Choline and trimethylamine N-oxide supplementation in normal chow diet and western diet promotes the development of atherosclerosis in Apoe -/- mice through different mechanisms.

Trimethylamine N-oxide (TMAO), a gut microbiota-dependent metabolite, has been shown to aggravate cardiovascular disease. However, the mechanisms of TMAO in the setting of cardiovascular disease progress remain unclear. Here, we aim to investigate the effects of TMAO on atherosclerosis (AS) development and the underlying mechanisms. Apoe -/- mice received choline or TMAO supplementation in a normal diet and a western diet for 12 weeks. Choline or TMAO supplementation in both normal diet and western diet significantly promoted plaque progression in Apoe-/- mice. Besides, serum lipids levels and inflammation response in the aortic root were enhanced by choline or TMAO supplementation. In particular, choline or TMAO supplementation in the western diet changed intestinal microbiota composition and bile acid metabolism. Therefore, choline or TMAO supplementation may promote AS by modulating gut microbiota in mice fed with a western diet and by other mechanisms in mice given a normal diet, even choline or TMAO supplementation in a normal diet can promote AS.

Ermin is a p116RIP -interacting protein promoting oligodendroglial differentiation and myelin maintenance.

Myelin sheaths, which insulate the axons and ensure saltatory conduction of the nerve impulse, are generated and maintained via largely uncharacterized mechanisms. Ermin is an oligodendrocyte-specific protein associated with the cytoskeleton, but how it regulates cytoskeletal remodeling during oligodendrocyte differentiation and its role in myelin maintenance are not clear. To address this, we generated mice constitutively deficient for Ermn, the Ermin-coding gene. We found that aged Ermn-knockout mice exhibit an aberrant myelin architecture, with splitting of myelin layers, peeling of the myelin sheath from axons, and breakdown of myelinated fibers. As a result, these mice had remarkably impaired motor coordination. Ermn knockout also accelerated cuprizone-induced demyelination and exacerbated the associated movement disorders. Ermin was found to contribute to oligodendrocyte morphogenesis by associating with the myosin phosphatase Rho interacting protein (Mprip/p116RIP ) and inactivating RhoA, a GTPase that controls cytoskeletal rearrangement in differentiating cells. These findings provide novel insights into the mechanisms regulating oligodendroglial differentiation, the maintenance of the myelin sheaths, and remyelination.

Research progress on 5hmC and TET dioxygenases in neurodevelopment and neurological diseases.

The development of the nervous system is coordinately regulated by multiple interacting factors. If a certain factor is altered or mutated, the coordinated developmental processes could be disrupted, resulting in neurological diseases. The 5-hydroxymethylcytosine (5hmC) is an intermediate product of the DNA demethylation processes. 5hmC and its metabolic enzymes, the ten-eleven translocation protein-TET family of dioxygenases, have recently been identified as new epigenetic players important in the regulation of the nervous system development, as well as in cognition, memory and other neurological functions. In various studies on neurodevelopment and neurodegeneration related diseases, the levels of 5hmC and TET proteins could be differentially regulated during development and/or disease pathogenesis, suggesting the potentially critical roles of 5hmC and TETs in these neural developmental and disease processes. In this review, we summarize the recent advances in research on 5hmC and TET dioxygenases in the regulation of neurodevelopment and neurological diseases, thereby providing significant insights on the involvements of 5hmC and TETs in neurodevelopment and on establishing new therapeutic strategies for human neurological diseases.

[Advances on the profiling of 5-hydroxymethylcytosine].

5-hydroxymethylcytosine (5hmC) is a naturally existing component in mammalian genomic DNA and is regarded as the sixth DNA base. Accumulating studies have revealed the essential role of 5hmC in embryonic development, brain function and cancer research. Compared to another well-known cytosine methylation derivate, 5-methylcytosine (5mC), the detection of 5hmC is difficult for its lower lever existing in most tissues. To distinguish 5hmC from other cytosine derivates, the methods using chemical or enzymatic DNA treatment, have been applied in targeted 5hmC detection or non-targeted 5hmC enrichment. Therefore, profiling DNA hydroxymethylcytosine by sensitive, accurate and reliable method is crucial for epigenetic study. This review discusses the principles behind recently developed techniques for 5hmC quantification and mapping. By comparing the advantages and shortcomings of these methods, the general guidelines were provided on how to select appropriate methods for specific experimental contexts.

The protective effects of inosine against chemical hypoxia on cultured rat oligodendrocytes.

