The use of melittin to enhance transgene expression mediated by recombinant adeno-associated virus serotype 2 vectors both in vitro and in vivo / 中西医结合学报

OBJECTIVE@#Melittin, a cell-penetrating peptide, improves the efficiency of many non-viral gene delivery vectors, yet its application in viral vectors has not been well studied. The non-pathogenic recombinant adeno-associated virus (rAAV) vector is an ideal in vivo gene delivery vector. However, its full potential will only be achieved after improvement of its transduction efficiency. To improve the transduction efficiency of rAAV2 vectors, we attempted to develop a melittin-based rAAV2 vector delivery strategy.@*METHODS@#The melittin peptide was inserted into the rAAV2 capsid either in the loop VIII of all viral proteins (VPs) or at the N terminus of VP2. Various rAAV2-gfp or -fluc vectors were subjected to quantitative real-time polymerase chain reaction and Western blot assays to determine their titers and integrity of capsid proteins, respectively. Alternatively, the vectors based on wild-type capsid were pre-incubated with melittin, followed by transduction of cultured cells or tail vein administration of the mixture to C57BL/6 and BALB/c nude mice. In vivo bioluminescence imaging was performed to evaluate the transgene expression.@*RESULTS@#rAAV2 vectors with melittin peptide inserted in the loop VIII of VPs had low transduction efficiency, probably due to dramatically reduced ability to bind to the target cells. Fusing the melittin peptide at the N-terminus of VP2 produced vectors without the VP2 subunit. Interestingly, among the commonly used rAAV vectors, pre-incubation of rAAV2 and rAAV6 vectors with melittin significantly enhanced their transduction efficiency in HEK293 and Huh7 cells in vitro. Melittin also had the ability to increase the rAAV2-mediated transgene expression in mouse liver in vivo. Mechanistically, melittin did not change the vector-receptor interaction. Moreover, cell counting kit-8 assays of cultured cells and serum transaminase levels indicated melittin had little cytotoxicity.@*CONCLUSION@#Pre-incubation with melittin, but not insertion of melittin into the rAAV2 capsid, significantly enhanced rAAV2-mediated transgene expression. Although further in vivo evaluations are required, this research not only expands the pharmacological potential of melittin, but also provides a new strategy to improve gene therapy mediated by rAAV vectors.

Evodiamine inhibits high-fat diet-induced colitis-associated cancer in mice through regulating the gut microbiota / 中西医结合学报

OBJECTIVE@#High-fat diet is one of the main risk factors that disrupt the balance of gut microbiota, which eventually will induce colorectal cancer (CRC). Evodiamine (EVO) is a wildly used multifunctional traditional Chinese medicine extract. In this study, we investigated the role of gut microbiota in high-fat diet-propelled CRC and the potential of EVO for CRC chemoprevention.@*METHODS@#Gut microbiota, serum d-lactic acid and endotoxin from 38 patients with colon cancer and 18 healthy subjects were detected by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA). In addition, body mass index, phospho-signal transducer and activator of transcription 3 (p-STAT3) expression in cancer tissues and paracancerous tissues were detected by immunohistochemistry. A mouse intestinal inflammatory tumor model was established by azomethane/sodium dextran sulfate, followed by treatment with EVO and 5-aminosalicylic acid (ASA). Gut microbiota and inflammatory factors were detected by quantitative polymerase chain reaction, while serum d-lactic acid and endotoxin were detected by ELISA. Furthermore, cell proliferation, cell apoptosis, and interleukin (IL)-6/STAT3/P65 pathway were evaluated by 5-ethynyl-2'-deoxyuridine, terminal-deoxynucleotidyl transferase-mediated nick-end labeling, and Western blot assays.@*RESULTS@#In patients with colon cancer, the numbers of Enterococcus faecalis and Escherichia coli were increased, while those of Bifidobacterium, Campylobacter and Lactobacillus were decreased. Serum endotoxin and d-lactic acid levels and p-STAT3 levels were significantly increased. In the mouse model, both EVO and ASA inhibited tumor formation, decreased the proliferation of tumor cells, and induced apoptosis of tumor cells. Compared with the control group, the numbers of E. faecalis and E. coli were decreased, while Bifidobacterium, Campylobacter and Lactobacillus numbers were increased. In the EVO group, serum endotoxin and d-lactic acid levels and inflammatory factors were significantly decreased. Further, the IL6/STAT3/P65 signaling pathway was inhibited in the EVO group.@*CONCLUSION@#EVO may inhibit the occurrence of colon cancer by regulating gut microbiota and inhibiting intestinal inflammation. The potential mechanism involves inhibition of the IL6/STAT3/P65 signaling pathway, revealing its potential therapeutic significance in clinical applications.

