Splicing abnormalities caused by a novel mutation in the PHKA2 gene in children with glycogen storage disease type IX / 中华肝脏病杂志

Objective: Glycogen storage disease type IX (GSD-IX) is a rare primary glucose metabolism abnormality caused by phosphorylase kinase deficiency and a series of pathogenic gene mutations. The clinical characteristics, gene analysis, and functional verification of a mutation in a child with hepatomegaly are summarized here to clarify the pathogenic cause of the disease. Methods: The clinical data of a child with GSD-IX was collected. Peripheral blood from the child and his parents was collected for genomic DNA extraction. The patient's gene diagnosis was performed by second-generation sequencing. The suspected mutations were verified by Sanger sequencing and bioinformatics analysis. The suspected splicing mutations were verified in vivo by RT-PCR and first-generation sequencing. Results: Hepatomegaly, transaminitis, and hypertriglyceridemia were present in children. Liver biopsy pathological examination results indicated glycogen storage disease. Gene sequencing revealed that the child had a c.285 + 2_285 + 5delTAGG hemizygous mutation in the PHKA2 gene. Sanger sequencing verification showed that the mother of the child was heterozygous and the father of the child was of the wild type. Software such as HSF3.1 and ESEfinder predicted that the gene mutation affected splicing. RT-PCR of peripheral blood from children and his mother confirmed that the mutation had caused the skipping of exon 3 during the constitutive splicing of the PHKA2 gene. Conclusion: The hemizygous mutation in the PHKA2 gene (c.285 + 2_285 + 5delTAGG) is the pathogenic cause of the patient's disease. The detection of the novel mutation site enriches the mutation spectrum of the PHKA2 gene and serves as a basis for the family's genetic counseling.

Severe COVID-19-associated sepsis is different from classical sepsis induced by pulmonary infection with carbapenem-resistant klebsiella pneumonia (CrKP) / 中华创伤杂志(英文版)

PURPOSE@#COVID-19 is also referred to as a typical viral septic pulmonary infection by 2019-nCoV. However, little is known regarding its characteristics in terms of systemic inflammation and organ injury, especially compared with classical bacterial sepsis. This article aims to investigate the clinical characteristics and prognosis between COVID-19-associated sepsis and classic bacterial-induced sepsis.@*METHODS@#In this retrospective cohort study, septic patients with COVID-19 in the intensive care unit (ICU) of a government-designed therapy center in Shenzhen, China between January 14, 2020 and March 10, 2020, and septic patients induced by carbapenem-resistant klebsiella pneumonia (CrKP) admitted to the ICU of the Second People's Hospital of Shenzhen, China between January 1, 2014 and October 30, 2019 were enrolled. Demographic and clinical parameters including comorbidities, critical illness scores, treatment, and laboratory data, as well as prognosis were compared between the two groups. Risk factors for mortality and survival rate were analyzed using multivariable logistic regression and survival curve, respectively.@*RESULTS@#A total of 107 patients with COVID-19 and 63 patients with CrKP were enrolled. A direct comparison between the two groups demonstrated more serious degrees of primary lung injury following 2019-nCoV infection (indicated by lower PaO@*CONCLUSION@#Critical COVID-19 shares clinical characteristics with classical bacterial sepsis, but the degree of systemic inflammatory response, secondary organ damage and mortality rate are less severe. However, following 2019-nCoV infection, the level of immunosuppression may be increased and thus induce in more death at the later stage of patients' hospitalstay.

