA-(66-80) peptide was estimated by mass spectrometry. The interaction of the peptide with native crystallin was studied by multi-angle light scattering and fluorescence methods. High molar ratios of peptide-to-crystallin were favourable for aggregation and precipitation. Time-lapse recordings showed that, in the presence of αA-(66-80) peptide, α-crystallin aggregates and functions as a nucleus for protein aggregation, attracting aggregation of additional α-, β- and γ-crystallins. Additionally, the αA-(66-80) peptide shares the principal properties of amyloid peptides, such as β-sheet structure and fibril formation.

Conclusions/Significance

These results suggest that crystallin-derived peptides such as αA-(66-80), generated in vivo, can induce age-related lens changes by disrupting the structure and organization of crystallins, leading to their insolubilization. The accumulation of such peptides in aging lenses may explain a novel mechanism for age-related crystallin aggregation and cataractogenesis.     

DNA Polymerase δ Is Required for Early Mammalian Embryogenesis

Background

In eukaryotic cells, DNA polymerase δ (Polδ), whose catalytic subunit p125 is encoded in the Pold1 gene, plays a central role in chromosomal DNA replication, repair, and recombination. However, the physiological role of the Polδ in mammalian development has not been thoroughly investigated.

Methodology/Principal Findings

To examine this role, we used a gene targeting strategy to generate two kinds of Pold1 mutant mice: Polδ-null (Pold1 ^−/−) mice and D400A exchanged Polδ (Pold1 ^exo/exo) mice. The D400A exchange caused deficient 3′–5′ exonuclease activity in the Polδ protein. In Polδ-null mice, heterozygous mice developed normally despite a reduction in Pold1 protein quantity. In contrast, homozygous Pold1 ^−/− mice suffered from peri-implantation lethality. Although Pold1 ^−/− blastocysts appeared normal, their in vitro culture showed defects in outgrowth proliferation and DNA synthesis and frequent spontaneous apoptosis, indicating Polδ participates in DNA replication during mouse embryogenesis. In Pold1 ^exo/exo mice, although heterozygous Pold1 ^exo/+ mice were normal and healthy, Pold1 ^exo/exo and Pold1 ^exo/− mice suffered from tumorigenesis.

Conclusions

These results clearly demonstrate that DNA polymerase δ is essential for mammalian early embryogenesis and that the 3′–5′ exonuclease activity of DNA polymerase δ is dispensable for normal development but necessary to suppress tumorigenesis.     

Sequence Conservation in Plasmodium falciparum α-Helical Coiled Coil Domains Proposed for Vaccine Development

Background

The availability of the P. falciparum genome has led to novel ways to identify potential vaccine candidates. A new approach for antigen discovery based on the bioinformatic selection of heptad repeat motifs corresponding to α-helical coiled coil structures yielded promising results. To elucidate the question about the relationship between the coiled coil motifs and their sequence conservation, we have assessed the extent of polymorphism in putative α-helical coiled coil domains in culture strains, in natural populations and in the single nucleotide polymorphism data available at PlasmoDB.

Methodology/Principal Findings

14 α-helical coiled coil domains were selected based on preclinical experimental evaluation. They were tested by PCR amplification and sequencing of different P. falciparum culture strains and field isolates. We found that only 3 out of 14 α-helical coiled coils showed point mutations and/or length polymorphisms. Based on promising immunological results 5 of these peptides were selected for further analysis. Direct sequencing of field samples from Papua New Guinea and Tanzania showed that 3 out of these 5 peptides were completely conserved. An in silico analysis of polymorphism was performed for all 166 putative α-helical coiled coil domains originally identified in the P. falciparum genome. We found that 82% (137/166) of these peptides were conserved, and for one peptide only the detected SNPs decreased substantially the probability score for α-helical coiled coil formation. More SNPs were found in arrays of almost perfect tandem repeats. In summary, the coiled coil structure prediction was rarely modified by SNPs. The analysis revealed a number of peptides with strictly conserved α-helical coiled coil motifs.

Conclusion/Significance

We conclude that the selection of α-helical coiled coil structural motifs is a valuable approach to identify potential vaccine targets showing a high degree of conservation.     

