Circulating Tumor Cell Transcriptomics as Biopsy Surrogates in Metastatic Breast Cancer.

BACKGROUND: Metastatic breast cancer (MBC) and the circulating tumor cells (CTCs) leading to macrometastases are inherently different than primary breast cancer. We evaluated whether whole transcriptome RNA-Seq of CTCs isolated via an epitope-independent approach may serve as a surrogate for biopsies of macrometastases. METHODS: We performed RNA-Seq on fresh metastatic tumor biopsies, CTCs, and peripheral blood (PB) from 19 newly diagnosed MBC patients. CTCs were harvested using the ANGLE Parsortix microfluidics system to isolate cells based on size and deformability, independent of a priori knowledge of cell surface marker expression. RESULTS: Gene expression separated CTCs, metastatic biopsies, and PB into distinct groups despite heterogeneity between patients and sample types. CTCs showed higher expression of immune oncology targets compared with corresponding metastases and PB. Predictive biomarker (n = 64) expression was highly concordant for CTCs and metastases. Repeat observation data post-treatment demonstrated changes in the activation of different biological pathways. Somatic single nucleotide variant analysis showed increasing mutational complexity over time. CONCLUSION: We demonstrate that RNA-Seq of CTCs could serve as a surrogate biomarker for breast cancer macrometastasis and yield clinically relevant insights into disease biology and clinically actionable targets.

A Pilot Study for the Feasibility of Exome-Sequencing in Circulating Tumor Cells Versus Single Metastatic Biopsies in Breast Cancer.

The comparison of the landscape of somatic alterations in circulating tumor cells (CTCs) versus metastases is challenging. Here, we comprehensively characterized the somatic landscape in bulk (amplified and non-amplified), spike-in breast cancer cells, CTCs, and metastases from breast cancer patients using whole-exome sequencing (WES). We determined the level of genomic concordance for somatic nucleotide variants (SNVs), copy number alterations (CNAs), and structural variants (SVs). The variant allele fractions (VAFs) of somatic variants were remarkably similar between amplified and non-amplified cell line samples as technical replicates. In clinical samples, a significant fraction of somatic variants had low VAFs in CTCs compared to metastases. The most frequently recurrent gene mutations in clinical samples were associated with an elevated C > T mutational signature. We found complex rearrangement patterns including intra- and inter-chromosomal rearrangements, singleton, and recurrent gene fusions, and tandem duplications. We observed high molecular discordance for somatic alterations between paired samples consistent with marked heterogeneity of the somatic landscape. The most prevalent copy number calls were focal deletion events in CTCs and metastases. Our results demonstrate the feasibility of an integrated workflow for the identification of a complete repertoire of somatic alterations and highlight the intrapatient genomic differences that occur between CTCs and metastases.

RNA-Seq of Circulating Tumor Cells in Stage II-III Breast Cancer.

BACKGROUND: We characterized the whole transcriptome of circulating tumor cells (CTCs) in stage II-III breast cancer to evaluate correlations with primary tumor biology. METHODS: CTCs were isolated from peripheral blood (PB) via immunomagnetic enrichment followed by fluorescence-activated cell sorting (IE/FACS). CTCs, PB, and fresh tumors were profiled using RNA-seq. Formalin-fixed, paraffin-embedded (FFPE) tumors were subjected to RNA-seq and NanoString PAM50 assays with risk of recurrence (ROR) scores. RESULTS: CTCs were detected in 29/33 (88%) patients. We selected 21 cases to attempt RNA-seq (median number of CTCs = 9). Sixteen CTC samples yielded results that passed quality-control metrics, and these samples had a median of 4,311,255 uniquely mapped reads (less than PB or tumors). Intrinsic subtype predicted by comparing estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) versus PAM50 for FFPE tumors was 85% concordant. However, CTC RNA-seq subtype assessed by the PAM50 classification genes was highly discordant, both with the subtype predicted by ER/PR/HER2 and by PAM50 tumors. Two patients died of metastatic disease, both of whom had high ROR scores and high CTC counts. We identified significant genes, canonical pathways, upstream regulators, and molecular interaction networks comparing CTCs by various clinical factors. We also identified a 75-gene signature with highest expression in CTCs and tumors taken together that was prognostic in The Cancer Genome Atlas and Molecular Taxonomy of Breast Cancer International Consortium datasets. CONCLUSION: It is feasible to use RNA-seq of CTCs in non-metastatic patients to discover novel tumor biology characteristics.

Natural variation of gene models in Drosophila melanogaster.

