Progress in research on B class CpG oligodeoxynucleotides in bacterial vaccines / 中国生物制品学杂志

@#CpG oligonucleotide(CpG ODN)is a synthetic oligodeoxynucleotide containing non-methylated CpG,which has broad application prospects in disease prevention and clinical treatment. Among them,B class CpG ODN is widely used in vaccine research because of its strong immune stimulation and some motifs have entered the clinical research stage. At the same time,the problem of bacterial resistance in clinical treatment has become increasingly serious,and the development of bacterial vaccine with B class CpG ODN as adjuvant has become a research hotspot. This paper reviewed the current research status and related progress of the existing B class CpG ODN 1826,2006,2007 and 1668 motifs in bacterial vaccines,with a view to providing a reference for the subsequent development and application of bacterial vaccines.

Progress in RNA-targeting and Gene Editing Therapies for Transthyretin Amyloidosis Cardiomyopathy / 罕见病研究

Transthyretin(TTR) protein is a tetramer protein, synthesized mainly by the liver. TTR can be misfolded and deposited as amyloid fibrilae and deposited in the myocardial interstroma leading to transthyroxin amyloidosis cardiomyopathy (ATTR-CM). ATTR-CM was included in China's First List of Rare Diseases. Therapeutic strategies for ATTR-CM include blocking TTR synthesis in the liver, stabilizing TTR tetramers and destroying TTR fibra. Small molecule drugs such as tafamidis and diflunisal offer new treatment options for patients. Chlorobenzolic acid became the first drug approved by the U.S. Food and Drug Administration for the treatment of ATTR-CM. Small interfering RNA(siRNA)patisiran and antisense oligonucleotide (ASO)inotersen block TTR expression in the liver and have been approved for the treatment of ATTR variant polyneuropathy (ATTRv-PN)and are in phase Ⅲ trials for the treatment of ATTR-CM. Other siRNA drugs, vutrisiran, and ASO, eplontersen, are being evaluated for clinical efficacy. This article reviews the development of RNA-targeted therapeutics and gene-editing drugs using CRISPR-Cas9.

Concordance of identifying the presence or absence of KIR genes by Flow-rSSO and PCR-SBT methods: a comparative study / 中国输血杂志

【Objective】 To study the concordance of identifying the presence or absence of KIR genes using flow reverse sequence-specific oligonucleotide probe (Flow-rSSO) hybridization and sequencing based typing-PCR (PCR-SBT) methods. 【Methods】 A total number of 131 cases of DNA samples from Han population were subjected to identify the presence or absence of all 16 KIR genes by Flow-rSSO method, and then sequenced at coding sequence for all 14 functional KIR genes using our in-house KIR PCR-SBT assay. The concordance of identifying the presence or absence of all functional KIR genes by Flow-rSSO and PCR-SBT was analyzed. Samples with inconsistent initial results were re-tested using the Flow-rSSO commercial kits with different Lot number, and further tested using the PCR-SSP commercial kit. 【Results】 The presence or absence of 14 functional KIR genes for 129 of 131 samples were completely in accordance via the PCR-SBT and Flow-rSSO methods. Two samples, one with 3DL1 negative, the other with both 2DS3 and 2DS5 negative initially-identified by Flow-rSSO, were actually all positive tested by PCR-SBT. Further retest by Flow-rSSO commercial kits with different Lot number and PCR-SSP commercial kit indicated that the two samples were all positive, which agreed well with PCR-SBT results. 【Conclusion】 In this paper, the initial test results of the presence or absence of KIR genes identified by Flow-rSSO for 2 samples were wrong, which indicated the importance of carrying out the quality control for reagents in KIR gene testing.

Therapeutics in paediatric genetic diseases: Current and future landscape

There are more than 7,000 paediatric genetic diseases (PGDs) but less than 5% have treatment options. Treatment strategies targeting different levels of the biological process of the disease have led to optimal health outcomes in a subset of patients with PGDs, where treatment is available. In the past 3 decades, there has been rapid advancement in the development of novel therapies, including gene therapy, for many PGDs. The therapeutic success of treatment relies heavily on knowledge of the genetic basis and the disease mechanism. Specifically, gene therapy has been shown to be effective in various clinical trials, and indeed, these trials have led to regulatory approvals, paving the way for gene therapies for other types of PGDs. In this review, we provide an overview of the treatment strategies and focus on some of the recent advancements in therapeutics for PGDs.