Influencing factors for the quality of bowel preparation with polyethylene glycol electrolyte powder combined with diet control before colonoscopy in children / 中国当代儿科杂志

OBJECTIVES@#To investigate the influencing factors for the quality of bowel preparation before colonoscopy in children and the association of the interval from the last administration of laxative to the start of colonoscopy (shortly referred to as waiting time) with the quality of bowel preparation.@*METHODS@#A retrospective analysis was performed for the children who were admitted to the Department of Gastroenterology, Children's Hospital of Nanjing Medical University, from January to November 2020, and received bowel preparation with polyethylene glycol electrolyte powder combined with diet control before colonoscopy. According to the score of Boston bowel preparation scale, they were divided into two groups: adequate bowel preparation group (n=337) and inadequate bowel preparation group (n=30). Related data were collected from the children in both groups, including general information, possible influencing factors for the quality of bowel preparation, adverse reactions associated with bowel preparation, duration of colonoscopy, and postoperative diagnosis. Univariate and multivariate analyses were used to explore the influencing factors for the quality of bowel preparation.@*RESULTS@#The univariate analysis showed that age, body weight, and waiting time were associated with inadequate bowel preparation (P<0.05). The multivariate analysis showed that older age (OR=2.155, 95%CI: 1.087-4.273, P=0.028) and longer waiting time (OR=1.559, 95% CI: 1.191-2.041, P=0.001) were independent risk factors for inadequate bowel preparation. The receiver operating characteristic (ROC) curve analysis showed that the cut-off value of waiting time was 5.5 hours in determining whether bowel preparation was adequate or not, with a sensitivity of 90.0%, a specificity of 50.7%, and an area under the ROC curve of 0.708. After grouping based on waiting time, it was found that the incidence rate of inadequate bowel preparation in the ≥5.5 hours group was significantly higher than that in the <5.5 hours group [14.0% (27/193) vs 1.7% (3/174), P<0.001].@*CONCLUSIONS@#For children who use polyethylene glycol electrolyte powder combined with diet control for bowel preparation, older age is an independent risk factor for inadequate bowel preparation before colonoscopy, which may be associated with an insufficient dose of polyethylene glycol in older children. Longer waiting time is also an independent risk factor for inadequate bowel preparation, and it is recommended that the waiting time should not exceed 5.5 hours.

YU Tian-yuan's experience in the clinical application of Danzhong (CV 17) for mental illness / 中国针灸

To summarize YU Tian-yuan's experience of applying Danzhong (CV 17) for mental illness in acupuncture and tuina. YU Tian-yuan uses Danzhong (CV 17) alone or in combination with other acupoints to treat mental illnesses such as insomnia, palpitation and chest distress. Professor YU emphasizes 4 tips when treating diseases, nourishing the heart to tranquilize by light stimulation; regulating spirit by combined stimulation; leaving the acupoints and holding on the meridian for a wide range of stimulation; using rubbing and pushing manipulation in several directions for regulating qi to soothe the chest. And in clinical practice, formed a unique therapy to treat mental illness.

A single-center retrospective analysis of 46 children with aerophagia / 中国当代儿科杂志

OBJECTIVE@#To study the clinical features of aerophagia in children.@*MEYJODS@#A retrospective analysis was performed on the medical data of 46 children with aerophagia who were diagnosed and treated in Children's Hospital Affiliated to Nanjing Medical University from October 2011 to September 2019.@*RESULTS@#Among these 46 children, 15 (33%) had Tourette syndrome. Abdominal distension was the most common symptom and was observed in 45 children (98%). The 24-hour esophageal multichannel intraluminal impedance monitoring showed a mean number of 341 times of air swallowing and a mean number of 212 times of gas reflux, and 95% of gas refluxes occurred in the upright body position. Compared with those without Tourette syndrome, the children with Tourette syndrome had a significantly higher incidence rate of air swallowing symptoms (67% vs 6%, P<0.001), but there were no significant differences in other symptoms and the results of 24-hour esophageal impedance. Dietary adjustment, psycho-behavioral therapy, and drug intervention significantly improved the scores of clinical symptoms and quality of life, among which psycho-behavioral therapy was an important intervention measure.@*CONCLUSIONS@#Some children with aerophagia may have Tourette syndrome, and such children are more likely to have air swallowing symptoms. Psycho-behavioral therapy is one of the most important treatment methods, and children with aerophagia tend to have a good prognosis after treatment.

