The living microarray: a high-throughput platform for measuring transcription dynamics in single cells

<p>Abstract</p> <p>Background</p> <p>Current methods of measuring transcription in high-throughput have led to significant improvements in our knowledge of transcriptional regulation and Systems Biology. However, endpoint measurements obtained from methods that pool populations of cells are not amenable to studying time-dependent processes that show cell heterogeneity.</p> <p>Results</p> <p>Here we describe a high-throughput platform for measuring transcriptional changes in real time in single mammalian cells. By using reverse transfection microarrays we are able to transfect fluorescent reporter plasmids into 600 independent clusters of cells plated on a single microscope slide and image these clusters every 20 minutes. We use a fast-maturing, destabilized and nuclear-localized reporter that is suitable for automated segmentation to accurately measure promoter activity in single cells. We tested this platform with synthetic drug-inducible promoters that showed robust induction over 24 hours. Automated segmentation and tracking of over 11 million cell images during this period revealed that cells display substantial heterogeneity in their responses to the applied treatment, including a large proportion of transfected cells that do not respond at all.</p> <p>Conclusions</p> <p>The results from our single-cell analysis suggest that methods that measure average cellular responses, such as DNA microarrays, RT-PCR and chromatin immunoprecipitation, characterize a response skewed by a subset of cells in the population. Our method is scalable and readily adaptable to studying complex systems, including cell proliferation, differentiation and apoptosis.</p

Blood Parasites in Owls with Conservation Implications for the Spotted Owl (Strix occidentalis)

The three subspecies of Spotted Owl (Northern, Strix occidentalis caurina; California, S. o. occidentalis; and Mexican, S. o. lucida) are all threatened by habitat loss and range expansion of the Barred Owl (S. varia). An unaddressed threat is whether Barred Owls could be a source of novel strains of disease such as avian malaria (Plasmodium spp.) or other blood parasites potentially harmful for Spotted Owls. Although Barred Owls commonly harbor Plasmodium infections, these parasites have not been documented in the Spotted Owl. We screened 111 Spotted Owls, 44 Barred Owls, and 387 owls of nine other species for haemosporidian parasites (Leucocytozoon, Plasmodium, and Haemoproteus spp.). California Spotted Owls had the greatest number of simultaneous multi-species infections (44%). Additionally, sequencing results revealed that the Northern and California Spotted Owl subspecies together had the highest number of Leucocytozoon parasite lineages (n = 17) and unique lineages (n = 12). This high level of sequence diversity is significant because only one Leucocytozoon species (L. danilewskyi) has been accepted as valid among all owls, suggesting that L. danilewskyi is a cryptic species. Furthermore, a Plasmodium parasite was documented in a Northern Spotted Owl for the first time. West Coast Barred Owls had a lower prevalence of infection (15%) when compared to sympatric Spotted Owls (S. o. caurina 52%, S. o. occidentalis 79%) and Barred Owls from the historic range (61%). Consequently, Barred Owls on the West Coast may have a competitive advantage over the potentially immune compromised Spotted Owls

Stable transmission of reversible modifications: maintenance of epigenetic information through the cell cycle

Even though every cell in a multicellular organism contains the same genes, the differing spatiotemporal expression of these genes determines the eventual phenotype of a cell. This means that each cell type contains a specific epigenetic program that needs to be replicated through cell divisions, along with the genome, in order to maintain cell identity. The stable inheritance of these programs throughout the cell cycle relies on several epigenetic mechanisms. In this review, DNA methylation and histone methylation by specific histone lysine methyltransferases (KMT) and the Polycomb/Trithorax proteins are considered as the primary mediators of epigenetic inheritance. In addition, non-coding RNAs and nuclear organization are implicated in the stable transfer of epigenetic information. Although most epigenetic modifications are reversible in nature, they can be stably maintained by self-recruitment of modifying protein complexes or maintenance of these complexes or structures through the cell cycle

