Sequencing the transcriptome of milk production: milk trumps mammary tissue

Background: Studies of normal human mammary gland development and function have mostly relied on cell culture, limited surgical specimens, and rodent models. Although RNA extracted from human milk has been used to assay the mammary transcriptome non-invasively, this assay has not been adequately validated in primates. Thus, the objectives of the current study were to assess the suitability of lactating rhesus macaques as a model for lactating humans and to determine whether RNA extracted from milk fractions is representative of RNA extracted from mammary tissue for the purpose of studying the transcriptome of milk-producing cells. Results: We confirmed that macaque milk contains cytoplasmic crescents and that ample high-quality RNA can be obtained for sequencing. Using RNA sequencing, RNA extracted from macaque milk fat and milk cell fractions more accurately represented RNA from mammary epithelial cells (cells that produce milk) than did RNA from whole mammary tissue. Mammary epithelium-specific transcripts were more abundant in macaque milk fat, whereas adipose or stroma-specific transcripts were more abundant in mammary tissue. Functional analyses confirmed the validity of milk as a source of RNA from milk-producing mammary epithelial cells. Conclusions: RNA extracted from the milk fat during lactation accurately portrayed the RNA profile of milk-producing mammary epithelial cells in a non-human primate. However, this sample type clearly requires protocols that minimize RNA degradation. Overall, we validated the use of RNA extracted from human and macaque milk and provided evidence to support the use of lactating macaques as a model for human lactation

Prediction of cell position using single-cell transcriptomic data: an iterative procedure

Single-cell sequencing reveals cellular heterogeneity but not cell localization. However, by combining single-cell transcriptomic data with a reference atlas of a small set of genes, it would be possible to predict the position of individual cells and reconstruct the spatial expression profile of thousands of genes reported in the single-cell study. To develop new algorithms for this purpose, the Dialogue for Reverse Engineering Assessments and Methods (DREAM) consortium organized a crowd-sourced competition known as DREAM Single Cell Transcriptomics Challenge (SCTC). In the spirit of this framework, we describe here the proposed procedures for adequate reference genes selection, and an iterative procedure to predict spatial expression profile of other genes.Fil: Alonso, Andrés Mariano. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Carrea, Alejandra. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Diambra, Luis Anibal. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentin

Molecular phylogeny of the dove genera Streptopelia and Columba

Journal ArticleEvolutionary history of the dove genus Streptopelia has not been examined with rigorous phylogenetic methods. We present a study of phylogenetic relationships of Streptopelia based on over 3,600 base pairs of nuclear and mitochondrial gene sequences

Genetic testing among high-risk individuals in families with hereditary nonpolyposis colorectal cancer

Hereditary nonpolyposis colorectal cancer (HNPCC) is frequently associated with constitutional mutations in a class of genes involved in DNA mismatch repair. We identified 32 kindreds, with germline mutations in one of three genes hMSH2, hMLH1 or hMSH6. In this study, we purposed to evaluate how many high-risk individuals in each family underwent genetic testing: moreover, we assessed how many mutation-positive unaffected individuals accepted colonoscopic surveillance and the main findings of the recommended follow-up. Families were identified through a population-based registry, or referred from other centres. Members of the families were invited for an education session with two members of the staff. When a kindred was consistent with HNPCC, neoplastic tissues were examined for microsatellite instability (MSI) and immunohistochemical expression of MSH2, MLH1 and MSH6 proteins. Moreover, constitutional mutations were searched by SSCP or direct sequencing of the whole genomic region. Of the 164 subjects assessed by genetic testing, 89 were gene carriers (66 affected - that is, with HNPCC-related cancer diagnosis - and 23 unaffected) and 75 tested negative. Among the 23 unaffected gene carriers, 18 (78.3%) underwent colonoscopy and four declined. On a total of 292 first degree at risk of cancer, 194 (66.4%) did not undergo genetic testing. The main reasons for this were: (a) difficulty to reach family members at risk, (b) lack of collaboration, (c) lack of interest in preventive medicine or 'fatalistic' attitude towards cancer occurrence. The number of colorectal lesions detected at endoscopy in gene carriers was significantly (P<0.01) higher than in controls (noncarriers). We conclude that a large fraction of high-risk individuals in mutation-positive HNPCC families does not undergo genetic testing, despite the benefits of molecular screening and endoscopic surveillance. This clearly indicates that there are still barriers to genetic testing in HNPCC, and that we are unable to provide adequate protection against cancer development in these families