Comparative genomics of methicillin-resistant Staphylococcus aureus ST239: distinct geographical variants in Beijing and Hong Kong

Background

The ST239 lineage is a globally disseminated, multiply drug-resistant hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA). We performed whole-genome sequencing of representative HA-MRSA isolates of the ST239 lineage from bacteremic patients in hospitals in Hong Kong (HK) and Beijing (BJ) and compared them with three published complete genomes of ST239, namely T0131, TW20 and JKD6008. Orthologous gene group (OGG) analyses of the Hong Kong and Beijing cluster strains were also undertaken.

Results

Homology analysis, based on highest-percentage nucleotide identity, indicated that HK isolates were closely related to TW20, whereas BJ isolates were more closely related to T0131 from Tianjin. Phylogenetic analysis, incorporating a total of 30 isolates from different continents, revealed that strains from HK clustered with TW20 into the ‘Asian clade’, whereas BJ isolates and T0131 clustered closely with strains of the ‘Turkish clade’ from Eastern Europe. HK isolates contained the typical φSPβ-like prophage with the SasX gene similar to TW20. In contrast, BJ isolates contained a unique 15 kb PT1028-like prophage but lacked φSPβ-like and φSA1 prophages. Besides distinct mobile genetic elements (MGE) in the two clusters, OGG analyses and whole-genome alignment of these clusters highlighted differences in genes located in the core genome, including the identification of single nucleotide deletions in several genes, resulting in frameshift mutations and the subsequent predicted truncation of encoded proteins involved in metabolism and antimicrobial resistance.

Conclusions

Comparative genomics, based on de novo assembly and deep sequencing of HK and BJ strains, revealed different origins of the ST239 lineage in northern and southern China and identified differences between the two clades at single nucleotide polymorphism (SNP), core gene and MGE levels. The results suggest that ST239 strains isolated in Hong Kong since the 1990s belong to the Asian clade, present mainly in southern Asia, whereas those that emerged in northern China were of a distinct origin, reflecting the complexity of dissemination and the dynamic evolution of this ST239 lineage.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-529) contains supplementary material, which is available to authorized users.     

Randomized Trial of Piperaquine with Sulfadoxine-Pyrimethamine or Dihydroartemisinin for Malaria Intermittent Preventive Treatment in Children

Background

The long terminal half life of piperaquine makes it suitable for intermittent preventive treatment for malaria but no studies of its use for prevention have been done in Africa. We did a cluster randomized trial to determine whether piperaquine in combination with either dihydroartemisin (DHA) or sulfadoxine-pyrimethamine (SP) is as effective, and better tolerated, than SP plus amodiaquine (AQ), when used for intermittent preventive treatment in children delivered by community health workers in a rural area of Senegal.

Methods

Treatments were delivered to children 3–59 months of age in their homes once per month during the transmission season by community health workers. 33 health workers, each covering about 60 children, were randomized to deliver either SP+AQ, DHA+PQ or SP+PQ. Primary endpoints were the incidence of attacks of clinical malaria, and the incidence of adverse events.

Results

1893 children were enrolled. Coverage of monthly rounds and compliance with daily doses was similar in all groups; 90% of children received at least 2 monthly doses. Piperaquine combinations were better tolerated than SP+AQ with a significantly lower risk of common, mild adverse events. 103 episodes of clinical malaria were recorded during the course of the trial. 68 children had malaria with parasitaemia >3000/µL, 29/671 (4.3%) in the SP+AQ group, compared with 22/604 (3.6%) in the DHA+PQ group (risk difference 0.47%, 95%CI −2.3%,+3.3%), and 17/618 (2.8%) in the SP+PQ group (risk difference 1.2%, 95%CI −1.3%,+3.6%). Prevalences of parasitaemia and the proportion of children carrying Pfdhfr and Pfdhps mutations associated with resistance to SP were very low in all groups at the end of the transmission season.

Conclusions

Seasonal IPT with SP+PQ in children is highly effective and well tolerated; the combination of two long-acting drugs is likely to impede the emergence of resistant parasites.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00529620","term_id":"NCT00529620"}}NCT00529620     

Altered Social Behaviours in Neurexin 1α Knockout Mice Resemble Core Symptoms in Neurodevelopmental Disorders

Background

Copy number variants have emerged as an important genomic cause of common, complex neurodevelopmental disorders. These usually change copy number of multiple genes, but deletions at 2p16.3, which have been associated with autism, schizophrenia and mental retardation, affect only the neurexin 1 gene, usually the alpha isoform. Previous analyses of neurexin 1α (Nrxn1α) knockout (KO) mouse as a model of these disorders have revealed impairments in synaptic transmission but failed to reveal defects in social behaviour, one of the core symptoms of autism.