Research progress of RNA drugs delivery / 药学学报

Ribonucleic acid (RNA) medicines have strong therapeutic potential for numerous rare genetic illnesses and malignancies because of its exact programmability based on Watson-Crick base pairing principle and unique ability to regulate gene expression. However, RNA medicines still have limitations in many areas, including stability, half-life time, immunogenicity, organ selectivity, cellular uptake and endosomal escape efficiency despite their great therapeutic potentials. This review briefly introduced numerous RNA medications [mostly messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA) and antisense oligonucleotide (ASO)] that have intrigued of researchers in recent years, as well as their action mechanism in vivo. A number of delivery techniques, such as chemical modification, ligands coupling and nanocarriers have been proposed. The manufacture and applications of lipid nanoparticle, polymer nanoparticle and exosomes were discussed in depth. The goal of this work is to give a theoretical foundation and design concepts for the development of effective and safe RNA delivery technology, as well as to facilitate RNA therapeutic clinical translation.

A Comprehensive Review of Duchenne Muscular Dystrophy: Genetics, Clinical Presentation, Diagnosis, and Treatment

Duchenne Muscular Dystrophy (DMD) is a genetic disorder involving progressive muscle deterioration leading to loss of mobility, cardiomyopathy, and respiratory complications leading to an early death by the fourth decade of life. Males are affected more often as DMD results from a mutation in the dystrophin gene residing on the X chromosome. The DMD genetic mutation results in a complete functional lack of dystrophin, which culminates as an inadequate connection between the intracellular actin filaments and the extracellular skeleton of muscle. Boys affected by DMD clinically present with muscle weakness before age five, are often wheelchair-bound by age 12, and rarely survive beyond the third decade of life. Traditional treatment strategies have focused primarily on quality-of-life improvement and have included the use of glucocorticoids and physical therapy. No cure currently exists, however many novel treatments for DMD are currently being explored. Some of these involve gene therapy, exon skipping, stop codon skipping, CRISPR technology interventions, and the use of a retinal dystrophin isoform. In this comprehensive review, we recapitulate the literature findings to summarize the history, epidemiology, genetics, clinical presentation, diagnosis, and current and future strategies for the treatment of Duchenne Muscular Dystrophy.

Preliminary study of ssDNA aptamer cytotoxicity on RhD+ red blood cells / 中国输血杂志

【Objective】 To investigate the effect of ssDNA aptamer of RhD blood group antigen on erythrocyte toxicity. 【Methods】 Two full-length ssDNA aptamers(82 bp) of RhD blood group antigen were obtained by gene synthesis.Five samples of whole blood with EDTA anticoagulant were collected to prepare red blood cell suspensions (4×107/mL), which were split into 10 tubes(100 μL/tube), corresponding to 5 experimental groups and 5 controls.Two monospecific full-length ssDNA sequences (100 pmol/μL, 5μL each) were added into the experimental group, while the same amount of normal saline into the control.After treatment, the experimental group and the control were incubated for 60 min at 37℃.After washing, they were suspended in LISS solution and stored at 4℃.The experimental group and the control were set according to different time point during storage (1 h, 1 d, 3 d, 10 d and 17 d), with 5 tubes in each group.For erythrocytes in LISS suspension at different storage time, Annexin V labeled with FITC was used as a probe to label the phosphatidylserine (PS) content and Fluo-4 to label Ca2+ .The eversion of PS and the change of Ca2+ concentration in red blood cells in LISS suspensions were determined by flow cytometry. 【Results】 After incubation, all groups were examined under the light microscope.No agglutination occurred in the experimental group, while agglutination occurred in the control.Flow cytometry showed that the number of Annexin V-FITC staining cells of suspended erythrocytes at the same storage time-point was similar between the experimental group and the control, with no significant differences.In the experimental group, apoptosis rate of Annexin V cells at 10-day storage(6.06±1.38) was significantly higher than that at 1-hour storage(P<0.05), so as at 17-day storage(7.77±1.23) than 1-hour, 1-day and 3-day storage(P<0.05). The apoptosis rate of Fluo-4 AM cell in suspended RBCs at the same storage time-point was similar between the two groups(P>0.05). In the experimental group, the apoptosis rate of Fluo-4 AM cell at the 3-day, 10-day and 17-day storage was 20.84±4.16, 22.35±3.37 and 27.06±2.81, respectively(P<0.05). 【Conclusion】 ssDNA aptamer was not found to have any cytotoxic effects on red blood cells, and RhD ssDNA aptamer may be used as a material for the detection and preparation of universal blood.

