Editing the cystine knot motif of MSTN enhances muscle development of Liang Guang Small Spotted pigs.

Myostatin (MSTN) is a member of the transforming growth factor-ß (TGF-ß) family, and functions as an inhibitor of muscle growth. Disrupting the inhibitory effect of MSTN on growth can provide an effective way to increase the muscle yield of livestock and poultry. The cysteine knot motif of TGF-ß can stabilize the structure of MSTN protein and plays an important regulatory role in the biological function of MSTN. Accordingly, in this study, we used the CRISRP/Cas9 to edit the exon 3 of MSTN in the kidney cells of Liang Guang Small Spotted pig (LPKCs), in order to disrupt the cysteine knot motif of MSTN and remove the inhibitory effect of MSTN on its target genes.MSTN-edited LPKCs were obtained through fluorescence-activated cell sorting (FACS) and used as donor cells for somatic cell nuclear transfer (SCNT) to generate cloned embryos, which were then transferred to surrogate sows to finally obtain eight MSTN-edited Liang Guang Small Spotted piglets. Among them, two survived to 10 days old. Genotyping revealed that these two piglets were gene edited heterozygotes with base deletion and substitution occurred within the coding sequence of C106 and C108 at the cystine knot motif of MSTN. These changes resulted in frameshift mutations, and conversion of C106 and C108 to other amino acids. More developments of muscles were observed at the shoulders and hips of the heterozygotes of MSTN-edited Liang Guang Small Spotted pigs. H&E analysis showed that the cross-sectional area (CSA) of myofiber inMSTN-edited pigs was significantly decreased, and the number of myofiber were significantly increased. Western blot analysis showed that the disruption of C106 and C108 did not affect the expression of MSTN protein, but significantly up-regulated the expression of its target genes such as Myf5, MyoD, Myogenin and other myogenic regulatory factors. In summary, the gene-edited pig model obtained in this study did not cause complete loss of MSTN expression, and could retain other biological functions of MSTN, thereby promoting muscle growth while minimizing the potential adverse effects on complete loss of MSTN in the Liang Guang Small Spotted pigs.

Bloodletting Puncture at Hand Twelve Jing-Well Points Improves Neurological Recovery by Ameliorating Acute Traumatic Brain Injury-Induced Coagulopathy in Mice.

Traumatic brain injury (TBI) contributes to hypocoagulopathy associated with prolonged bleeding and hemorrhagic progression. Bloodletting puncture therapy at hand twelve Jing-well points (BL-HTWP) has been applied as a first aid measure in various emergent neurological diseases, but the detailed mechanisms of the modulation between the central nervous system and systemic circulation after acute TBI in rodents remain unclear. To investigate whether BL-HTWP stimulation modulates hypocoagulable state and exerts neuroprotective effect, experimental TBI model of mice was produced by the controlled cortical impactor (CCI), and treatment with BL-HTWP was immediately made after CCI. Then, the effects of BL-HTWP on the neurological function, cerebral perfusion state, coagulable state, and cerebrovascular histopathology post-acute TBI were determined, respectively. Results showed that BL-HTWP treatment attenuated cerebral hypoperfusion and improve neurological recovery post-acute TBI. Furthermore, BL-HTWP stimulation reversed acute TBI-induced hypocoagulable state, reduced vasogenic edema and cytotoxic edema by regulating multiple hallmarks of coagulopathy in TBI. Therefore, we conclude for the first time that hypocoagulopathic state occurs after acute experimental TBI, and the neuroprotective effect of BL-HTWP relies on, at least in part, the modulation of hypocoagulable state. BL-HTWP therapy may be a promising strategy for acute severe TBI in the future.

Study on Umbilical Cord-Matrix Stem Cells Transplantation for Treatment of Acute Traumatic Brain Injury in Rats.

