Accelerometer-measured sedentary volume and bouts during the segmented school day among Chinese school students.

Background: This study examined sedentary volume and bouts of Chinese primary and middle school students during different segments of a school day and determined whether gender and school level are associated with their sedentary volume and bouts. Methods: A total of 472 students participated in this study. Accelerometers were used to measure the sedentary volume and sedentary bouts of different durations (i.e., 1-4 min, 5-9 min and ≥10 min) during all segments. Results: The participants spent the majority of their time in sitting (61.7%) and sitting bouts of ≥10 min (37.3%). They spent higher percentages of time in sitting during regular classes (76.7%) and out-of-school time (54.5%), and lower during physical education (PE) classes (32.2%), lunch break (35.4%) and recess (38.0%). The highest proportions of time were in sedentary bouts of ≥10 min during regular classes (50.2%), out-of-school time (28.0%) and lunch break (18.8%), while the greatest percentages occurred in sitting bouts of 1-4 min during PE class (16.4%) and recess (18.6%). Girls and middle school students had higher percentages of sedentary volume than boys and primary school students during most segments. They spent greater proportions of time in sitting bouts of ≥10 min during regular classes, lunch break, and out-of-school time, and higher proportions in sedentary bouts of 1-4 min than boys and primary students during PE classes. Conclusion: Regular class and out-of-school time were identified as key segments for reducing sedentary volume and breaking up prolonged sitting. Interventions on interrupting prolonged sitting during lunch break should also be explored. Girls and middle school students should receive more attention in future interventions.

Cool and hot chitosan/platelet-derived growth factor nanofibers for outdoors burns.

Burns and scalds are thermal injuries caused by a large amount of heat accumulation in local tissues. The first cooling emergency is a key step. However, it is hard that in outdoors to find clean water to cool the scald tissue sites. Moreover, most dressings are concentrated on the treatment process today, neglecting the emergency treatment of temperature reduction. In this study, we imported refrigeration in the electrospinning process while using dirty water, rainwater and even urine of outdoors, so that the cooled sterile fibers were directly deposited on the burn and scald wounds, and the cooling emergency was achieved through the dual cooling mechanism. Since this fiber which is made up of cheap fish gelatin contains CuS adopting the green method, it can generate heat and effectively kill bacteria under the irradiation of an illumination lamp at the front end of a spinning device. As a result of the direct deposition, there is an excellent fit between the fibrous membrane and the skin, which reduces the air gap to achieve a better and quick cooling and heating effects. On the same Chitosan/Platelet-derived Growth Factor fiber membrane, this method of cooling first and heating second can shorten the recovery time from 30 days to 21 days. Thus, this treatment strategy has a great potential application prospect in the field of outdoor burn treatment.

Role of exosomes in the pathogenesis of inflammation in Parkinson's disease.

Inflammatory responses, including glial cell activation and peripheral immune cell infiltration, are involved in the pathogenesis of Parkinson's disease (PD). These inflammatory responses appear to be closely related to the release of extracellular vesicles, such as exosomes. However, the relationships among different forms of glial cell activation, synuclein dysregulation, mitochondrial dysfunction, and exosomes are complicated. This review discusses the multiple roles played by exosomes in PD-associated inflammation and concludes that exosomes can transport toxic α-synuclein oligomers to immature neurons and into the extracellular environment, inducing the oligomerization of α-synuclein in normal neurons. Misfolded α-synuclein causes microglia and astrocytes to activate and secrete exosomes. Glial cell-derived exosomes participate in communications between glial cells and neurons, triggering anti-stress and anti-inflammatory responses, in addition to axon growth. The production and release of mitochondrial vesicles and exosomes establish a new mechanism for linking mitochondrial dysfunction to systemic inflammation associated with PD. Given the relevance of exosomes as mediators of neuron-glia communication in neuroinflammation and neuropathogenesis, new targeted treatment strategies are currently being developed that use these types of extracellular vesicles as drug carriers. Exosome-mediated inflammation may be a promising target for intervention in PD patients.

cGAS knockdown promotes microglial M2 polarization to alleviate neuroinflammation by inhibiting cGAS-STING signaling pathway in cerebral ischemic stroke.

