The utility of the Ion Torrent PGM next generation sequencing for analysis of the most commonly mutated genes among patients with colorectal cancer in India

Background: The requirement for the mutation analysis for Kirsten rat sarcoma viral oncogene (KRAS) in colorectal cancer (CRC) is rapidly increasing as it is a predictive biomarker and also, its absence signifies response to anti?epidermal growth factor receptor (anti?EGFR) antibody treatment. The aim of our study was to investigate the pathological diagnosis and distribution of KRAS mutations in colorectal cancer with the use of next generation sequencing platform (Ion Torrent). Methods: A total of 56 CRC samples were tested to identify the genetic mutations, especially KRAS using the primers which included ~2800 COSMIC mutations of 50 oncogenes. Ion Torrent personal genome machine (semiconductor?based sequencing) was used for the sequencing and analysis. Along with KRAS, other 49 genes were also studied for COSMIC mutations. Results: KRAS mutation 25 (44.6%) had the highest frequency, followed by TP53 10 (17.9%) and PIK3CA mutation 4 (7.1%). Of all the KRAS mutations identified, mutations in codon 12 were most frequent followed by mutations in codon 13 and 61. The most frequent substitution was glycine to aspartate mutation in codon 12 (p.Gly12Asp) followed by glycine to valine (p.Gly12Val). Combinations of mutations were also studied. Our study revealed that seven cases (12.5%) had both KRAS and TP53 mutations (highest of all the combinations). Conclusion: The analysis of KRAS mutation frequency and its mutational subtype analysis in human CRCs by using semiconductor?based platform in routine clinical practices have been performed in Indian population. The findings were similar to earlier published reports from the Western literature.

From genes to ecosystems: a synthesis of amphibian biodiversity research in Brazil / De genes a ecossistemas: uma síntese da pesquisa em biodiversidade de anfíbios no Brasil

Abstract Here, we summarize examples of significant advances in amphibian research supported by the São Paulo Research Foundation (FAPESP), focusing on recent discoveries in the fields of community ecology, habitat change, infection diseases, and multipurpose DNA sequencing. We demonstrated that FAPESP has been fundamental not only by directly funding research projects and scholarships, but also through its science training policy, fostering international collaborations with world-class research institutions, improving and consolidating new lines of research that often depended on a synergetic combination of different knowledge and complex tools. We emphasized that future studies will continue to focus on basic questions, such as description of new species, as well as taxonomic and systematic corrections. Furthermore, we also expect that there will be a strong integration among different disciplines using novel bioinformatics tools and modeling approaches, such as machine learning. These new approaches will be critical to further develop our understanding of foundational questions of amphibian life-history trait variation, disease transmission, community assembly, biogeography, and population forecasts under different global change scenarios such as agricultural expansion, agrochemical use, habitat loss, and climate change.

Resumo No presente estudo apresentamos exemplos de avanços significativos nas pesquisas com anfíbios financiadas pela Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), focando em descobertas recentes nos campos de ecologia de comunidades, modificação do habitat, doenças infecciosas e o sequenciamento de DNA com múltiplos propósitos. Demonstramos que a FAPESP tem sido fundamental não somente pelo financiamento direto de projetos de pesquisa e bolsas de estudo, mas também através de sua política de formação científica, fomentando colaborações internacionais com instituições de pesquisa de excelência mundial, melhorando e consolidando novas linhas de pesquisa que frequentemente dependem da combinação sinérgica entre diferentes linhas de conhecimento e ferramentas complexas. Enfatizamos que futuros estudos continuem com foco em questões básicas, como a descrição de novas espécies, bem como correções taxonômicas e sistemáticas. Além disso, esperamos uma forte integração entre diferentes disciplinas usando novas ferramentas de bioinformática e abordagens de modelagem, como o aprendizado de máquina. Essas novas abordagens serão críticas para desenvolver ainda mais nossa compreensão a respeito de questões fundamentais sobre as características da história de vida dos anfíbios, transmissão de doenças, estrutura de comunidades, biogeografia e previsões populacionais em diferentes cenários de mudanças globais, como a expansão da agricultura, uso de agrotóxicos, perda de habitat e mudanças climáticas.