Inosine is a purine nucleoside and is considered protective to neural cells including neurons and astrocytes against hypoxic injury. However, whether oligodendrocytes (OLs) could also be protected from hypoxia by inosine is not known. Here we investigated the effects of inosine on primarily cultured rat OLs injured by rotenone-mediated chemical hypoxia, and the mechanisms of the effects using ATP assay, MTT assay, PI-Hoechst staining, TUNEL, and immunocytochemistry. Results showed that rotenone exposure for 24 h caused cell death and impaired viability in both immature and mature OLs, while pretreatment of 10 mM inosine 30 min before rotenone administration significantly reduced cell death and improved the viability of OLs. The same concentration of inosine given 120 min after rotenone exposure also improved viability of injured mature OLs. Immunocytochemistry for nitrotyrosine and cellular ATP content examination indicated that inosine may protect OLs by providing ATP and scavenging peroxynitrite for cells. In addition, immature OLs were more susceptible to hypoxia than mature OLs; and at the similar degree of injury, inosine protected immature and mature OLs differently. Quantitative real-time PCR revealed that expression of adenosine receptors was different between these two stages of OLs. These data suggest that inosine protect OLs from hypoxic injury as an antioxidant and ATP provider, and the protective effects of inosine on OLs vary with cell differentiation, possibly due to the adenosine receptors expression profile. As OLs form myelin in the central nervous system, inosine could be used as a promising drug to treat demyelination-involved disorders.

Analysis of probenazole-responsiveness of rice RPR1 upstream fragments.

RPR1 (rice probenazole-responsive) is a rice gene, the expression of which is responsive to probenazole (PBZ), a synthetic compound that may act as a plant defense activator. It has been shown that RPR1 gene may be involved in disease resistance responses. In this study, a series of amplified fragments from the rice RPR1 promoter region, including 2,416 bp, 1,574 bp, 819 bp, 568 bp and 208 bp fragments upstream to the ATG translation start site, were prepared and linked to the coding region of beta-glucuronidase (GUS) gene. Analysis of GUS gene transient expression in rice calli demonstrated that the 568 bp fragment was sufficient for probenazole responsiveness. Analysis of GUS gene stable expression in Arabidopsis thaliana indicated that the 2,416 bp and 1,574 bp fragments drove GUS expression only in shoot apical meristem and petiole. Identification of these PBZ-responsive fragments provides a basis on which PBZ-inducible gene regulatory systems can be constructed for experimental analysis of gene expression and for field application.

miR-342-5p Regulates Neural Stem Cell Proliferation and Differentiation Downstream to Notch Signaling in Mice.

Notch signaling is critically involved in neural development, but the downstream effectors remain incompletely understood. In this study, we cultured neurospheres from Nestin-Cre-mediated conditional Rbp-j knockout (Rbp-j cKO) and control embryos and compared their miRNA expression profiles using microarray. Among differentially expressed miRNAs, miR-342-5p showed upregulated expression as Notch signaling was genetically or pharmaceutically interrupted. Consistently, the promoter of the miR-342-5p host gene, the Ena-vasodilator stimulated phosphoprotein-like (Evl), was negatively regulated by Notch signaling, probably through HES5. Transfection of miR-342-5p promoted the differentiation of neural stem cells (NSCs) into intermediate neural progenitors (INPs) in vitro and reduced the stemness of NSCs in vivo. Furthermore, miR-342-5p inhibited the differentiation of neural stem/intermediate progenitor cells into astrocytes, likely mediated by targeting GFAP directly. Our results indicated that miR-342-5p could function as a downstream effector of Notch signaling to regulate the differentiation of NSCs into INPs and astrocytes commitment.

Nicotinic acid supplementation in diet favored intramuscular fat deposition and lipid metabolism in finishing steers.

Nicotinic acid (NA) acting as the precursor of NAD(+)/NADH and NADP(+)/NADPH, participates in many biochemical processes, e.g. lipid metabolism. The main purpose of this study was to investigate the effects of dietary NA on carcass traits, meat quality, blood metabolites, and fat deposition in Chinese crossbred finishing steers. Sixteen steers with the similar body weight and at the age of 24 months were randomly allocated into control group (feeding basal diet) and NA group (feeding basal diet + 1000 mg/kg NA). All experimental cattle were fed a 90% concentrate diet and 10% forage straw in a 120-day feeding experiment. The results showed that supplemental NA in diet increased longissimus area, intramuscular fat content (17.14% vs. 9.03%), marbling score (8.08 vs. 4.30), redness (a*), and chroma (C*) values of LD muscle, but reduced carcass fat content (not including imtramuscular fat), pH24 h and moisture content of LD muscle, along with no effect on backfat thickness. Besides, NA supplementation increased serum HDL-C concentration, but decreased the serum levels of LDL-C, triglyceride, non-esterified fatty acid, total cholesterol, and glycated serum protein. In addition, NA supplementation increased G6PDH and ICDH activities of LD muscle. These results suggested that NA supplementation in diet improves the carcass characteristics and beef quality, and regulates the compositions of serum metabolites. Based on the above results, NA should be used as the feed additive in cattle industry.

