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Rare circulating tumor cells (CTCs) present in the bloodstream of patients with cancer provide a potentially accessible source for detection, characterization, and monitoring of nonhematological cancers. We previously demonstrated the effectiveness of a microfluidic device, the CTC-Chip, in capturing these epithelial cell adhesion molecule (EpCAM)-expressing cells using antibody-coated microposts. Here, we describe a high-throughput microfluidic mixing device, the herringbone-chip, or “HB-Chip,” which provides an enhanced platform for CTC isolation. The HB-Chip design applies passive mixing of blood cells through the generation of microvortices to significantly increase the number of interactions between target CTCs and the antibody-coated chip surface. Efficient cell capture was validated using defined numbers of cancer cells spiked into control blood, and clinical utility was demonstrated in specimens from patients with prostate cancer. CTCs were detected in 14 of 15 (93%) patients with metastatic disease (median = 63 CTCs/mL, mean = 386 ± 238 CTCs/mL), and the tumor-specific TMPRSS2-ERG translocation was readily identified following RNA isolation and RT-PCR analysis. The use of transparent materials allowed for imaging of the captured CTCs using standard clinical histopathological stains, in addition to immunofluorescence-conjugated antibodies. In a subset of patient samples, the low shear design of the HB-Chip revealed microclusters of CTCs, previously unappreciated tumor cell aggregates that may contribute to the hematogenous dissemination of cancer.

A new method allows the collection of circulating tumour cells (CTCs) despite their rarity; transcriptome sequencing of CTCs could allow identification of pathways involved in metastasis. WNT implicated in metastasis Circulating tumour cells (CTCs) shed from primary tumours are thought to initiate metastasis in distant organs. They are increasingly appreciated as potential biomarkers of disease progression and therapeutic response in patients with cancer. Their low frequency, in comparison with normal blood cells, means that it is a major technological challenge to characterize them fully. Daniel Haber and colleagues have optimized previous microfluidic approaches to capture CTCs from an endogenous mouse pancreatic cancer model. Using RNA sequencing, RNA transcripts enriched in CTCs compared with the primary tumours were identified. The proto-oncogene protein Wnt2 was also identified. The authors were also able to capture CTCs from patients with pancreatic cancer patients, which, like their mouse counterparts, show evidence of upregulation of WNT signalling. Circulating tumour cells (CTCs) shed into blood from primary cancers include putative precursors that initiate distal metastases 1 . Although these cells are extraordinarily rare, they may identify cellular pathways contributing to the blood-borne dissemination of cancer. Here, we adapted a microfluidic device 2 for efficient capture of CTCs from an endogenous mouse pancreatic cancer model 3 and subjected CTCs to single-molecule RNA sequencing 4 , identifying Wnt2 as a candidate gene enriched in CTCs. Expression of WNT2 in pancreatic cancer cells suppresses anoikis, enhances anchorage-independent sphere formation, and increases metastatic propensity in vivo . This effect is correlated with fibronectin upregulation and suppressed by inhibition of MAP3K7 (also known as TAK1) kinase. In humans, formation of non-adherent tumour spheres by pancreatic cancer cells is associated with upregulation of multiple WNT genes, and pancreatic CTCs revealed enrichment for WNT signalling in 5 out of 11 cases. Thus, molecular analysis of CTCs may identify candidate therapeutic targets to prevent the distal spread of cancer.

Nature 487, 510–513 (2012); doi:10.1038/nature11217 In this Letter, we omitted the following accession information. The digital gene expression matrix and the Helicos single-molecule sequence data from which it was derived are available at the NCBI GEO database with accession number GSE40176. A total of 44 samples were uploaded including 12 mouse and 32 human samples.