Gene Mutation and Overexpression of Newly Diagnosed Multiple Myeloma Patients / 中国实验血液学杂志

OBJECTIVE@#To analyze the characteristics of gene mutation and overexpression in newly diagnosed multiple myeloma (NDMM) patients.@*METHODS@#Bone marrow cells from 208 NDMM patients were collected and analyzed. The gene mutation of 28 genes and overexpression of 6 genes was detected by DNA sequencing. Chromosome structure abnormalities were detected by fluorescence in situ hybridization (FISH).@*RESULTS@#Gene mutations were detected in 61 (29.33%) NDMM patients. Some mutations occurred in 5 or more cases, such as NRAS, PRDM1, FAM46C, MYC, CCND1, LTB, DIS3, KRAS, and CRBN. Overexpression of six genes (CCND1, CCND3, BCL-2, CCND2, FGFR3, and MYC) were detected in 83 (39.9%) patients, and cell cycle regulation gene was the most common. Single nucleotide polymorphisms (SNP) changes were detected in 169 (81.25%) patients, the TP53 P72R gene SNP (70.17%) was the most common. Abnormality in chromosome structure was correlated to gene overexpression. Compared to the patients with normal chromosome structure, patients with 14q32 deletion showed higher proportion of CCND1 overexpression. Similarly, patients with 13q14 deletion showed higher proportion of FGFR3 overexpression, whereas patients with 1q21 amplification showed higher proportion of CCND2, BCL-2 and FGFR3 overexpression.@*CONCLUSION@#There are multiple gene mutations and overexpression in NDMM. However, there is no dominated single mutation or overexpression of genes. The most common gene mutations are those in the RAS/MAPK pathway and the genes of cyclin family CCND are overexpression.

Prediction of bloodstream infection pathogens in hematopoietic stem cell transplantation patients by plasma microbial cell-free DNA sequencing / 中华检验医学杂志

Objective:To predict the pathogens of bloodstream infection (BSI) in hematopoietic stem cell transplantation (HSCT) patients by plasma microbial cell-free DNA (mcfDNA) sequencing with and without additional amplification.Methods:A total of 978 HSCT patients were enrolled in Peking University People′s Hospital from March to July 2021, and the 7 428 blood samples were prospectively collected from pretransplant conditioning period to 4 months after transplantation. The plasma samples were separated and then cryopreserved. According to blood culture results and whether there were plasma samples before BSI onset, twenty-eight HSCT patients with positive blood culture (39 plasma samples within 1-8 days before BSI onset) and 9 HSCT patients with negative blood culture (9 plasma samples) were filtered. The 39 samples were performed with mcfDNA additional and non-additional amplification sequencing, and the 9 samples were only performed with additional amplification sequencing. With the blood culture results as the gold standard, the consistency between the sequencing and the blood culture results was observed. Student t test and Wilcoxon test were used for statistical analysis. Results:Without additional amplification sequencing, only 7 samples sequencing results were consistent with the blood culture results, and the total pathogen detection rate was 17.95% (7/39). The rates within 3 days and 4-8 days were 23.81% (5/21) and 2/18, respectively. The main pathogenic type detected was gram-negative bacteria (5/7). With additional amplification sequencing, the total pathogen detection rate was 59.26% (16/27) and the rate within 3 days was 8/13. The number of gram-positive bacteria detected was elevated (13/16) and the number of additional microorganisms in additional amplification sequencing was increased significantly ( P=0.001 0), compared with non-additional amplification sequencing. Moreover, additional sequencing analysis of 9 samples from patients with negative culture result showed that no pathogen was detected in six samples, and the common Torque teno virus in HSCT patients was detected in only three samples. Conclusion:The pathogen detection rate of plasma mcfDNA additional amplification sequencing was better than that of non-additional amplification sequencing in HSCT patients before BSI onset, especially in the first three days, which has the potential to predict BSI pathogens.

