Linking abnormal mitosis to the acquisition of DNA damage

Cellular defects that impair the fidelity of mitosis promote chromosome missegregation and aneuploidy. Increasing evidence reveals that errors in mitosis can also promote the direct and indirect acquisition of DNA damage and chromosome breaks. Consequently, deregulated cell division can devastate the integrity of the normal genome and unleash a variety of oncogenic stimuli that may promote transformation. Recent work has shed light on the mechanisms that link abnormal mitosis with the development of DNA damage, how cells respond to such affronts, and the potential impact on tumorigenesis

The KinI kinesin Kif2a is required for bipolar spindle assembly through a functional relationship with MCAK

Although the microtubule-depolymerizing KinI motor Kif2a is abundantly expressed in neuronal cells, we now show it localizes to centrosomes and spindle poles during mitosis in cultured cells. RNAi-induced knockdown of Kif2a expression inhibited cell cycle progression because cells assembled monopolar spindles. Bipolar spindle assembly was restored in cells lacking Kif2a by treatments that altered microtubule assembly (nocodazole), eliminated kinetochore–microtubule attachment (loss of Nuf2), or stabilized microtubule plus ends at kinetochores (loss of MCAK). Thus, two KinI motors, MCAK and Kif2a, play distinct roles in mitosis, and MCAK activity at kinetochores must be balanced by Kif2a activity at poles for spindle bipolarity. These treatments failed to restore bipolarity to cells lacking the activity of the kinesin Eg5. Thus, two independent pathways contribute to spindle bipolarity, with the Eg5-dependent pathway using motor force to drive spindle bipolarity and the Kif2a-dependent pathway relying on microtubule polymer dynamics to generate force for spindle bipolarity

Transdermal Delivery of Cytochrome C—A 12.4kDa Protein—Across Intact Skin by Constant-Current Iontophoresis

Purpose: To demonstrate the transdermal iontophoretic delivery of a small (12.4kDa) protein across intact skin. Materials and Methods: The iontophoretic transport of Cytochrome c (Cyt c) across porcine ear skin in vitro was investigated and quantified by HPLC. The effect of protein concentration (0.35 and 0.7mM), current density (0.15, 0.3 or 0.5mA.cm−2 applied for 8h) and competing ions was evaluated. Co-iontophoresis of acetaminophen was employed to quantify the respective contributions of electromigration (EM) and electroosmosis (EO). Results: The data confirmed the transdermal iontophoretic delivery of intact Cyt c. Electromigration was the principal transport mechanism, accounting for ∼90% of delivery; correlation between EM flux and electrophoretic mobility was consistent with earlier results using small molecules. Modest EO inhibition was observed at 0.5mA.cm−2. Cumulative permeation at 0.3 and 0.5mA.cm−2 was significantly greater than that at 0.15mA.cm−2; fluxes using 0.35 and 0.7mM Cyt c in the absence of competing ions (J tot  = 182.8 ± 56.8 and 265.2 ± 149.1μg.cm−2.h−1, respectively) were statistically equivalent. Formulation in PBS (pH8.2) confirmed the impact of competing charge carriers; inclusion of ∼170mM Na+ resulted in a 3.9-fold decrease in total flux. Conclusions: Significant amounts (∼0.9mg.cm−2 over 8h) of Cyt c were delivered non-invasively across intact skin by transdermal electrotranspor

Mechanical Properties of Austenitic Stainless Steel After Exposure to Elevated Temperature

Stainless steel has been widely used in the building industry as load-bearing elements such as beams and columns. Many fire accidents in stainless steel buildings were recorded, but only a few have collapsed entirely. These buildings can be rehabilitated and the undamaged parts can be reused, which reduces the economic losses in buildings exposed to fire. The residual mechanical properties of stainless steel after the fire, are the primary determinant of the validity of the stainless steel structure. In this paper, the effect of high temperatures and the time of exposure and cooling method on the mechanical properties of S304 stainless steel was studied. The specimens were heated to 800°C and 1000°C for different heating times (30, 60,90 and 120 minutes) and cooling methods (air-cooled and water-cooled). Results showed that the post-fire yield stress was reduced by 24% and 18% after heating to 800°C for 120minutes and cooled in water and air respectively. However, heating to 1000°C showed a marginal effect on the yield stress of air-cooled specimens and a clear reduction (29%) in the water-cooled specimens. Elongation capacity increased with heating time for 1000oC specimens but decreased for 800oC specimens in both cooling methods