Preparation of folate-targeted anti-miR- 221 anionic liposome and its in vitro anti-hepatocellular carcinoma effect / 中国药房

OBJECTIVE To prep are folate-targeted miR- 221 antisense oligonucleotide （anti-miR-221）delivery system ，and to preliminarily evaluate its in vitro anti-cancer effect on hepatocellular carcinoma. METHODS Folate-targeted anti-miR- 221 liposomes（FRL）were prepared by thin-film dispersion method ；the particle size ，Zeta potential and encapsulation efficiency were determined. The delivery efficiency of folate-targeted anionic liposome in human hepatoma HepG 2 cells was determined by in vitro cellular uptake experiment using calcein as the model drug. Flow cytometry was used to detect the effects of FRL on the apoptosis and cell cycle of HepG 2 cells. RESULTS The particle size of prepared FRL was （172.70±3.76）nm，Zeta potential was （－1.16± 0.15）mV and encapsulation efficiency was （83.53±1.85）％. In vitro cellular uptake experiments showed that folate-targeted anionic liposome successfully delivered calcein to HepG 2 cells，and the delivery efficiency in targeted group was higher than that of non-targeted liposome group （P＜0.01）. Apoptosis experiment results showed that the apoptotic rate of HepG 2 cells treated with FRL was significantly higher than that of non-targeted liposome （P＜0.01）. In cell cycle experiment ，FRL could shorten the S phase fraction of HepG 2 cells and induced arrest in the G 0/G1 and G 2/M phases. CONCLUSIONS FRL can encapsulate anti-miR-221 well and deliver it to hepatocellular carcinoma HepG 2 cells successfully ，and has a good in vitro anti-hepatoma effect in inducing apoptosis and cell cycle regulation.

Identification of genomic imbalances in oral clefts

Abstract Objective This article presents a clinical and cytogenomic approach that focuses on the diagnosis of syndromic oral clefts (OCs). Methods The inclusion criteria were individuals with OC presenting four or more minor signs and no major defects (non-syndromic oral clefts [NSOCs]) as well as individuals with OC presenting at least another major defect, regardless of the number of minor signs (syndromic oral clefts [SOCs]). The exclusion criteria included NSOC with less than four minor signs, SOC with known etiology, as well as atypical oral clefts. Results Of 1647 individuals with OC recorded in the Brazilian Database of Craniofacial Anomalies, 100 individuals were selected for chromosome microarray analysis (CMA). Among these, 44 individuals were clinically classified as NSOC and 56 as SOC. CMA was performed for both groups, and abnormal CMA was identified in 9%, all previously classified as SCO. The clinical and CMA data analyses showed a significant predominance of abnormal CMA in individuals classified as SOC (p = 0.0044); prematurity, weight, length, and head circumference at birth were significantly lower in the group with abnormal CMA. Besides, minor signs were significantly higher in this group (p = 0.0090). Conclusion The rigorous selection of cases indicates that the significant variables could help in early recognition of SOC. This study reinforces the importance of applying the CMA technique to establish the diagnosis of SOC. This is an important and universal issue in clinical practice for intervention, care, and genetic counseling.

Managing intrathecal administration of nusinersen in adolescents and adults with 5q-spinal muscular atrophy and previous spinal surgery / Manejo da administração intratecal de nusinersena em adolescentes e adultos com atrofia muscular espinhal 5q e cirurgia prévia de coluna

ABSTRACT Background: Spinal muscular atrophy (SMA) is a neurodegenerative disease of lower motor neurons associated with frequent occurrence of spinal deformity. Nusinersen is an antisense oligonucleotide that increases SMN protein level and is administrated by frequent intrathecal lumbar injections. Thus, spinal deformities and previous spinal surgery are important challenges for drug delivery in SMA. Objective: To report imaging methods used for Nusinersen injection in SMA patients. Methods: Nusinersen injection procedures in SMA types 2 and 3 patients who had previous spinal surgery were analyzed retrospectively to describe the imaging and puncture procedures, as well as the occurrence of complications. Results: Nine SMA patients (14 to 50 years old) underwent 57 lumbar punctures for nusinersen injection. Six patients had no interlaminar space available; in five of them, a transforaminal approach was used, and another one underwent a surgery to open a posterior bone window for the injections. Transforaminal puncture was performed using CT scan in three cases and fluoroscopy in the other two, with a similar success rate. One patient in the transforaminal group had post-procedure radiculitis, and another one had vagal reaction (hypotension). In three cases, with preserved interlaminar space, injections were performed by posterior interlaminar puncture, and only one adverse event was reported (post-puncture headache). Conclusion: In SMA patients with previous spinal surgery, the use of imaging-guided intervention is necessary for administering intrathecal nusinersen. Transforaminal technique is indicated in patients for whom the interlaminar space is not available, and injections should always be guided by either CT or fluoroscopy.