Methods

We performed a detailed investigation of the behavioural effects of Nrxn1α deletion in mice bred onto a pure genetic background (C57BL/6J) to gain a better understanding of its role in neurodevelopmental disorders. Wildtype, heterozygote and homozygote Nrxn1α KO male and female mice were tested in a battery of behavioural tests (n = 9–16 per genotype, per sex).

Results

In homozygous Nrxn1α KO mice, we observed altered social approach, reduced social investigation, and reduced locomotor activity in novel environments. In addition, male Nrxn1α KO mice demonstrated an increase in aggressive behaviours.

Conclusions

These are the first experimental data that associate a deletion of Nrxn1α with alterations of social behaviour in mice. Since this represents one of the core symptom domains affected in autism spectrum disorders and schizophrenia in humans, our findings suggest that deletions within NRXN1 found in patients may be responsible for the impairments seen in social behaviours, and that the Nrxn1α KO mice are a useful model of human neurodevelopmental disorder.     

Elevated Levels of Dickkopf-1 Are Associated with β-Catenin Accumulation and Poor Prognosis in Patients with Chondrosarcoma

Background

Dickkopf-1 (DKK1) is an antagonist of Wnt/β-catenin signaling implicated in tumorigenesis. However, the biological role of DKK1 and β-catenin involved in chondrosarcoma has not been sufficiently investigated. This study was designed to investigate the expression profiles of DKK1 and β-catenin, and to clarify their clinical values in chondrosarcoma.

Methods

The mRNA and protein levels of DKK1 and β-catenin in fresh chondrosarcoma and the corresponding non-tumor tissues were analyzed by Real-time PCR and Western blot, respectively. The protein expression patterns of DKK1 and β-catenin were investigated by immunohistochemistry. The associations among DKK1 level, β-catenin accumulation, clinicopathological factors and the overall survival were separately evaluated.

Results

Both DKK1 and β-catenin levels were remarkably elevated in chondrosarcoma compared with the corresponding non-tumor tissues. High DKK1 level and positive β-catenin accumulation in chondrosarcoma specimens were 58.7% and 53.9%, respectively. Elevated DKK1 level significantly correlated with positive β-catenin accumulation, and they were remarkably associated with histological grade and Musculoskeletal Tumor Society stage. Furthermore, DKK1 level and β-catenin accumulation had significant impacts on the prognosis of chondrosarcoma patients. Multivariate analysis revealed that DKK1 level was an independent prognostic factor for overall survival.

Conclusions

Elevated DKK1 levels associated with β-catenin accumulation play a crucial role in chondrosarcoma. DKK1 can serve as a novel predictor of poor prognosis in patients with chondrosarcoma.     

Insights in 17β-HSD1 Enzyme Kinetics and Ligand Binding by Dynamic Motion Investigation

Background

Bisubstrate enzymes, such as 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), exist in solution as an ensemble of conformations. 17β-HSD1 catalyzes the last step of the biosynthesis of estradiol and, thus, it is a potentially attractive target for breast cancer treatment.

Methodology/Principal Findings

To elucidate the conformational transitions of its catalytic cycle, a structural analysis of all available crystal structures was performed and representative conformations were assigned to each step of the putative kinetic mechanism. To cover most of the conformational space, all-atom molecular dynamic simulations were performed using the four crystallographic structures best describing apoform, opened, occluded and closed state of 17β-HSD1 as starting structures. With three of them, binary and ternary complexes were built with NADPH and NADPH-estrone, respectively, while two were investigated as apoform. Free energy calculations were performed in order to judge more accurately which of the MD complexes describes a specific kinetic step.

Conclusions/Significance

Remarkably, the analysis of the eight long range trajectories resulting from this multi-trajectory/-complex approach revealed an essential role played by the backbone and side chain motions, especially of the βFαG′-loop, in cofactor and substrate binding. Thus, a selected-fit mechanism is suggested for 17β-HSD1, where ligand-binding induced concerted motions of the FG-segment and the C-terminal part guide the enzyme along its preferred catalytic pathway. Overall, we could assign different enzyme conformations to the five steps of the random bi-bi kinetic cycle of 17β-HSD1 and we could postulate a preferred pathway for it. This study lays the basis for more-targeted biochemical studies on 17β-HSD1, as well as for the design of specific inhibitors of this enzyme. Moreover, it provides a useful guideline for other enzymes, also characterized by a rigid core and a flexible region directing their catalysis.     

