The tumor suppressor PTEN is a phosphatase with sequence similarity to the cytoskeletal protein tensin. Here the cellular roles of PTEN were investigated. Overexpression of PTEN inhibited cell ...migration, whereas antisense PTEN enhanced migration. Integrin-mediated cell spreading and the formation of focal adhesions were down-regulated by wild-type PTEN but not by PTEN with an inactive phosphatase domain. PTEN interacted with the focal adhesion kinase FAK and reduced its tyrosine phosphorylation. Overexpression of FAK partially antagonized the effects of PTEN. Thus, PTEN phosphatase may function as a tumor suppressor by negatively regulating cell interactions with the extracellular matrix.
Synergistic sites in the central cell-adhesive domain of fibronectin (FN) substantially enhance cell adhesion mediated by
the alpha 5 beta 1 integrin receptor for fibronectin. We characterized a ...critical minimal sequence needed for synergistic
activity using site-directed mutagenesis and homology scanning using intramolecular chimeras. The minimal cell-binding domain
of FN consisting of the 9th and 10th type III FN repeat was expressed in an Escherichia coli expression system. This protein
retained high biological activity when assayed using a competitive inhibition assay for FN-mediated adhesion of baby hamster
kidney or HT-1080 cells. In contrast, a construct consisting of the 8th and 10th repeat displayed very low biological activity.
By replacing various portions of the 8th repeat with homologous 9th repeat segments, we mapped the synergistic region to the
center of the 9th repeat. When a very short peptide sequence, Pro-His-Ser-Arg-Asn (PHSRN), from the 9th repeat was substituted
for the homologous pentapeptide site in the 8th repeat sequence, the recombinant protein showed markedly enhanced activity.
Further mutagenesis analysis suggested that the arginine residue of this pentapeptide sequence is important for function.
We also identified a weaker adjacent synergy region other than the PHSRN region. Epitope mapping of an anti-FN monoclonal
antibody that inhibits FN-mediated adhesion identified the same critical regions. A synthetic peptide containing the PHSRN
sequence showed neither competitive inhibitory activity in solution nor synergy with a soluble RGD-containing peptide. However,
when the same synthetic peptide was positioned via a covalent bond at the corresponding site of the normally inactive 8th
repeat, it mediated an enhancement of adhesive activity. These results identify a pentapeptide site that synergistically enhances
the cell-adhesive activity of the FN RGD sequence.
The early stages of corrosion occurring at liquid-solid interfaces controls the evolution of the material's degradation process, yet due to their transient state, their analysis remains a formidable ...challenge. Here we perform corrosion tests on a MgCa alloy, a candidate material for biodegradable implants using pure water as a model system. The corrosion reaction was suspended by plunge freezing into liquid nitrogen. We studied the evolution of the early-stage corrosion process on the nanoscale by correlating cryo-atom probe tomography with transmission-electron microscopy and spectroscopy. We observed the outward growth of Mg hydroxide and the inward growth of an intermediate corrosion layer consisting of hydroxides of different compositions, mostly monohydroxide Mg(OH) instead of the expected MgO layer. In addition, Ca partitions to these newly formed hydroxides and oxides. Density-functional theory calculations suggest a domain of stability for this previously experimental unreported Mg(OH) phase. These new approach and findings advance the understanding of the early stages of magnesium corrosion, and in general reactions and processes at liquid-solid interfaces, which can further facilitate the development of corrosion-resistant materials or better control of the biodegradation rate of future implants. This article is protected by copyright. All rights reserved.
Background
Although prestorage leucoreduction (LR) of blood components for transfusion has gained favour around the world, evidence of its beneficial clinical effects is ambiguous.
