Molecular analysis of pancreatic cystic neoplasm in routine clinical practice

BACKGROUND Cystic pancreatic lesions consist of a wide variety of lesions that are becoming increasingly diagnosed with the growing use of imaging techniques.Of these,mucinous cysts are especially relevant due to their risk of malignancy.However,morphological findings are often suboptimal for their differentiation.Endoscopic ultrasound fine-needle aspiration(EUS-FNA)with molecular analysis has been suggested to improve the diagnosis of pancreatic cysts.AIM To determine the impact of molecular analysis on the detection of mucinous cysts and malignancy.METHODS An 18-month prospective observational study of consecutive patients with pancreatic cystic lesions and an indication for EUS-FNA following European clinical practice guidelines was conducted.These cysts included those>15 mm with unclear diagnosis,and a change in follow-up or with concerning features in which results might change clinical management.EUS-FNA with cytological,biochemical and glucose and molecular analyses with next-generation sequencing were performed in 36 pancreatic cysts.The cysts were classified as mucinous and non-mucinous by the combination of morphological,cytological and biochemical analyses when surgery was not performed.Malignancy was defined as cytology positive for malignancy,high-grade dysplasia or invasive carcinoma on surgical specimen,clinical or morphological progression,metastasis or death related to neoplastic complications during the 6-mo follow-up period.Next-generation sequencing results were compared for cyst type and malignancy.RESULTS Of the 36 lesions included,28(82.4%)were classified as mucinous and 6(17.6%)as non-mucinous.Furthermore,5(13.9%)lesions were classified as malignant.The amount of deoxyribonucleic acid obtained was sufficient for molecular analysis in 25(69.4%)pancreatic cysts.The amount of intracystic deoxyribonucleic acid was not statistically related to the cyst fluid volume obtained from the lesions.Analysis of KRAS and/or GNAS showed 83.33%[95%confidence interval(CI):63.34-100]sensitivity,60%(95%CI:7.06-100)specificity,88.24%(95%CI:69.98-100)positive predictive value and 50%(95%CI:1.66-98.34)negative predictive value(P=0.086)for the diagnosis of mucinous cystic lesions.Mutations in KRAS and GNAS were found in 2/5(40%)of the lesions classified as non-mucinous,thus recategorizing those lesions as mucinous neoplasms,which would have led to a modification of the follow-up plan in 8%of the cysts in which molecular analysis was successfully performed.All 4(100%)malignant cysts in which molecular analysis could be performed had mutations in KRAS and/or GNAS,although they were not related to malignancy(P>0.05).None of the other mutations analyzed could detect mucinous or malignant cysts with statistical significance(P>0.05).CONCLUSION Molecular analysis can improve the classification of pancreatic cysts as mucinous or non-mucinous.Mutations were not able to detect malignant lesions.

Advances in microfluidic-based DNA methylation analysis

DNA methylation has been extensively investigated in recent years,not least because of its known relationship with various diseases.Progress in analytical methods can greatly increase the relevance of DNA methylation studies to both clinical medicine and scientific research.Microflu-idic chips are excellent carriers for molecular analysis,and their use can provide improvements from multiple aspects.On-chip molecular analysis has received extensive attention owing to its advantages of portability,high throughput,low cost,and high efficiency.In recent years,the use of novel microfluidic chips for DNA methylation analysis has been widely reported and has shown obvious superiority to conventional methods.In this review,wefirst focus on DNA methylation and its applications.Then,we discuss advanced microfluidic-based methods for DNA methylation analysis and describe the great progress that has been made in recent years.Finally,we summarize the advantages that microfluidic technology brings to DNA methylation analysis and describe several challenges and perspectives for on-chip DNA methylation analysis.This review should help researchers improve their understanding and make progress in developing microfluidic-based methods for DNA methylation analysis.

Cloning, E. coil Expression and Molecular Analysis of Amorpha-4,11-Diene Synthase from a High-Yield Strain of Artemisia annua L.

increasing demand of artemisinin in the treatment of malaria has placed substantial stress on the total artemisinin supplies world-wide, so more attention has been paid to increasing the content of artemisinin in the Artemisia annua L. plant. In this study, amorpha-4, 11-diene synthase （ADS） cDNA （ads1） and genomics gene （gads1） were cloned from a high-yield A. annua strain 001. The activity of ADS1 was confirmed by heterogeneous overexpression of ads I and in vitro enzymatic incubation. Reverse transcript-polymerase chain reaction results demonstrated that ads1 expressed in leaves, flowers and young stems, but not in roots. This organ-specific expression pattern of ads1 is consistent with that of artemisinin accumulation in the plant. The gads1 has a complex organization including seven exons and six introns, and belongs to class III terpene synthase. DNA gel blotting revealed that the ADS gene has at least four copies in the genome of strain 001. The higher copy numbers might be one of the reasons for its high artemisinin content.