AIM: To evaluate the effect of umbilical cord derived mesenchymal stem cells (UC-MSCs) transplantation on traumatic brain injury (TBI). MATERIAL AND METHODS: UC-MSCs were isolated from human umbilical cord and TBI rat model was constructed. 30 male SD rats were randomly divided into 3 groups: control group, TBI group and MSCs transplantation group. Rats in MSCs group received the injection of a total of 1.5 C- 106 MSCs (25 I»l) via ventricle at operated ventricular coordinates (0 at bregma, 1.5 mm at lateral, 1.1 mm at behind, 4.5 mm in depth). RESULTS: 80% confluence of cells was formed from tissue at day 10 and the amount of CD90, CD73, CD105 positive cells increased correspondingly. In TBI model, clear hyperemia, edema and obvious infiltration of inflammatory cells in brain tissue were found. However, the manifestations were alleviated after the treatment of MSCs. In MSCs group, GFP in the brain tissue and the area around the vessels were found after the injection, while the expression levels of micro-vessel density (MVD), brain-derived neurotrophic factor (BDNF) and glial fibrillary acidic protein (GFAP) were elevated. CONCLUSION: UC-MSCs transplantation for treatment of acute TBI could effectively reduce the injury and improve the vascular reconstruction.

Magnetic resonance imaging-three-dimensional printing technology fabricates customized scaffolds for brain tissue engineering.

Conventional fabrication methods lack the ability to control both macro- and micro-structures of generated scaffolds. Three-dimensional printing is a solid free-form fabrication method that provides novel ways to create customized scaffolds with high precision and accuracy. In this study, an electrically controlled cortical impactor was used to induce randomized brain tissue defects. The overall shape of scaffolds was designed using rat-specific anatomical data obtained from magnetic resonance imaging, and the internal structure was created by computer-aided design. As the result of limitations arising from insufficient resolution of the manufacturing process, we magnified the size of the cavity model prototype five-fold to successfully fabricate customized collagen-chitosan scaffolds using three-dimensional printing. Results demonstrated that scaffolds have three-dimensional porous structures, high porosity, highly specific surface areas, pore connectivity and good internal characteristics. Neural stem cells co-cultured with scaffolds showed good viability, indicating good biocompatibility and biodegradability. This technique may be a promising new strategy for regenerating complex damaged brain tissues, and helps pave the way toward personalized medicine.

Endothelial protein C receptor polymorphisms and risk of sepsis in a Chinese population.

Objective To examine the potential relationship of EPCR polymorphisms and the risk of sepsis in a Chinese population. Methods Snapshot SNP genotyping assays and DNA sequencing methods were used to detect polymorphisms of the EPCR gene, rs2069948C/T (2532C/T) and rs867186A/G (6936A/G), in 64 patients with sepsis and in 113 controls. Soluble EPCR (sEPCR) was measured by ELISA. Results There were significant differences in the allele and genotype frequencies of EPCR gene rs2069948C/T and allele frequencies of rs867186A/G between male and female patients and controls. Females carrying rs2069948 C/T genotype or T allele and males carrying rs867186 A allele were associated with a significantly increased risk of sepsis. Plasma sEPCR levels of sepsis patients were higher than controls and showed no correlation with EPCR gene polymorphisms. Conclusions EPCR polymorphisms may be associated with increased risk of sepsis, but this has no effect on the release of sEPCR in patients with sepsis.

Intraoperative Perfusion Computed Tomography in Carotid Endarterectomy: Initial Experience in 16 Cases.

BACKGROUND This study aimed to evaluate the changes in perfusion computed tomography (PCT) parameters in carotid endarterectomy (CEA), and to discuss the use of intraoperative PCT in CEA. MATERIAL AND METHODS Sixteen patients with carotid stenosis who also underwent CEA with intraoperative CT were recruited in this study. We calculated quantitative data on cerebral blood flow (CBF), cerebral blood volume (CBV), time to peak (TTP), and the relative parameter values, including relative CBF (rCBF), relative CBV (rCBV), and relative TTP (rTTP). The role of PCT was assessed and compared to conventional monitoring methods. RESULTS There were no significant differences in any of the parameters in the anterior cerebral artery (ACA) territory (P>0.05). In the middle cerebral artery (MCA) territory, the CBF and CBV increased and TTP decreased in the operated side during CEA; the rCBF and rCBV increased and the rTTP decreased significantly (P<0.05). In 16 patients, CT parameters were improved, SSEP was normal, and MDU was abnormal. In 3 patients, CBF increased by more than 70% during CEA. Relative PCT parameters are sensitive indicators for detecting early cerebral hemodynamic changes during CEA. Cerebral hemodynamics changed significantly in the MCA territory during CEA. CONCLUSIONS Intraoperative PCT could be an important adjuvant monitoring method in CEA.