Identification of Cannabis Sativa L. Based on rbcL Sequence / 法医学杂志

Objective To assess the feasibility of the rbcL sequence of chloroplast DNA as a genetic marker to identify Cannabis sativa L. Methods The rbcL sequences in 62 Cannabis sativa L. samples, 10 Humulus lupulus samples and 10 Humulus scandens DNA samples were detected, and 96 rbcL sequences of the Cannabaceae family were downloaded from Genbank. Sequence alignment was performed by MEGA X software, the intraspecific and interspecific Kimura-2-Parameter （K2P） genetic distances were calculated, and the system clustering tree was constructed. Results The rbcL sequence length acquired by sequencing of Cannabis sativa L. and Humulus scandens were 617 bp and 649 bp, respectively, and two haplotypes of Cannabis sativa L. were observed in the samples. The BLAST similarity search results showed that the highest similarity between the sequences acquired by sequencing and Cannabis sativa L. rbcL sequences available from Genbank was 100%. The genetic distance analysis showed that the maximum intraspecific genetic distance （0.004 9） of Cannabis sativa L. was less than the minimum interspecific genetic distance （0.012 9）. The results of median-joining network and system clustering tree analysis showed that Cannabis sativa L. and other members of the Cannabaceae family were located in different branches. Conclusion The rbcL sequence could be used as a DNA barcode for identifying Cannabis sativa L., and combined with comparative analysis of the rbcL sequence and system cluster analysis could be a reliable and effective detection method for Cannabis sativa L. identification in forensic investigation.

Absceso frío en una mano por un hongo dematiáceo. Primera comunicación en Chile de Pleurostomophora richardsiae / Hand cold abscess by a dematiaceous fungus. First report in Chile of Pleurostomophora richardsiae

Resumen Los hongos dematiáceos son un grupo heterogéneo de microorganismos capaces de sintetizar melanina. Las infecciones de este grupo que producen hifas en tejidos se denominan feohifomicosis y generalmente afectan la piel y tejidos vecinos. Presentamos el caso de un varón de 86 años con un tumor quístico blando progresivo en su mano y muñeca derecha, no asociado a dolor o signos inflamatorios. Se demostró una tenosinovitis de los flexores con pseudocapsula y sinovitis adherida a los tendones. El cultivo demostró un hongo dematiáceo compatible con Pleurostomophora richardsiae que se confirmó por secuenciación de la región ITS. La biopsia mostró una inflamación crónica granulomatosa e hifas. Después del drenaje quirúrgico, el paciente fue dado de alta sin terapia antifúngica, pero falleció por causas no relacionadas, tres meses después. Esta es la primera descripción de P. richardsiae como causa de feohifomicosis en Chile. Esta patología se puede sospechar cuando una lesión quística cutánea crónica involucra extremidades sin signos inflamatorios. Puede afectar a pacientes inmunocompetentes o inmunocomprometidos. El tratamiento contempla la escisión quirúrgica con o sin terapia antifúngica.

Abstract Dematiaceous fungi are a heterogeneous group of microorganisms able to synthesize melanin. Infections by this group that provoke tissular hyphae are called phaeohyphomycosis and usually involve skin and neighbor tissues. We present the case of a 86 years old men with a progressive soft cystic tumor in his right hand and wrist not associated to pain or inflammatory signs. A surgical intervention demonstrated flexor tenosynovitis with serous secretion, pseudocapsule and synovitis. Fungal culture demonstrated a dematiaceous fungi compatible with Pleurostomophora richardsiae that was confirmed by sequencing of the ITS region. Biopsy showed chronic inflammation with granuloma and hyphae. After surgical drainage, the patient was discharged without antifungal therapy but died of unrelated causes three month later. This is the first description of P. richardsiae as a cause of phaeohyphomycosis in Chile, a country with a template climate. Phaeohyphomycosis can be suspected when a chronic skin cystic lesion involves extremities without inflammatory signs, sometimes with an associated fistula. It may affect immunocompetent or immunosuppressed patients. Treatment involves surgical excision with or without antifungal therapy and prognosis is favorable.

Detection of 3243 A/G and 3316 G/A mitochondrial DNA mutations in Nagpur population

The present study aims to detect 3243 A/G and 3316 G/A mitochondrial DNA (mtDNA) mutations in Nagpurpopulation. Total of 142 patients of type 2 diabetes mellitus and 142 healthy control individuals were selected forthe study from Nagpur city. Selected mutations studied using restriction fragment length polymorphism methodand confirmed by DNA sequencing. Results showed that 3316 G/A mt DNA mutation found in seven patientsof type 2 diabetes mellitus with a 4.92% prevalence, however, found absent in healthy control individuals. ChiSquare and Fisher's exact test showed a significant association between healthy control individuals and type 2diabetes mellitus patients detected with 3316 G/A mutation (p ≤ 0.01). ODDS ratio found significant (for 95%CI; p = 0.05) for 3316 G/A mutation. Furthermore, we did not find 3243 A/G mtDNA mutation in the studiedpopulation. Among studied mutations, 3316 G/A mutation in the ND1 gene is a pathogenic mutation that mayresponsible for type 2 diabetes mellitus in the Nagpur population.

