Cellular expansion of MSCs: Shifting the regenerative potential

Mesenchymal-derived stromal or progenitor cells, commonly called “MSCs,” have attracted significant clinical interest for their remarkable abilities to promote tissue regeneration and reduce inflammation. Recent studies have shown that MSCs' therapeutic effects, originally attributed to the cells' direct differentiation capacity into the tissue of interest, are largely driven by the biomolecules the cells secrete, including cytokines, chemokines, growth factors, and extracellular vesicles containing miRNA. This secretome coordinates upregulation of endogenous repair and immunomodulation in the local microenvironment through crosstalk of MSCs with host tissue cells. Therapeutic applications for MSCs and their secretome-derived products often involve in vitro monolayer expansion. However, consecutive passaging of MSCs significantly alters their therapeutic potential, inducing a broad shift from a pro-regenerative to a pro-inflammatory phenotype. A consistent by-product of in vitro expansion of MSCs is the onset of replicative senescence, a state of cell arrest characterized by an increased release of proinflammatory cytokines and growth factors. However, little is known about changes in the secretome profile at different stages of in vitro expansion. Some culture conditions and bioprocessing techniques have shown promise in more effectively retaining the pro-regenerative and anti-inflammatory MSC phenotype throughout expansion. Understanding how in vitro expansion conditions influence the nature and function of MSCs, and their associated secretome, may provide key insights into the underlying mechanisms driving these alterations. Elucidating the dynamic and diverse changes in the MSC secretome at each stage of in vitro expansion is a critical next step in the development of standardized, safe, and effective MSC-based therapies.     

Use of 31P magnetisation transfer magnetic resonance spectroscopy to measure ATP changes after 670 nm transcranial photobiomodulation in older adults

Mitochondrial function declines with age, and many pathological processes in neurodegenerative diseases stem from this dysfunction when mitochondria fail to produce the necessary energy required. Photobiomodulation (PBM), long-wavelength light therapy, has been shown to rescue mitochondrial function in animal models and improve human health, but clinical uptake is limited due to uncertainty around efficacy and the mechanisms responsible. Using ³¹P magnetisation transfer magnetic resonance spectroscopy (MT-MRS) we quantify, for the first time, the effects of 670 nm PBM treatment on healthy ageing human brains. We find a significant increase in the rate of ATP synthase flux in the brain after PBM in a cohort of older adults. Our study provides initial evidence of PBM therapeutic efficacy for improving mitochondrial function and restoring ATP flux with age, but recognises that wider studies are now required to confirm any resultant cognitive benefits.     

Knockdown resistance (kdr) to DDT and pyrethroid insecticides maps to a sodium channel gene locus in the housefly (Musca domestica)

The voltage-sensitive sodium channel is generally regarded as the primary target site of dichlorodiphenyl-trichloro-ethane (DDT) and pyrethroid insecticides, and has been implicated in the widely reported mechanism of nerve insensitivity to these compounds. This phenomenon is expressed as knockdown resistance (kdr) and has been best characterised in the housefly where several putative alleles, including the more potent super-kdr factor, have been identified. We report the isolation of cDNA clones containing part of a housefly sodium channel gene, designated Msc, which show close homology to the para sodium channel of Drosophila (99% amino acid identity within the region of overlap). Using Southern blots of insect DNA, restriction fragment length polymorphisms (RFLPs) at the Msc locus were identified in susceptible, kdr and super-kdr housefly strains. These RFLPs showed tight linkage to resistance in controlled crosses involving these strains, thus providing clear genetic evidence that kdr, and hence pyrethroid mode of action, is closely associated with the voltage-sensitive sodium channel.     

Apoptosis: suicide, execution or murder?

Apoptosis, a controlled form of cell death, appears to be regulated in several ways. Early studies indicated that de novo protein synthesis was required for apoptosis of thymocytes, but more recent studies have found that other cells can undergo apoptosis when protein synthesis is blocked or that inhibition of protein or RNA synthesis can induce apoptosis. Whether these findings reflect distinct forms of apoptosis or variations on a single pathway is not yet known. In this article the case for a single pathway to apoptosis, accessible at multiple points, is discussed.     

Protein folding in the cell: molecular chaperones pave the way

In vitro, many unfolded polypeptides are able to fold to the native state spontaneously, indicating that the amino acid sequence of a protein contains all the information necessary to specify its three-dimensional conformation. It had been assumed that protein folding in vivo also generally occurs in a spontaneous process. This view has changed only recently due to the discovery of a number of proteins, now commonly called 'molecular chaperones', which are essential for cellular protein folding and occur ubiquitously in eubacteria, archaebacteria and in eukaryotic cells.     

Abundance,horizontal and vertical distribution of fish in eastern Weddell Sea micronekton

Summary The spatial distribution and species composition of high-Antarctic ichthyonekton was investigated during the EPOS 3 cruise by RV Polarstern in the eastern Weddell Sea during January–February 1989. A multiple rectangular midwater trawl was used to collect samples from the surface to near the sea floor at 11 stations along a 245 kra transect off Halley Bay. Early larval stages of 18 species, representing about 24% of the known Weddell Sea ichthyofauna, were present in the water column. The Antarctic silver-fish, Pleuragramma antarcticum, over-whelmingly dominated the catches comprising 84.5% of the 5022 specimens caught. The abundance of this species markedly increased towards the offshore end of the transect with the highest numbers occurring near the shelf-break front associated with the westerly current of the southern limb of the Weddell Gyre. The increased abundance of P. antarcticum in continental slope waters was attributed to deflection of the East Weddell Coastal Current beyond the shelf/slope break by fringing ice shelves. Most larval and juvenile fish were found in the seasonally warmed upper 0–70 m layer of the Antarctic Surface Water where conditions occurred that appeared to be favourable to both feeding and growth. Cluster analysis indicated that inner-, central-and outer-shelf assemblages were represented and that the species composition was most effectively described by reference to water mass and depth.     