[The surgery of giant symptomatic cavernous sinus aneurysms].

OBJECTIVE: To evaluate the management and outcomes in patients with giant symptomatic cavernous sinus aneurysms who underwent aneurysms trapping with bypass, proximal carotid occlusion and aneurysms trapping. METHODS: Twenty-three patients with giant symptomatic cavernous sinus aneurysms underwent surgery between February 2007 and March 2013, 3 cases were male and 20 cases were female patients, the age of the patients ranged between 24 and 68 years, mean age was 54.7 years. The pre-operative digital subtraction angiography (DSA) and ballon occlusion test (BOT) were performed to confirm the diagnosis and identify hemodynamic reserve with carotid occlusion, and the aneurysms trapping with bypass, aneurysms trapping and proximal occlusion of the internal carotid artery were performed according to BOT results. During the surgery, the neurophysiological monitoring and the intraoperative CT perfusion were used. The follow-up by DSA or CT angiography were made. RESULTS: Seventeen patients underwent aneurysms trapping with bypass, 1 underwent aneurysms trapping and 5 underwent proximal occlusion of the internal carotid artery. After surgery, symptom improved in 4 cases, did not change in 10 cases, and new neural function deficit developed in 9 cases. The follow-up period were 3 months to 75 months. Two patients were lost. The Glasgow Outcome Scale of last follow-up were 5 in 19 patients, 3 in 1 patient and 1 in 1 patient. CONCLUSIONS: The aneurysms trapping with bypass and proximal occlusion of the internal carotid artery are effective and reliable procedure for treatment of giant symptomatic cavernous sinus aneurysms in selected patients after evaluation of the pre-operative BOT, intra-operative neurophysiological monitoring and the intraoperative CT perfusion.

Curcumin attenuates ischemia-like injury induced IL-1ß elevation in brain microvascular endothelial cells via inhibiting MAPK pathways and nuclear factor-κB activation.

Inflammatory reactions play a key role in the cerebral injury after stroke or other ischemic brain diseases. Curcumin, which is extracted from herb turmeric, has been reported to have anti-inflammatory effects. The present study was aimed to investigate the anti-inflammatory effects of curcumin on oxygen-glucose deprivation (OGD) injured brain microvascular endothelial cells (BMECs). Rat BMECs were used and the results showed that OGD induced a significant elevation of the leakage of lactate dehydrogenase and the secretion of the proinflammation cytokine, IL-1ß. Activation of p38, JNK MAPKs, and NF-κB in BMECs was also observed after OGD. The treatment of curcumin (20 µM) inhibited the increased production of IL-1ß both at the protein and mRNA levels. The increased phosphorylation of p38 and JNK induced by OGD was decreased under the treatment of curcumin, whereas the p38 inhibitor, SB203580, significantly inhibited OGD-induced IL-1ß production, but the JNK inhibitor, SP600125, failed to do so. These results suggest that the inhibition of IL-1ß by curcumin may dependent on the p38 signaling pathway. The OGD-induced IL-1ß production was also inhibited by the NF-κB inhibitor, and curcumin suppressed OGD-induced NF-κB activation. Furthermore, the NF-κB activation was attenuated by the SB203580, indicating that NF-κB activation was dependent on p38 signaling pathway. The present study suggests that curcumin displays an anti-inflammatory effect on OGD-injured BMECs via down-regulating of MAPK and NF-κB signaling pathways and might have therapeutic potential for the ischemic brain diseases.