Clathrin Is Spindle-Associated but Not Essential for Mitosis

Clathrin is a multimeric protein involved in vesicle coat assembly. Recently clathrin distribution was reported to change during the cell cycle and was found to associate with the mitotic spindle. Here we test whether the recruitment of clathrin to the spindle is indicative of a critical functional contribution to mitosis.Previously a chicken pre-B lymphoma cell line (DKO-R) was developed in which the endogenous clathrin heavy chain alleles were replaced with the human clathrin heavy chain under the control of a tetracycline-regulatable promoter. Receptor-mediated and fluid-phase endocytosis were significantly inhibited in this line following clathrin knockout, and we used this to explore the significance of clathrin heavy chain expression for cell cycle progression. We confirmed using confocal microscopy that clathrin colocalised with tubulin at mitotic spindles. Using a propidium iodide flow cytometric assay we found no statistical difference in the cell cycle distribution of the knockout cells versus the wild-type. Additionally, we showed that the ploidy and the recovery kinetics following cell cycle arrest with nocodazole were unchanged by repressing clathrin heavy chain expression.We conclude that the association of clathrin with the mitotic spindle and the contribution of clathrin to endocytosis are evolutionarily conserved. However we find that the contribution of clathrin to mitosis is less robust and dependent on cellular context. In other cell-lines silencing RNA has been used by others to knockdown clathrin expression resulting in an increase in the mitotic index of the cells. We show an effect on the G2/M phase population of clathrin knockdown in HEK293 cells but show that repressing clathrin expression in the DKO-R cell-line has no effect on the size of this population. Consequently this work highlights the need for a more detailed molecular understanding of the recruitment and function of clathrin at the spindle, since the localisation but not the impact of clathrin on mitosis appears to be robust in plants, mammalian and chicken B-cells

The Mitotic Arrest Deficient Protein MAD2B Interacts with the Clathrin Light Chain A during Mitosis

Contains fulltext : 87811.pdf (publisher's version ) (Open Access)BACKGROUND: Although the mitotic arrest deficient protein MAD2B (MAD2L2) is thought to inhibit the anaphase promoting complex (APC) by binding to CDC20 and/or CDH1 (FZR1), its exact role in cell cycle control still remains to be established. METHODOLOGY/PRINCIPAL FINDINGS: Using a yeast two-hybrid interaction trap we identified the human clathrin light chain A (CLTA) as a novel MAD2B binding protein. A direct interaction was established in mammalian cells via GST pull-down and endogenous co-immunoprecipitation during the G2/M phase of the cell cycle. Through subsequent confocal laser scanning microscopy we found that MAD2B and CLTA co-localize at the mitotic spindle. Clathrin forms a trimeric structure, i.e., the clathrin triskelion, consisting of three heavy chains (CLTC), each with an associated light chain. This clathrin structure has previously been shown to be required for the function of the mitotic spindle through stabilization of kinetochore fibers. Upon siRNA-mediated MAD2B depletion, we found that CLTA was no longer concentrated at the mitotic spindle but, instead, diffusely distributed throughout the cell. In addition, we found a marked increase in the percentage of misaligned chromosomes. CONCLUSIONS/SIGNIFICANCE: Previously, we identified MAD2B as an interactor of the renal cell carcinoma (RCC)-associated protein PRCC. In addition, we found that fusion of PRCC with the transcription factor TFE3 in t(X;1)(p11;q21)-positive RCCs results in an impairment of this interaction and a concomitant failure to shuttle MAD2B to the nucleus. Our current data show that MAD2B interacts with CLTA during the G2/M phase of the cell cycle and that depletion of MAD2B leads to a marked increase in the percentage of misaligned chromosomes and a redistribution of CLTA during mitosis

TrkB is highly expressed in NSCLC and mediates BDNF-induced the activation of Pyk2 signaling and the invasion of A549 cells