Inflammation plays a pivotal role in promoting the pathophysiology of ischemic stroke (IS). Microglia is the major immunocompetent cells involved in different neuropathologies. The activation of cyclic GMP-AMP synthase (cGAS) and its downstream signaling protein-stimulator of interferon genes (STING) is increasingly recognized as a crucial determinant of neuropathophysiology. However, the mechanisms underlying cGAS-STING signaling regulating inflammatory response during IS remains to be elucidated. In this study, HT22 cells was used to establish an oxygen-glucose deprivation (OGD) cell model in vitro, and then this cell culture supernatant containing OGD-induced DAMPs (OIDs) was employed to stimulate BV2 microglia. Furthermore, a middle cerebral artery occlusion (MCAO) mouse model was established. Cells and MCAO mice were treated with si-cGAS or si-NC lentivirus. The expression levels of STING, cGAS and p-IRF3 in BV2 cells or MCAO mouse brain; the microglial M1/M2 polarization of BV2 microglia or isolated microglial cells from MCAO mouse brain; the contents of iNOS, TNF-α, TGF-ß and IL-10 in the culture medium of BV2 cells or in murine brain homogenates, were all detected. In addition, the severity of cerebral infarction with or without the knockdown of cGAS in a MCAO mouse model was also determined by TTC staining. Results showed that OGD-induced DAMPs strongly activated cGAS-STING pathway and triggered microglia polarization in BV2 cells, reflecting as the accumulation of a plethora of pro-inflammatory factors in activated microglia. However, these effects could be inhibited by cGAS knockdown. In the MCAO mouse model, the inhibition of cGAS-STING pathway resulted from cGAS knockdown could effectively diminish cell apoptosis in mouse brain stimulated by MIDs (MCAO-induced DAMPs), reduced the area ratio of cerebral infarction and ultimately improved the injured nerve function during IS. Taken together, our elucidation of underlying mechanisms involved in the microglial inflammatory response, triggered by cGAS-STING signaling, highlights this pathway as a potential therapeutic target in IS.

Non-motor symptoms are associated with midline tremor in essential tremor.

OBJECTIVES: Essential tremor (ET) patients presenting tremor in the midline structures may be a distinct subtype of the syndrome. Therefore, we sought to explore the clinical manifestations, especially non-motor symptoms (NMS) of Chinese ET patients with midline tremor (MT). METHODS: In the cross-sectional study, we grouped 290 definite or probable ET patients based on their MT conditions. The NMS in ET patients were evaluated using the NMS scale (NMSS). NMS and other clinical correlates were then compared among subgroups with, and without MT. RESULTS: We revealed that 39.0%, 27.6%, and 6.9% of the patients respectively had neck, voice, and facial tremors. With the accumulation of tremor in midline structures, NMS became more severe and prevalent. Logistic regression analyses revealed that factors such as: female gender (OR = 2.164, 95% CI: 1.307-3.583), having least or highest action arm tremor (OR = 2.512, 95% CI: 1.520-4.151), having higher score of sleep/fatigue domain (OR = 1.692, 95% CI: 1.004-2.850) and mood/apathy (OR = 1.926, 95% CI: 1.143-3.246) domain, to be independently associated with MT manifestation. CONCLUSIONS: Our study demonstrates the heterogeneity of symptoms in ET patients with MT, especially in prominent NMS. In addition, the discrepancy of NMS between patients with, and without MT provides novel insight into the underlying pathophysiology and therapeutic of ET.

[Generation of a new strain of NOD/SCID/IL2Rγ-/- mice with targeted disruption of Prkdc and IL2Rγ genes using CRISPR/Cas9 system].