Caracterización fenotípica y molecular, e influencia de medios de cultivo, en el crecimiento y emisión de luz de bacterias del litoral de La Habana, Cuba

Introducción: El complejo enzimático emisor de luz de las bacterias luminiscentes es una poderosa herramienta bioquímica, con una amplia variedad de aplicaciones, incluyendo el control de la calidad ambiental. Objetivos: Identificar taxonómicamente dos bacterias luminiscentes de las aguas de la plataforma cubana, así como seleccionar los medios de cultivo que favorezcan su crecimiento y luminiscencia. Métodos: La identificación taxonómica de las bacterias luminiscentes se llevó a cabo utilizando métodos tradicionales y moleculares. Cuatro medios de cultivo (LM, Boss, Chalk, ZoBell) fueron evaluados en función de la tasa de crecimiento específico (μ) y la luminiscencia utilizando un espectrofotómetro Genesys 10UV y un espectro fluorómetro Shimadzu RF-5301pc, respectivamente. Resultados: La caracterización bioquímica y fisiológica de los aislamientos de CBM-976 y CBM-992 mostró similitudes con las especies de Vibrio harveyi. El análisis del posicionamiento taxonómico confirmó una alta correspondencia con las cepas de V. harveyi aisladas de entornos acuáticos, utilizando secuencias parciales de los genes 16S rRNA, gyrB y pyrH. Se seleccionaron los medios de cultivo LM y ZoBell por tener una alta tasa de crecimiento específico de las cepas CBM-976 y CBM-992; así como por mostrar altos valores de luminiscencia. Los resultados permitirán profundizar en la caracterización fisiológica y son el punto de partida para el desarrollo de métodos de detección de contaminantes. Conclusiones: La combinación de las características fisiológicas y bioquímicas, así como las técnicas de biología molecular contribuyeron a determinar la posición taxonómica de las cepas CBM-976 y CBM-992 aisladas de las aguas marinas cubanas como Vibrio harveyi. Además, se seleccionaron los medios de cultivo LM y ZoBell como los más adecuados para el crecimiento y la emisión de luminiscencia de ambas cepas.

Introduction: The light-emitting enzyme complex of luminescent bacteria is a powerful biochemical tool, with a wide variety of applications including environmental quality monitoring. Objectives: To identify taxonomically two luminescent bacteria from Cuban shelf waters, as well as select the culture media that favor their growth and luminescence. Methods: The taxonomic location of the luminescent bacteria was carried out using traditional and molecular methods. Four culture media (LM, Boss, Chalk, ZoBell) were evaluated as a function of specific growth rate (μ) and luminescence, using a Genesys 10UV spectrophotometer and a Shimadzu RF-5301pc spectrofluorometer, respectively. Results: Biochemical and physiological characterization of CBM-976 and CBM-992 isolates showed similarities with Vibrio harveyi species. Phylogenetic positioning analysis confirmed a high correspondence with V. harveyi strains isolated from aquatic environments, using partial sequences of 16S rRNA, gyrB and pyrH genes. LM and ZoBell culture media were selected for having a high specific growth rate of CBM-976 and CBM-992 strains, as well as for showing high luminescence values. The results will allow deepening the physiological characterization and are the starting point for the development of contaminant detection methods. Conclusions: The rational combination of physiological and biochemical characteristics, as well as the molecular approach, contributed to determine the taxonomic position of CBM-976 and CBM-992 strains isolated from Cuban marine waters as Vibrio harveyi. Furthermore, LM and ZoBell culture media were selected as the most suitable for growth and luminescence emission for both strains.

Caracterización molecular de los microorganismos presentes durante el proceso fermentativo de los granos de cacao (Theobroma cacao) / Molecular characterization of microorganisms during cocoa-bean fermentation process (Theobroma cacao)

La fermentación de granos de cacao es un proceso espontáneo de post cosecha muy importante para el desarrollo de aroma y sabor a chocolate el cual involucra un sin número de actividades microbianas complejas. En el presente estudio se identifican los microorganismos presentes en granos de cacao antes, durante y después del proceso de fermentación aplicando dos métodos: el análisis de secuenciamiento de ADN y la espectrometría de masas MALDI TOF/TOF. Dentro del grupo de bacterias y levaduras predominantes identificadas por el primer método se encontro a Lactobacillus plantarum (29%), L. brevis (18%), Bacillus cereus (15%), Pediococcus acidilactici (12%), y Pichia kudriavzevii (100%). Asimismo se caracterizó por huella de masas las secuencias peptídicas más importantes de cada cepa identificada. Por otro lado, aplicando el segundo método, se identificaron 57 especies de microorganismos, siendo el 73.7% especies bacterianas y el 26.3% especies de levaduras. Adicionalmente se detectaron secuencias peptídicas de la proteína vicilina responsable del aroma característico de los granos de cacao fermentados y a la proteína albumina de 21KDa.