Genetic diagnosis in chronic diarrhea in children / 中国实用儿科杂志

Chronic diarrhea in children is a common digestive system disease with many types,and the clinical symptoms are complex. The symptoms of various chronic diarrhea diseases often overlap,so it is difficult to diagnose. Gene detection is an effective method for making diagnosis. In recent years,the progress of molecular diagnosis technology,especially the wide application of second generation gene sequencing(NGS)technology,has reduced the cost and time of gene detection. Therefore,selecting the appropriate gene diagnosis strategies and interpreting the reports are very important for clinicians.

Comparison of plasma radiofrequency ablation to the total inferior turbinate with its anterior extremity / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To compare the curative effect and complications between plasma radiofrequency ablation to the anterior extremity of the inferior turbinate and the treatment to the total of the inferior turbinate for the treatment of hypertrophic inferior turbinate.@*METHOD@#One hundred and fifty-three patients were randomly divided into two groups, and all of the patients had obviously nasal obstruction due to isolated inferior turbinate hypertrophy. In the experimental group, plasma radiofrequency ablation was only applied to the anterior extremity of the inferior turbinate, whereas the whole concha was ablated in the control group. Visual analogue scale (VAS) for nasal obstruction were obtained both before the treatment and 1 year after surgery, and during the follow-up period, their recovery of nasal mucosa and complications were evaluated. RESULT. VAS scores before and after treatment were statistically significant in both groups (P 0.05). Complications between the two groups were statistically significant (P < 0.01).@*CONCLUSION@#Compare to radiofrequency ablation to the total of the inferior turbinate, the application to the anterior extremity of the inferior turbinate could provide adequate curative effect. The treatment to the anterior extremity of the inferior turbinate is simpler, which induce less complications, and which is safer for there is less risk of damage of continuity of the nasal mucosa. So it has a higher clinical application value.

Rac1+ cells distributed in accordance with CD 133+ cells in glioblastomas and the elevated invasiveness of CD 133+ glioma cells with higher Rac1 activity / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Recent studies have suggested that cancer stem cells are one of the major causes for tumor recurrence due to their resistance to radiotherapy and chemotherapy. Although the highly invasive nature of glioblastoma (GBM) cells is also implicated in the failure of current therapies, it is not clear how glioma stem cells (GSCs) are involved in invasiveness. Rac1 activity is necessary for inducing reorganization of actin cytoskeleton and cell movement. In this study, we aimed to investigate the distribution characteristics of CD133+ cells and Rac1+ cells in GBM as well as Rac1 activity in CD133+ GBM cells, and analyze the migration and invasion potential of these cells.</p><p><b>METHODS</b>A series of 21 patients with GBM were admitted consecutively and received tumor resection in Tianjin Medical University General Hospital during the first half of the year 2011. Tissue specimens were collected both from the peripheral and the central parts for each tumor under magnetic resonance imaging (MRI) navigation guidance. Immunohistochemical staining was used to detect the CD133+ cells and Rac1+ cells distribution in GBM specimens. Double-labeling immunofluorescence was further used to analyze CD133 and Rac1 co-expression and the relationship between CD133+ cells distribution and Rac1 expression. Serum-free medium culture and magnetic sorting were used to isolate CD133+ cells from U87 cell line. Rac1 activation assay was conducted to assess the activation of Rac1 in CD133+ and CD133 - U87 cells. The migration and invasive ability of CD133+ and CD133 - U87 cells were determined by cell migration and invasion assays in vitro. Student's t-test and one-way analysis of variance (ANOVA) test were used to determine statistical significance in this study.</p><p><b>RESULTS</b>In the central parts of GBMs, CD133+ cells were found to cluster around necrosis and occasionally cluster around the vessels under the microscope by immunohistological staining. In the peripheral parts of the tumors, CD133+ cells were lined up along the basement membrane of the vessels and myelinated nerve fibers. Rac1 expression was high and diffused in the central parts of the GBMs, and the Rac1+ cells were distributed basically in accordance with CD133+ cells both in the central and peripheral parts of GBMs. In double-labeling immunofluorescence, Rac1 was expressed in (83.14 ± 4.23)% of CD133+ cells, and CD133 and Rac1 co-expressed cells were located around the vessels in GBMs. Significantly higher amounts of Rac1-GTP were expressed in the CD133+ cells (0.378 ± 0.007), compared to CD133- cells (0.195 ± 0.004) (t = 27.81; P < 0.05). CD133+ cells had stronger ability to migrate (74.34 ± 2.40 vs. 38.72 ± 2.60, t = 42.71, P < 0.005) and invade (52.00 ± 2.28 vs. 31.26 ± 1.82, t = 30.76, P < 0.005), compared to their counterpart CD133- cells in transwell cell migration/invasion assay.</p><p><b>CONCLUSIONS</b>These data suggest that CD133+ GBM cells highly express Rac1 and have greater potential to migrate and invade through activated Rac1-GTP. The accordance of distribution between Rac1+ cells and CD133+ cells in GBMs implies that Rac1 might be an inhibited target to prevent invasion and migration and to avoid malignant glioma recurrence.</p>

