Discovery of specific mutations in spinal muscular atrophy patients by next-generation sequencing.

Spinal muscular atrophy (SMA) is a type of autosomal recessive genetic disease, which seriously threatens the health and lives of children and adolescents. We attempted to find some genes and mutations related to the onset of SMA. Eighty-three whole-blood samples were collected from 28 core families, including 28 probands with clinically suspected SMA (20 SMA patients, 5 non-SMA children, and 3 patients with unknown etiology) and their parents. The multiplex ligation probe amplification (MLPA) was performed for preliminary diagnosis. The high-throughput sequencing technology was used to conduct the whole-exome sequencing analysis. We analyzed the mutations in adjacent genes of SMN1 gene and the unique mutations that only occurred in SMA patients. According to the MLPA results, 20 probands were regarded as experimental group and 5 non-SMA children as control group. A total of 10 mutations were identified in the adjacent genes of SMN1 gene. GUSBP1 g.[69515863G>A], GUSBP1 g.[69515870C>T], and SMA4 g.[69515738C>A] were the top three most frequent sites. SMA4 g.[69515726A>G] and OCLN c.[818G>T] have not been reported in the existing relevant researches. Seventeen point mutations in the DYNC1H1 gene were only recognized in SMA children, and the top two most common mutations were c.[2869-34A>T] and c.[345-89A>G]; c.[7473+105C>T] was the splicing mutation that might change the mRNA splicing site. The mutations of SMA4 g.[69515726A>G], OCLN c.[818G>T], DYNC1H1 c.[2869-34A>T], DYNC1H1 c.[345-89A>G], and DYNC1H1 c.[7473+105C>T] in the adjacent genes of SMN1 gene and other genes might be related to the onset of SMA.

[Changes in plasma high mobility group box-1 protein levels and its relationship with sepsis in severely burned patients].

OBJECTIVE: To investigate the significance of changes in plasma high mobility group box-1 protein (HMGB1) levels and its relationship with sepsis and endotojemia in severely burned patients. METHODS: Totally 25 large area burned patients ( > 30% total body surface area) were included in this study, and 8 healthy volunteers served as normal controls. The plasma levels of HMGB1 were measured by ELISA, and endotoxin concentrations was determined by the modified chromogenic limulus amebocyte lysate (LAL) assay on posthurn days 1, 3, 5, 7, 14, 21, and 28. RESULTS: The plasma HMGBL levels were markedly elevated on postburn day 1 in severely burned patients, and they were significantly higher in septic patients than those without sepsis on days 7, 21, and 28 after burns (P<0.05). Among septic patients, plasma HMGBI levels in the survival group were significantly lower than those with fatal outcome on days 3 and 21 (P<0.05, P<0.01). No significant correlations were found between HMGB1 levels and the sizes of total body surface area (P>0.05). In addition, the plasma HMGB1 levels were positively correlated with endotoxin concentrations on days 3, 5, 7, 21 after major burns (P<0.05, P<0.01). CONCLUSIONS: HMGB1, as an important late mediators of inflammation, may be involved in the development of sepsis following extensive burns, and it can be markedly induced by endotoxemia secondary to acute insults. Dynamic measurements of circulating HMGB1 levels should be helpful to monitor the disease course and judge the prognosis of burned patients.

[In vitro culture and identification of tolerogenic dendritic cells from mouse bone marrow].

OBJECTIVE: To establish a simple and economic method for in vitro culture of tolerogenic dendritic cells (Tol-DC) from mouse bone marrow to facilitate further research of the application of Tol-DC in autoimmune disease. METHODS: The dendritic cells were derived from in vitro culture of the precursor cells from mouse bone marrow with recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and interleukin (IL)-4, respectively. Eighteen hours before completion of cell culture, lipopolysaccharide (LPS) was added in the medium for stimulating rmGM-CSF-treated cells, but not IL-4-treated cells. The cells were collected on day 6, and the cell morphology, phenotype and immunofunction were analyzed. RESULTS: DC cultured in vitro had a CD11c(+) expression rate of 76.67% with typical morphology. The DCs without LPS treatment were characterized by moderate expression of class II major histocompatibility complex (MHCII) and low expression of costimulatory molecules, while those with LPS treatment had high expressions of MHCIIand costimulatory molecules. CONCLUSION: Immature tolerogenic DCs can be derived in large quantity using this method with the potential for use in the treatment of autoimmune diseases.

Electroacupuncture at Zusanli (ST36) promotes gastric emptying and mucosal blood flow during oral resuscitation of scalded rats with a pyruvate-enriched ORS.