Cocoa beans fermentation is a spontaneous process of post-harvest very important for the development of chocolate aroma and flavor, which involves a number of complex microbial activities. In this work, we identify the microorganisms present in cocoa beans before, during and after the fermentation process, applying DNA sequencing analysis and MALDI TOF / TOF mass spectrometry. With the first method, the predominant bacteria and yeast identified were Lactobacillus plantarum (29%), L. brevis (18%), Bacillus cereus (15%), Pediococcus acidilactici (12%), and Pichia kudriavzevii (100%). The most important peptide sequences of each identified strain by mass fingerprint were characterized too. By the second method, 51 species of microorganisms being 73.7% bacterial species and 26.3% yeast species were identified. Additionally peptide sequences responsible Vicilin protein characteristic aroma of the fermented cocoa beans and the albumin protein of 21KDa were detected.

Association Of Eda Gene Polymorphisms Rs 372024, Rs 3764746, Rs 3795170 In Skeletal Class I Cases With Dental Crowding

The EDA gene, associated with X linked hydrotic form of Ectodermal Dysplasia, its mutations could potentially lead to differential gene expression that causes large tooth phenotype, which has been suggested to cause dental crowding. We analyzed the association of genetic polymorphisms in EDA gene variants rs 372024, rs 3764746, and rs 3795170 among Skeletal Class I crowding cases using blood samples of 30 cases and 30 controls, which were subjected to PCR amplication and DNA sequencing. Based on the statistical analysis using the Z test we found CG and GG genotype for rs3764746 and GT and TT genotype for rs3795170 showed a statistically signicant result. These results suggest that EDA gene variants rs3764746 and rs3795170 could be genetic markers for dental crowding in our population while EDA gene variant rs372024 did not show any signicant association in our population. These ndings can provide in-depth knowledge, regarding the genetic inuences on the incidence of crowding of teeth.

Mutational profile by targeted next generation sequencing of non-small cell lung cancer in the Mexican population / Perfil mutacional por secuenciación de nueva generación en cáncer de pulmón de células no pequeñas en población mexicana

Abstract: Objective: Targeted next-generation sequencing (t-NGS) has revolutionized clinical diagnosis allowing multiplexed detection of genomic alterations. This study evaluated the profile of somatic mutations by t-NGS in Mexican patients with non-small cell lung cancer (NSCLC). Materials and methods: Genomic DNA was extracted from 90 lung adenocarcinomas and sequences were generated for a panel of 48 cancer genes. Epidermal Growth Factor Receptor (EGFR) mutations were detected in parallel by quantitative PCR. Results: The mutational profile of NSCLC revealed alterations in 27 genes, where TP53 (47.8%) and EGFR (36.7%) exhibited the highest mutation rates. EGFR Q787 mutations were present in 14 cases (15.6%), 10 cases had exon 19 deletions (11.1%), seven cases had L858R (7.8%). The mutational frequency for genes like EGFR, MET, HNF1A, HER2 and GUSB was different compared to caucasian population. Conclusion: t-NGS improved NSCLC treatments efficacy due to its sensitivity and specificity. A distinct pattern of somatic mutations was found in Mexican population.

Resumen: Objetivo: La secuenciación dirigida de nueva generación (SNG) permite la detección múltiple de mutaciones. Este estudio evalúa el perfil de mutaciones somáticas por SNG en pacientes mexicanos con cáncer de pulmón de células no pequeñas (CPCNP). Material y métodos: Se aisló ADN de 90 muestras de pacientes con CPCNP y se analizarón 48 genes relacionados con cáncer. Las mutaciones del receptor del factor de crecimiento epidérmico (EGFR) se detectaron por PCR cuantitativa. Resultados. Se detectaron alteraciones en 27 genes. Las mutaciones más frecuentes fueron TP53 (47.8%) y EGFR (36.7%). En el gen EGFR, 14 casos fueron mutaciones Q787 (15.6%), 10 presentaron microdeleciones en el exón 19 (11.1%), y siete en L858R (7.8%). La frecuencia de mutación en EGFR, MET, HNF1A, HER2 y GUSB fue diferente en comparación con población caucásica. Conclusión: NGS modifica el tratamiento del paciente con CPCNP por su sensibilidad y especificidad para detectar mutaciones. La población mexicana presenta un perfil mutacional particular.