Molecular Analysis of Legume Nodule Development and Autoregulation

Legumes are highly important food, feed and biofuel crops. With few exceptions, they can enter into an intricate symbiotic relationship with specific soil bacteria called rhizobia. This interaction results in the formation of a new root organ called the nodule in which the rhizobia convert atmospheric nitrogen gas into forms of nitrogen that are useable by the plant. The plant tightly controls the number of nodules it forms, via a complex root-to-shoot-to-root signaling loop called autoregulation of nodulation （AON）. This regulatory process involves peptide hormones, receptor kinases and small metabolites. Using modern genetic and genomic techniques, many of the components required for nodule formation and AON have now been isolated. This review addresses these recent findings, presents detailed models of the nodulation and AON processes, and identifies gaps in our understanding of these process that have yet to be fully explained.

Molecular analysis for diagnosis of Marfan syndrome and Marfan-associated disorders

Marfan syndrome is a systemic disorder of connective tissue, caused by mutations in the FBN1, TGFBR1 or TGFBR2 genes. This syndrome is characterized by involvement of three major systems, skeletal, ocular, and cardiovascular. The continuing improvements in molecular biology and increasing availability of molecular diagnosis in clinical practice allow recognition of Marfan syndrome in patients with incomplete phenotypes. Additionally, molecular analyses could also be used for preimplantation genetic diagnosis. The identification of a mutation allows for early diagnosis, prognosis, genetic counseling, preventive management of carriers and reassurance for unaffected relatives. The importance of knowing in advance the location of the putative family mutation is highlighted by its straightforward application to prenatal and postnatal screening.

Integration of molecular testing for the personalized management of patients with diffuse large B-cell lymphoma and follicular lymphoma

Diffuse large B-cell lymphoma(DLBCL)and follicular lymphoma(FL)are the most common forms of aggressive and indolent lymphoma,respectively.The majority of patients are cured by standard R-CHOP immunochemotherapy,but 30%–40%of DLBCL and 20%of FL patients relapse or are refractory(R/R).DLBCL and FL are phenotypically and genetically hereterogenous B-cell neoplasms.To date,the diagnosis of DLBCL and FL has been based on morphology,immunophenotyping and cytogenetics.However,next-generation sequencing(NGS)is widening our understanding of the genetic basis of the B-cell lymphomas.In this review we will discuss how integrating the NGS-based characterization of somatic gene mutations with diagnostic or prognostic value in DLBCL and FL could help refine B-cell lymphoma classification as part of a multidisciplinary pathology work-up.We will also discuss how molecular testing can identify candidates for clinical trials with targeted therapies and help predict therapeutic outcome to currently available treatments,including chimeric antigen receptor T-cell,as well as explore the application of circulating cell-free DNA,a non-invasive method for patient monitoring.We conclude that molecular analyses can drive improvements in patient outcomes due to an increased understanding of the different pathogenic pathways affected by each DLBCL subtype and indolent FL vs R/R FL.

Molecular Epidemiological Analysis of Group A Streptococci Isolated from Children in Chaoyang District of Beijing, 2011:emm Types, Virulence Factor Genes and Erythromycin Resistant Genes

Group A streptococcus （GAS） causes a wide range of diseases in the human population. GAS diseases are more common in children than in adults, with clinical manifestations ranging from pharyngitis and impetigo to invasive infections and post streptococcal sequelae, such as acute rheumatic fever and acute post-streptococcal glomerulonephritis[1]. GAS harbors a host of virulence factors that contribute to its complex pathogenicity and differences in the disease severity and frequency. M protein, one of the major virulence factors, is encoded by the emm gene induces a type of specific host immune response and confers antiphagocytic properties.