BACKGROUND: Variation within splicing regulatory sequences often leads to differences in gene models among individuals within a species. Two alleles of the same gene may express transcripts with different exon/intron structures and consequently produce functionally different proteins. Matching genomic and transcriptomic data allows us to identify putative regulatory variants associated with changes in splicing patterns. RESULTS: Here we analyzed natural variation of splicing patterns in the transcriptomes of 81 natural strains of Drosophila melanogaster with known genotypes. We identified dozens of genotype-specific splicing patterns associated with putative cis-splicing quantitative trait loci (sQTL). The majority of changes can be explained by mutations in splice sites. Allelic-imbalance in splicing patterns confirmed that the majority are regulated mainly by cis-genetic effects. Remarkably, allele-specific splicing changes often lead to qualitative changes in gene models, yielding many isoforms not previously annotated. The observed alterations are typically outside protein-coding regions or affect only very short protein segments. CONCLUSIONS: Overall, the sets of gene models appear to be flexible within D. melanogaster populations. The observed variation in splicing patterns are predicted to have limited effects on the encoded protein sequences. To our knowledge, this is the first sQTL mapping study in Drosophila.

Population genomic analysis uncovers African and European admixture in Drosophila melanogaster populations from the south-eastern United States and Caribbean Islands.

Drosophila melanogaster is postulated to have colonized North America in the past several 100 years in two waves. Flies from Europe colonized the east coast United States while flies from Africa inhabited the Caribbean, which if true, make the south-east US and Caribbean Islands a secondary contact zone for African and European D. melanogaster. This scenario has been proposed based on phenotypes and limited genetic data. In our study, we have sequenced individual whole genomes of flies from populations in the south-east US and Caribbean Islands and examined these populations in conjunction with population sequences from the west coast US, Africa, and Europe. We find that west coast US populations are closely related to the European population, likely reflecting a rapid westward expansion upon first settlements into North America. We also find genomic evidence of African and European admixture in south-east US and Caribbean populations, with a clinal pattern of decreasing proportions of African ancestry with higher latitude. Our genomic analysis of D. melanogaster populations from the south-east US and Caribbean Islands provides more evidence for the Caribbean Islands as the source of previously reported novel African alleles found in other east coast US populations. We also find the border between the south-east US and the Caribbean island to be the admixture hot zone where distinctly African-like Caribbean flies become genomically more similar to European-like south-east US flies. Our findings have important implications for previous studies examining the generation of east coast US clines via selection.

Evolution in the block: common elements of 5S rDNA organization and evolutionary patterns in distant fish genera.

The 5S rDNA is organized in the genome as tandemly repeated copies of a structural unit composed of a coding sequence plus a nontranscribed spacer (NTS). The coding region is highly conserved in the evolution, whereas the NTS vary in both length and sequence. It has been proposed that 5S rRNA genes are members of a gene family that have arisen through concerted evolution. In this study, we describe the molecular organization and evolution of the 5S rDNA in the genera Lepidorhombus and Scophthalmus (Scophthalmidae) and compared it with already known 5S rDNA of the very different genera Merluccius (Merluccidae) and Salmo (Salmoninae), to identify common structural elements or patterns for understanding 5S rDNA evolution in fish. High intra- and interspecific diversity within the 5S rDNA family in all the genera can be explained by a combination of duplications, deletions, and transposition events. Sequence blocks with high similarity in all the 5S rDNA members across species were identified for the four studied genera, with evidences of intense gene conversion within noncoding regions. We propose a model to explain the evolution of the 5S rDNA, in which the evolutionary units are blocks of nucleotides rather than the entire sequences or single nucleotides. This model implies a "two-speed" evolution: slow within blocks (homogenized by recombination) and fast within the gene family (diversified by duplications and deletions).

Urinary arsenic levels and risk of renal injury in a cross-sectional study in open population.

INTRODUCTION: Arsenic (As) is one of the most ubiquitous elements in nature, and a prolonged exposure has been associated with an increase in the risk of cancer, diabetes mellitus, hypertension and cardiovascular disease. There are few studies addressing the effects of As on albuminuria, tubular injury and biochemical variables as uric acid. AIM. To analyze the association between urinary As levels, albuminuria, and al-microglobulin as marker of tubular injury. MATERIAL AND METHODS: This is a cross-sectional, and comparative study done in 5 communities localized close to Queretaro City. Subjects with no antecedents of renal disease, diabetes, hypertension, or industrial exposure to As were included. A questionnaire about risk factors for arsenic exposure was done, blood was taken for biochemical analysis and a spot urine sample was collected for albumin, alpha1-microglobulin, and As measurements. RESULTS: A total of 90 adult persons were included with no antecedents of renal disease, diabetes or hypertension; the mean age was 40.9 +/- 12.9 years and the median for urinary As levels was 15 microg/gr Cr (range 0.56-89.2 microg/gr Cr), 10 (11.1%) persons had critical levels > 50 microg/gr Cr. Age more than 50 years old [OR 2.48 IC95 (0.9-6.6)] and place of residence were the most important risk factors associated with higher levels of As. There was association between urinary As levels and al-microglobulin urinary excretion (r2 = 0.07, p = 0.01) but not with albuminuria or other biochemical variables. CONCLUSIONS: This is the first study in Mexico to show an association between As and urinary excretion of al-microglobulin as marker of early renal injury. We did not found association with albuminuria or other serum biochemical variables. Arsenic may be considered as a risk factor for tubular injury.