Glioma stem cells enhanced angiogenesis and its relationship with microvessel / 中华外科杂志

<p><b>OBJECTIVES</b>To dynamically observe how glioma stem cells promote the tumor formation and angiogenesis, and to study the correlation between the distribution of glioma stem cells and microvessels within different growth stages of subcutaneous tumor.</p><p><b>METHODS</b>Stem cell medium culture and magnetic activated cell sorting were carried out to obtain CD133+ cells from C6 rat glioma cell line. Sprague Dawley (SD) rat ears model were established to observe glioma stem cells promoting blood vessel formation. Subcutaneous glioma model of C6 and immunohistochemical staining of hypoxia inducible factor-1α (HIF-1α) and CD133 were used to investigate the relationship between distribution of glioma stem cells and microvessels. Expressions of CD133 protein in each stage of the subcutaneous tumor were detected by Western blot.</p><p><b>RESULTS</b>Isolation and identification of glioma stem cells deprived from C6 glioma cell line successfully, the establishment of ears model showed real-time dynamic observation of CD133+ cells involved in angiogenesis and tumor formation. SD rat model of subcutaneous glioma showed the initial of tumor formation, CD133+ cells scattered. With tumor growth, CD133+ cells began to tend to capillaries, in late distributed clusters in perivascular. Meanwhile as tumor growth, CD133 protein expression was gradually increased: the values of Western blot analysis of CD133 expression on 6, 9, 12, 15, 20 d were 0.208±0.004, 0.282±0.003, 0.360±0.004, 0.564±0.135, 0.756±0.007, the differences were significant between different groups (F=2601.681, P<0.01). At a high magnification, the CD133 scores with immunohistochemical staining on 6, 9, 12, 15 d were 0.8±0.4, 2.4±0.5, 4.0 ± 0.7, 6.0±0.7; HIF-1α scores were 0.8±0.4, 2.8±0.8, 5.0±0.7, 6.8±0.4. By Spearman rank correlation analysis found that the relationship between CD133 and HIF-1α expression was positively correlated (r=0.921, P<0.01).</p><p><b>CONCLUSIONS</b>Glioma stem cells promote angiogenesis more than non-stem cells; HIF-1α and its downstream gene product might mediate the distribution of glioma stem cells around the perivascular.</p>