AIM: The aim of this study was to investigate the effect of electroacupuncture at ST36 (EA ST36) on gastric emptying and mucosal blood flow during intragastric resuscitation with pyruvate-enriched oral rehydration solution (Pyr-ORS) in scalded rats. METHODS: The rats were subjected to a 35% total body surface area (TBSA) of scald injury and randomly divided into five groups (N=24) and two subgroups (n=12) in each group. The Pyr-ORS was delivered intragastrically according to the Parkland formula immediately after scalding at a dose of 1 mL kg(-1) %TBSA(-1) in 1 h. In these animals, the bilateral Zusanli points (ST36) were electroacupunctured at a constant voltage (2 mA and 2-100 HZ) for 0.5 h immediately after intragastric resuscitation. At 2 and 4 h after scalding, the gastric emptying rate (GER) and gastric mucosal blood flow (GMBF) were determined, and the motilin levels of the plasma and gastric tissues were also analyzed at two time points, respectively. RESULTS: GER and GMBF were markedly decreased in groups with scalding and resuscitation, compared with the sham groups at two time points (P<0.05), but they were greatly improved in groups byEAST36 at 2 and 4 h after sustaining scald injuries (P<0.05). Bilateral vagotomy further aggravated the reduction of GER and GMBF in scalded rats. EA after gastric vagotomy failed to raise GER and GMBF. Neither EA nor vagotomy had effects on the reduced motilin levels of plasma and gastric tissues in animals after scalding. CONCLUSION: EA ST36 has a significant effect on improving gastric emptying and mucosal ischemia in the oral resuscitation of burn injury, possibly through the activation of a cholinergic nerve-dependent mechanism. In addition, EA ST36 showed no effects on motilin levels, but requires further investigations.

Electroacupuncture activates enteric glial cells and protects the gut barrier in hemorrhaged rats.

AIM: To investigate whether electroacupuncture ST36 activates enteric glial cells, and alleviates gut inflammation and barrier dysfunction following hemorrhagic shock. METHODS: Sprague-Dawley rats were subjected to approximately 45% total blood loss and randomly divided into seven groups: (1) sham: cannulation, but no hemorrhage; (2) subjected to hemorrhagic shock (HS); (3) electroacupuncture (EA) ST36 after hemorrhage; (4) vagotomy (VGX)/EA: VGX before hemorrhage, then EA ST36; (5) VGX: VGX before hemorrhage; (6) α-bungarotoxin (BGT)/EA: intraperitoneal injection of α-BGT before hemorrhage, then EA ST36; and (7) α-BGT group: α-BGT injection before hemorrhage. Morphological changes in enteric glial cells (EGCs) were observed by immunofluorescence, and glial fibrillary acidic protein (GFAP; a protein marker of enteric glial activation) was evaluated using reverse transcriptase polymerase chain reaction and western blot analysis. Intestinal cytokine levels, gut permeability to 4-kDa fluorescein isothiocyanate (FITC)-dextran, and the expression and distribution of tight junction protein zona occludens (ZO)-1 were also determined. RESULTS: EGCs were distorted following hemorrhage and showed morphological abnormalities. EA ST36 attenuated the morphological changes in EGCs at 6 h, as compared with the VGX, α-BGT and HS groups. EA ST36 increased GFAP expression to a greater degree than in the other groups. EA ST36 decreased intestinal permeability to FITC-dextran (760.5 ± 96.43 ng/mL vs 2466.7 ± 131.60 ng/mL, P < 0.05) and preserved ZO-1 protein expression and localization at 6 h after hemorrhage compared with the HS group. However, abdominal VGX and α-BGT treatment weakened or eliminated the effects of EA ST36. EA ST36 reduced tumor necrosis factor-α levels in intestinal homogenates after blood loss, while vagotomy or intraperitoneal injection of α-BGT before EA ST36 abolished its anti-inflammatory effects. CONCLUSION: EA ST36 attenuates hemorrhage-induced intestinal inflammatory insult, and protects the intestinal barrier integrity, partly via activation of EGCs.

[RelB gene expression in murine bone marrow-derived mature and immature dendritic cells].

AIM: To explore the expression of avian reticuloendotheliosis viral(v-rel) oncogene-related B (RelB) mRNA in vitro in murine mature and immature myeloid dendritic cells (DCs). METHODS: The bone marrow was collected from the femur and tibias of C57BL/6 mice in sterile condition. Bone marrow precursors were cultured with recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and interleukin-4 (rmIL-4) to produce immature DCs. Immature DCs were stimulated by LPS 18 h before the end of culture to become mature. DCs' phenotype was detected by flow cytometry (FCM). RelB expression and protein level were determined by RT-PCR and immunofluorescence staining. RESULTS: The expression level of co-stimulatory molecules (CD86 and CD40) and MHC-II class molecule were low in immature DCs, whereas high in mature DCs. RelB expression was significantly higher in mature DCs than in immature DCs (P<0.01). CONCLUSION: RelB expression is closely associated with the maturative situation of DCs. Inhibition of RelB expression in DCs may induce tolerogenic DCs.