Analysis of the yeast DEAD-box nucleic acid helicase Mss116p provides a structural model for how DEAD-box proteins recognize and unwind RNA duplexes. DEAD-box protein recognition of duplex RNA Alan Lambowitz and colleagues have solved the structure of Mss116, a yeast DEAD-box protein, bound to double-stranded RNA and a DNA–RNA hybrid. DEAD-box proteins are nucleic acid helicases that function to unwind and remodel RNAs and RNA-protein complexes. The structure shows the enzyme in a pre-unwound state, with ATP and RNA bound to different domains; it is proposed that a conformational change brings them together during unwinding. The structure also reveals how the enzyme discriminates between A-form RNA and B-form DNA. DEAD-box proteins are the largest family of nucleic acid helicases, and are crucial to RNA metabolism throughout all domains of life 1 , 2 . They contain a conserved ‘helicase core’ of two RecA-like domains (domains (D)1 and D2), which uses ATP to catalyse the unwinding of short RNA duplexes by non-processive, local strand separation 3 . This mode of action differs from that of translocating helicases and allows DEAD-box proteins to remodel large RNAs and RNA–protein complexes without globally disrupting RNA structure 4 . However, the structural basis for this distinctive mode of RNA unwinding remains unclear. Here, structural, biochemical and genetic analyses of the yeast DEAD-box protein Mss116p indicate that the helicase core domains have modular functions that enable a novel mechanism for RNA-duplex recognition and unwinding. By investigating D1 and D2 individually and together, we find that D1 acts as an ATP-binding domain and D2 functions as an RNA-duplex recognition domain. D2 contains a nucleic-acid-binding pocket that is formed by conserved DEAD-box protein sequence motifs and accommodates A-form but not B-form duplexes, providing a basis for RNA substrate specificity. Upon a conformational change in which the two core domains join to form a ‘closed state’ with an ATPase active site, conserved motifs in D1 promote the unwinding of duplex substrates bound to D2 by excluding one RNA strand and bending the other. Our results provide a comprehensive structural model for how DEAD-box proteins recognize and unwind RNA duplexes. This model explains key features of DEAD-box protein function and affords a new perspective on how the evolutionarily related cores of other RNA and DNA helicases diverged to use different mechanisms.

Growth faltering in children (low length for age or low weight for length) during the first 1,000 days of life (from conception to 2 years of age) influences short-term and long-term health and survival 1 , 2 . Interventions such as nutritional supplementation during pregnancy and the postnatal period could help prevent growth faltering, but programmatic action has been insufficient to eliminate the high burden of stunting and wasting in low- and middle-income countries. Identification of age windows and population subgroups on which to focus will benefit future preventive efforts. Here we use a population intervention effects analysis of 33 longitudinal cohorts (83,671 children, 662,763 measurements) and 30 separate exposures to show that improving maternal anthropometry and child condition at birth accounted for population increases in length-for-age z -scores of up to 0.40 and weight-for-length z -scores of up to 0.15 by 24 months of age. Boys had consistently higher risk of all forms of growth faltering than girls. Early postnatal growth faltering predisposed children to subsequent and persistent growth faltering. Children with multiple growth deficits exhibited higher mortality rates from birth to 2 years of age than children without growth deficits (hazard ratios 1.9 to 8.7). The importance of prenatal causes and severe consequences for children who experienced early growth faltering support a focus on pre-conception and pregnancy as a key opportunity for new preventive interventions. Analysis of data from 33 longitudinal cohorts from low- and middle-income countries indicates that conditions during pre-conception, pregnancy and the first few months of life are crucial in determining the risk of growth faltering in young children.

The Transatlantic Slave Trade transported more than 9 million Africans to the Americas between the early 16th and the mid-19th centuries. We performed a genome-wide analysis using 6,267 individuals from 25 populations to infer how different African groups contributed to North-, South-American, and Caribbean populations, in the context of geographic and geopolitical factors, and compared genetic data with demographic history records of the Transatlantic Slave Trade. We observed that West-Central Africa and Western Africa-associated ancestry clusters are more prevalent in northern latitudes of the Americas, whereas the South/East Africa-associated ancestry cluster is more prevalent in southern latitudes of the Americas. This pattern results from geographic and geopolitical factors leading to population differentiation. However, there is a substantial decrease in the between-population differentiation of the African gene pool within the Americas, when compared with the regions of origin from Africa, underscoring the importance of historical factors favoring admixture between individuals with different African origins in the New World. This between-population homogenization in the Americas is consistent with the excess of West-Central Africa ancestry (the most prevalent in the Americas) in the United States and Southeast-Brazil, with respect to historical-demography expectations. We also inferred that in most of the Americas, intercontinental admixture intensification occurred between 1750 and 1850, which correlates strongly with the peak of arrivals from Africa. This study contributes with a population genetics perspective to the ongoing social, cultural, and political debate regarding ancestry, admixture, and the mestizaje process in the Americas.