Report of a case with Johanson-Blizzard syndrome and literatures review / 中华儿科杂志

<p><b>OBJECTIVE</b>To study the clinical characteristics and diagnosis of the Johanson-Blizzard syndrome.</p><p><b>METHOD</b>The clinical characteristics and diagnosing procedure of 1 case with Johanson-Blizzard syndrome were analyzed, and genetic analysis was made in diagnosing procedure, and 28 cases of Johanson-Blizzard syndrome with detailed clinical data were reviewed and analyzed.</p><p><b>RESULT</b>A one year and nine months old girl, who was initially admitted to the hospital because of fatty diarrhea and increased frequency of defecation. Imperforate anus, and aplastic alae nasi was noticed after birth. On physical examination, short stature, mental retardation, tooth abnormalities and scalp defects were observed. Fat globule was found by routine stool test. Serum biochemistry showed an exocrine and endocrine pancreatic insufficiency, CT scan of the abdomen demonstrated fatty replacement of the pancreas, UBR1 gene analysis showed heterozygous for two missense changes. In all 29 cases, exocrine pancreatic insufficiency (72.4%) and hypoplasia of the alae nasi (93%) were the most common clinical manifestations, and sensorineural hearing loss (59%), scalp defects (69%) and hair thinning or upsweep of the hair (44.8%), hypothyroidism (44.8%), absence of permanent teeth (44.8%) and imperforate anus (21%) were also very common, but did not include consanguineous marriage of parents (10.3%).</p><p><b>CONCLUSION</b>Johanson-Blizzard syndrome is a rare autosomal recessive multisystem disorder, it is characterized by the association of congenital exocrine pancreatic insufficiency and hypoplasia or aplasia of the nasal wings, and can be diagnosed by clinical characteristics and UBR1 gene analysis.</p>

Enhanced invasion in vitro and the distribution patterns in vivo of CD133+ glioma stem cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Recent studies have suggested that cancer stem cells cause tumor recurrence based on their resistance to radiotherapy and chemotherapy. Although the highly invasive nature of glioblastoma cells is also implicated in the failure of current therapies, it is not clear whether cancer stem cells are involved in invasiveness. This study aimed to assess invasive ability of glioma stem cells (GSCs) derived from C6 glioma cell line and the distribution patterns of GSCs in Sprague-Dawley (SD) rat brain tumor.</p><p><b>METHODS</b>Serum-free medium culture and magnetic isolation were used to gain purely CD133(+) GSCs. The invasive ability of CD133(+) and CD133(-) C6 cells were determined using matrigel invasion assay. Immunohistochemical staining for stem cell markers and luxol fast blue staining for white matter tracts were performed to show the distribution patterns of GSCs in brain tumor of rats and the relationship among GSCs, vessels, and white matter tracts. The results of matrigel invasion assay were estimated using the Student's t test and the analysis of Western blotting was performed using the one-way analysis of variance (ANOVA) test.</p><p><b>RESULTS</b>CD133(+) GSCs (number: 85.3 ± 4.0) were significantly more invasive in vitro than matched CD133(-) cells (number: 25.9 ± 3.1) (t = 14.5, P < 0.005). GSCs invaded into the brain diffusely and located in perivascular niche of tumor-brain interface or resided within perivascular niche next to white fiber tracts. The polarity of glioma cells containing GSCs was parallel to the white matter tracts.</p><p><b>CONCLUSIONS</b>Our data suggest that CD133(+) GSCs exhibit more aggressive invasion in vitro and GSCs in vivo probably disseminate along the long axis of blood vessels and transit through the white matter tracts. The therapies targeting GSCs invasion combined with traditional glioblastoma multiforme therapeutic paradigms might be a new approach for avoiding malignant glioma recurrence.</p>

