Innate-like control of human iNKT cell autoreactivity via the hypervariable CDR3beta loop

Invariant Natural Killer T cells (iNKT) are a versatile lymphocyte subset with important roles in both host defense and immunological tolerance. They express a highly conserved TCR which mediates recognition of the non-polymorphic, lipid-binding molecule CD1d. The structure of human iNKT TCRs is unique in that only one of the six complementarity determining region (CDR) loops, CDR3beta, is hypervariable. The role of this loop for iNKT biology has been controversial, and it is unresolved whether it contributes to iNKT TCR:CD1d binding or antigen selectivity. On the one hand, the CDR3beta loop is dispensable for iNKT TCR binding to CD1d molecules presenting the xenobiotic alpha-galactosylceramide ligand KRN7000, which elicits a strong functional response from mouse and human iNKT cells. However, a role for CDR3beta in the recognition of CD1d molecules presenting less potent ligands, such as self-lipids, is suggested by the clonal distribution of iNKT autoreactivity. We demonstrate that the human iNKT repertoire comprises subsets of greatly differing TCR affinity to CD1d, and that these differences relate to their autoreactive functions. These functionally different iNKT subsets segregate in their ability to bind CD1d-tetramers loaded with the partial agonist alpha-linked glycolipid antigen OCH and structurally different endogenous beta-glycosylceramides. Using surface plasmon resonance with recombinant iNKT TCRs and different ligand-CD1d complexes, we demonstrate that the CDR3beta sequence strongly impacts on the iNKT TCR affinity to CD1d, independent of the loaded CD1d ligand. Collectively our data reveal a crucial role for CDR3beta for the function of human iNKT cells by tuning the overall affinity of the iNKT TCR to CD1d. This mechanism is relatively independent of the bound CD1d ligand and thus forms the basis of an inherent, CDR3beta dependent functional hierarchy of human iNKT cells.

Assessment of letrozole and tamoxifen alone and in sequence for postmenopausal women with steroid hormone receptor-positive breast cancer: the BIG 1-98 randomised clinical trial at 8·1 years median follow-up

Postmenopausal women with hormone receptor-positive early breast cancer have persistent, long-term risk of breast-cancer recurrence and death. Therefore, trials assessing endocrine therapies for this patient population need extended follow-up. We present an update of efficacy outcomes in the Breast International Group (BIG) 1-98 study at 8·1 years median follow-up.

Natural variations at position 93 of the invariant Vα24-Jα18 α chain of human iNKT-cell TCRs strongly impact on CD1d binding

Human invariant natural killer T (NKT) cell TCRs bind to CD1d via an "invariant" Vα24-Jα18 chain (iNKTα) paired to semi-invariant Vβ11 chains (iNKTβ). Single-amino acid variations at position 93 (p93) of iNKTα, immediately upstream of the "invariant" CDR3α region, have been reported in a substantial proportion of human iNKT-cell clones (4-30%). Although p93, a serine in most human iNKT-cell TCRs, makes no contact with CD1d, it could affect CD1d binding by altering the conformation of the crucial CDR3α loop. By generating recombinant refolded iNKT-cell TCRs, we show that natural single-nucleotide variations in iNKTα, translating to serine, threonine, asparagine or isoleucine at p93, exert a powerful effect on CD1d binding, with up to 28-fold differences in affinity between these variants. This effect was observed with CD1d loaded with either the artificial α-galactosylceramide antigens KRN7000 or OCH, or the endogenous glycolipid β-galactosylceramide, and its importance for autoreactive recognition of endogenous lipids was demonstrated by the binding of variant iNKT-cell TCR tetramers to cell surface expressed CD1d. The serine-containing variant showed the strongest CD1d binding, offering an explanation for its predominance in vivo. Complementary molecular dynamics modeling studies were consistent with an impact of p93 on the conformation of the CDR3α loop.

Obesity and risk of recurrence or death after adjuvant endocrine therapy with letrozole or tamoxifen in the breast international group 1-98 trial

To examine the association of baseline body mass index (BMI) with the risk of recurrence or death in postmenopausal women with early-stage breast cancer receiving adjuvant tamoxifen or letrozole in the Breast International Group (BIG) 1-98 trial at 8.7 years of median follow-up.