Molecular phylogenetic analysis of sulfate-reducing bacteria from deep sediment layers of the tropical West Pacific warm pool

The diversity of sulfate-reducing bacteria （SRB） from deep layers of deep-sea sediments [ more than 2 m bsf （below seafloor） ] of two sites （WO1 -3 and WPO1 -4） in a tropical West Pacific warm pool region was characterized by using molecular phylogenetic analysis. The results of culture-independent samples demonstrated that the dominant clones from both sites were related to Grampositive spore forming genus, Desulfotomaculum, which accounted for 36.8% of all the sequencing clones from Site WP01 - 3 and 62.8% from Site WP01 -4. However, the other SRB group which was generally reported to be predominant in the deep-sea sediments of other regions, δ- subclass of the proteobacteria was found to be in very low percentages. Therefore, it could be speculated that there existed a unique chemical environment in the deep-sea sediment of this warm pool region. When comparing the Desulfotomaculum sp. related sequences from both sites, it was revealed that though the Desulfotomaculum-like sequences from Site WP01 -3 were more diverse than those from Site WP01 -4, all these sequences from both sites showed high similarity and formed a new phylogenetically homogeneous cluster in the Desulfotomaculum genus which had never been reported before. Successful enrichment of SRB was only achieved from samples of Site WP01 -4 and the sequence analysis of culture-dependent samples further confirmed the dominance of Desulfotomaculum genus. But Desulfotomaculum-related sequences from culture-dependent and culture-independent samples belonged to two different clusters respectively. This difference showed the choice of cultivation to the microorganisms.

Surface functionalization of SPR chip for specific molecular interaction analysis under flow condition

Surface functionalization of sensor chip for probe immobilization is crucial for the biosensing applications of surface plasmon resonance(SPR)sensors.In this paper,we report a method circulating the dopamine aqueous solution to coat polydopamine film on sensing surface for surface functionalization of SPR chip.The polydopamine film with available thickness can be easily prepared by controlling the circulation time and the biorecognition elements can be immobilized on the polydopamine film for specific molecular interaction analysis.These opera-tions are all performed under flow condition in the fuidic system,and have the advantages of easy implementation,less time consuming,and low cost,because the reagents and devices used in the operations are routinely applied in most laboratories.In this study,the specific absorption between the protein A probe immobilized on the sensing surface and human immunoglobulin G in the buffer is monitored based on this surface functionalization strategy to demonstrated its feasibility for SPR biosensing applications.

Molecular Genetic Analysis of Partial 9p Trisomy in Two Chinese Families with Mental Retardation and Facial Anomaly

Mental retardation is defined by significant limitations in intellectual function and adaptive behavior that occur before 18 years of age.Many chromosomal diseases come with mental retardation.We reported two Chinese families with partial trisomy 9p and other chromosome partial monosomy,clinical features of mental retardation and mild facial and pinkie anomalies.In the family 1,we showed that the proband carried a trisomy 9p21.3→pter and monosomy 21q22.3→qter by using fluorescence in situ hybridization analysis.Molecular genetic analysis defined the precise breakpoint on chromosome 9p between markers D9S1846 and D9S171,an interval of about 2.9 Mb on 9p21.3,and the breakpoint on chromosome 21q between markers D21S1897 and D21S1446,a region of about 1.5 Mb on 21q22.3.In the family 2,a patient with trisomy 9p21.3→pter and monosomy 5p15.33→pter,and a de novo maternal balanced translocation between chromosomes 5 and 9 was identified in his mother.Cytogenetic and molecular genetic analysis defined the precise breakpoints on chromosome 9p21.3 and chromosome 5p15.33.Further clinical investigation found that any individual had no refractoriness eczema disease except the proband in this family.These results further implicate that trisomy 9p is associated with mental retardation,and that there may be key gene duplication on chromosome 9p21.3→9pter responsible for mental retardation and mild facial anomaly.This result has been applied successfully in prenatal diagnosis of the second family.

An Enhanced Comparative Molecular Field Analysis Method Using Genetic Algorithm

In this study. an automated conformer selection procedure using generic algorithm (GA) has been applied in comparative molecular field analysis (CoMFA) method. Using genetic algorithm. the 3D-QSAR model is optimized to an optimal one. From the calculation results, a group of QSAR models with high predictive ability can be obtained, which is superior than using conventional CoMFA: meanwhile. the active conformers for these compounds in data set can be determined fi om the best model.