Loss of microRNA-124 expression in neurons in the peri-lesion area in mice with spinal cord injury.

MicroRNA-124 (miR-124) is abundantly expressed in neurons in the mammalian central nervous system, and plays critical roles in the regulation of gene expression during embryonic neurogenesis and postnatal neural differentiation. However, the expression profile of miR-124 after spinal cord injury and the underlying regulatory mechanisms are not well understood. In the present study, we examined the expression of miR-124 in mouse brain and spinal cord after spinal cord injury using in situ hybridization. Furthermore, the expression of miR-124 was examined with quantitative RT-PCR at 1, 3 and 7 days after spinal cord injury. The miR-124 expression in neurons at the site of injury was evaluated by in situ hybridization combined with NeuN immunohistochemical staining. The miR-124 was mainly expressed in neurons throughout the brain and spinal cord. The expression of miR-124 in neurons significantly decreased within 7 days after spinal cord injury. Some of the neurons in the peri-lesion area were NeuN(+)/miR-124(-). Moreover, the neurons distal to the peri-lesion site were NeuN(+)/miR-124(+). These findings indicate that miR-124 expression in neurons is reduced after spinal cord injury, and may reflect the severity of spinal cord injury.

Daidzein enhances intramuscular fat deposition and improves meat quality in finishing steers.

An experiment was conducted to determine the effects of soy isoflavone daidzein on carcass characteristics, fat deposition, meat quality, and blood metabolites in finishing steers. Fourteen crossbred steers were used in a 120-d finishing study. These steers were stratified by weight into groups and randomly allotted by group to one of two dietary treatments: (1) control and (2) daidzein (500 mg/kg concentrate). The steers were fed a 90% concentrate diet. Supplemental daidzein did not affect slaughter weight, hot carcass weight, and dressing percentage, but tended to reduce fat proportion (not including intramuscular fat) in carcass and backfat thickness of steers. The carcass bone proportion was greater in steers fed daidzein diets than those fed control diets. Daidzein supplementation reduced pH at 24 h after slaughtered and moisture content and increased isocitrate dehydrogenase activity, fat content (16.28% and 7.94%), marbling score (5.29 and 3.36), redness (a*), and chroma (C*) values in longissimus muscle relative to control treatment. The concentrations of blood metabolites including glucose, blood urea nitrogen, triglyceride, total cholesterol, non-esterified fatty acid, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were all lower in steers fed daidzein diets than those fed control diets. Current results suggest that supplemental daidzein can affect lipid metabolism, increase intramuscular fat content and marbling score, and improve meat quality in finishing steers. Daidzein should be a promising feed additive for production of high-quality beef meat.

Development of an electrochemical micromachining instrument for the confined etching techniques.

This study proposes an electrochemical micromachining instrument for two confined etching techniques, namely, confined etchant layer technique (CELT) and electrochemical wet stamping (E-WETS). The proposed instrument consists of a granite bridge base, a Z-axis coarse/fine dual stage, and a force sensor. The Z-axis coarse/fine dual stage controls the vertical movement of the substrate with nanometer accuracy. The force sensor measures the contact force between the mold and the substrate. A contact detection method based on a digital lock-in amplifier is developed to make the mold-substrate contact within a five-nanometer range in CELT, and a force feedback controller is implemented to keep the contact force in E-WETS at a constant value with a noise of less than 0.2 mN. With the use of the confined etching techniques, a microlens array and a curvilinear ridge microstructure are successfully fabricated with high accuracy, thus demonstrating the promising performance of the proposed micromachining instrument.

Two monoclonal antibodies recognising aa 634-668 and aa 1026-1055 of NogoA enhance axon extension and branching in cultured neurons.