Implementing a Novel Workplace Smoking Cessation Intervention Targeting Hispanic/Latino Construction Workers: A Pilot Cluster Randomized Trial.

BACKGROUND: U.S. Hispanic/Latino construction workers constitute a large and historically underserved group in terms of smoking cessation services. Using formative research, we developed a worksite smoking cessation intervention tailored to the life/work circumstances of these workers. AIMS: This study aims to examine the feasibility, acceptability, and potential efficacy of the developed intervention "Enhanced Care" (EC; one group behavioral counseling session provided around the food truck + fax referral to tobacco quitline [QL] + 8-week nicotine replacement treatment [NRT]) compared with "Standard Care" (SC; fax referral to tobacco QL + 8-week NRT) in a pilot, two-arm, cluster randomized controlled trial. METHOD: In collaboration with construction site safety managers, a sample of 17 construction sites (EC: nine sites/65 smokers; SC: eight sites/69 smokers) was enrolled. Participants received two follow-ups at 3 and 6 months after enrollment. Feasibility outcomes were enrollment rate, adherence to treatment, and 6-month retention rates. The primary efficacy outcome was 6 months prolonged abstinence verified by expired carbon monoxide <10 ppm. RESULTS: Enrollment rate was high (85.9%). Six-month follow-up rates were acceptable (EC = 76.9%, SC = 66.6%). Adherence to treatment was better in the EC group (received worksite intervention: EC = 93.8%, SC = 88.4%; contacted by QL: EC = 49.2%, SC = 40.6%). Abstinence rates were 27.7% for the EC and 20.3% for the SC (p = .315). DISCUSSION: The developed intervention was feasible and acceptable, and it substantially improved abstinence among Hispanic/Latino workers. The involvement of safety managers was essential to the implementation of the intervention. Training safety managers to deliver the intervention has great potential to implement a sustainable smoking cessation service in the construction sector.

Molecular organization and evolution of 5S rDNA in the genus Merluccius and their phylogenetic implications.

The molecular organization of the 5S rRNA gene family has been studied in a wide variety of animal taxa, including many bony fish species. It is arranged in tandemly repeated units consisting of a highly conserved 120 base pair-long region, which encodes for the 5S rRNA, and a nontranscribed spacer (NTS) of variable length, which contains regulatory elements for the transcription of the coding sequence. In this work, a comparative analysis of 5S ribosomal DNA (rDNA) organization and evolution in the 12 species of the genus Merluccius, which are distributed in the Atlantic and Pacific oceans, was carried out. Two main types of 5S rDNA (types A and M) were identified, as differentiated by the absence or presence of a simple sequence repeat within the NTS. Four species exhibited the 2 types of 5S rDNA, whereas the rest showed only 1 type. In addition, the species M. albidus and M. bilinearis showed 2 variants (S and L) of type-M 5S rDNA, which differentiated by length. The results obtained here support the hypothesis of a 5S rRNA dual system as an ancient condition of the Piscine genome. In contrast, some inconsistencies were found between the phylogeny of the genus Merluccius based on mitochondrial genes and that obtained from nuclear markers (5S rDNA, microsatellite loci, and allozyme data). Hybrid origin of the American species M. australis is suggested based on these results.

A20 regulates canonical wnt-signaling through an interaction with RIPK4.

A20 is a ubiquitin-editing enzyme that is known to regulate inflammatory signaling and cell death. However, A20 mutations are also frequently found in multiple malignancies suggesting a potential role as a tumor suppressor as well. We recently described a novel role for A20 in regulating the wnt-beta-catenin signaling pathway and suppressing colonic tumor development in mice. The underlying mechanisms for this phenomenon are unclear. To study this, we first generated A20 knockout cell lines by genome-editing techniques. Using these cells, we show that loss of A20 causes dysregulation of wnt-dependent gene expression by RNAseq. Mechanistically, A20 interacts with a proximal signaling component of the wnt-signaling pathway, receptor interacting protein kinase 4 (RIPK4), and regulation of wnt-signaling by A20 occurs through RIPK4. Finally, similar to the mechanism by which A20 regulates other members of the receptor interacting protein kinase family, A20 modifies ubiquitin chains on RIPK4 suggesting a possible molecular mechanism for A20's control over the wnt-signaling pathway.