DFT Calculations and NBO Analysis of 2-Chloroethylethyldichlorosilane Unimolecular Elimination Kinetics in the Gas Phase

Ab initio molecular orbital calculations have been used to investigate the thermal decomposition kinetics of 2-chloroethylethyldichlorosilane at the B3LYP/6-311＋G^＊＊,B3PW91/6-311＋G^＊＊,and MPW1PW91/6-311＋G^＊＊ levels of theory.Among these methods,the results（activation parameters） obtained using the B3LYP/6-311＋G＊＊ level are in good agreement with the available experimental data.The calculated data imply that in the unimolecular β-elimination reactions of the studied compound in the gas phase,the polarization of C（1）-Cl（3） and C（1）-H（4） bonds in the sense of C（1）^δ＋-Cl（3）^δ-and C（1）^δ＋-H（4）^δ-,respectively,is a determining factor in the gas phase elimination reactions 1,2 and 3.Analysis of bond order,natural bond orbital charges,bond indexes,synchro-nicity parameters,and IRC calculations suggest the elimination of 2-chloroethylethyldichlorosilane via reactions 1～3 can be described as concerted and slightly asynchronous.The transition state structures of these reactions are a four-membered cyclic structure.

Molecular Cloning and Expression Analysis of a Cys2/His2 Type Zinc Finger Protein Gene in Upland Cotton

The zinc finger proteins belong to the largest family of transcription factors.But there is little research of Cys2/His2 type zinc finger proteins in cotton,and there is no submission of correlating

Genetic Analysis of Cotton Fiber Traits by Molecular Markers

1.Development of EST-SSRs derived from G.barbadense:One hundred and nineteen EST-SSRs were developed based on 98 unique ESTs from a cDNA library constructed in our laboratory using developing fibers from G.barbadense cv.3-79.Among the SSRs,trinucleotide AAG appeared

Collision tumor of primary malignant lymphoma and adenocarcinoma in the colon diagnosed by molecular pathology:A case report and literature review

BACKGROUND Collision tumors of primary malignant lymphoma and adenocarcinoma in the colon are rare.Primary diffuse large B-cell lymphoma(DLBCL)–adenocarcinoma collision tumors are especially rare.CASE SUMMARY A 74-year-old woman presented with abdominal pain of 1 mo duration.Biopsy under colonoscopy revealed adenocarcinoma of the ascending colon.Subsequently,the patient underwent laparoscopic radical resection of right colon cancer with lymph node dissection.A collision tumor was found incidentally through postoperative pathological sampling.Genetic analysis showed a collision tumor of DLBCL with germinal center B-cell subtype and TP53 mutation,and adenocarcinoma arising in a tubulovillous adenoma in the colon,with BRAF mutation and mutL homolog 1 promoter methylation.The patient died 3 mo after surgery.To our knowledge,this is the 23rd reported case of collision tumor of colorectal adenocarcinoma and lymphoma.The mean age of the 23 patients was 73 years.The most common site was the cecum.There were 15 cases with followup data including 11 living and four dead with a 3-year overall survival rate of 71.5%.CONCLUSION Based on pathological and genetic analysis,surgery combined with chemotherapy or chemoradiotherapy may have good therapeutic effects for collision tumor.

Identification of Fusarium wilt resistance gene SiRLK1 in Sesamum indicum L.

Sesame Fusarium wilt(SFW),caused by Fusarium oxysporum f.sp.sesami(Fos),is one of the most devastating diseases affecting sesame cultivation.Deciphering the genetic control of SFW resistance is pivotal for effective disease management in sesame.An inheritance study on a cross between the highly resistant variety Yuzhi 11 and the highly susceptible accession Sp1 using a Fos pathogenicity group 1 isolate indicated that resistance was conferred by a single dominant allele.The target locus was located in a 1.24 Mb interval on chromosome 3 using a combination of cross-population association mapping and bulked segregant analysis.Fine genetic mapping further narrowed the interval between 21,350 and 21,401 kb.The locus Sindi_0812400 was identified as the SFW resistance gene and officially designated SiRLK1.This gene encodes a specific malectin/receptor-like protein kinase with three putative tandem kinase domains and is considered a kinase fusion protein.Sequence analysis revealed that a high proportion(49.44%)of variants within the locus was located within the kinase domainⅢ,and several of which were evidently associated with the diversity in SFW response,indicating the critical role of kinase domainⅢin expression of disease resistance.These findings provide valuable information for further functional analysis of SFW resistance genes and marker-assisted resistance breeding in sesame.