In a previous study, we generated two monoclonal antibodies (mAbs) in mice, aNogoA-N and aNogo-66 mAb, which were raised against recombinant N-terminal fragments of rat NogoA and Nogo-66, respectively. When compared with the commercial rabbit anti-rat NogoA polyclonal antibody (pAb), which can specifically recognise NogoA, the two mAbs were also specific for the NogoA antigen in immunofluorescence histochemical (IHC) staining and Western blot (WB) analysis. Serial truncations of NogoA covering the N-terminal region of NogoA (aa 570-691) and Nogo-66 (aa 1026-1091) were expressed in E. coli. The epitopes recognised by aNogoA-N and aNogo-66 are located in the aa 634-668 and aa 1026-1055 regions of NogoA, respectively. Both mAbs remarkably enhanced the axon growth and branching of cultured hippocampal neurons in vitro. These results suggest that the antibodies that bind to aa 634-668 and aa 1026-1055 of NogoA may have stimulatory effects on axon growth and branching. Additionally, the two mAbs that we generated are specific for NogoA and significantly block NogoA function. In conclusion, two sites in NogoA located within aa 634-668 and aa 1026-1055 are recognised by our two antibodies and are novel and potentially promising targets for repair after central nervous system (CNS) injury.

The effect of simvastatin on chemotactic capability of SDF-1α and the promotion of bone regeneration.

The purpose of this study was to investigate the cooperative effects of simvastatin (SIM) and stromal cell-derived factor-1α (SDF-1α) on the osteogenic and migration capabilities of mesenchymal stem cells (MSCs), and construct a cell-free bone tissue engineering system comprising SIM, SDF-1α and scaffold. We found that 0.2 µm SIM significantly increased alkaline phosphatase activity (P < 0.05) of mouse bone marrow MSCs with no inhibition of cell proliferation, and enhanced the chemotactic capability of SDF-1α (P < 0.05). Next, we constructed a novel cell-free bone tissue engineering system using PLGA loaded with SIM and SDF-1α, and applied it in critical-sized calvarial defects in mice. New bone formation in the defect was evaluated by micro-CT, HE staining and immunohistochemistry. The results showed that PLGA loaded with SIM and SDF-1α promoted bone regeneration significantly more than controls. We investigated possible mechanisms, and showed that SDF-1α combined with SIM increased MSC migration and homing in vivo, promoted angiogenesis and enhanced the expression of BMP-2 in newly-formed bone tissue. In conclusion, SIM enhanced the chemotactic capability of SDF-1α and the cell-free bone tissue engineering system composed of SIM, SDF-1α and scaffold promoted bone regeneration in mouse critical-sized calvarial defects.

[Identification of six kinds of monoclonal antibodies against rat Nogo-A molecule in the immunofluorescent histochemical staining].

AIM: To detect and characterize of 6 monoclonal antibodies (mAbs) against different epitopes of rat Nogo-A molecule in immunohistochemistry to decide their applications in futrue. Four mAbs against Nogo66 fragment are named Nogo66-1, Nogo66-2, Nogo66-3 and Nogo66-4. The rest of 2 mAbs against N-termial 570-691aa fragment are named NogoN-1 and NogoN-2. METHODS: The immunofluorescence staining was used to detect the reactivity and specificity of those 6 mAbs in spinal tissue sections of rat. RESULTS: All 6 mAbs were double-labelled with commercial rabbit anti-Rat Nogo-A polyclonal antibody (PcAb) in spinal cord sections respecitvely. All 6 mAbs were colocalization with MBP respectively. However Nogo66-3 and NogoN-1 could also be double-staining with GFAP respectively. CONCLUSION: Nogo66-1, Nogo66-2, Nogo66-4 and NogoN-2 could recognize specifically in Nogo-A protein of tissues in immunohistochemical methods.

Improved biological performance of microarc-oxidized low-modulus Ti-24Nb-4Zr-7.9Sn alloy.

Ti-24Nb-4Zr-7.9Sn (TNZS) is a newly developed beta-titanium alloy with low modulus and it has been considered as good material for dental or orthopedic implant. The purpose of the current study is to evaluate the effect of micro-arc oxidation (MAO) treatment on the biological performance of TNZS surface. The phases, morphology and chemical composition of the MAO-treated surface were characterized by X-ray diffraction, energy dispersive spectroscope and scanning electron microscopy analysis respectively. Then we tested the biocompatibility by examining the cell morphology and viability of osteoblast cells growing on MAO-TNZS surface. The bone binding strength of the specimens was evaluated by removal torque test after implantation in rabbit tibiae for 6 weeks. Compared with the none-treated titanium and TNZS specimens, MAO treated TNZS specimens showed a significant increase (p<0.05) in hydrophilicity, roughness, cell viability and removal torque forces. In summary, MAO treatment helps to form a porous surface with a biologically active bone-like apatite layer on TNZS specimens, which may improve the biological response of MAO-TNZS implants.