RESUMO Introdução: A atrofia muscular espinal (AME) é uma desordem neurodegenerativa dos motoneurônios inferiores frequentemente associada à ocorrência de deformidade da coluna vertebral. Nusinersena é um oligonucleotídeo antisense que aumenta os níveis da proteína SMN, sendo administrado através de injeções lombares intratecais frequentes. Assim, deformidades da coluna vertebral e abordagem cirúrgica prévia são desafios importantes para a administração de medicamentos na AME. Objetivo: descrever os métodos de imagens utilizados para administração do Nusinersena nos pacientes com AME. Métodos: Os procedimentos de administração de nusinersena em pacientes com AME dos tipos 2 e 3 submetidos à cirurgia prévia da coluna foram analisados retrospectivamente para descrever os métodos de imagem e punção, e a ocorrência de complicações. Resultados: Nove pacientes com AME (14 a 50 anos) foram submetidos a 57 punções lombares para administração de nusinersena. Seis pacientes tinham enxerto ósseo ou nenhum espaço interlaminar disponível; em cinco deles foi utilizada uma abordagem transforaminal, e outra paciente foi submetida à abertura cirúrgica de janela óssea para as injeções. A punção transforaminal foi realizada usando tomografia computadorizada (TC) em três casos e fluoroscopia nos outros dois, com taxa de sucesso semelhante. Um paciente no grupo de abordagem transforaminal apresentou radiculite pós-procedimento e outro apresentou reação vagal (hipotensão). Em três casos, com espaço interlaminar preservado, foram realizadas técnica de punção interlaminar posterior e apenas um evento adverso foi relatado (cefaleia pós-punção). Conclusão: Em pacientes com AME e cirurgia prévia, o uso de intervenção guiada por imagem é necessário para a administração de nusinersena. A técnica transforaminal é indicada nos casos onde o espaço interlaminar não está disponível, devendo ser guiada por TC ou técnicas de imagem fluoroscópica.

Long non-coding RNAs: From disease code to drug role

Enormous studies have corroborated that long non-coding RNAs (lncRNAs) extensively participate in crucial physiological processes such as metabolism and immunity, and are closely related to the occurrence and development of tumors, cardiovascular diseases, nervous system disorders, nephropathy, and other diseases. The application of lncRNAs as biomarkers or intervention targets can provide new insights into the diagnosis and treatment of diseases. This paper has focused on the emerging research into lncRNAs as pharmacological targets and has reviewed the transition of lncRNAs from the role of disease coding to acting as drug candidates, including the current status and progress in preclinical research. Cutting-edge strategies for lncRNA modulation have been summarized, including the sources of lncRNA-related drugs, such as genetic technology and small-molecule compounds, and related delivery methods. The current progress of clinical trials of lncRNA-targeting drugs is also discussed. This information will form a latest updated reference for research and development of lncRNA-based drugs.

Analysis of full-length sequence and 18 point mutations of HLA-B in a leukemia patient and her family / 中国组织工程研究

