Biological activity differences between TGF-β1 and TGF-β3 correlate with differences in the rigidity and arrangement of their component monomers

[Image: see text] TGF-β1, -β2, and -β3 are small, secreted signaling proteins. They share 71–80% sequence identity and signal through the same receptors, yet the isoform-specific null mice have distinctive phenotypes and are inviable. The replacement of the coding sequence of TGF-β1 with TGF-β3 and TGF-β3 with TGF-β1 led to only partial rescue of the mutant phenotypes, suggesting that intrinsic differences between them contribute to the requirement of each in vivo. Here, we investigated whether the previously reported differences in the flexibility of the interfacial helix and arrangement of monomers was responsible for the differences in activity by generating two chimeric proteins in which residues 54–75 in the homodimer interface were swapped. Structural analysis of these using NMR and functional analysis using a dermal fibroblast migration assay showed that swapping the interfacial region swapped both the conformational preferences and activity. Conformational and activity differences were also observed between TGF-β3 and a variant with four helix-stabilizing residues from TGF-β1, suggesting that the observed changes were due to increased helical stability and the altered conformation, as proposed. Surface plasmon resonance analysis showed that TGF-β1, TGF-β3, and variants bound the type II signaling receptor, TβRII, nearly identically, but had small differences in the dissociation rate constant for recruitment of the type I signaling receptor, TβRI. However, the latter did not correlate with conformational preference or activity. Hence, the difference in activity arises from differences in their conformations, not their manner of receptor binding, suggesting that a matrix protein that differentially binds them might determine their distinct activities

A multimodal cell census and atlas of the mammalian primary motor cortex

ABSTRACT We report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex (MOp or M1) as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties, and cellular resolution input-output mapping, integrated through cross-modal computational analysis. Together, our results advance the collective knowledge and understanding of brain cell type organization: First, our study reveals a unified molecular genetic landscape of cortical cell types that congruently integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a unified taxonomy of transcriptomic types and their hierarchical organization that are conserved from mouse to marmoset and human. Third, cross-modal analysis provides compelling evidence for the epigenomic, transcriptomic, and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types and subtypes. Fourth, in situ single-cell transcriptomics provides a spatially-resolved cell type atlas of the motor cortex. Fifth, integrated transcriptomic, epigenomic and anatomical analyses reveal the correspondence between neural circuits and transcriptomic cell types. We further present an extensive genetic toolset for targeting and fate mapping glutamatergic projection neuron types toward linking their developmental trajectory to their circuit function. Together, our results establish a unified and mechanistic framework of neuronal cell type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties

Cell migration modulating compounds

Migration Stimulating Factor (MSF) is a stress-response molecule expressed by epithelial and stromal cells in fetal skin and common human tumours. MSF is not normally present in healthy adult skin, but is transiently re-expressed during wound healing. Human recombinant MSF displays a number of potent bioactivities relevant to wound healing and cancer progression, including the stimulation of cell migration (target: carcinoma cells, keratinocytes, dermal fibroblasts, endothelial cells), stimulation of hyaluronan synthesis by fibroblasts, and angiogenesis in vivo and in vitro. MSF is a truncated isoform of fibronectin produced from the primary fibronectin gene transcript by a bypass of normal alternative splicing involving read-through of the intron separating exons III-1a and -1b. Intron retention results in the inclusion of a unique 30 bp coding sequence. MSF protein is consequently identical to the 70 kDa N-terminus of fibronectin, including nine type I and two type II modules, and terminates with the sequence coded by module III-1a and a unique decamer not present in any previously described "full-length" fibronectin isoform. The IGD (isoleucine, glycine, aspartate) tripeptide motif, a highly conserved feature of the fibronectin type I module, is present within the third, fifth, seventh and ninth constituent type I modules of MSF. Interestingly, synthetic trimer and tetramer peptides containing the IGD motif exhibit the same range of biological activities as those displayed by MSF. Furthermore, in vitro mutagenesis and analysis of IGDrecombinant constructs has demonstrated that the motogenic activity of MSF on target fibroblasts is mediated by the IGD sequences. Studies have implicated the related RGDamino acid motif (located in the tenth type III repeat module) in mediating the cell migrating stimulating effects of both native fibronectin and its cell-binding domain. Significantly, small RGD-containing synthetic peptides did not stimulate cell migration; indeed, these peptides inhibited the adhesive and migration stimulatory activity of larger protein domains contining the RGD motif by competition for receptor ligation. International patent application published under number WO 99/02674, relates to peptides containing the IGD motif and their use as cell migration modulators. There is a need to provide molecules which express MSF/IGD bioactivities and show improved stability compared to the IGD tripeptide. Such molecules may be useful as therapeutic agents for the management of patients with impaired wound healing and other pathologies requiring the stimulation of cell migration and angiogenesis. It is an object of the present invention to provide IGD mimetic molecules having MSF and/or IGD bioactivity, such as stimulatory and/or inhibitory activity