Structure and binding kinetics of three different human CD1d-alpha-galactosylceramide-specific T cell receptors

Invariant human TCR Valpha24-Jalpha18+/Vbeta11+ NKT cells (iNKT) are restricted by CD1d-alpha-glycosylceramides. We analyzed crystal structures and binding characteristics for an iNKT TCR plus two CD1d-alpha-GalCer-specific Vbeta11+ TCRs that use different TCR Valpha chains. The results were similar to those previously reported for MHC-peptide-specific TCRs, illustrating the versatility of the TCR platform. Docking TCR and CD1d-alpha-GalCer structures provided plausible insights into their interaction. The model supports a diagonal orientation of TCR on CD1d and suggests that complementarity determining region (CDR)3alpha, CDR3beta, and CDR1beta interact with ligands presented by CD1d, whereas CDR2beta binds to the CD1d alpha1 helix. This docking provides an explanation for the dominant usage of Vbeta11 and Vbeta8.2 chains by human and mouse iNKT cells, respectively, for recognition of CD1d-alpha-GalCer.

Radiotherapy and temozolomide for newly diagnosed glioblastoma: recursive partitioning analysis of the EORTC 26981/22981-NCIC CE3 phase III randomized trial

PURPOSE: The European Organisation for Research and Treatment of Cancer and National Cancer Institute of Canada trial on temozolomide (TMZ) and radiotherapy (RT) in glioblastoma (GBM) has demonstrated that the combination of TMZ and RT conferred a significant and meaningful survival advantage compared with RT alone. We evaluated in this trial whether the recursive partitioning analysis (RPA) retains its overall prognostic value and what the benefit of the combined modality is in each RPA class. PATIENTS AND METHODS: Five hundred seventy-three patients with newly diagnosed GBM were randomly assigned to standard postoperative RT or to the same RT with concomitant TMZ followed by adjuvant TMZ. The primary end point was overall survival. The European Organisation for Research and Treatment of Cancer RPA used accounts for age, WHO performance status, extent of surgery, and the Mini-Mental Status Examination. RESULTS: Overall survival was statistically different among RPA classes III, IV, and V, with median survival times of 17, 15, and 10 months, respectively, and 2-year survival rates of 32%, 19%, and 11%, respectively (P < .0001). Survival with combined TMZ/RT was higher in RPA class III, with 21 months median survival time and a 43% 2-year survival rate, versus 15 months and 20% for RT alone (P = .006). In RPA class IV, the survival advantage remained significant, with median survival times of 16 v 13 months, respectively, and 2-year survival rates of 28% v 11%, respectively (P = .0001). In RPA class V, however, the survival advantage of RT/TMZ was of borderline significance (P = .054). CONCLUSION: RPA retains its prognostic significance overall as well as in patients receiving RT with or without TMZ for newly diagnosed GBM, particularly in classes III and IV.

The European Modern Pollen Database (EMPD) project

Modern pollen samples provide an invaluable research tool for helping to interpret the quaternary fossil pollen record, allowing investigation of the relationship between pollen as the proxy and the environmental parameters such as vegetation, land-use, and climate that the pollen proxy represents. The European Modern Pollen Database (EMPD) is a new initiative within the European Pollen Database (EPD) to establish a publicly accessible repository of modern (surface sample) pollen data. This new database will complement the EPD, which at present holds only fossil sedimentary pollen data. The EMPD is freely available online to the scientific community and currently has information on almost 5,000 pollen samples from throughout the Euro-Siberian and Mediterranean regions, contributed by over 40 individuals and research groups. Here we describe how the EMPD was constructed, the various tables and their fields, problems and errors, quality controls, and continuing efforts to improve the available data.

Erratum to: The European Modern Pollen Database (EMPD) project

Unfortunately, the list of authors contains a number of duplications, omissions and other errors in the original publication of the article. The correct list appears in this erratum.