Study design and ...methods
To reveal whether leucocytes and/or platelets in transfused blood are related to transfusion‐related adverse effects, a prospective randomized crossover study was performed on patients who donated autologous blood prior to elective surgery. Among 1487 primary enrolees, a total of 192 patients undergoing two‐stage, bilateral total hip arthroplasty were randomized to receive autologous blood that was either prestorage leucoreduced, or not, for the first procedure. For the second procedure, each patient was crossed over to receive alternatively processed autologous blood. Length of hospital stay served as a primary end‐point, with perioperative infectious/thrombotic complications, pre‐ and postoperative laboratory values, and body temperature serving as secondary endpoints.
Results
No significant differences emerged between prestorage LR and non‐LR cohorts in length of hospital stay, as well as perioperative infectious/thrombotic complications, postoperative body temperature and duration of fever. Postoperative laboratory values including white blood cell counts and C‐reactive protein levels had no significant differences.
Conclusion
This study could not prove any superiority of prestorage LR over non‐LR for autologous whole blood among patients who underwent total hip arthroplasty.
Alloys processed by laser powder-bed fusion show distinct microstructures composed of dislocation cells, dispersed nanoparticles, and columnar grains. Upon post-build annealing, such alloys show ...sluggish recrystallization kinetics compared to the conventionally processed counterpart. To understand this behavior, AISI 316L stainless steel samples were constructed using the island scan strategy. Rhodonite-like (MnSiO
3
) nanoparticles and dislocation cells are found within weakly-textured grains in the as-built condition. Upon isothermal annealing at 1150 °C (up to 2880 min), the nucleation of recrystallization occurs along the center of the melt pool, where nuclei sites, high stored elastic energy, and local large misorientation are found in the as-built condition. The low value of the Avrami coefficient (
n
= 1.16) can be explained based on the non-random distribution of nucleation sites. The local interaction of the recrystallization front with nanoparticles speeds up their coarsening causing the decrease of the Zener-Smith pinning force. This allows the progression of recrystallization in LPBF alloys, although sluggish. These results allow us to understand the progress of recrystallization in LPBF 316L stainless steel, shedding light on the nucleation mechanisms and on the competition between driving and dragging pressures in non-conventional microstructures. They also help to understand the most relevant microstructural aspects applicable for tuning microstructures and designing new LPBF alloys.
Graphical abstract
The
Pax6 gene plays crucial roles in eye development and encodes a transcription factor containing both a paired domain and a homeodomain. During embryogenesis,
Pax6 is expressed in restricted ...tissues under the direction of distinct
cis-regulatory regions. The head surface ectoderm-specific enhancer of mouse
Pax6 directs reporter expression in the derivatives of the ectoderm in the eye, such as lens and cornea, but the molecular mechanism of its control remains largely unknown. We identified a Pax6 protein-responsive element termed LE9 (52 bp in length) within the head surface ectoderm-specific enhancer. LE9, a sequence well conserved across vertebrates, acted as a highly effective enhancer in reporter analyses. Pax6 protein formed in vitro a complex with the distal half of LE9 in a manner dependent on the paired domain. The proximal half of the LE9 sequence contains three plausible sites of HMG domain recognition, and HMG domain-containing transcription factors Sox2 and Sox3 activated LE9 synergistically with Pax6. A scanning mutagenesis experiment indicated that the central site is most important among the three presumptive HMG domain recognition sites. Furthermore, Pax6 and Sox2 proteins formed a complex when they were expressed together. Based on these findings, we propose a model in which Pax6 protein directly and positively regulates its own gene expression, and Sox2 and Sox3 proteins interact with Pax6 protein, resulting in modification of the transcriptional activation by Pax6 protein.