<p>Abstract</p> <p>Background</p> <p>Aberrant regulation in the invasion of cancer cells is closely associated with their metastatic potentials. TrkB functions as a receptor tyrosine kinase and is considered to facilitate tumor metastasis. Pyk2 is a non-receptor tyrosine kinase and integrates signals in cell invasion. However, little is known about the expression of TrkB in NSCLC and whether Pyk2 is involved in TrkB-mediated invasion of A549 cells.</p> <p>Methods</p> <p>The expression of TrkB was investigated in NSCLC by immunohistochemical staining. Both HBE and A549 cells were treated with BDNF. The expression of TrkB, Pyk2 and ERK phosphorylations were assessed by western blot. Besides, A549 cells were transfected with TrkB-siRNA or Pyk2-siRNA, or treated with ERK inhibitor where indicated. Transwell assay was performed to evaluate cell invasion.</p> <p>Results</p> <p>40 cases (66.7%) of NSCLC were found higher expression of TrkB and patients with more TrkB expression had significant metastatic lymph nodes (p = 0.028). BDNF facilitated the invasion of A549 cells and the activations of Pyk2 in Tyr402 and ERK. However, the effects of BDNF were not observed in HBE cells with lower expression of TrkB. In addition, the increased Pyk2 and ERK activities induced by BDNF were significantly inhibited by blocking TrkB expression, so was the invasion of A549 cells. Knockdown studies revealed the essential role of Pyk2 for BDNF-induced cell invasion, since the invasion of A549 cells was abolished by Pyk2-siRNA. The application of ERK inhibitor also showed the suppressed ERK phosphorylation and cell invasion.</p> <p>Conclusion</p> <p>These data indicated that higher expression of TrkB in NSCLC was closely correlated with lymph node metastasis, and BDNF probably via TrkB/Pyk2/ERK promoted the invasion of A549 cells.</p

Preoperative muscle weakness as defined by handgrip strength and postoperative outcomes: a systematic review

<p>Abstract</p> <p>Background</p> <p>Reduced muscle strength- commonly characterized by decreased handgrip strength compared to population norms- is associated with numerous untoward outcomes. Preoperative handgrip strength is a potentially attractive real-time, non-invasive, cheap and easy-to-perform "bedside" assessment tool. Using systematic review procedure, we investigated whether preoperative handgrip strength was associated with postoperative outcomes in adults undergoing surgery.</p> <p>Methods</p> <p>PRISMA and MOOSE consensus guidelines for reporting systematic reviews were followed. MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Clinical Trials (1980-2010) were systematically searched by two independent reviewers. The selection criteria were limited to include studies of preoperative handgrip strength in human adults undergoing non-emergency, cardiac and non-cardiac surgery. Study procedural quality was analysed using the Newcastle-Ottawa Quality Assessment score. The outcomes assessed were postoperative morbidity, mortality and hospital stay.</p> <p>Results</p> <p>Nineteen clinical studies (17 prospective; 4 in urgent surgery) comprising 2194 patients were identified between1980-2010. Impaired handgrip strength and postoperative morbidity were defined inconsistently between studies. Only 2 studies explicitly ensured investigators collecting postoperative outcomes data were blinded to preoperative handgrip strength test results. The heterogeneity of study design used and the diversity of surgical procedures precluded formal meta-analysis. Despite the moderate quality of these observational studies, lower handgrip strength was associated with increased morbidity (n = 10 studies), mortality (n = 2/5 studies) and length of hospital stay (n = 3/7 studies).</p> <p>Conclusions</p> <p>Impaired preoperative handgrip strength may be associated with poorer postoperative outcomes, but further work exploring its predictive power is warranted using prospectively acquired, objectively defined measures of postoperative morbidity.</p

Sequential and Coordinated Actions of c-Myc and N-Myc Control Appendicular Skeletal Development

