Glycine binding primes NMDA receptor internalization

NMDA (N-methyl-d-aspartate) receptors (NMDARs) are a principal subtype of excitatory ligand-gated ion channel with prominent roles in physiological and disease processes in the central nervous system. Recognition that glycine potentiates NMDAR-mediated currents as well as being a requisite co-agonist of the NMDAR subtype of 'glutamate' receptor profoundly changed our understanding of chemical synaptic communication in the central nervous system. The binding of both glycine and glutamate is necessary to cause opening of the NMDAR conductance pore. Although binding of either agonist alone is insufficient to cause current flow through the channel, we report here that stimulation of the glycine site initiates signalling through the NMDAR complex, priming the receptors for clathrin-dependent endocytosis. Glycine binding alone does not cause the receptor to be endocytosed; this requires both glycine and glutamate site activation of NMDARs. The priming effect of glycine is mimicked by the NMDAR glycine site agonist d-serine, and is blocked by competitive glycine site antagonists. Synaptic as well as extrasynaptic NMDARs are primed for internalization by glycine site stimulation. Our results demonstrate transmembrane signal transduction through activating the glycine site of NMDARs, and elucidate a model for modulating cell-cell communication in the central nervous system.     

A recombinant dromedary antibody fragment (VHH or nanobody) directed against human Duffy antigen receptor for chemokines

Fy blood group antigens are carried by the Duffy antigen receptor for chemokines (DARC), a red cells receptor for Plasmodium vivax broadly implicated in human health and diseases. Recombinant VHHs, or nanobodies, the smallest intact antigen binding fragment derivative from the heavy chain-only antibodies present in camelids, were prepared from a dromedary immunized against DARC N-terminal extracellular domain and selected for DARC binding. A described VHH, CA52, does recognize native DARC on cells. It inhibits P. vivax invasion of erythrocytes and displaces interleukin-8 bound to DARC. The targeted epitope overlaps the well-defined DARC Fy6 epitope. K _D of CA52?CDARC equilibrium is sub-nanomolar, hence ideal to develop diagnostic or therapeutic compounds. Immunocapture by immobilized CA52 yielded highly purified DARC from engineered K562 cells. This first report on a VHH with specificity for a red blood cell protein exemplifies VHHs?? potentialities to target, to purify, and to modulate the function of cellular markers.     

Poly(ADP-ribosyl)ation regulates CTCF-dependent chromatin insulation

Chromatin insulators demarcate expression domains by blocking the cis effects of enhancers or silencers in a position-dependent manner. We show that the chromatin insulator protein CTCF carries a post-translational modification: poly(ADP-ribosyl)ation. Chromatin immunoprecipitation analysis showed that a poly(ADP-ribosyl)ation mark, which exclusively segregates with the maternal allele of the insulator domain in the H19 imprinting control region, requires the bases that are essential for interaction with CTCF. Chromatin immunoprecipitation-on-chip analysis documented that the link between CTCF and poly(ADP-ribosyl)ation extended to more than 140 mouse CTCF target sites. An insulator trap assay showed that the insulator function of most of these CTCF target sites is sensitive to 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase activity. We suggest that poly(ADP-ribosyl)ation imparts chromatin insulator properties to CTCF at both imprinted and nonimprinted loci, which has implications for the regulation of expression domains and their demise in pathological lesions.     

The clonal and mutational evolution spectrum of primary triple-negative breast cancers

Primary triple-negative breast cancers (TNBCs), a tumour type defined by lack of oestrogen receptor, progesterone receptor and ERBB2 gene amplification, represent approximately 16% of all breast cancers. Here we show in 104 TNBC cases that at the time of diagnosis these cancers exhibit a wide and continuous spectrum of genomic evolution, with some having only a handful of coding somatic aberrations in a few pathways, whereas others contain hundreds of coding somatic mutations. High-throughput RNA sequencing (RNA-seq) revealed that only approximately 36% of mutations are expressed. Using deep re-sequencing measurements of allelic abundance for 2,414 somatic mutations, we determine for the first time-to our knowledge-in an epithelial tumour subtype, the relative abundance of clonal frequencies among cases representative of the population. We show that TNBCs vary widely in their clonal frequencies at the time of diagnosis, with the basal subtype of TNBC showing more variation than non-basal TNBC. Although p53 (also known as TP53), PIK3CA and PTEN somatic mutations seem to be clonally dominant compared to other genes, in some tumours their clonal frequencies are incompatible with founder status. Mutations in cytoskeletal, cell shape and motility proteins occurred at lower clonal frequencies, suggesting that they occurred later during tumour progression. Taken together, our results show that understanding the biology and therapeutic responses of patients with TNBC will require the determination of individual tumour clonal genotypes.     

Effects of heat treatment on the intermetallic compounds and mechanical properties of the stainless steel 321-aluminum 1230 explosive-welding interface

The effects of heat treatment on the microstructure and mechanical properties of intermetallic compounds in the interface of stainless steel 321 explosively bonded to aluminum 1230 were investigated in this study. Experimental investigations were performed by optical microscopy, scanning electron microscopy, and microhardness and shear tensile strength testing. Prior to heat treatment, increasing the stand-off distance between samples from 1 to 2.5 mm caused their interface to become wavy and the thickness of intermetallic layers to increase from 3.5 to 102.3 μm. The microhardness increased from HV 766 in the sample prepared at a stand-off distance of 1 mm to HV 927 in the sample prepared at a stand-off distance of 2.5 mm; in addition, the sample strength increased from 103.2 to 214.5 MPa. Heat treatment at 450℃ for 6 h increased the thickness of intermetallic compound layers to 4.4 and 118.5 μm in the samples prepared at stand-off distances of 1 and 2.5 mm, respectively. These results indicated that increasing the duration and temperature of heat treatment decreased the microhardness and strength of the interface of explosively welded stainless steel 321-Al 1230 and increased the thickness of the intermetallic region.