3D dSTORM imaging reveals novel detail of ryanodine receptor localization in rat cardiac myocytes

Cardiomyocyte contraction is dependent on Ca2+ release from ryanodine receptors (RyRs). However, the precise localization of RyRs remains unknown, due to shortcomings of imaging techniques which are diffraction limited or restricted to 2D. We aimed to determine the 3D nanoscale organization of RyRs in rat cardiomyocytes by employing direct stochastic optical reconstruction microscopy (dSTORM) with phase ramp technology. Initial observations at the cell surface showed an undulating organization of RyR clusters, resulting in their frequent overlap in the z‐axis and obscured detection by 2D techniques. Non‐overlapping clusters were imaged to create a calibration curve for estimating RyR number based on recorded fluorescence blinks. Employing this method at the cell surface and interior revealed smaller RyR clusters than 2D estimates, as erroneous merging of axially aligned RyRs was circumvented. Functional groupings of RyR clusters (Ca2+ release units, CRUs), contained an average of 18 and 23 RyRs at the surface and interior, respectively, although half of all CRUs contained only a single ‘rogue’ RyR. Internal CRUs were more tightly packed along z‐lines than surface CRUs, contained larger and more numerous RyR clusters, and constituted ∼75% of the roughly 1 million RyRs present in an average cardiomyocyte. This complex internal 3D geometry was underscored by correlative imaging of RyRs and t‐tubules, which enabled quantification of dyadic and non‐dyadic RyR populations. Mirroring differences in CRU size and complexity, Ca2+ sparks originating from internal CRUs were of longer duration than those at the surface. These data provide novel, nanoscale insight into RyR organization and function across cardiomyocytes

Simulation-based characterization of electrolyte and small molecule diffusion in oriented mesoporous silica thin films

We developed a new workflow for simulating ion reaction-adsorption-diffusion in nanoporous silica-based materials that are resolved through electron microscopy. Firstly, we propose a matched filtering procedure to identify and segment unique porous regions of the material that will be subject to PDE simulation. Secondly, we perform reaction-adsorption-diffusion PDE simulations on representative material regions that are then applied to characterize the entire microscopy-resolved film surface. Using this model, we examine the capacity of a recently synthesized mesoporous film to tune small molecule permeation through modulating the material permeability, surface chemistryincluding buffering and adsorption, as well as electrolyte composition. Specifically, we find that our proposed matched filtering approach reliably discriminates hexagonal close packed (HCP) porous regions (bulk) from characterized defect regions in transmission electron microscopy (EM) data for nanoporous silica films. Further, based on our implementation of a pH-/surface-chemistry dependent Poisson-Nernst-Planck (PNP) model that is consistent with existing experimental measurements of KCl and CaCl2 conductance, we characterize ion and 5(6)-Carboxyfluorescein (CF) dye permeability in silica-based nanoporous materials over a broad range of ionic strengths, pHs, and surface chemistries. Using this protocol, we probe conditions for selectively tuning small molecule permeability based on mesoporous film pore size, surface charge, ionic strength and surface reactions in the rapid-equilibrium limit. <br /

Simulation-based characterization of electrolyte and small molecule diffusion in oriented mesoporous silica thin films

We developed a new workflow for simulating ion reaction-adsorption-diffusion in nanoporous silica-based materials that are resolved through electron microscopy. Firstly, we propose a matched filtering procedure to identify and segment unique porous regions of the material that will be subject to PDE simulation. Secondly, we perform reaction-adsorption-diffusion PDE simulations on representative material regions that are then applied to characterize the entire microscopy-resolved film surface. Using this model, we examine the capacity of a recently synthesized mesoporous film to tune small molecule permeation through modulating the material permeability, surface chemistry<br>including buffering and adsorption, as well as electrolyte composition. Specifically, we find that our proposed matched filtering approach reliably discriminates hexagonal close packed (HCP) porous regions (bulk) from characterized defect regions in transmission electron microscopy (EM) data for nanoporous silica films. Further, based on our implementation of a pH-/surface-chemistry dependent Poisson-Nernst-Planck (PNP) model that is consistent with existing experimental measurements of KCl and CaCl2 conductance, we characterize ion and 5(6)-Carboxyfluorescein (CF) dye permeability in silica-based nanoporous materials over a broad range of ionic strengths, pHs, and surface chemistries. Using this protocol, we probe conditions for selectively tuning small molecule permeability based on mesoporous film pore size, surface charge, ionic strength and surface reactions in the rapid-equilibrium limit. <br><br