Birds of Two Oceans? Trans-Andean and Divergent Migration of Black Skimmers (Rynchops niger cinerascens) from the Peruvian Amazon

We are grateful for assistance from John Terborgh, Antonio Guerra Rosas, Marcos Maguiña, Inés Nole, John Takekawa, Lisa Ferguson, Juan Kapeshi, Nikanor Kapeshi, Cathy Bykowsky, Chi (Tim) Lam, Scott Robinson, Fabrice Schmitt, and Cesar Flores. Alex Jahn, Ugo Mellone, Sergio Lambertucci and one anonymous reviewer provided comments that helped improve the manuscript.Seasonal flooding compels some birds that breed in aquatic habitats in Amazonia to undertake annual migrations, yet we know little about how the complex landscape of the Amazon region is used seasonally by these species. The possibility of trans-Andes migration for Amazonian breeding birds has largely been discounted given the high geographic barrier posed by the Andean Cordillera and the desert habitat along much of the Pacific Coast. Here we demonstrate a trans-Andes route for Black Skimmers (Rynchops niger cinerascens) breeding on the Manu River (in the lowlands of Manu National Park, Perú), as well as divergent movement patterns both regionally and across the continent. Of eight skimmers tracked with satellite telemetry, three provided data on their outbound migrations, with two crossing the high Peruvian Andes to the Pacific. A third traveled over 1800 km to the southeast before transmissions ended in eastern Paraguay. One of the two trans-Andean migrants demonstrated a full round-trip migration back to its tagging location after traveling down the Pacific Coast from latitude 9° South to latitude 37° S, spending the austral summer in the Gulf of Arauco, Chile. This is the first documentation of a trans-Andes migration observed for any bird breeding in lowland Amazonia. To our knowledge, this research also documents the first example of a tropical-breeding waterbird migrating out of the tropics to spend the non-breeding season in the temperate summer, this being the reverse pattern with respect to seasonality for austral migrants in general.Yeshttp://www.plosone.org/static/editorial#pee

“Completely out-at-sea” with “two-gender medicine”: A qualitative analysis of physician-side barriers to providing healthcare for transgender patients

<p>Abstract</p> <p>Background</p> <p>Members of the transgender community have identified healthcare access barriers, yet a corresponding inquiry into healthcare provider perspectives has lagged. Our aim was to examine physician perceptions of barriers to healthcare provision for transgender patients.</p> <p>Methods</p> <p>This was a qualitative study with physician participants from Ontario, Canada. Semi-structured interviews were used to capture a progression of ideas related to barriers faced by physicians when caring for trans patients. Qualitative data were then transcribed verbatim and analysed with an emergent grounded theory approach.</p> <p>Results</p> <p>A total of thirteen (13) physician participants were interviewed. Analysis revealed healthcare barriers that grouped into five themes: Accessing resources, medical knowledge deficits, ethics of transition-related medical care, diagnosing vs. pathologising trans patients, and health system determinants. A centralising theme of “not knowing where to go or who to talk to” was also identified.</p> <p>Conclusions</p> <p>The findings of this study show that physicians perceive barriers to the care of trans patients, and that these barriers are multifactorial. Access barriers impede physicians when referring patients to specialists or searching for reliable treatment information. Clinical management of trans patients is complicated by a lack of knowledge, and by ethical considerations regarding treatments—which can be unfamiliar or challenging to physicians. The disciplinary division of responsibilities within medicine further complicates care; few practitioners identify trans healthcare as an interest area, and there is a tendency to overemphasise trans status in mental health evaluations. Failure to recognise and accommodate trans patients within sex-segregated healthcare systems leads to deficient health policy. The findings of this study suggest potential solutions to trans healthcare barriers at the informational level—with increased awareness of clinical guidelines and by including trans health issues in medical education—and at the institutional level, with support for both trans-focused and trans-friendly primary care models.</p