Inactivation of glycogen synthase kinase-3beta and up-regulation of LINGO-1 are involved in LINGO-1 antagonist regulated survival of cerebellar granular neurons.

LINGO-1 has been critically implicated in the central regulation of CNS axon regeneration and oligodendrocyte maturation. We have recently demonstrated that pretreatment with LINGO-1 antagonist (LINGO-1-Fc) inhibited low potassium-induced cerebellar granular neurons (CGNs) apoptosis. In the present study, we examined the neuroprotective mechanism of LINGO-1-Fc by Western blot and in situ GST pull-down assay. CGN cultures were preincubated in medium with LINGO-1-Fc or control protein at the concentration of 10 mug/ml for 2 h and then switched to low potassium medium in the presence of corresponding proteins. Cultures were harvested at indicated time intervals for successive analysis. Several apoptosis-associated signaling factors, GSK-3beta, ERK1/2, and Rho GTPases, were observed to be activated in response to potassium deprivation and the activation/dephosphorylation of GSK-3beta was suppressed by LINGO-1-Fc pretreatment compared with control group. Besides, the endogenous LINGO-1 expression level of CGN cultures was augmented by low potassium stimuli and restrained by LINGO-1 antagonist treatment. Although the protein level of p75(NTR) and Nogo-A were down-regulated in different patterns during apoptosis, neither of them was affected by LINGO-1-Fc application. Taken together, these results suggest a new mechanism of LINGO-1 antagonist regulated neuronal survival involving protein synthesis of LINGO-1 and inactivation of GSK-3 pathway.

[A comparative study on the reliability of grating projection measuring system in three-dimensional reconstruction of dental cast].

OBJECTIVE: To investigate the reliability of a newly developed grating projection system 3DSS-STD-II by three-dimensional reconstruction of dental cast, so as to offer some evidence for dental computer aided design and computer aided manufacturing (CAD/CAM). METHODS: Five groups' data of mandibular dentition cast from different angle: Occlusion, right lateral dentition, anterior dentition, left lateral dentition and posterior of the cast were scanned and acquired by 3DSS-STD-II new measuring system. The five groups of acquired data were then under simplification and combination process and the digital dental cast was finally reconstructed by the reverse engineering software Geomagic 6.0. After the reconstruction process, the plaster dental cast and digital reconstructed dental cast were then manually and digitally measured respectively by different items: Width of incisors, width of anterior dental arch, width of buccal segment, length of anterior dental arch and length of buccal segment. The manual process was undergone by vernier caliper and the digital process was by reverse engineering software. The statistical analysis was then undergone in order to evaluate the reliability, repeatability and scan-precision of the new grating projection system. RESULTS: With the statistical analysis results, the grating projection system 3DSS-STD-II showed its good reliability and repeatability in three-dimensional reconstruction of dental plaster cast. There were no significant differences between the data acquired by 3DSS-STD-II digital scanning system and manual measurement by vernier caliper in the precision. CONCLUSION: The new grating projection system of 3DSS-STD- II equipped with high reliability and fast speed can meet the need of the fast data acquisition and three-dimensional reconstruction of dental cast and CAD process.

An in vitro study on the involvement of LINGO-1 and Rho GTPases in Nogo-A regulated differentiation of oligodendrocyte precursor cells.

Nogo-A has been considered as one of the most important myelin-associated axonal regeneration inhibitors in the central nervous system. Recent studies have demonstrated various additional physiological roles of Nogo family members. To understand the possible effect of Nogo-A on the differentiation of oligodendrocytes, antibodies against distinct extracellular domains of Nogo-A were applied in cell cultures. Oligodendrocyte precursor cells from P2 rat cortex were grown in the presence of monoclonal antibody against the N-terminal inhibitory domain of Nogo-A or the C-terminal 66 amino acid loop of Nogo-A for 3 days, and the antibody treatment resulted in stunted process extension and inhibited differentiation of oligodendrocytes. Concomitant with morphology changes, Rho GTPases activity was greatly increased upon the antibody treatment and the expression level of LINGO-1, which was recently shown to be a negative regulator for the oligodendrocyte maturation, was upregulated in the process of antibody treatment. These results indicate that endogenous Nogo-A expressed in oligodendrocyte may act though Rho GTPase and LINGO-1 to influence the morphological differentiation of oligodendrocytes and will help us to understand the physiology role of Nogo-A in oligodendrocyte biology.