Molecular phylogenies in angiosperm evolution

We have cloned and sequenced cDNAs for the glyceraldehyde-3-phosphate dehydrogenase of glycolysis, gapC, from a bryophyte, a gymnosperm, and three angiosperms. Phylogenetic analyses are presented for these data in the context of other gapC sequences and in parallel with published nucleotide sequences for the chloroplast encoded gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL). Relative-rate tests were performed for these genes in order to assess variation in substitution rate for coding regions, along individual plant lineages studied. The results of both gene analyses suggest that the deepest dichotomy within the angiosperms separates not magnoliids from remaining angiosperms, but monocotyledons from dicotyledons, in sharp contrast to prediction from the Euanthial theory for angiosperm evolution. Furthermore, these chloroplast and nuclear sequence data taken together suggest that the separation of monocotyledonous and dicotyledonous lineages took place in late Carboniferous times [approximately 300 Myr before the present (Mybp)]. This date would exceed but be compatible with the late-Triassic (approximately 220 Mybp) occurrence of fossil reproductive structures of the primitive angiosperm Sanmiguelia lewisii.      

Cytoskeleton modifications induced by phenobarbital, 2-acetylaminofluorene and 4-acetylaminofluorene in normal and initiated/selected hepatocytes: Relation with the “resistant” phenotype

Initiatedlselected (ISH) and normal (NH) rat hepatocytes were used to study cytoskeleton modifications induced by three liver acting chemicals: 2-AAF, a liver complete carcinogen; PB, a liver tumor promoter; and 4-AAF, a noncarcinogen analogue of 2-AAF. Cytoskeleton alterations were visualized by disappearance of F-actin fibers and tubulin depolymerization. The three drugs induced actin fragmentation in normal hepatocytes; a net loss of actin protein was observed with PB. They also induced varied tubulin depolymerization. The principal difference between chemicals is that 2-AAF led to non-reversible effects, in comparison with PB and 4-AAF which induced reversible damages on cytoskeleton. By contrast to normal hepatocytes, the cytoskeleton of ISH obtained from rats subjected to the resistant hepatocyte protocol was much less susceptible to the effect of the three chemicals. Moreover, we observed a lack of LDH release in the culture medium and a very rapid inducibility of GST activity after exposure of ISH to drugs. The moderate effect of the three chemicals on actin and tubdin in ISH could thus be explained by the resistant metabolic profile of these cells.Abbreviations TPA 12-O-tetradecanoyl-phorbol-13-acetate - PB phenobarbital - 2-AAF 2-acetylaminofluorene - 4-AAF 4-acetylaminofluorene - GSH reduced glutathione - GST glutathione-S-transferase - LDH lactatedehydrogenase - NH normal hepatocytes - ISH initiated/selected hepatocytes - BSA bovine serum albumin     

1,25-dihydroxyvitamin D3 and macrophage colony-stimulating factor-1 synergistically phosphorylate talin

Macrophage colony stimulating factor (CSF-1) and 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) are potent inducers of macrophage differentiation. Both appear to modulate protein phosphorylation, at least in part, through protein kinase C (PKC) raising the question as to whether they concurrently impact on macrophage-like cells. In this regard, we utilized the CSF-1 dependent murine macrophage-like line BAC 1.25F5. CSF-1 treatment of these cells for 30 min leads to particular phosphorylation of a 165 kDa protein, the putative CSF-1 receptor, and a 210 kDa moiety. 1,25(OH)₂D₃ exposure for 24 h prior to addition of CSF-1 enhances phosphorylation of the 165 kDa species and, especially, the 210 kDa protein. Phosphorylation of the latter protein is 1,25(OH)₂D₃ dose- and time-dependent and the molecule is specifically immunoprecipitated with a rabbit polyclonal anti-talin antibody. Experiments with okadaic acid show that the enhanced phosphorylation of talin does not result from serine phosphatase inhibition. CSF-1 and 1,25(OH)₂D₃, alone or in combination, do not increase talin protein expression. The tyrosine kinase inhibitor, genestein, blocks 1,25(OH)₂D₃/CSF-1 induced phosphorylation of the putative CSF-1 receptor but has no effect on talin phosphorylation which occurs exclusively on serine. In contrast to genestein, staurosporin, an inhibitor of PKC, inhibits phosphorylation of talin. Moreover, exposure of 1,25(OH)₂D₃ pretreated cells to phorbol 12-myristate 13-acetate (PMA) in place of CSF-1 also prompts talin phosphorylation. Finally, 1,25(OH)₂D₃ enhances ³[H]PDBu binding, indicating that the steroid increases PMA receptor capacity. Thus, CSF-1 and 1,25(OH)₂D₃ act synergistically via PKC to phosphorylate talin, a cytoskeletal-associated protein.