Humanin Prevents Age-Related Cognitive Decline in Mice and is Associated with Improved Cognitive Age in Humans.

Advanced age is associated with a decline in cognitive function, likely caused by a combination of modifiable and non-modifiable factors such as genetics and lifestyle choices. Mounting evidence suggests that humanin and other mitochondrial derived peptides play a role in several age-related conditions including neurodegenerative disease. Here we demonstrate that humanin administration has neuroprotective effects in vitro in human cell culture models and is sufficient to improve cognition in vivo in aged mice. Furthermore, in a human cohort, using mitochondrial GWAS, we identified a specific SNP (rs2854128) in the humanin-coding region of the mitochondrial genome that is associated with a decrease in circulating humanin levels. In a large, independent cohort, consisting of a nationally-representative sample of older adults, we find that this SNP is associated with accelerated cognitive aging, supporting the concept that humanin is an important factor in cognitive aging.

Phylogeny of the genus Merluccius based on mitochondrial and nuclear genes.

Genus Merluccius is considered one of the most important groups within the Teleostei, and comprises 12 extant species distributed along the coasts of America, Europe and Africa, being its fisheries very important in these continents. Despite their noticeable economical importance for humans, to date the phylogeny of hakes has not been clearly established. In this study we used mitochondrial sequences (the ribosomal genes 12S rDNA and 16S rDNA, the coding gene cytochrome b and the control region) and the nuclear 5S rDNA conserved region in order to determine the phylogenetic and biogeographical relationships within the genus Merluccius. This is the first time that all the species of this genus recognized by the FAO are included in a phylogeny. Maximum Parsimony, Maximum Likelihood and Bayesian analyses of the mitochondrial sequences suggest that the geographical origin of the genus was the North Atlantic Ocean, and indicate that two main clades, early separated in the evolution, exist within the genus: one American (7 species) and one Euro-African (5 species). Among the American species, M. bilinearis seems to be the most ancient one, and the rise of the Panama Isthmus could act as a physical barrier leading to further processes of speciation. Within the Euro-African clade, successive events of geographical differentiation could explain the observed pattern of species distribution. Therefore, we propose both vicariant speciation and geographical dispersion as main mechanisms to explain the evolutionary history of the genus Merluccius.

The association between social support and cognitive function in Mexican adults aged 50 and older.

Social support networks are crucial for the health of older adults; however, personal characteristics and time of life may diminish the protective effect of social support. OBJECTIVE: to determine if the presence of social support networks were associated with cognitive impairment among Mexican adults aged 50 or older and if this relationship was different based on age. METHOD: This study analyzed data from the National Representation Survey performed in Mexico, Study on Global Ageing (SAGE) wave 1. Cognitive function was evaluated by a standardized test, social support was evaluated through latent class analysis (LCA). The LCA was run to obtain three subgroups of different Social Support Levels (SSL): low, medium, and high. Logistic regression models, stratified by age, were performed to analyze the association between SSL and cognitive function. RESULTS: For respondents ages 71-80 y/o, there was an inverse relationship with cognitive impairment for those with medium (OR 0.23, p=0.020) and high (OR 0.07, p=0.000) SSL in comparison with low SSL. While social support helped to improve cognitive function in older adults aged 71-80, this same association was not observed in adults of other ages. Those younger than 70 y/o may not need such a strong support network as a result of being more self-sufficient. After 80, social networks were not enough to help diminish the negative impact of cognitive impairment. CONCLUSION: Social support could improve the cognitive function of adults ages 71 and 80; suggesting there could be a window of opportunity to improve cognitive functioning for this group.

Genetic utility of natural history museum specimens: endangered fairy shrimp (Branchiopoda, Anostraca).