OBJECTIVE: The NOD/SCID/IL2Rγ- /- (NSG) mouse strain is the most widely used immunodeficient strain for xenograft transplantation. However, the existing SCID mutation is a spontaneous mutation of the Prkdc gene, which leads to leaky T cell developmental block and difficulty in genotyping. It is therefore important to develop a new strain of NSG mice with targeted disruption of Prkdc and IL2Rγ genes. METHODS: Targeted disruption of Prkdc and IL2Rγ genes was achieved using the CRISPR/Cas9 system. By intercrossing the knockout and NOD mice, we obtained a novel strain of NOD/SCID/IL2Rγ- /-(NSG) mice, denoted as cNSG (Chinese NSG) mice. RESULTS: In addition to the NOD mutation, cNSG mice exhibited a complete absence of T cells, B cells and NK cells. cNSG mice allowed more efficient engraftment of human cancer cells than the commonly used immunodeficient nude mice. CONCLUSION: cNSG mice will provide an important xenotransplantation model for biomedical research.

Nuclear ß-catenin accumulation is associated with increased expression of Nanog protein and predicts poor prognosis of non-small cell lung cancer.

BACKGROUND: Although the prognostic roles of ß-catenin expression in non-small cell lung cancer (NSCLC) have been reported in several immunohistochemical (IHC) studies, the results were not consistent because some studies lack sufficient number of the positive cases or did not evaluate the subcellular localization features of the protein. METHOD: In this study, we have evaluated the expression levels and subcellular localization of ß-catenin and Nanog proteins IHC staining in tissue specimens from 309 patients with NSCLC, and explored their association with clinicopathological features and patient outcome. RESULTS: We showed that patients with negative expression of membranous beta-catenin had a trend towards shorter survival (p=0.064) than those with positive expression. In contrast to previous studies, we found that increased expression of either cytoplasmic or nuclear ß-catenin was strongly associated with poor prognosis and was an independent prognosticator for overall survival (p <0.01). We further found that NSCLC cells frequently exhibited an abundance of nuclear Nanog protein which was significantly correlated with nuclear ß-catenin expression (p <0.01) and poor prognosis (p <0.01). Interestingly, immunofluorescent staining results revealed that increased expression of Nanog and nuclear translocation of ß-catenin occurred concomitantly in response to epidermal growth factor receptor(EGFR) signaling in A549 and H23 cells. Furthermore, western blot analysis show that nuclear ß-catenin rather than cytoplasmic ß-catenin expression in the A549 and H23 cells can be enhanced by adding EGF, Nanog expression in the A549 and H23 cells with knockdown of ß-catenin can not be obviously enhanced by adding EGF. CONCLUSION: We propose that evaluation of subcellular localization of ß-catenin and Nanog expression is of clinical significance for patients with NSCLC.

[Detection of interferon-induced transmembrane-1 gene expression for clinical diagnosis of colorectal cancer].