BACKGROUND: During limb development, chondrocytes and osteoblasts emerge from condensations of limb bud mesenchyme. These cells then proliferate and differentiate in separate but adjacent compartments and function cooperatively to promote bone growth through the process of endochondral ossification. While many aspects of limb skeletal formation are understood, little is known about the mechanisms that link the development of undifferentiated limb bud mesenchyme with formation of the precartilaginous condensation and subsequent proliferative expansion of chondrocyte and osteoblast lineages. The aim of this study was to gain insight into these processes by examining the roles of c-Myc and N-Myc in morphogenesis of the limb skeleton. METHODOLOGY/PRINCIPAL FINDINGS: To investigate c-Myc function in skeletal development, we characterized mice in which floxed c-Myc alleles were deleted in undifferentiated limb bud mesenchyme with Prx1-Cre, in chondro-osteoprogenitors with Sox9-Cre and in osteoblasts with Osx1-Cre. We show that c-Myc promotes the proliferative expansion of both chondrocytes and osteoblasts and as a consequence controls the process of endochondral growth and ossification and determines bone size. The control of proliferation by c-Myc was related to its effects on global gene transcription, as phosphorylation of the C-Terminal Domain (pCTD) of RNA Polymerase II, a marker of general transcription initiation, was tightly coupled to cell proliferation of growth plate chondrocytes where c-Myc is expressed and severely downregulated in the absence of c-Myc. Finally, we show that combined deletion of N-Myc and c-Myc in early limb bud mesenchyme gives rise to a severely hypoplastic limb skeleton that exhibits features characteristic of individual c-Myc and N-Myc mutants. CONCLUSIONS/SIGNIFICANCE: Our results show that N-Myc and c-Myc act sequentially during limb development to coordinate the expansion of key progenitor populations responsible for forming the limb skeleton

Differences in Cell Division Rates Drive the Evolution of Terminal Differentiation in Microbes

Multicellular differentiated organisms are composed of cells that begin by developing from a single pluripotent germ cell. In many organisms, a proportion of cells differentiate into specialized somatic cells. Whether these cells lose their pluripotency or are able to reverse their differentiated state has important consequences. Reversibly differentiated cells can potentially regenerate parts of an organism and allow reproduction through fragmentation. In many organisms, however, somatic differentiation is terminal, thereby restricting the developmental paths to reproduction. The reason why terminal differentiation is a common developmental strategy remains unexplored. To understand the conditions that affect the evolution of terminal versus reversible differentiation, we developed a computational model inspired by differentiating cyanobacteria. We simulated the evolution of a population of two cell types –nitrogen fixing or photosynthetic– that exchange resources. The traits that control differentiation rates between cell types are allowed to evolve in the model. Although the topology of cell interactions and differentiation costs play a role in the evolution of terminal and reversible differentiation, the most important factor is the difference in division rates between cell types. Faster dividing cells always evolve to become the germ line. Our results explain why most multicellular differentiated cyanobacteria have terminally differentiated cells, while some have reversibly differentiated cells. We further observed that symbioses involving two cooperating lineages can evolve under conditions where aggregate size, connectivity, and differentiation costs are high. This may explain why plants engage in symbiotic interactions with diazotrophic bacteria

Structural aspects and physiological consequences of APP/APLP trans-dimerization

The amyloid precursor protein (APP) is one of the key proteins in Alzheimer’s disease (AD), as it is the precursor of amyloid β (Aβ) peptides accumulating in amyloid plaques. The processing of APP and the pathogenic features of especially Aβ oligomers have been analyzed in detail. Remarkably, there is accumulating evidence from cell biological and structural studies suggesting that APP and its mammalian homologs, the amyloid precursor-like proteins (APLP1 and APLP2), participate under physiological conditions via trans-cellular dimerization in synaptogenesis. This offers the possibility that loss of synapses in AD might be partially explained by dysfunction of APP/APLPs cell adhesion properties. In this review, structural characteristics of APP trans-cellular interaction will be placed critically in context with its putative physiological functions focusing on cell adhesion and synaptogenesis

Patient-directed self-management of pain (PaDSMaP) compared to treatment as usual following total knee replacement; a randomised controlled trial