The use of miR122 and its target sequence in adeno-associated virus-mediated trichosanthin gene therapy / 中西医结合学报

OBJECTIVE@#Plant-derived cytotoxic transgene expression, such as trichosanthin (tcs), regulated by recombinant adeno-associated virus (rAAV) vector is a promising cancer gene therapy. However, the cytotoxic transgene can hamper the vector production in the rAAV producer cell line, human embryonic kidney (HEK293) cells. Here, we explored microRNA-122 (miR122) and its target sequence to limit the expression of the cytotoxic gene in the rAAV producer cells.@*METHODS@#A miR122 target (122T) sequence was incorporated into the 3' untranslated region of the tcs cDNA sequence. The firefly luciferase (fluc) transgene was used as an appropriate control. Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells. The effects of miR122 overexpression on cell growth, transgene expression, and rAAV production were determined.@*RESULTS@#The presence of 122T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line (in vitro), fresh human hepatocytes (ex vivo), and mouse liver (in vivo). Also, the normal liver physiology was unaffected by delivery of 122T sequence by rAAV vectors. Compared with the parental cells, the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122T, as well as the ability to produce liver-targeting rAAV vectors. Fascinatingly, the yield of rAAV vectors carrying the tcs-122T gene was increased by 77.7-fold in HEK293-mir122 cells. Moreover, the tcs-122T-containing rAAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells.@*CONCLUSION@#HEK293-mir122 cells along with the 122T sequence provide a potential tool to attenuate the cytotoxic transgene expression, such as tcs, during rAAV vector production.

Fifty-two cases analysis of cliff suicide / 法医学杂志

OBJECTIVE@#To analyze suicide scene, damage morphology and case investigation of mountain type scenic spot for giving the references to determine the nature of cliff suicide cases.@*METHODS@#The suicide cases collected from 2002 to 2012 in scenic spot of Mount Huangshan. The age, gender, native place, case location, damage inspection and behavior were analyzed.@*RESULTS@#In the 52 suicide cases, the suicide rate of male was higher than that of female. The numbers from other provinces were higher than that of local province. The age was mainly range from 19 to 50. The time of suicide cases mostly happened between 16:00 to 24:00. The major damage was compound injury with varying degrees of traumatic brain injury, organic injury of pleuroperitoneal cavity and surface bruise and scratch.@*CONCLUSION@#In order to determine the nature of cliff suicide cases, it needs to work synthetically in the investigation of crime scene and interview.

Gene analysis in four inherited coagulation FⅫ deficiency pedigree / 中华血液学杂志

<p><b>OBJECTIVE</b>To identify the genotype and pathogenesis in four Chinese pedigrees with Factor Ⅻ deficiency.</p><p><b>METHODS</b>Activated partial thromboplastin time (APTT), FⅫ procoagulant activity (FⅫ∶C), FⅫ antigen（FⅫ∶Ag）and other coagulant parameters were detected. The FⅫ deficiency Pedigree members,all exons,boundary introns including the splice junctions of the FⅫ gene were amplified with Polymerase chain reaction (PCR). Expression plasmids were constructed by mutagenesis based on the wild-type and transfected into COS7 cells. FⅫ∶C and FⅫ∶Ag of the expression levels were tested in the supernatant and cell lysate.</p><p><b>RESULTS</b>The four probands presented prolonged APTT with all the values of FⅫ∶C and FⅫ∶Ag were low to 2% and 1%, respectively. There were common 46C/T polymorphism in the promoter regions of FⅫ gene in four pedigrees. Proband A was heterozygous for two mutations, g.5741-5742delCA (His101Gln) and g.7142insertC (Lys346Gln). Proband B was a heterozygous deletion mutation g.6800-6808del9bp. The results of the transfection revealed that FⅫ∶Ag in cell lysates and conditioned media protein FⅫ6800-6808del9bp were 85.6% and 51.9%. The FⅫ∶C in the conditioned media was 56.4%. Proband C was a heterozygous mutation g.8699G>A(Gly542Ser). Proband D was a homozygous mutation 8699G>A, whose parents with consanguineous marriage.</p><p><b>CONCLUSION</b>Four mutations, g.5741-5742delCA, g.7142insertC, g.6800-6808del9bp and g.8699G>A with 46C/T polymorphism in the promoter regions of FⅫ gene, were identified in the four Factor Ⅻ deficiency pedigrees. The two mutations g.5741-5742delCA and g.6800-6808del9bp were first found in China. FⅫ 6800-6808del9bp expressed in vitro suggested that almost normal proteinum synthesis but defect proteinum secretion.</p>