Effect of ultrasound on osteoprotegerin and receptor activator nuclear factor kappaB ligand expression during root resorption in rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the effect of pulsed ultrasound on the expressions of osteoprotegerin (OPG) and receptor activator nuclear factor kappaB ligand (RANKL) during root resorption in a mouse model of orthodontic tooth movement.</p><p><b>METHODS</b>Thirty-two male Wistar rats (6-8 weeks old) were randomly assigned into 4 equal groups, including the blank control group, two ultrasound exposure groups with daily local LIPUS stimulation (100 and 150 MW/cm(2)) for 10 days during mechanical loading, and the control group with mechanical loading but not LIPUS exposure. Nickel-titanium closed-coil springs were used to generate 100 g mesial force for 10 days to move the maxillary right first molars. The expression of OPG and RANKL proteins at the compression sites was detected by immunohistochemistry.</p><p><b>RESULTS</b>Ultrasound stimulation significantly up-regulated the expression of OPG and down-regulated RANKL expression (P<0.05). The expressions of OPG and RANKL showed significant differences between the two ultrasound exposure groups (P<0.05).</p><p><b>CONCLUSION</b>Ultrasound stimulation might be useful to protect against root resorption and accelerate its repair by regulating the expressions of OPG and RANKL.</p>

Proteomic analysis of chronic restraint stress-induced Gan (肝)-stagnancy syndrome in rats / 中国结合医学杂志

<p><b>OBJECTIVE</b>To analyze the proteomic characteristics of Gan (肝)-stagnancy syndrome (GSS) by seeking the differential protein in blood and tissues of GSS model rats.</p><p><b>METHODS</b>GSS model rats were established by chronic restraint stress, keeping rats in restrain chamber for 6 h every day for 21 successive days. Their blood and liver samples were collected at the end of experiment for differential protein detection with methods of isoelectrofocusing and polyacrylamide SDS-PAGE, silver staining, and scanning. The gel images were analyzed with Imagemaster 2D Elite software, and the excavated differential protein spots were identified with matrix assistant laser resolving TOF mass spectrometry, Western blot, ELISA, and RT-PCR, respectively.</p><p><b>RESULTS</b>A method for isolating the protein in blood serum and tissues by two-dimensional gel electrophoresis was established and optimized. Six serum proteins and three liver proteins that differentially expressed were identified. The down-regulated differential proteins in serum of GSS model rats were serum albumin precursor, beta 1 globin, antibody against muscle acetylcholine receptor, Ig lambda-2 C region, and transthyretin (TTR), and those in liver tissue were aryl sulfotransferase, enoyl-CoA hydratase, and TTR. TTR down-regulation was found in both serum and liver. Preliminary biological information analysis showed that these differential proteins involved in immune, neuroendocrine, nutrition, and substance metabolism.</p><p><b>CONCLUSION</b>Proteomic analysis of differential proteins showed that TTR, aryl sulfotransferase, and enoyl-CoA hydratase expressions are downregulated in the GSS model rats, suggesting that the susceptibility of cancer could be enhanced by chronic stress.</p>

Reconstruction of an intracellular transduction system based on HIV-1 TAT protein transduction domain / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the reasons for the low intracellular transduction efficiency of a previously constructed His-TAT-Flag recombinant protein and establish a more efficient transduction system.</p><p><b>METHODS</b>The Flag tag of pET14b-His- Tat-Flag vector was deleted with PCR mutant kit, and enhanced green fluorescent protein (EGFP) coding sequence was inserted into the new pET14b-His-TAT recombinant vector. Enzyme digestion and DNA sequencing were performed for identification of pET14b-His-TAT-EGFP vector, which was then transformed into E. coli BL21(DE(3)). After IPTG induction, the recombinant protein of His-TAT-EGFP was isolated and analyzed with SDS-PAGE. Purified His-TAT-EGFP recombinant protein was added to ECV304 cells and the fluorescence was observed to evaluate the transduction efficiency.</p><p><b>RESULTS</b>pET14b-His-TAT vector and pET14b-His-TAT- EGFP vector were successfully constructed, which was identified with enzyme digestion and DNA sequencing. His-TAT-EGFP fusion protein was expressed and purified successfully and showed cellular transduction activity.</p><p><b>CONCLUSION</b>The prokaryotic expression vector has been successfully constructed by modifying pET14b-His-TAT-Flag, and the expressed and purified recombinant protein of His-TAT-EGFP possesses high efficiency of intracellular transduction activity.</p>