Background Self-administration of medicines by patients whilst in hospital is being increasingly promoted despite little evidence to show the risks and benefits. Pain control after total knee replacement (TKR) is known to be poor. The aim of the study was to determine if patients operated on with a TKR who self-medicate their oral analgesics in the immediate post-operative period have better pain control than those who receive their pain control by nurse-led drug rounds (Treatment as Usual (TAU)). Methods A prospective, parallel design, open-label, randomised controlled trial comparing pain control in patient-directed self-management of pain (PaDSMaP) with nurse control of oral analgesia (TAU) after a TKR. Between July 2011 and March 2013, 144 self-medicating adults were recruited at a secondary care teaching hospital in the UK. TAU patients (n = 71) were given medications by a nurse after their TKR. PaDSMaP patients (n = 73) took oral medications for analgesia and co-morbidities after two 20 min training sessions reinforced with four booklets. Primary outcome was pain (100 mm visual analogue scale (VAS)) at 3 days following TKR surgery or at discharge (whichever came soonest). Seven patients did not undergo surgery for reasons unrelated to the study and were excluded from the intention-to-treat (ITT) analysis. Results ITT analysis did not detect any significant differences between the two groups’ pain scores. A per protocol (but underpowered) analysis of the 60% of patients able to self-medicate found reduced pain compared to the TAU group at day 3/discharge, (VAS -9.9 mm, 95% CI -18.7, − 1.1). One patient in the self-medicating group over-medicated but suffered no harm. Conclusion Self-medicating patients did not have better (lower) pain scores compared to the nurse-managed patients following TKR. This cohort of patients were elderly with multiple co-morbidities and may not be the ideal target group for self-medication

Ergocalciferol and Microcirculatory Function in Chronic Kidney Disease and Concomitant Vitamin D Deficiency: An Exploratory, Double Blind, Randomised Controlled Trial

Vitamin D deficiency and endothelial dysfunction are non-traditional risk factors for cardiovascular events in chronic kidney disease. Previous studies in chronic kidney disease have failed to demonstrate a beneficial effect of vitamin D on arterial stiffness, left ventricular mass and inflammation but none have assessed the effect of vitamin D on microcirculatory endothelial function.We conducted a randomised controlled trial of 38 patients with non diabetic chronic kidney disease stage 3-4 and concomitant vitamin D deficiency (<16 ng/dl) who received oral ergocalciferol (50,000 IU weekly for one month followed by 50,000 IU monthly) or placebo over 6 months. The primary outcome was change in microcirculatory function measured by laser Doppler flowmetry after iontophoresis of acetylcholine. Secondary endpoints were tissue advanced glycation end products, sublingual functional capillary density and flow index as well as macrovascular parameters. Parallel in vitro experiments were conducted to determine the effect of ergocalciferol on cultured human endothelial cells.Twenty patients received ergocalciferol and 18 patients received placebo. After 6 months, there was a significant improvement in the ergocalciferol group in both endothelium dependent microcirculatory vasodilatation after iontophoresis of acetylcholine (p = 0.03) and a reduction in tissue advanced glycation end products (p = 0.03). There were no changes in sublingual microcirculatory parameters. Pulse pressure (p = 0.01) but not aortic pulse wave velocity was reduced. There were no significant changes in bone mineral parameters, blood pressure or left ventricular mass index suggesting that ergocalciferol improved endothelial function independently of these parameters. In parallel experiments, expression of endothelial nitric oxide synthase and activity were increased in human endothelial cells in a dose dependent manner.Ergocalciferol improved microcirculatory endothelial function in patients with chronic kidney disease and concomitant vitamin D deficiency. This process may be mediated through enhanced expression and activity of endothelial nitric oxide synthase.Clinical trials.gov NCT00882401

Schwann-Spheres Derived from Injured Peripheral Nerves in Adult Mice - Their In Vitro Characterization and Therapeutic Potential

Multipotent somatic stem cells have been identified in various adult tissues. However, the stem/progenitor cells of the peripheral nerves have been isolated only from fetal tissues. Here, we isolated Schwann-cell precursors/immature Schwann cells from the injured peripheral nerves of adult mice using a floating culture technique that we call “Schwann-spheres." The Schwann-spheres were derived from de-differentiated mature Schwann cells harvested 24 hours to 6 weeks after peripheral nerve injury. They had extensive self-renewal and differentiation capabilities. They strongly expressed the immature-Schwann-cell marker p75, and differentiated only into the Schwann-cell lineage. The spheres showed enhanced myelin formation and neurite growth compared to mature Schwann cells in vitro. Mature Schwann cells have been considered a promising candidate for cell-transplantation therapies to repair the damaged nervous system, whereas these “Schwann-spheres" would provide a more potential autologous cell source for such transplantation