OBJECTIVE: To investigate the expression of the interferon-induced transmembrane-1 (IFITM1) gene in colorectal cancer (CRC) tissue and the serum anti-IFITM1 antibody responses of the patients and assess their value in clinical diagnosis of CRC. METHODS: Semi-quantitative RT-PCR was performed to detect IFITM1 mRNA expression in the specimens of normal colonic mucosa, CRC tissue, inflammatory polyps, adenomatous polyps, gastric cancer, esophageal carcinoma and liver cancer tissues. Serum samples were collected from the patients to detect anti-IFITM1 antibody responses using Western blotting. The clinicopathological features of the carcinoma expressing IFITM1 gene were analyzed. RESULTS: IFITM1 mRNA was expressed in 47.4 % (18/38) of the CRC specimens, a rate significantly higher than that in adenomatous polyps [15% (3/20)] and gastric cancer [4.8% (1/21)]; no obvious IFITM1 expression was found in normal colonic mucosa, inflammatory polyp, esophageal carcinoma or liver cancer tissues (P<0.001 or P<0.05). IFITM1 mRNA was strongly expressed in CRC at the expression level of 0.8048-/+0.2273, which was significantly higher than that in adenomatous polyps (0.4447-/+0.0989, P<0.001). No anti-IFITM1 antibody response was detected in healthy human sera, but in the CRC patients, the serum antibody response was detected at the rate of 36.8% (14/38), significantly higher than the rate of 9.5% (2/21) in gastric cancer (P<0.05). No antibody response was detected in esophageal carcinoma, liver cancer, inflammatory polyp or adenomatous polyps. Most of the IFITM1-expressing CRC had a diameter exceeding 5 cm, often invading the serous membrane with metastasis to the lymph nodes and the distant organs; these tumors were identified mostly as well-differentiated adenocarcinoma in Dukes stage C or D. CONCLUSION: IFITM1 gene may play an important role in the pathogenesis, development and metastasis of CRC, and may serve as a potential biomarker for clinical diagnosis of CRC.

Oral immunization of mice with vaccine of attenuated Salmonella typhimurium expressing Helicobacter pylori urease B subunit.

OBJECTIVE: To prepare the live recombinant vaccine of attenuated Salmonella typhimurium SL3261 expressing Helicobacter pylori (H. pylori) B subunit (UreB) and to determine whether it could be used as an oral vaccine against H. pylori infection. METHODS: Using genomic DNA of H. pylori Sydney strain (SSI) as template, the H. pylori UreB gene fragment was amplified by PCR and subcloned into the expression vector pTC01. The recombinant plasmid pTC01-UreB was then transferred into LB5000 to obtain modified forms, and further conversed into the attenuated Salmonella typhimurium SL3261 to obtain recombinant SL3261/pCT01-UreB as an oral immunization reagent, which was then used to orally immunize Balb/c mice twice at a three-week interval. Twelve weeks later, anti-UreB IgA antibodies in intestinal fluid and IgG antibodies in sera were determined by ELISA. The relating data in control groups (including body weight, gastric inflammation, etc.) were also collected. RESULTS: The sequencing analysis showed that the UreB gene fragment amplified by PCR was consistent with the sequence of the H. pylori UreB gene. The restriction enzyme digestion revealed that the correct pTC01-UreB was obtained. SDS-PAGE and Western blot showed that a 61KD protein was expressed in SL3261/pTC01-UreB, which could be recognized by anti-H. pylori UreB antiserum and was absent in the control containing only Salmonella typhimurium SL3261 strain. The multiple oral immunization with SL3261/pTC01-UreB could significantly induce H. pylori specific mucosal IgA response as well as serum IgG responses. IFN-gamma and IL-10 levels were significantly increased in SL3261/pTC01-UreB group, and no obvious side effect and change in gastric inflammation were observed. CONCLUSION: The attenuated vaccine of Salmonella typhimurium expressing H. pylori UreB can be used as an oral vaccine against H. pylori infection.

[Construction and identification of the cDNA phage expression library for human colorectal cancer antigens].

OBJECTIVE: To construct a cDNA phage expression library for human colorectal carcinoma antigens. METHODS: After the total RNA was extracted from human colorectal cancer tissues, the single-strand and double-strand cDNA were synthesized through reverse transcriptase PCR and long-distance PCR, with the cDNA fragments smaller than 500 bp removed and the remaining cDNA combined with the right and left arms of dephosphorylated lambdaTriplEx2 phage vector. The recombinant phage were then packaged in vitro by MaxPlax Packaging extract, and a small portion of the packaged phage was used to infect E.coli XL1-Blue. Titer measurement was performed so as to determine the capacity of the library. SfiI restriction endonucleases was used to cut the recombined phage DNA in order to identify the size of inserted cDNA. RESULTS: The constructed cDNA phage expression library for human colorectal cancer antigens consisted of 2.39 x 10(6) pfu/ml bacteriophages with a recombination rate of 97.5% and the length of the inserted cDNA fragment ranged from 600 to 4,000 bp with an average of 1,400 bp. CONCLUSION: The cDNA phage expression library of human colorectal cancer antigens is successfully constructed to meet the currently recognized standards, and can be well applicable in screening cDNA-cloned genes of human colorectal cancer-associated antigens by immunoscreening.