BACKGROUND: The human leukocyte antigen (HLA) has undergone long-term evolution to form diverse polymorphisms. In recent years, due to the increase in the number of examinees and the rapid development of HLA typing technology, novel HLA alleles have been discovered constantly. OBJECTIVE: To analyze the full-length sequence and 18 point mutations of HLA-B gene in a leukemia patient and her family using the next-generation sequencing technology. METHODS: Polymerase chain reaction and sequence-specific oligonucleotide probes (PCR-SSOP) and polymerase chain reaction-sequence based typing (PCR-SBT) revealed abnormalities in the patient’s HLA-B. To identify the genotype, we sequenced the full length of the gene by next-generation sequencing technology and collected blood samples from the patient’s father, mother and two sisters for genetic analysis of HLA genes. RESULTS AND CONCLUSION: Both PCR-SSOP and PCR-SBT indicated that the HLA-B sample had no perfectly matched genotype. Further detection using the next-generation sequencing technology revealed that the novel allele had 18 base mutations in the exon, intron and 3’UTR compared to the most homologous allele B*15:09:01. Five exon base mutations were located in the exons 3 and 4, which were: 486G→C, 583T→C, 636T→C, 652A→G, 756C→T, resulting in changes in the five corresponding codons, including 171 tyrosine (Tyr) → histidine (His) and 194 isoleucine (Ile) → valine (Val). A pedigree survey found that the patient’s novel HLA B allele was inherited from her father. The novel allele sequence was submitted to the Genbank database (MG595995). A novel HLA-B allele was confirmed by the next-generation sequencing, which was officially named HLA-B*15:435 by the World Health Organization HLA Factor Nomenclature Committee in December 2017.

Gene expression analysis identifies hypothetical genes that may be critical during the infection process of Xanthomonas citri subsp. citri

Background: Gene expression analysis via microarray is widely used in phytobacteria to validate differential gene expression associated with virulence or to compare biological profiles of wild type and mutant strains. Here, we employed DNA microarrays to study the early stages of the infection process (24, 72 and 120 h post-inoculation) of Xanthomonas citri subsp. citri (Xac) infecting Citrus sinensis to interrogate the expression profiles of hypothetical genes. Results: Under infective conditions, 446 genes were up- and 306 downregulated. Outstanding among genes upregulated during infection were those involved in synthesizing the Type 3 Secretion System and effectors, xanthan gum and quorum-sensing induction, and flagellum synthesis and regulation. Additionally, 161 hypothetical genes were up- and 100 were downregulated, 49 of which are known to have a significant biological role. To understand hypothetical gene co-regulation or -expression, nine expression profiles including 158 genes were identified during the three infection phases. Of these, 47 hypothetical genes were identified as having expression profiles associated with at least one connected to a gene associated with adaptation and virulence. Conclusions: Expression patterns of six differentially expressed genes were validated by quantitative reverse transcription polymerase chain reaction, thus demonstrating the effectiveness of this tool in global gene expression analysis in Xac.

Effect of emodin on renal tubular epithelial cell injury and inflammatory respons / 国际中医中药杂志

Objective@#To investigate the effects of emodin on lipopolysaccharide (LPS)-induced inflammation and damage in HK-2 cells.@*Methods@#The HK-2 cells were divided into blank group, emodin group, LPS group, and LPS+emodin group. The emodin group was treated with culture medium containing 0.5 μg/ml emodin, the LPS group was treated with culture medium containing 10 μg/ml LPS, the LPS+emodin group was treated with culture medium containing both 0.5 μg/ml emodin and 10 μg/ml LPS, and the blank group was cultured with fresh medium. Twelve hours later, HK-2 cells from each group were harvested for the analysis. The mRNA levels of oligonucleotide receptor protein 3 (NLRP3), IL-1β, IL-18, TNF-α, and neutrophil gelatinase-associated lipocalin (NGAL) in each group were determined by RT-PCR.@*Results@#Compared to the blank group, the mRNA levels of NLRP3 (2.74 ± 0.38 vs. 1.00 ± 0.14), IL-1β (2.40 ± 0.33 vs. 1.00 ± 0.19), and IL-18 (3.00 ± 0.42 vs. 1.00 ± 0.07), NGAL (31.73 ± 3.41 vs. 1.00 ± 0.07), and TNF-α (9.73±1.60 vs. 1.00 ± 0.11) significantly increased by LPS stimulation (P<0.05). Compared to the LPS group, the mRNA expression of NLRP3 (1.98 ± 0.24 vs. 2.74 ± 0.39), IL-1β (1.54 ± 0.24 vs. 2.40 ± 0.33), and IL-18 (2.09 ± 0.53 vs. 3.00 ± 0.42), NGAL (16.76 ± 1.72 vs. 31.73 ± 3.41) and TNF-α (9.73 ± 1.60 vs. 4.65 ± 1.09) in HK-2 cells of LPS+emodin group significantly decreased (P<0.05).@*Conclusions@#The emodin protects HK-2 cells from LPS-induced damage and inflammation by inhibiting NLRP3 inflammasome activation.