Cell-penetrating peptide-based functional study of p38 MAPK / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a p38 MAPK protein delivery system based on TAT protein and study its functions in eukaryotic cells.</p><p><b>METHODS</b>Recombinant vectors pHis-TAT-p38 and pHis-TAT-p38(AF) were constructed, and two recombinant proteins, His-TAT-p38 and His-TAT-p38(AF), were expressed and purified in E. coli. The two fusion proteins were then incubated with ECV304 cells, respectively. The phosphorylation of ATF2 was detected to assay the effect of His-TAT-p38 on endogeneious p38 activity after the cells were stimulated by sorbitol.</p><p><b>RESULTS</b>The results of restriction enzyme digestion and DNA sequencing showed that the two recombinant vectors were correctly constructed. The recombinant proteins of His-TAT-p38 and His-TAT-p38(AF) were isolated and purified by SDS-PAGE, and Western blotting suggested that His-TAT-p38 and its mutant with dual phosphorylation sites could enter the cells efficiently in a time- and concentration-dependent manner. His-TAT-p38 was found capable of increasing the activity of endogenous p38 in ECV304 cells, but His-TAT-p38(AF) inhibited the phosphorylation of ATF2 so as to block the transduction of p38 signal pathway when the cells were stimulated with sorbitol.</p><p><b>CONCLUSION</b>p38 MAPK protein delivery system based on TAT protein has been constructed successfully. It is confirmed that TAT can transfer the proteins into the cells in a time- and dose-depended manner. TAT-p38 and its dominant negative form possess high biological activity after transduction into ECV304 cells by TAT protein delivery system, and the former can increase the activity of endogenous ATF2, but the latter inhibits the transduction of endogeneious p38 signal pathway in ECV304 cells with high osmotic stress.</p>

Construction and functional study of a cell penetrating peptide-based expression vector for targeted delivery of proteins into the cell nuclei / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct an cell penetrating peptide-based expression vector capable of targeted delivery of proteins into the cell nuclei and study its function of protein transduction.</p><p><b>METHODS</b>The fusion protein expression vector pET14b-HC(L)NE (pET14-b-His-CPP-Linker-NLS-EGFP) incorporating cell penetrating peptide (CPP), nuclear localization signal(NLS), linker and enhanced green fluorescent protein (EGFP) was constructed based on His-tagged pET14b-HE (pET14b-His-EGFP) by site-directed mutagenesis PCR method. After identification by enzyme digestion and DNA sequencing, the recombinant plasmid was transformed into BL21(DE(3)) strain. The HC(L)NE fusion protein was expressed following IPTG induction and purified with Ni(2+)-NTA affinity chromatography. After dialysis and filtration, the HC(L)NE fusion protein was added into cultured eukaryotic cells. The protein transduction in the living cells was observed under fluorescence microscope and analyzed by Western blotting.</p><p><b>RESULTS</b>Enzyme digestion and DNA sequencing confirmed successful construction of the pET14b-HC(L)NE vector, and the fusion protein efficiently expressed in E. coli. Protein transduction experiments in eukaryotic cells revealed that the fusion protein could rapidly penetrate the cell membrane and reach the cell nucleus, and this internalization was time- and concentration-dependent.</p><p><b>CONCLUSION</b>The cell penetrating peptide-based expression vector for targeted protein delivery to the cell nucleus has been successfully constructed, and a transport system that can delivery exogenous proteins or polypeptides into the cytoplasm and cell nucleus is established, which provides an economical and efficient means for functional study of the proteins and polypeptide in cells and targeted drug delivery.</p>