Analysis of a hereditary protein C deficient consanguineous pedigree caused by Phe139Val homozygous mutation / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze genetic mutation and explore its molecular pathogenesis for an hereditary protein C (PC) deficient consanguineous pedigree.</p><p><b>METHODS</b>The pedigree included three generations and contained eight members. PC activity (PC:A), PC antigen (PC:Ag) and other coagulant parameters were detected for all family members. Protein C gene (PROC) include all the exons and intron exon boundaries were amplified by PCR for the proband, then analyzed by direct sequencing. Mutation sites were detected for the other family members.</p><p><b>RESULTS</b>The PC:A and PC:Ag in the proband plasma were 20% (normal range 70% -140%) and 13.2% (normal range 70%-130%). A homozygous missense mutation g.6128T>G in exon 7 resulting in Phe139Val was identified in the proband. The PC:A and PC:Ag in her younger brother were 31% and 18.90%, Phe139Val homozygous was also found. The left family members were heterozygous for Phe139Val.</p><p><b>CONCLUSION</b>Phe139Val homozygous missense mutation in exon 7 of PROC caused serious hereditary protein C deficiency. We speculated that homozygous mutation might be resulted from this consanguineous marriage.</p>

Analysis of a consanguineous pedigree featuring hereditary coagulation factor Ⅴ deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To screen potential mutation and explore the underlying mechanism for a consanguineous pedigree featuring hereditary coagulation factor Ⅴ (FⅤ) deficiency.</p><p><b>METHODS</b>Clinical diagnosis was validated by coagulant parameter assays of prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), FⅤ procoagulant activity (FⅤ:C) and FⅤ antigen (FⅤ:Ag). Potential mutations of the F5 gene in the proband and his family members were analyzed by direct DNA sequencing of PCR products of all exons, exon-intron boundaries and 3', 5' untranslated regions. Suspected mutation was confirmed by reverse sequencing.</p><p><b>RESULTS</b>The PT and APTT in the proband were significantly prolonged, which measured 23.5 s (reference range 11.8-14.8 s) and 50.5 s (reference range 27.0-41.0 s), respectively. FⅤ activity and FⅤ antigen of the proband were significantly reduced to 8% and <1%, respectively. PT and APTT in the younger sister of the proband were also significantly prolonged (24.1 s and 62.4 s, respectively). Her FⅤ activity and FⅤ antigen were also significantly decreased (7% and <1%, respectively). PT and APTT of other family members were within the normal range. The homozygous missence mutation causing T→C transition at position 29170 in exon 5 of F5 gene has resulted in a Phe190Ser substitution in the proband. His younger sister was also homozygous for Phe190Ser. Heterozygosity for Phe190Ser was confirmed in his elder brother, elder sister, two daughters and niece, and their FⅤ activity were slightly decreased (57%, 73%, 72%, 66% and 75%, respectively). A normal wild type was observed in two younger brothers of the proband, and their FⅤ activity and FⅤ antigen were in the normal range.</p><p><b>CONCLUSION</b>Homozygous missence mutation of Phe190Ser has been found in above family featuring hereditary FⅤ deficiency. The homozygous missence mutation was inherited from the parents by consanguineous marriage. Phe190Ser probably underlies may underlie the pathogenesis of hereditary FⅤ deficiency in this pedigree.</p>

Analysis of an hereditary protein C deficiency pedigree with compound heterozygous gene mutations / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze genetic mutations and explore its molecular pathogenesis for an hereditary protein C (PC) deficiency pedigree.</p><p><b>METHODS</b>The pedigree has included 15 individuals from 4 generations. Plasma levels of PC activity (PC:A), PC antigen (PC:Ag) and other coagulant parameters were determined for members of the family. The 9 exons and intron-exon boundaries of protein C gene (PROC) of the proband were amplified with PCR and analyzed with direct sequencing. Detected mutations were confirmed with reverse sequencing. Corresponding PCR fragments from the family members were also directly sequenced.</p><p><b>RESULTS</b>Plasma PC:A and PC:Ag for the proband was 26% and 18.60%, respectively, both being lower than normal references. Seven members from the pedigree also had lower PC:A, six had lower PC:Ag. A compound heterozygous missense mutation, including a T to G transition at position 6128 of exon 7, which results in Phe139Val, and a G to C transition at position 8478 in exon 9, which results in Asp255His, were identified in the proband. The paternal grandma, father and two aunts were heterozygous for g.6128 T to G, whilst the mother, the second uncle, sister and son were heterozygous for g.8478 G to C. There were lower PC:A in family members with g.8478 G to C.</p><p><b>CONCLUSION</b>The proband had inherited two independent mutations of the PROC gene including g.6128 T to G in exon 7 and g.8478 G to C in exon 9 from her father and mother, respectively. The resulting compound heterozygous mutation has caused a serious hereditary protein C deficiency.</p>