Effect of emodin on renal tubular epithelial cell injury and inflammatory respons / 国际中医中药杂志

Objective To investigate the effects of emodin on lipopolysaccharide (LPS)-induced inflammation and damage in HK-2 cells. Methods The HK-2 cells were divided into blank group, emodin group, LPS group, and LPS+emodin group. The emodin group was treated with culture medium containing 0.5 μg/ml emodin, the LPS group was treated with culture medium containing 10 μg/ml LPS, the LPS+emodin group was treated with culture medium containing both 0.5 μg/ml emodin and 10 μg/ml LPS, and the blank group was cultured with fresh medium. Twelve hours later, HK-2 cells from each group were harvested for the analysis. The mRNA levels of oligonucleotide receptor protein 3 (NLRP3), IL-1β, IL-18, TNF-α, and neutrophil gelatinase-associated lipocalin (NGAL) in each group were determined by RT-PCR. Results Compared to the blank group, the mRNA levels of NLRP3 (2.74 ± 0.38 vs. 1.00 ± 0.14), IL-1β (2.40 ± 0.33 vs. 1.00 ± 0.19), and IL-18 (3.00 ± 0.42 vs. 1.00 ± 0.07), NGAL (31.73 ± 3.41 vs. 1.00 ± 0.07), and TNF-α (9.73±1.60 vs. 1.00 ± 0.11) significantly increased by LPS stimulation (P<0.05). Compared to the LPS group, the mRNA expression of NLRP3 (1.98 ± 0.24 vs. 2.74 ± 0.39), IL-1β (1.54 ± 0.24 vs. 2.40 ± 0.33), and IL-18 (2.09 ± 0.53 vs. 3.00 ± 0.42), NGAL (16.76 ± 1.72 vs. 31.73 ± 3.41) and TNF-α (9.73 ± 1.60 vs. 4.65 ± 1.09) in HK-2 cells of LPS+emodin group significantly decreased (P<0.05). Conclusions The emodin protects HK-2 cells from LPS-induced damage and inflammation by inhibiting NLRP3 inflammasome activation.

Development of an oligonucleotide microarray for simultaneous detection of two canine MDR1 genotypes and association between genotypes and chemotherapy side effects

Canine MDR1 gene mutations produce translated P-glycoprotein, an active drug efflux transporter, resulting in dysfunction or over-expression. The 4-base deletion at exon 4 of MDR1 at nucleotide position 230 (nt230[del4]) in exon 4 makes P-glycoprotein lose function, leading to drug accumulation and toxicity. The G allele of the c.-6-180T>G variation in intron 1 of MDR1 (single nucleotide polymorphism [SNP] 180) causes P-glycoprotein over-expression, making epileptic dogs resistant to phenobarbital treatment. Both of these mutations are reported to be common in collies. This study develops a more efficient method to detect these two mutations simultaneously, and clarifies the genotype association with the side effects of chemotherapy. Genotype distribution in Taiwan was also investigated. An oligonucleotide microarray was successfully developed for the detection of both genotypes and was applied to clinical samples. No 4-base deletion mutant allele was detected in dogs in Taiwan. However, the G allele variation of SNP 180 was spread across all dog breeds, not only in collies. The chemotherapy adverse effect percentages of the SNP 180 T/T, T/G, and G/G genotypes were 16.7%, 6.3%, and 0%, respectively. This study describes an efficient way for MDR1 gene mutation detection, clarifying genotype distribution, and the association with chemotherapy.

Identify the molecular target of nasopharyngeal carcinoma by bioinformatics analysis / 天津医药

Objective To study the pathogenesis of nasopharyngeal carcinoma and identify potential biomarkers or therapeutic targets. Methods Microarray data (GSE12452 and GSE13597) were downloaded from Gene Expression Omnibus. Processing of original microarray data and screening of differentially expressed genes were performed through bioinformatics analysis. Then, GO and KEGG pathway enrichment analysis was performed for these genes using DAVID database. Real time-PCR and Western blot assay were used to detect the expression levels of the identified genes. Results A total of 260 overlap DEGs were obtained including 16 GO entries and 4 signal pathways. Eighteen potential therapeutic targets that relative to cell cycle were identified by gene enrichment analysis. Expression levels of 12 selected genes were confirmed by real-time PCR. Finally, 4 selected genes were confirmed by Western blot assay. Conclusion By bioinformatics analysis of two sets of microarray data and molecular biology research, four genes were found including CDC6, CDK1,MCM2 and CCNB1, which might be potential key genes that can be developed for therapy targets of NPC in the future.