Pleckstrin homology domain of G protein-coupled receptor kinase-2 binds to PKC and affects the activity of PKC kinase.

AIM: To study the detail mechanism of interaction between PKC and GRK(2) and the effect of GRK(2) on activity of PKC. METHODS: The cDNA of pleckstrin homology (PH) domain located in GRK(2) residue 548 to 660 was amplified by PCR with the mRNA of human GRK(2) (beta1-adrenergic receptor kinase) as template isolated from human fresh placenta, the expression vector pGEX-PH inserted with the aboved cDNA sequence for GRK(2) PH domain protein and the expression vectors for GST (glutathion-s-transferase) -GRK(2) PH domain fusion protein, BTK (Bruton's tyrosine kinase) PH domain and GST protein were constructed. The expression of GRK(2) in culture mammalian cells (6 cell lines: PC-3, MDCK, SGC7901, Jurkat cell etc.) was determined by SDS-PAGE and Co-immunoprecipitation. The binding of GRK(2) PH domain, GST-GRK(2) PH domain fusion protein and BTK PH domain to PKC in Vitro were detected by SDS-PAGE and Western blot, upon prolonged stimulation of epinephrine, the binding of GRK(2) to PKC was also detected by western blot and Co-immunoprecipitation. RESULTS: The binding of GRK(2) PH domain to PKC in Vitro was confirmed by western blot, as were the binding upon prolonged stimulation of epinephrine and the binding of BTK PH domain to PKC. In the present study, GRK(2) PH domain was associated with PKC and down-regulated PKC activity, but Btk PH domain up-regulated PKC activity as compared with GRK(2) PH domain. CONCLUSION: GRK(2) can bind with PKC and down-regulated PKC activity.

[Cloning, sequencing and bioinformatics analysis of a new tumor suppressor gene ndr2 from mouse].

BACKGROUND & OBJECTIVE: Ndr2 (N-myc down stream regulator) gene in human is a new gene cloned with the human adult whole brain cDNA as template in 1999, which accession number is AF159092 in GenBank. Locating backward position of the N-myc gene in human chromosome, this gene was named Ndr2 gene. The previous experimental results showed Ndr2 gene probably is a tumor suppressor gene. To research the function of Ndr2 gene, the authors cloned the genomic sequence of ndr2 from mouse. METHODS: To clone Ndr2 genomic sequence by reverse transcription-polymerase chain reaction(RT-PCR) with the mouse genome library as template; automatic sequencing was performed using 310 Genetic Analyzer; homogeneous analysis was made using GenBank BLAST; open reading fragment(ORF) analysis was made using PC Gene and ORF Finder; domain analysis was made using ProDom system. RESULTS: A fragment (about 3310bp,identified by agarose gel electrophoresis) was obtained using RT-PCR with the mouse genome library as template. The fragment was cloned in pMD18-T vector. BLAST analysis showed that the sequence was highly homogeneous (with the homogeneity rate of 91.4%) with Ndr2 gene in human and non-homogeneous with genomic sequence database in mouse. ORF analysis showed that there was a complete coding region in it, which including 8 extrons and 7 introns; it can interpret a protein containing about 200 amino acid residuals. ProDom analysis showed there was a domain like acyl carrier protein(ACP) in it. CONCLUSION: The authors cloned Ndr2 gene in mouse and proved that the sequence is a new genome sequence in mouse genomic sequence database. At present, the genome sequence has been submitted to GenBank(the accession number: AY151387).