Four novel mutations of ADAR1 in Chinese patients with dyschromatosis symmetrica hereditaria

Background: Novel mutations in adenosine deaminase acting on RNA 1 gene (ADAR1) are responsible for dyschromatosis symmetrica hereditaria (DSH). DSH patients display a mixture of hyperpigmented and hypopigmented macules on the dorsal aspects of the extremities, and freckle-like macules on the face. Aims: To provide new evidence for further study of the etiopathogenisis of DSH. Methods: Genomic DNA was extracted and used as a template for the polymerase chain reaction (PCR) amplification of all 15 coding exons as well as intron-exon boundaries of ADAR1. The PCR products were sequenced directly. Results: We identified eight mutations of ADAR1 in four Chinese pedigrees and four individual patients, which were c.2722G>T, p.(Asp908Tyr), c.1657delA, p.(Ser553fs), c.2563_2564delCT, p.(Leu855fs), c.526T>G, p.(Leu176Val) as well as four previously reported mutations c. 3363_3364insT, p.(Lys1122fs), c. 2865_2866delGT, p.(Val955fs), c.1630C>T, p.(Arg544X), and c.2894C>T, p.(Pro965Leu). In silico analysis predicted that all the mutations reported were pathogenic. Limitations: We did not study how ADAR1 played its role in DSH. So, the exact pathogenic mechanism of ADAR1 in DSH patients wasn't clarified in this study. Conclusion: We found four novel ADAR1 mutations in this study. Our results enlarge the database on ADAR1 mutations associated with DSH.

Pythium keratitis in South India: Incidence, clinical profile, management, and treatment recommendation

Purpose: To study the demographic profile, clinical features, treatment outcome, and ocular morbidity of microbiologically proven Pythium keratitis in South India. Methods: A retrospective analysis of clinical records of microbiologically proven Pythium keratitis at a tertiary eye care referral center in South India from January 2016 to November 2017 was performed. Demographic details, predisposing risk factors, microbiological investigations, clinical course, and visual outcome were analyzed. Results: Seventy-one patients with microbiologically proven Pythium keratitis were identified. The mean age was 44(±18.2) years with an increase in male preponderance and 50% were farmers. Duration of delay at time of presentation to the hospital was a mean of 14(±7.2) days. The visual acuity at baseline ranged from 6/6 to no light perception (median 2.1 logMAR). A combination of 5% natamycin and 1% voriconazole was given to 42% patients, and natamycin alone was given to 39.4% patients. 1% itraconazole eye drops alone was initiated in 7 (10%) patients and 3 among this group responded. Therapeutic keratoplasty (TPK) was performed in 48 (67.6%) patients. None of the primary grafts remained clear after a period of 1 month. Twenty-six eyes (54.2%) had graft reinfection and all these eyes either developed anterior staphyloma (4) or were eviscerated (3) and 13 eyes became phthisical. The remaining 22 patients who had TPK resulted in failed graft. Among these, re-grafts were performed in 6 patients, of which 5 were doing well at the last follow-up. Conclusion: We report a large series of patients with Pythium keratitis. Promoting early and differential diagnosis, awareness of clinicians and specific treatment options are needed for this devastating corneal disease.

Genetic polymorphism of rsl051169 G/C and rs9984765 T/C in S100B gene in Chinese Guangxi population / 解剖学报