The comparison of Mycobacterium tuberculosis bacterial genotypes with phenotypic, demographic, geospatial and clinical data improves our understanding of how strain lineage influences the development of drug-resistance and the spread of tuberculosis. To investigate the association of Mycobacterium tuberculosis bacterial genotype with drug-resistance. Drug susceptibility testing together with genotyping using both 15-loci MIRU-typing and spoligotyping, was performed on 2,139 culture positive isolates, each from a different patient in Lima, Peru. Demographic, geospatial and socio-economic data were collected using questionnaires, global positioning equipment and the latest national census. The Latin American Mediterranean (LAM) clade (OR 2.4, p<0.001) was significantly associated with drug-resistance and alone accounted for more than half of all drug resistance in the region. Previously treated patients, prisoners and genetically clustered cases were also significantly associated with drug-resistance (OR's 2.5, 2.4 and 1.8, p<0.001, p<0.05, p<0.001 respectively). Tuberculosis disease caused by the LAM clade was more likely to be drug resistant independent of important clinical, genetic and socio-economic confounding factors. Explanations for this include; the preferential co-evolution of LAM strains in a Latin American population, a LAM strain bacterial genetic background that favors drug-resistance or the \"founder effect\" from pre-existing LAM strains disproportionately exposed to drugs.

Cyclosporin A (CsA) has recently been proposed for use in the early phase after traumatic brain injury (TBI), for its ability to preserve mitochondrial integrity in experimental brain injury models, and thereby provide improved behavioral outcomes as well as significant histological protection. The aim of this prospective, randomized, double-blind, dual-center, placebo-controlled trial was to evaluate the safety, tolerability, and pharmacokinetics of a single intravenous infusion of CsA in patients with severe TBI. Fifty adult severe TBI patients were enrolled over a 22-month period. Within 12 h of the injury patients received 5 mg/kg of CsA infused over 24 h, or placebo. Blood urea nitrogen (BUN), creatinine, hemoglobin, platelets, white blood cell count (WBC), and a hepatic panel were monitored on admission, and at 12, 24, 36, and 48 h, and on days 4 and 7. Potential adverse events (AEs) were also recorded. Neurological outcome was recorded at 3 and 6 months after injury. This study revealed only transient differences in BUN levels at 24 and 48 h and for WBC counts at 24 h between the CsA and placebo patients. These modest differences were not clinically significant in that they did not negatively impact on patient course. Both BUN and creatinine values, markers of renal function, remained within their normal limits over the entire monitoring period. There were no significant differences in other mean laboratory values, or in the incidence of AEs at any other measured time point. Also, no significant difference was demonstrated for neurological outcome. Based on these results, we report a good safety profile of CsA infusion when given at the chosen dose of 5 mg/kg, infused over 24 h, during the early phase after severe head injury in humans, with the aim of neuroprotection.

Adherence by Helicobacter pylori increases the risk of gastric disease. Here, we report that more than 95% of strains that bind fucosylated blood group antigen bind A, B, and O antigens (generalists), whereas 60% of adherent South American Amerindian strains bind blood group O antigens best (specialists). This specialization coincides with the unique predominance of blood group O in these Amerindians. Strains differed about 1500-fold in binding affinities, and diversifying selection was evident in babA sequences. We propose that cycles of selection for increased and decreased bacterial adherence contribute to babA diversity and that these cycles have led to gradual replacement of generalist binding by specialist binding in blood group O-dominant human populations.