A novel additive manufacturing approach is proposed to produce an electrical steel (Fe-3.5%Si) by laser powder bed fusion (LPBF) followed by conventional thermomechanical processing. The aim of this ...proof-of-concept study is to develop a new processing route for grain-oriented electrical steels from LPBF-processed plates with strong texture and SiO
2
nanoparticles followed by cold rolling and long-term annealing to trigger abnormal grain growth. The slabs were processed with two different scanning strategies; e.g., with (90R) and without 90° rotation (0R) between layers aiming at intensifying the as-built textures near-Goss and/or cube components. The as-built slabs were cold rolled to 83% reduction and annealed for subsequent monitoring of primary recrystallization and abnormal grain growth. Goss or near-Goss nuclei were identified for both strategies after cold rolling. Abnormal grain growth occurred more intensely in samples with a 90° rotation between layers. Goss-oriented grains are bounded by high-angle boundaries with peak misorientations of 50° in 90R and 37.5° in 0R strategy. Porosity in 0R is three times higher than in 90R, while the total fraction of CSL boundaries is similar for both strategies (about 6%). Boundary mobility seems to be higher in 90R, which explains easier grain boundary depinning from oxide nanoparticles than in 0R strategy. In comparison with grain-oriented commercial products, increased total magnetic losses can be explained by thickness effects, porosity and deviation from ideal Goss orientation.
Graphical Abstract
Aerospace and automotive industries utilize advanced high strength steels due to their exceptional mechanical strength and ductility. Laser beam welding has shown potential in reducing the melted ...zone, heat affected zone, and process time for these steels. This study focused on dissimilar welding between DP 780 and 300M steel sheets, commonly used in the automotive and aerospace industries, respectively. The aim was to expand the range of possibilities and innovations by enabling the use of these steels in both applications. The study investigated the optimal process parameters, microstructure, and mechanical properties for the laser welding process. It also examined the influence of intercritical quenching and tempering on the microstructure and mechanical properties of the laser welded steels. The materials underwent dilution and different phase transformations due to the welding process and heat treatments, as revealed by microstructural characterization. The weld showed a notable increase in hardness, however without compromising toughness. The fractures during tensile testing occurred in the DP 780 steel, far from the MZ and HAZ. Heat treatments increased ultimate tensile strength, but lowered ductility. Welding affected the fatigue life, especially in the intercritically quenched joint, which showed a quasi-cleavage crack growth mechanism and a decrease in fracture toughness.
The secretion of fibronectin by differentiating osteoblasts and its accumulation at sites of osteogenesis suggest that fibronectin participates in bone formation. To test this directly, we determined ...whether fibronectin-cell interactions regulate progressive differentiation of cultured fetal rat calvarial osteoblasts. Spatial distributions of alpha 5 integrin subunit, fibronectin, osteopontin (bone sialoprotein I) and osteocalcin (bone Gla-protein) were similar in fetal rat calvaria and mineralized, bone-like nodules formed by cultured osteoblasts. Addition of anti-fibronectin antibodies to cultures at confluence reduced subsequent formation of nodules to less than 10% of control values, showing that fibronectin is required for normal nodule morphogenesis. Anti-fibronectin antibodies selectively inhibited steady-state expression of mRNA for genes associated with osteoblast differentiation; mRNA levels for alkaline phosphatase and osteocalcin were suppressed, whereas fibronectin, type I collagen and osteopontin were unaffected. To identify functionally relevant domains of fibronectin, we treated cells with soluble fibronectin fragments and peptides. Cell-binding fibronectin fragments (type III repeats 6-10) containing the Arg-Gly-Asp (RGD) sequence blocked both nodule initiation and maturation, whether or not they contained a functional synergy site. In contrast, addition of the RGD-containing peptide GRGDSPK alone did not inhibit nodule initiation, although it did block nodule maturation. Thus, in addition to the RGD sequence, other features of the large cell-binding fragments contribute to the full osteogenic effects of fibronectin. Nodule formation and osteoblast differentiation resumed after anti-fibronectin antibodies or GRGDSPK peptides were omitted from the media, showing that the inhibition was reversible and the treatments were not cytotoxic. Outside the central cell-binding domain, peptides from the IIICS region and antibodies to the N terminus did not inhibit nodule formation. We conclude that osteoblasts interact with the central cell-binding domain of endogenously produced fibronectin during early stages of differentiation, and that these interactions regulate both normal morphogenesis and gene expression.
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