Objective: To explore the distribution characteristics of S100B gene rsl051169 G/C and rs9984765 T/C polymorphisms in Guangxi population, and to compare the distribution differences among different races and districts population. Methods: Polymerase chain reaction-single base extension (SBE-PCR) and DNA sequencing were used to detect the genotype of rslOSl 169 G/C and rs9984765 T/C among 398 individuals in Guangxi. The results were compared with the allele and genotype of other four populations (HapMap-CEU, HapMap-YRI, HapMap-JPT, HapMap-HCB) from Haplotype Map(Hap Map). Results: There were GG, CG and CC genotypes at the rs 1051169 G/C of S100B gene in Guangxi population, with frequencies of 41.2%, 44.7% and 14. 1%, respectively, and G and C allele frequencies were 63.6% and 36.4%, respectively. TT, CT and CC genotypes were found at rs9984765 T/C, with frequencies of 47. 7%, 44.5% and 7. 8%.respectively, and allele frequencies of T and C were 70.0% and 30. 0%, respectively. There were no significant differences in genotype and allele frequencies of rsl051169 G/C and rs9984765 T/C among male and female in Guangxi population (P>0. 05). The genotype and allele frequency of rs 1051169 G/C in Guangxi population showed significant difference as compared with HapMap-CEU, HapMap-YRI and HapMap-JPT (P0. 05). The genotype and allele frequency of rs9984765 T/C in Guangxi population showed significant difference as compared with HapMap-YRI, HapMap- JPT and HapMap-HBC (1 0. 05). Conclusion: The polymorphisms of rs 1051169 G/C and rs9984765 T/C in S100B gene in Guangxi populations, and their polymorphisms are different from different races and district populations.

Sampling and storage methods of fecal samples in human intestinal microbiome study / 国际儿科学杂志

With the rapid development of high-throughput sequencing ( NGS) technique, intestinal microbiome could be studied more deeply. Intestinal contents and fecal samples have the characteristics of con-venient sampling and strong representation, so they are often used as the main research objects in the study of intestinal microbiota. The method of collecting and storage of samples are very important to affect the internal flora structure and diversity, which determines the accuracy of subsequent sequencing analysis. This review summarizes the sampling and storage methods of fecal samples in the study of intestinal microbiome.

Hypermethylation Analysis of p16INK4a and p15INK4b Promoters in Chronic Lymphocytic Leukaemia Patients and Normal Individuals

@#Introduction: Chronic Lymphocytic Leukaemia (CLL) is a common type of leukaemia in persons of predominantly European descent but is rare in the Asian population. Disparities in CLL incidence among people of Asian and European descent may be related to the genetic make-up of the two different populations. Hypermethylation event might be one of the silencing mechanisms that inactivate the tumour suppressor genes in CLL. The aim of this study was to determine the hypermethylation status of p16INK4a and p15INK4b among CLL patients and normal individuals. Materials & Methods: A total of 25 CLL patients and 25 normal individuals were recruited for this study and their genomic DNA were extracted from the peripheral blood. The hypermethylation status of p16INK4a and p15INK4b were determined using Methylation Specific-PCR (MS-PCR) whereas DNA sequencing method was applied to selected samples for validation of the MS-PCR results. We also evaluated the association between hypermethylation of these genes with the clinical and demographic characteristics of each group of subjects. Results: Among the CLL patients, p15INK4b partialmethylation occurred in 6 (24%) subjects while methylation occurred in 1 (4%) subject. All the remaining patients were unmethylated at p15INK4b. All the samples showed unmethylation at p16INK4a. Statistically significant associations were found between p15INK4b hypermethylation with the presence of CLL (p=0.01) and with race (p=0.02). Conclusion: Further study using a larger sample size is warranted to explore the significance of DNA methylation incidence among the CLL patients of the Malaysian population. Hence, we suggest that hypermethylation at p15INK4b has a huge influence that kick-starts CLL disease among Malaysians and MS-PCR technique is applicable to be used in methylation study.

Recent Update in Pathologic Diagnosis for Pancreatic Cystic Neoplasm / 대한췌담도학회지

Pancreas cystic neoplasm is a relatively common disease. However, its' pathologic diagnosis is not easy. The most frequent problem is low cellularity when compared to another organ cytology or biopsy material. Considering the procedure and anatomic difficulty, it is not uncommon to observe a low cellular smear or scanty volume of cells in the biopsy specimen. In this case, the molecular pathology test, including next-generation sequencing, may be helpful. If pathologist can identify some mutation in cells or cystic fluid, differential diagnosis of cystic neoplasm may be possible. These are KRAS and GNAS, VHL, and CTNNB1 mutation in mucinous cystic neoplasm, intraductal papillary-mucinous neoplasm, serous cystic neoplasm, and solid pseudopapillary neoplasm, respectively. The next-generation sequencing is an emerging molecular test that can detect multiple biomarkers for diagnosis, including pancreas cystic neoplasm. It has been reported that next-generation sequencing test can be applied for differential diagnosis of pancreas cystic neoplasm. However, these molecular pathology tests were not all-around; it needs to be properly managed with pathologist's quality control. It should be remembered that even if it goes through quality control, it may show a failure rate of around 30%. Despite the advances in molecular methods of high techniques, it should be remembered that the most important thing in pathologic diagnosis of pancreas cystic neoplasm is an endoscopist's skill and pathologist's expertise those provide adequate specimen and accurate diagnosis.