Whole-genome characterization and resistance-associated substitutions in a new HCV genotype 1 subtype

HCV; Direct-acting antivirals; Genotype 1VHC; Antivirals d'acció directa; Genotip 1VHC; Antivirales de acción directa; Genotipo 1Hepatitis C virus (HCV) is a highly variable infectious agent, classified into 8 genotypes and 86 subtypes. Our laboratory has implemented an in-house developed high-resolution HCV subtyping method based on next-generation sequencing (NGS) for error-free classification of the virus using phylogenetic analysis and analysis of genetic distances in sequences from patient samples compared to reference sequences. During routine diagnostic, a sample from an Equatorial Guinea patient could not be classified into any of the existing subtypes. The whole genome was analyzed to confirm that the new isolate could be classified as a new HCV subtype. In addition, naturally occurring resistance-associated substitutions (RAS) were analyzed by NGS. Whole-genome analysis based on p-distances suggests that the sample belongs to a new HCV genotype 1 subtype. Several RAS in the NS3 (S122T, D168E and I170V) and NS5A protein (Q(1b)24K, R(1b)30Q and Y93L+Y93F) were found, which could limit the use of some inhibitors for treating this subtype. RAS studies of new subtypes are of great interest for tailoring treatment, as no data on treatment efficacy are reported. In our case, the patient has not yet been treated, and the RAS report will be used to design the most effective treatment

Mutations in the LKB1 tumour suppressor are frequently detected in tumours from Caucasian but not Asian lung cancer patients

Somatic mutations of LKB1 tumour suppressor gene have been detected in human cancers including non-small cell lung cancer (NSCLC). The relationship between LKB1 mutations and clinicopathological characteristics and other common oncogene mutations in NSCLC is inadequately described. In this study we evaluated tumour specimens from 310 patients with NSCLC including those with adenocarcinoma, adenosquamous carcinoma, and squamous cell carcinoma histologies. Tumours were obtained from patients of US (n=143) and Korean (n=167) origin and screened for LKB1, KRAS, BRAF, and EGFR mutations using RT—PCR-based SURVEYOR-WAVE method followed by Sanger sequencing. We detected mutations in the LKB1 gene in 34 tumours (11%). LKB1 mutation frequency was higher in NSCLC tumours of US origin (17%) compared with 5% in NSCLCs of Korean origin (P=0.001). They tended to occur more commonly in adenocarcinomas (13%) than in squamous cell carcinomas (5%) (P=0.066). LKB1 mutations associated with smoking history (P=0.007) and KRAS mutations (P=0.042) were almost mutually exclusive with EGFR mutations (P=0.002). The outcome of stages I and II NSCLC patients treated with surgery alone did not significantly differ based on LKB1 mutation status. Our study provides clinical and molecular characteristics of NSCLC, which harbour LKB1 mutations

Frequent downregulation of 14-3-3 σ protein and hypermethylation of 14-3-3 σ gene in salivary gland adenoid cystic carcinoma

14-3-3 σ, a target gene of the p53 tumour suppressor protein, has been shown to regulate the cell cycle at the G2/M checkpoint. Recent studies have demonstrated that 14-3-3 σ is downregulated by hypermethylation of the CpG island in several types of cancer. In this study, we investigated the expression and methylation status of 14-3-3 σ in human salivary gland adenoid cystic carcinoma (ACC) and mucoepidermoid carcinoma (MEC). Immunohistochemical analysis revealed that the positive expression rate of 14-3-3 σ in ACC (one out of 14) was markedly lower than that in MEC (ten out of 10). Since most of the ACCs carried the wild-type p53 protein, downregulation of 14-3-3 σ in ACC may not be due to the dysfunction of p53 pathway. Microdissection–methylation-specific PCR revealed that frequent hypermethylation of the 14-3-3 σ gene was observed in ACC when compared to that in MEC. In cultured-ACC cells, we confirmed the downregulation of 14-3-3 σ via hemimethylation of the gene by sequencing analysis after sodium bisulphite treatment. Furthermore, re-expression of 14-3-3 σ in the ACC cells was induced by the treatment with DNA demethylating agent, 5-aza-2′-deoxycytidine. Irradiation apparently induced the enhanced expression of 14-3-3 σ and G2/M arrest in normal salivary gland cells; however, in the ACC cells, neither induction of 14-3-3 σ nor G2/M arrest was induced by irradiation. These results suggest that downregulation of 14-3-3 σ might play critical roles in the neoplastic development and radiosensitivity of ACC