We examined the potential utility of museum specimens as a source for genetic analysis of fairy shrimp. Because of loss of their vernal pool habitat, some fairy shrimp (including Branchinectasandiegonensis and Branchinectalynchi) are listed as threatened or endangered in Southern California by the United States Fish and Wildlife Service. Management of those species requires extensive population genetics studies and the resolution of important genetic complexity (e.g. possible hybridization between endangered and non-endangered species). Regulations mandating deposition of specimens of listed species have resulted in thousands of specimens accessioned into the Natural History Museum of Los Angeles County that have been preserved in a variety of solutions. We subsampled those specimens, as well as other Anostraca with known collection and preservation histories, to test their potential for genetic analysis by attempting DNA extraction and amplification for mt16SrDNA. Fixation and preservation in not denatured ethanol had a far greater sequencing success rate than other (and unknown) fixatives and preservatives. To maximize scientific value we recommend field preservation in 95% not denatured ethanol (or, if pure ethanol is unavailable, high-proof drinking spirits, e.g. Everclear™, or 151 proof white rum), followed by storage in 95% not denatured ethanol.

Identifying Fishes through DNA Barcodes and Microarrays.

BACKGROUND: International fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection. METHODOLOGY/PRINCIPAL FINDINGS: This study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S), cytochrome b (cyt b), and cytochrome oxidase subunit I (COI) for the identification of 50 European marine fish species by combining techniques of "DNA barcoding" and microarrays. In a DNA barcoding approach, neighbour Joining (NJ) phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the "position of label" effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridisation experiments (>90%) renders the DNA barcoding marker as rather unsuitable for this high-throughput technology. CONCLUSIONS/SIGNIFICANCE: Based on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products.

Una aproximación a la medición de la violencia familiar en Santiago de Cali, 2003-2005 / An approximation to family violence measurement in Santiago of Cali, 2003-2005

Antecedentes. La violencia ha sido declarada como un problema de salud pública, en violencia interpersonal se incluye maltrato a menores, violencia contra la pareja y maltrato a personas mayores. La medición de la violencia familiar es objeto de investigación, los resultados muestran como el maltrato conyugal es el más frecuente; los autores coinciden en el bajo reporte de los casos ante las instituciones. Objetivo. Identificar el tipo de violencia familiar y sus protagonistas en Cali (Colombia) entre 2003-2005. Material y métodos. Se realizó un estudio descriptivo utilizando los datos del Observatorio de Violencia de familiar entre los años 2003 a 2005. Se realizó análisis por múltiple respuesta, se conformaron dos grupos de análisis: niños y niñas menores de 10 años, mujeres y hombres mayores de nueve años. Resultados. Se encontró un mayor reporte de casos en el año 2005. En los niños, el grupo de cinco a nueve años reportó el mayor número de casos, el 35,3 por ciento por maltrato físico, los padres fueron los mayores victimarios. El 31,5 por ciento de las mujeres reportó maltrato físico, los cónyuges fueron los principales agresores. En los hombres se encontró mayor reporte entre 10-14 y 15-29 años, el maltrato físico fue ejercido en el 27,9 por ciento por madres y el 18,2 por ciento por los padres. Conclusión. Los datos obtenidos fueron similares a los encontrados en otros estudios con otras metodologías. Se requiere cualificar el estudio de la problemática para fortalecer la información del observatorio de violencia familiar en la ciudad.

Cáncer de cérvix metastásico y gestación: una situación poco frecuente que invita a la reflexión / Metastatic cervical cancer and pregnancy: an unusual and thought-provoking association

Introducción. La coexistencia de una gestación y cáncer de cérvix con diseminación metastásica es una situación excepcional que marcará, en el caso que presentamos, la evolución de la misma. Caso clínico. Paciente de 34 años, gestante de 13 semanas, acude a urgencias por metrorragia. Es diagnosticada de carcinoma invasivo de cérvix. La evolución se complica con tromboembolismo pulmonar masivo y detección de metástasis pulmonares y supraclaviculares. La paciente decide interrupción de la gestación en la semana 17 de la misma. Discusión. Este es un caso muy poco frecuente, no tanto así la presencia de cáncer de cérvix en estadio precoz en una gestante. Debe recordarnos la necesidad de examen con espéculo y citología en la primera visita de control del embarazo, muchas veces omitida en nuestra práctica habitual (AU)

Background. Coexistence of pregnancy and metastasic cervical cancer in an exceptional situation that will mark, in the presented case, the evolution of the pregnancy. Case. A 34-year-old woman, gravida 2 para 1, at 13 weeks’ gestation was taken to our emergency service with menorragia, and was admitted under the suspicion of invasive cervical cancer. Patient¿s evolution was complicated by the detection of supraclavicular metastasis and massive pulmonary tromboembolism. The patient decides voluntary interruption of pregnancy in the 17th week. Conclusions. This is a very unusual case, not as much the presence of an early stage cervical cancer in a pregnant woman. It reminds us of the need to carry out the mandatory gynecological exploration and smear in the first visit of prenatal care, often omitted in our dayly practice (AU)