Complete nuclear ribosomal DNA sequence amplification and molecular analyses of Bangia (Bangiales, Rhodophyta) from China

Filamentous Bangia,which are distributed extensively throughout the world,have simple and similar morphological characteristics.Scientists can classify these organisms using molecular markers in combination with morphology.We successfully sequenced the complete nuclear ribosomal DNA.approximately 13 kb in length,from a marine Bangia population.We further analyzed the small subunit ribosomal DNA gene(nrSSU) and the internal transcribed spacer(ITS) sequence regions along with nine other marine,and two freshwater Bangia samples from China.Pairwise distances of the nrSSU and 5.8S ribosomal DNA gene sequences show the marine samples grouping together with low divergences(0-0.003;0-0.006,respectively) from each other,but high divergences(0.123-0.126;0.198,respectively) from freshwater samples.An exception is the marine sample collected from Weihai,which shows high divergence from both other marine samples(0.063-0.065;0.129,respectively) and the freshwater samples(0.097;0.120,respectively).A maximum likelihood phylogenetic tree based on a combined SSU-ITS dataset with maximum likelihood method shows the samples divided into three clades,with the two marine sample clades containing Bangia spp.from North America,Europe,Asia,and Australia;and one freshwater clade,containing Bangia atropurpurea from North America and China.

Morphological,Molecular and Pathogenicity of Alternaria gaisen Associated with Leaf Brown-Spot on‘Gonggan’Mandarin in China

[Objective]This paper was to identify the pathogen of leaf brown-spot on‘gonggan’mandarin(Citrus reticulate var.gonggan)in Zhaoqing,Guangdong Province.[Method]The pathogen was determined based on sequence analysis of ITS,endoPG,tef1,gapdh,Alt a1,rpb2 and opa10-2 genes;the morphological characteristics were recorded on PDA and PCA;and its pathogenicity on excised and intact host leaves of citrus‘gonggan’was tested.[Result]A detailed description of Alternaria gaisen was obtained based on morphological,molecular and pathogenic characterization,which was the causal agent of brown-spot disease on leaves of‘gonggan’mandarin orchard trees in Zhaoqing,Guangdong,China.[Conclusion]This study provides a scientific basis for the effective control of leaf brown-spot on‘gonggan’mandarin.

A novel pathogen Fusarium cuneirostrum causing common bean(Phaseolus vulgaris)root rot in China

Several fungal pathogens cause root rot of common bean,among which Fusarium spp.are the most common pathogens causing Fusarium root rot(FRR)worldwide.FRR has been becoming an increasingly severe disease of common bean in China,but the species of Fusarium spp.have remained unclear.Thus,this study was performed to identify the pathogen causing common bean root rot in Liangcheng County,Inner Mongolia,China.Nineteen Fusarium-like isolates were obtained after pathogen isolation and purification.The pathogenicity test indicated that eight isolates caused severe disease symptoms on common bean,while 11 other isolates were not pathogenic.The eight pathogenic isolates,FCL1–FCL8,were identified as Fusarium cuneirostrum by morphological characterization and phylogenetic analysis using partial sequences of EF-1α,ITS,28S,and IGS regions.Host range test showed that the representative F.cuneirostrum isolate FCL3 was also pathogenic to mung bean,while not pathogenic to adzuki bean,chickpea,cowpea,faba bean,pea,and soybean.Moreover,50 common bean and 50 mung bean cultivars were screened for resistance to FRR,and seven highly resistant or resistant cultivars of common bean were identified,while no resistant cultivars of mung bean were screened.This study revealed that F.cuneirostrum was one of common bean FRR pathogens in Inner Mongolia and it could induce mung bean root rot as well.To our knowledge,this is the first report of F.cuneirostrum causing FRR of common bean in China.

Report and Phylogeny of a Novel Euplotes Species from China:E.mazeii n.sp.,and Review of E.balteatus(Alveolata,Ciliophora)

The freshwater species,Euplotes mazeii n.sp.,and the marine species,E.balteatus(Dujardin,1841)Diesing,1850 were collected from China and investigated based on their living morphology,ciliary pattern,and small subunit ribosomal RNA(SSU rRNA)gene sequence data.Euplotes mazeii n.sp.was characterized by its small cell size((40-55)μm×(25-35)μm),nine frontoventral cirri,one marginal and two caudal cirri,seven dorsal kineties,and a double-eurystomus type of dorsal silverline system.Phylogenetic analyses based on molecular data confirmed the branching position of this new taxa within the genus Euplotes,with 5.01%(84 nucleotides)sequence variation,supporting the identification of a new species.We re-examined all described and supposedly conspecific populations and give a review of Euplotes balteatus.E.balteatus has been reported frequently;however,E.balteatus sensu Agamaliev,1968 is different from other populations in the number of marginal cirri or the type of silverline system,which suggests there might be a misidentification.The SSU rRNA gene sequences of two populations of E.balteatus from the studies of Chen et al.(2013)and Pan et al.(2012)differ from each other by more than 135 nucleotides,implying that the identity of the species currently associated with the SSU rRNA gene sequence in molecular databases requires further confirmation.