Electroelasticity of charged black branes

We present the first-order corrected dynamics of fluid branes carrying higher-form charge by obtaining the general form of their equations of motion to pole-dipole order in the absence of external forces. Assuming linear response theory, we characterize the corresponding effective theory of stationary bent charged (an)isotropic fluid branes in terms of two sets of response coefficients, the Young modulus and the piezoelectric moduli. We subsequently find large classes of examples in gravity of this effective theory, by constructing stationary strained charged black brane solutions to first order in a derivative expansion. Using solution generating techniques and bent neutral black branes as a seed solution, we obtain a class of charged black brane geometries carrying smeared Maxwell charge in Einstein-Maxwell-dilaton gravity. In the specific case of ten-dimensional space-time we furthermore use T-duality to generate bent black branes with higher-form charge, including smeared D-branes of type II string theory. By subsequently measuring the bending moment and the electric dipole moment which these geometries acquire due to the strain, we uncover that their form is captured by classical electroelasticity theory. In particular, we find that the Young modulus and the piezoelectric moduli of our strained charged black brane solutions are parameterized by a total of 4 response coefficients, both for the isotropic as well as anisotropic cases.

Direct observation of liquid crystals using cryo-TEM: Specimen preparation and low-dose imaging

Liquid crystals (LCs) represent a challenging group of materials for direct transmission electron microscopy (TEM) studies due to the complications in specimen preparation and the severe radiation damage. In this paper, we summarize a series of specimen preparation methods, including thin film and cryo-sectioning approaches, as a comprehensive toolset enabling high-resolution direct cryo-TEM observation of a broad range of LCs. We also present comparative analysis using cryo-TEM and replica freeze-fracture TEM on both thermotropic and lyotropic LCs. In addition to the revisits of previous practices, some new concepts are introduced, e.g., suspended thermotropic LC thin films, combined high-pressure freezing and cryo-sectioning of lyotropic LCs, and the complementary applications of direct TEM and indirect replica TEM techniques. The significance of subnanometer resolution cryo-TEM observation is demonstrated in a few important issues in LC studies, including providing direct evidences for the existence of nanoscale smectic domains in nematic bent-core thermotropic LCs, comprehensive understanding of the twist-bend nematic phase, and probing the packing of columnar aggregates in lyotropic chromonic LCs. Direct TEM observation opens ways to a variety of TEM techniques, suggesting that TEM (replica, cryo, and in situ techniques), in general, may be a promising part of the solution to the lack of effective structural probe at the molecular scale in LC studies. Microsc. Res. Tech. 77:754-772, 2014. © 2014 Wiley Periodicals, Inc.

Cell force measurements in 3D microfabricated environments based on compliant cantilevers.

We report the fabrication, functionalization and testing of microdevices for cell culture and cell traction force measurements in three-dimensions (3D). The devices are composed of bent cantilevers patterned with cell-adhesive spots not lying on the same plane, and thus suspending cells in 3D. The cantilevers are soft enough to undergo micrometric deflections when cells pull on them, allowing cell forces to be measured by means of optical microscopy. Since individual cantilevers are mechanically independent of each other, cell traction forces are determined directly from cantilever deflections. This proves the potential of these new devices as a tool for the quantification of cell mechanics in a system with well-defined 3D geometry and mechanical properties.

Prehistoric genomes reveal the genetic foundation and cost of horse domestication

The domestication of the horse revolutionized warfare, trade, and the exchange of people and ideas. This at least 5,500-y-long process, which ultimately transformed wild horses into the hundreds of breeds living today, is difficult to reconstruct from archeological data and modern genetics alone. We therefore sequenced two complete horse genomes, predating domestication by thousands of years, to characterize the genetic footprint of domestication. These ancient genomes reveal predomestic population structure and a significant fraction of genetic variation shared with the domestic breeds but absent from Przewalski’s horses. We find positive selection on genes involved in various aspects of locomotion, physiology, and cognition. Finally, we show that modern horse genomes contain an excess of deleterious mutations, likely representing the genetic cost of domestication.

Reclassification of Actinobacillus muris as Muribacter muris gen. nov. comb. nov.