Construction of a recombinant adenovirus vector for interferon-gamma-inducible protein 10 and the adenovirus preparation / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a recombinant adenovirus vector for expressing interferon-gamma-inducible protein 10 (IP-10) by homogenous bacterial recombination.</p><p><b>METHODS</b>IP-10 gene was cloned into the shuttle plasmid pAdTrack-CMV that contained the coding sequence of enhanced green fluorescent protein (EGFP). The shuttle plasmid was then transformed into E. coli BJ5183 with pAdEasy-1 vector by chemical transformation. The recombinant adenovirus vector pAd/IP-10 was identified by enzyme digestion with Pac I and the linearized plasmid was transfected into HEK293 cells.</p><p><b>RESULTS</b>The positive clones were identified with enzyme digestion and polymerase chain reaction (PCR) and were further verified by DNA sequencing. The recombinant adenovirus of high titration was obtained after transfection and packaging in HEK293 cells.</p><p><b>CONCLUSION</b>A recombinant adenovirus vector for expression of IP-10 has been constructed successfully and high-titer active adenovirus is obtained for functional study of IP-10 protein.</p>

Effects of allitridin on the expression of human cytomegalovirus immediate early antigens-IE72 and IE86 in human embryonic lung cells / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the effect of allitridin injection on the expression of human cytomegalovirus (HCMV) immediate-early antigens (IEAs including IE72 and IE86) in human embryonic lung cells.</p><p><b>METHOD</b>HCMV AD 169 Virus strain infected cell model (MOI = 2.5 and 0.25, respectively) were established, and then treated with ICm5 and MTC doses of allitridin. Western blot was used to analyze the of IE72 and IE86 expression after the treatment, ganciclovir(GCV, IC50 and 2.3 x IC50) treatment as control.</p><p><b>RESULT</b>No matter what kind of MOI was used, both IE86 and IE72 antigens' expression was effectively suppressed by allitridin treatment, and the inhibitory rate of IE86 was almost twice of IE72's. Compared with GCV, allitridin had stronger inhibitory effect on IE86 expressing, although its efficacy on IE72 was weaker than GCV.</p><p><b>CONCLUSION</b>Allitridin could suppress the expression of IE72 and IE86, especially for IE86 expressing, maybe it is ore of key role in the mechanism of allitridin against HCMV.</p>

Study Progress of Effect of Curcuma on Anti-Helicobacter Pylori / 实用儿科临床杂志

Helicobacter pylori(Hp) is an important pathogenic factor in chronic gastritis,peptic ulcer and gastric cancer.With the extensive use of broad-spectrum atibiotics in recent years,the antibiotic resistence of Hp was increasing,together with many side effets and low patient compliance,so the study of new anti-Hp drugs became a research hotspot.Domestic and foreign scholars had extracted active anti-Hp ingredients from the traditional Chinese drug curcuma,they hope to open a new way for the treatment of Hp infection.This paper was to make a brief overview on the study progress of effect of curcuma on anti-Hp.

Influence of Helicobacter Pylori Infection on Gastric Mucin Expression in Children / 实用儿科临床杂志

Objective To investigate the influence of Helicobacter pylori(Hp) infection on gastric mucin MUC5AC and MUC6 expression in children.Methods Sixty-six cases with gastric biopsy specimens were obtained from 66 children undergone gastroscopy from Jan.2005 to Jun.2006 for episodic or continuous abdominal pain,nausea,vomiting,abdominal distension,retching and dyspepsia,and so on.Among these children,39 cases were male and 27 cases were female,owning a average age(8.8?3.0)years.These specimens were divided into 2 groups followed by the presence of Hp,which was detected by rapid urease tests and Hp-PCR.Gastric mucin MUC5AC and MUC6 mRNA were also measured by reverse transcription PCR(RT-PCR),and Hematoxylin and Eosin staining were used for pathology observation.Comparisons between every groups were performed using t test and ?2 test,and statistical significance was defined as P