Sequence analysis of the HBV S protein in patients with chronic hepatitis B / 中华检验医学杂志

Objective To analysis the frequency and mutational patterns of HBV(hepatitis B virus) S protein in the different phases of chronic hepatitis B patients, exploring the clinical implication of these mutations in CHB. Methods The difference of S protein mutation rates and mutation site among each groups were analyzed by cases-comparison. 112 cases of chronic hepatitis B patients in Shanghai Shu-guang hospital were enrolled in this study; they were divided into four groups, immune-tolerance group [IT, 36 cases, male 20 cases,female 16 cases,age 28.00 (25.00,30.75)years], immune-clearance group [IC, 28 cases, male 17 cases,female 11 cases,age 29.00 (25.25,31.75) years], low-replication group [LR, 25 cases, male 17 cases,female 8 cases,age 39.00 (35.00,45.50) years], reactivation group [RE, 23 cases, male 15 cases,female 8 cases, age 43.00 (36.00, 48.00) years]. DNA sequencing was used to detection HBV S protein gene mutation. Difference between categorical variables were analyzed using Chi-square test, Non-parametric test (kruskal-wallis H and Mann-Whitney U) was used for non-normal distribution data. Results Naturally occurring MHR (major hydrophilic region) variants was 16.67％ (6/36 cases) in IT group, preferentially within"a"determinant in the first loop, compared with IC (21.43％,6/28 cases), LR (20.00％,5/25 cases) and RE (34.78％,8/23 cases), there was no statistics Significance (χ2=2.814, P=0.421), while variant frequencies outside the MHR were 11.11％ (4/36 cases) and 14.29％ (4/28 cases) in IT and IC group, viral diversity gradually increased during LR phase with 24.00％(6/25 cases) of mutation frequency, and peak at RE phase (52.17％, 12/23 cases) with a statistics significance (χ2=15.041, P=0.002). Conclusion Amino acid substitutions is not only the consequence of the pressure of host immunity, it may be a probably cause of disease reactivation.

The First Acanthamoeba keratitis Case of Non-Contact Lens Wearer with HIV Infection in Thailand

Acanthamoeba keratitis (AK) is a rare sight-threatening corneal infection, often reporting from contact lens wearers. An asymptomatic human immunodeficiency virus (HIV)-infected Thai male without history of contact lens use complained foreign body sensation at his left eye during motorbike riding. He had neither specific keratitis symptoms nor common drugs responding, which contributed to delayed diagnosis. By corneal re-scraping, Acanthamoeba-like cysts were detected by calcofluor white staining and agar culture. The etiological agent obtained from the culture was molecularly confirmed by Acanthamoeba spp.-specific PCR, followed by DNA sequencing. The results from BLAST and phylogenetic analysis based on the DNA sequences, revealed that the pathogen was Acanthamoeba T4, the major genotype most frequently reported from clinical isolates. The infection was successfully treated with polyhexamethylene biguanide resulting in corneal scar. This appears the first reported AK case from a non-contact lens wearer with HIV infection in Thailand. Although AK is sporadic in developing countries, a role of free-living Acanthamoeba as an opportunistic pathogen should not be neglected. The report would increase awareness of AK, especially in the case presenting unspecific keratitis symptoms without clinical response to empirical antimicrobial therapy.

Fungal keratitis caused by Podospora austroamericana: A first case report

We report a case of keratitis caused by a rare fungus Podospora austroamericana. Clinical and microbiological evaluation of the corneal ulcer was done and the treatment outcome was studied. The fungus was grown from the corneal scraping, and it was identified as P. austroamericana based on DNA sequence and analysis of the internal transcribed spacer region. The patient was treated with topical azithromycin, natamycin and voriconazole. Despite maximum medical therapy, the ulcer progressed very rapidly and the patient developed panophthalmitis and evisceration of the eye had to be done. This is the first reported case of keratitis caused by P. Austroamericana.