To reinvestigate the taxonomy of [Actinobacillus] muris, 474 strains mainly from mice and rats were characterized by phenotype and 130 strains selected for genotypic characterization by 16S rRNA and partial rpoB gene sequencing. The type strain was further investigated by whole genome sequencing. Phylogenetic analysis of the DNA sequences showed one monophyletic group with intra group similarities of 96.7 % and 97.2 % for 16S rRNA and rpoB genes, respectively. The lowest 16S rRNA similarity to the closest related valid named taxon outside the group was 95.9 % to the type strain of [Pasteurella] pneumotropica. The closest related taxon based on rpoB sequence comparison was 'Haemophilus influenzae-murium' with 88.4 %. A new genus, Muribacter is proposed based on a distinct phylogenetic position based on 16S rRNA and rpoB gene sequence comparisons with major divergence to the existing genera of Pasteurellaceae. The new genus includes the characteristics of [Actinobacillus] muris with the emendation that acid formation from (-)-D-mannitol is variable as well the hydrolysis of esculin while the α-glucosidase test is positive. There is no requirement for exogenously supplied nicotinamide adenine dinucleotide (V factor) for the majority of strains investigated, however, one strain was found positive. The major fatty acids of the type strain of Muribacter muris were C 14:0, C 14:0 3OH/C 16:1 ISOI, C 16:1 ω7c and C 16:0 which is in line with most genera of Pasteurellaceae. The type strain of Muribacter muris is CCUG 16938T ( = NCTC 12432T = ATCC 49577T).

Evolutionary Genomics and Conservation of the Endangered Przewalski's Horse.

Przewalski's horses (PHs, Equus ferus ssp. przewalskii) were discovered in the Asian steppes in the 1870s and represent the last remaining true wild horses. PHs became extinct in the wild in the 1960s but survived in captivity, thanks to major conservation efforts. The current population is still endangered, with just 2,109 individuals, one-quarter of which are in Chinese and Mongolian reintroduction reserves [1]. These horses descend from a founding population of 12 wild-caught PHs and possibly up to four domesticated individuals [2-4]. With a stocky build, an erect mane, and stripped and short legs, they are phenotypically and behaviorally distinct from domesticated horses (DHs, Equus caballus). Here, we sequenced the complete genomes of 11 PHs, representing all founding lineages, and five historical specimens dated to 1878-1929 CE, including the Holotype. These were compared to the hitherto-most-extensive genome dataset characterized for horses, comprising 21 new genomes. We found that loci showing the most genetic differentiation with DHs were enriched in genes involved in metabolism, cardiac disorders, muscle contraction, reproduction, behavior, and signaling pathways. We also show that DH and PH populations split ∼45,000 years ago and have remained connected by gene-flow thereafter. Finally, we monitor the genomic impact of ∼110 years of captivity, revealing reduced heterozygosity, increased inbreeding, and variable introgression of domestic alleles, ranging from non-detectable to as much as 31.1%. This, together with the identification of ancestry informative markers and corrections to the International Studbook, establishes a framework for evaluating the persistence of genetic variation in future reintroduced populations.

Tracking the origins of Yakutian horses and the genetic basis for their fast adaptation to subarctic environments

Yakutia, Sakha Republic, in the Siberian Far East, represents one of the coldest places on Earth, with winter record temperatures dropping below -70 °C. Nevertheless, Yakutian horses survive all year round in the open air due to striking phenotypic adaptations, including compact body conformations, extremely hairy winter coats, and acute seasonal differences in metabolic activities. The evolutionary origins of Yakutian horses and the genetic basis of their adaptations remain, however, contentious. Here, we present the complete genomes of nine present-day Yakutian horses and two ancient specimens dating from the early 19th century and ∼5,200 y ago. By comparing these genomes with the genomes of two Late Pleistocene, 27 domesticated, and three wild Przewalski's horses, we find that contemporary Yakutian horses do not descend from the native horses that populated the region until the mid-Holocene, but were most likely introduced following the migration of the Yakut people a few centuries ago. Thus, they represent one of the fastest cases of adaptation to the extreme temperatures of the Arctic. We find cis-regulatory mutations to have contributed more than nonsynonymous changes to their adaptation, likely due to the comparatively limited standing variation within gene bodies at the time the population was founded. Genes involved in hair development, body size, and metabolic and hormone signaling pathways represent an essential part of the Yakutian horse adaptive genetic toolkit. Finally, we find evidence for convergent evolution with native human populations and woolly mammoths, suggesting that only a few evolutionary strategies are compatible with survival in extremely cold environments.