Volatile Anesthetics,Not Intravenous Anesthetic Propofol Bind to and Attenuate the Activation of Platelet Receptor Integrin αIIbβ3

Background

In clinical reports, the usage of isoflurane and sevoflurane was associated with more surgical field bleeding in endoscopic sinus surgeries as compared to propofol. The activation of platelet receptor αIIbβ3 is a crucial event for platelet aggregation and clot stability. Here we studied the effect of isoflurane, sevoflurane, and propofol on the activation of αIIbβ3.

Methods

The effect of anesthetics on the activation of αIIbβ3 was probed using the activation sensitive antibody PAC-1 in both cell-based (platelets and αIIbβ3 transfectants) and cell-free assays. The binding sites of isoflurane on αIIbβ3 were explored using photoactivatable isoflurane (azi-isoflurane). The functional implication of revealed isoflurane binding sites were studied using alanine-scanning mutagenesis.

Results

Isoflurane and sevoflurane diminished the binding of PAC-1 to wild-type αIIbβ3 transfectants, but not to the high-affinity mutant, β3-N305T. Both anesthetics also impaired PAC-1 binding in a cell-free assay. In contrast, propofol did not affect the activation of αIIbβ3. Residues adducted by azi-isoflurane were near the calcium binding site (an important regulatory site termed SyMBS) just outside of the ligand binding site. The mutagenesis experiments demonstrated that these adducted residues were important in regulating integrin activation.

Conclusions

Isoflurane and sevoflurane, but not propofol, impaired the activation of αIIbβ3. Azi-isoflurane binds to the regulatory site of integrin αIIbβ3, thereby suggesting that isoflurane blocks ligand binding of αIIbβ3 in not a competitive, but an allosteric manner.     

Interleukin-1β-31C/T and -511T/C Polymorphisms Were Associated with Preeclampsia in Chinese Han Population

Objective

The purpose of our study is to investigate the relationship between IL-1β -31C/T (rs1143627) and -511T/C (rs16944) polymorphisms and the preeclampsia (PE), and analyze the Linkage disequilibrium (LD) and haplotype frequency of the two polymorphism loci.

Methods

Polymorphisms at -31C/T and -511T/C of IL-1β were genotyped with the method of polymerase chain reaction-restriction fragment length polymorphism (PCR- RFLP) in 232 PE and 447 control subjects. Genotype and allele frequencies between case-control groups were compared by chi-square(X²) tests. Two-point LD and haplotype frequency analyses were done with the software Haploview4.2.

Results

Significant statistical differences were found between PE and control groups regarding genotype and allele frequencies of the two polymorphisms of IL-1β (For IL-1β -31C/T: X² = 11.478, P = 0.003; For IL-1β-511T/C: X² = 9.687, P = 0.008). LD analysis revealed that the IL-1β -31C/T SNP was in high LD with the IL-1β-511C/T SNP(D′ = 0.92, r² = 0.79). Both CT and TC haplotypes showed significant differences between case and control groups. Only the plasma level of Prothrombin Time had a significantly statistical difference among TT, CT and CC groups of the preeclamptic two polymorphisms of IL-1β-31C/T and -511T/C (for IL-1β-31C/T, F = 1.644, P = 0.01; F = 1.587, P = 0.016).

Conclusion

Our results revealed IL-1β was associated with the PE in Chinese Han population. The CT haplotype may increase the risk of PE, while haplotype TC could be considered as a protective haplotype of PE.     

Extra Cellular Matrix Derived Metabolite Regulates Angiogenesis by FasL Mediated Apoptosis

Object

Antiangiogenic treatments are beginning to give promising outcomes in many vascular diseases including tumor angiogenesis. In this current study the antiangiogenic and pro-apoptotic actions of α1(IV)NC1 and its N- and C- peptides α1S1(IV)NC1, α1S2(IV)NC1 were investigated in-vitro and in-vivo.

Study Method

Endothelial cells (ECs) were treated with α1(IV)NC1, α1S1(IV)NC1, α1S2(IV)NC1 and in-vitro proliferation, migration, tube formation and apoptotic assays were executed. FasL, Fas, Caspase-8, -3 and PARP activations were studied using immunoblotting analysis using specific antibodies. Also the in-vivo antiangiogenic and pro-apoptotic effects were tested using α1(IV)NC1 in a mice model.