Sequencing analysis of HPV-other type on an HPV DNA chip

OBJECTIVES: To identify the specific human papillomavirus (HPV) genotypes from HPV-other type on an HPV DNA chip test by sequencing. METHODS: Among 13,600 women undergoing a routine gynecology examination including Pap smear and/or HPV test by DNA chip test in the healthcare system at Gangnam Center from July 2012 to February 2013, we prospectively collected and performed sequencing for a total of 351 consecutive cervicovaginal samples consisting of 180 samples that tested positive for HPV-other type and 171 samples that tested positive for either high-risk HPV or low-risk HPV. RESULTS: Of a total of 351 samples, individual HPV genotypes were successfully sequenced in 215 cases: 119 HPV-other type, 82 HPV-high-risk, and 14 HPV-low-risk. Based on the sequencing for 119 HPV-other type samples, 91.6% were detected as HPV types that were not included on the DNA chip; however, 7.6% (9/119) were proven to be high-risk HPV types: HPV 18 (n=4), HPV 33 (n=3), HPV 35 (n=1), and HPV 59 (n=1). For correlation analysis of all high-risk and HPV 16/18, the correlation rate was 76.2% and 86.6% with kappa-value of 0.38 and 0.69, respectively. CONCLUSION: HPV-other type on DNA chip test may still have possibility of high-risk HPV, i.e., HPV 18 and thus the significance of HPV-other type in detecting cervical disease remains to be investigated.

Cortical Axonal Secretion of BDNF in the Striatum Is Disrupted in the Mutant-huntingtin Knock-in Mouse Model of Huntington's Disease

Deficient BDNF signaling is known to be involved in neurodegenerative diseases such as Huntington's disease (HD). Mutant huntingtin (mhtt)-mediated disruption of either BDNF transcription or transport is thought to be a factor contributing to striatal atrophy in the HD brain. Whether and how activity-dependent BDNF secretion is affected by the mhtt remains unclear. In the present study, I provide evidence for differential effects of the mhtt on cortical BDNF secretion in the striatum during HD progression. By two-photon imaging of fluorescent BDNF sensor (BDNF-pHluorin and -EGFP) in acute striatal slices of HD knock-in model mice, I found deficient cortical BDNF secretion regardless of the HD onset, but antisense oligonucleotide (ASO)-mediated reduction of htts only rescues BDNF secretion in the early HD brain before the disease onset. Although secretion modes of individual BDNF-containing vesicle were not altered in the pre-symptomatic brain, the full-fusion and partial-fusion modes of BDNF-containing vesicles were significantly altered after the onset of HD symptoms. Thus, besides abnormal BDNF transcription and transport, our results suggest that mhtt-mediated alteration in activity-dependent BDNF secretion at corticostriatal synapses also contributes to the development of HD.

Subclassification and Detection of New Markers for the Discrimination of Primary Liver Tumors by Gene Expression Analysis Using Oligonucleotide Arrays

BACKGROUND/AIMS: The failure to correctly differentiate between intrahepatic cholangiocarcinoma (CC) and hepatocellular carcinoma (HCC) is a significant clinical problem, particularly in terms of the different treatment goals for both cancers. In this study a specific gene expression profile to discriminate these two subgroups of liver cancer was established and potential diagnostic markers for clinical use were analyzed. METHODS: To evaluate the gene expression profiles of HCC and intrahepatic CC, Oligonucleotide arrays (AffymetrixU133A) were used. Overexpressed genes were checked for their potential use as new markers for discrimination and their expression values were validated by reverse transcription polymerase chain reaction and immunohistochemistry analyses. RESULTS: 695 genes/expressed sequence tags (ESTs) in HCC (245 up-/450 down-regulated) and 552 genes/ESTs in CC (221 up-/331 down-regulated) were significantly dysregulated (p 2, ≥70%). Using a supervised learning method, and one-way analysis of variance a specific 270-gene expression profile that enabled rapid, reproducible differentiation between both tumors and non-malignant liver tissues was established. A panel of 12 genes (e.g., HSP90β, ERG1, GPC3, TKT, ACLY, and NME1 for HCC; SPT2, T4S3, CNX43, TTD1, HBD01 for CC) were detected and partly described for the first time as potential discrimination markers. CONCLUSIONS: A specific gene expression profile for discrimination of primary liver cancer was identified and potential marker genes with feasible clinical impact were described.