VarGoats project: a dataset of 1159 whole-genome sequences to dissect Capra hircus global diversity

International audienceBackground Since their domestication 10,500 years ago, goat populations with distinctive genetic backgrounds have adapted to a broad variety of environments and breeding conditions. The VarGoats project is an international 1000-genome resequencing program designed to understand the consequences of domestication and breeding on the genetic diversity of domestic goats and to elucidate how speciation and hybridization have modeled the genomes of a set of species representative of the genus Capra . Findings A dataset comprising 652 sequenced goats and 507 public goat sequences, including 35 animals representing eight wild species, has been collected worldwide. We identified 74,274,427 single nucleotide polymorphisms (SNPs) and 13,607,850 insertion-deletions (InDels) by aligning these sequences to the latest version of the goat reference genome (ARS1). A Neighbor-joining tree based on Reynolds genetic distances showed that goats from Africa, Asia and Europe tend to group into independent clusters. Because goat breeds from Oceania and Caribbean (Creole) all derive from imported animals, they are distributed along the tree according to their ancestral geographic origin. Conclusions We report on an unprecedented international effort to characterize the genome-wide diversity of domestic goats. This large range of sequenced individuals represents a unique opportunity to ascertain how the demographic and selection processes associated with post-domestication history have shaped the diversity of this species. Data generated for the project will also be extremely useful to identify deleterious mutations and polymorphisms with causal effects on complex traits, and thus will contribute to new knowledge that could be used in genomic prediction and genome-wide association studies

Conservation status of the world's skinks (Scincidae): taxonomic and geographic patterns in extinction risk

Our knowledge of the conservation status of reptiles, the most diverse class of terrestrial vertebrates, has improved dramatically over the past decade, but still lags behind that of the other tetrapod groups. Here, we conduct the first comprehensive evaluation (~92% of the world's ~1714 described species) of the conservation status of skinks (Scincidae), a speciose reptile family with a worldwide distribution. Using International Union for Conservation of Nature (IUCN) criteria, we report that ~20% of species are threatened with extinction, and nine species are Extinct or Extinct in the Wild. The highest levels of threat are evident in Madagascar and the Neotropics, and in the subfamilies Mabuyinae, Eugongylinae and Scincinae. The vast majority of threatened skink species were listed based primarily on their small geographic ranges (Criterion B, 83%; Criterion D2, 13%). Although the population trend of 42% of species was stable, 14% have declining populations. The key threats to skinks are habitat loss due to agriculture, invasive species, and biological resource use (e.g., hunting, timber harvesting). The distributions of 61% of species do not overlap with protected areas. Despite our improved knowledge of the conservation status of the world's skinks, 8% of species remain to be assessed, and 14% are listed as Data Deficient. The conservation status of almost a quarter of the world's skink species thus remains unknown. We use our updated knowledge of the conservation status of the group to develop and outline the priorities for the conservation assessment and management of the world's skink species

Geographical contrasts of Y‐chromosomal haplogroups from wild and domestic goats reveal ancient migrations and recent introgressions

International audienceBy their paternal transmission, Y-chromosomal haplotypes are sensitive markers of population history and male-mediated introgression. Previous studies identified biallelic single-nucleotide variants in the SRY, ZFY and DDX3Y genes, which in domestic goats identified four major Y-chromosomal haplotypes, Y1A, Y1B, Y2A and Y2B, with a marked geographical partitioning. Here, we extracted goat Y-chromosomal variants from whole-genome sequences of 386 domestic goats (75 breeds) and seven wild goat species, which were generated by the VarGoats goat genome project. Phylogenetic analyses indicated domestic haplogroups corresponding to Y1B, Y2A and Y2B, respectively, whereas Y1A is split into Y1AA and Y1AB. All five haplogroups were detected in 26 ancient DNA samples from southeast Europe or Asia. Haplotypes from present-day bezoars are not shared with domestic goats and are attached to deep nodes of the trees and networks. Haplogroup distributions for 186 domestic breeds indicate ancient paternal population bottlenecks and expansions during migrations into northern Europe, eastern and southern Asia, and Africa south of the Sahara. In addition, sharing of haplogroups indicates male-mediated introgressions, most notably an early gene flow from Asian goats into Madagascar and the crossbreeding that in the 19th century resulted in the popular Boer and Anglo-Nubian breeds. More recent introgressions are those from European goats into the native Korean goat population and from Boer goat into Uganda, Kenya, Tanzania, Malawi and Zimbabwe. This study illustrates the power of the Y-chromosomal variants for reconstructing the history of domestic species with a wide geographical range