Results

Like α1(IV)NC1, its N- and C- terminal α1S2(IV)NC1 and α1S1(IV)NC1 domains posses anti-proliferative, pro-apoptotic activity and inhibit ECs migration and tube formation in-vitro. Both α1S1(IV)NC1 and α1S2(IV)NC1 domains promote apoptosis by activating FasL and down stream apoptotic events including activation of caspase-8, -3 and PARP cleavage in a dose dependent manner in-vitro in ECs. Tumors in mice showed apoptotic TUNEL positive microvasculature upon α1(IV)NC1 treatment, indicating inhibition of tumor angiogenesis and tumor growth. Further, the antitumor activity of α1(IV)NC1 was abrogated when caspase-3 inhibitor was used. These results conform additional properties of α1(IV)NC1 as an endogenous angioinhibitor that induces apoptosis in-vitro and in-vivo by activating FasL mediated caspase-3.

Significance

α1(IV)NC1 and its N- and C- terminal α1S1(IV)NC1 and α1S2(IV)NC1 domains also posses pro-apoptotic and angioinhibitory activity in-vitro and in-vivo. α1(IV)NC1 regulates tumor angiogenesis by activating FasL mediated apoptosis in-vitro and in-vivo. These results demonstrate that α1(IV)NC1 and its peptides inhibit neo-vascular diseases.     

The HSP70 Molecular Chaperone Is Not Beneficial in a Mouse Model of α-synucleinopathy

Background

Aggregation and misfolded α-synuclein is thought to be central in the pathogenesis of Parkinson''s disease (PD). Heat-shock proteins (HSPs) that are involved in refolding and degradation processes could lower the aggregate load of α-synuclein and thus be beneficial in α-synucleinopathies.

Methodology/Principal Findings

We co-overexpressed human A53T point-mutated α-synuclein and human HSP70 in mice, both under the control of Thy1 regulatory sequences. Behavior read-outs showed no beneficial effect of HSP70 expression in mice. In contrast, motor coordination, grip strength and weight were even worse in the α-synucleinopathy model in the presence of HSP70 overexpression. Biochemical analyses revealed no differences in α-synuclein oligomers/aggregates, truncations and phosphorylation levels and α-synuclein localization was unchanged in immunostainings.

Conclusion/Significance

Overexpressing HSP70 in a mouse model of α-synucleinopathy did not lower the toxic load of α-synuclein species and had no beneficial effect on α-synuclein-related motor deficits.     

Simvastatin inhibits TGFβ1-induced fibronectin in human airway fibroblasts

Background

Bronchial fibroblasts contribute to airway remodelling, including airway wall fibrosis. Transforming growth factor (TGF)-β1 plays a major role in this process. We previously revealed the importance of the mevalonate cascade in the fibrotic response of human airway smooth muscle cells. We now investigate mevalonate cascade-associated signaling in TGFβ1-induced fibronectin expression by bronchial fibroblasts from non-asthmatic and asthmatic subjects.

Methods

We used simvastatin (1-15 μM) to inhibit 3-hydroxy-3-methlyglutaryl-coenzyme A (HMG-CoA) reductase which converts HMG-CoA to mevalonate. Selective inhibitors of geranylgeranyl transferase-1 (GGT1; GGTI-286, 10 μM) and farnesyl transferase (FT; FTI-277, 10 μM) were used to determine whether GGT1 and FT contribute to TGFβ1-induced fibronectin expression. In addition, we studied the effects of co-incubation with simvastatin and mevalonate (1 mM), geranylgeranylpyrophosphate (30 μM) or farnesylpyrophosphate (30 μM).

Results

Immunoblotting revealed concentration-dependent simvastatin inhibition of TGFβ1 (2.5 ng/ml, 48 h)-induced fibronectin. This was prevented by exogenous mevalonate, or isoprenoids (geranylgeranylpyrophosphate or farnesylpyrophosphate). The effects of simvastatin were mimicked by GGTI-286, but not FTI-277, suggesting fundamental involvement of GGT1 in TGFβ1-induced signaling. Asthmatic fibroblasts exhibited greater TGFβ1-induced fibronectin expression compared to non-asthmatic cells; this enhanced response was effectively reduced by simvastatin.

Conclusions

We conclude that TGFβ1-induced fibronectin expression in airway fibroblasts relies on activity of GGT1 and availability of isoprenoids. Our results suggest that targeting regulators of isoprenoid-dependent signaling holds promise for treating airway wall fibrosis.