Efficient processing of an antigenic sequence for presentation by MHC class I molecules depends on its neighboring residues in the protein

Processing of endogenously synthesized proteins generates short peptides that are presented by MHC class I molecules to CD8 T lymphocytes. Here it is documented that not only the sequence of the presented peptide but also the residues by which it is flanked in the protein determine the efficiency of processing and presentation. This became evident when a viral sequence of proven antigenicity was inserted at different positions into an unrelated carrier protein. Not different peptides, but different amounts of the antigenic insert itself were retrieved by isolation of naturally processed peptides from cells expressing the different chimeric proteins. Low yield of antigenic peptide from an unfavorable integration site could be overcome by flanking the insert with oligo-alanine to space it from disruptive neighboring sequences. Notably, the degree of protection against lethal virus disease related directly to the amount of naturally processed antigenic peptide

Complete genome sequence of an astrovirus identified in a domestic rabbit (\u3cem\u3eOryctolagus cuniculus\u3c/em\u3e) with gastroenteritis

A colony of domestic rabbits in Tennessee, USA, experienced a high-mortality (~90%) outbreak of enterocolitis. The clinical characteristics were one to six days of lethargy, bloating, and diarrhea, followed by death. Heavy intestinal coccidial load was a consistent finding as was mucoid enteropathy with cecal impaction. Preliminary analysis by electron microscopy revealed the presence of virus-like particles in the stool of one of the affected rabbits. Analysis using the Virochip, a viral detection microarray, suggested the presence of an astrovirus, and follow-up PCR and sequence determination revealed a previously uncharacterized member of that family. Metagenomic sequencing enabled the recovery of the complete viral genome, which contains the characteristic attributes of astrovirus genomes. Attempts to propagate the virus in tissue culture have yet to succeed. Although astroviruses cause gastroenteric disease in other mammals, the pathogenicity of this virus and the relationship to this outbreak remains to be determined. This study therefore defines a viral species and a potential rabbit pathogen

Identification of a Novel Gammaretrovirus in Prostate Tumors of Patients Homozygous for R462Q RNASEL Variant

Ribonuclease L (RNase L) is an important effector of the innate antiviral response. Mutations or variants that impair function of RNase L, particularly R462Q, have been proposed as susceptibility factors for prostate cancer. Given the role of this gene in viral defense, we sought to explore the possibility that a viral infection might contribute to prostate cancer in individuals harboring the R462Q variant. A viral detection DNA microarray composed of oligonucleotides corresponding to the most conserved sequences of all known viruses identified the presence of gammaretroviral sequences in cDNA samples from seven of 11 R462Q-homozygous (QQ) cases, and in one of eight heterozygous (RQ) and homozygous wild-type (RR) cases. An expanded survey of 86 tumors by specific RT-PCR detected the virus in eight of 20 QQ cases (40%), compared with only one sample (1.5%) among 66 RQ and RR cases. The full-length viral genome was cloned and sequenced independently from three positive QQ cases. The virus, named XMRV, is closely related to xenotropic murine leukemia viruses (MuLVs), but its sequence is clearly distinct from all known members of this group. Comparison of gag and pol sequences from different tumor isolates suggested infection with the same virus in all cases, yet sequence variation was consistent with the infections being independently acquired. Analysis of prostate tissues from XMRV-positive cases by in situ hybridization and immunohistochemistry showed that XMRV nucleic acid and protein can be detected in about 1% of stromal cells, predominantly fibroblasts and hematopoietic elements in regions adjacent to the carcinoma. These data provide to our knowledge the first demonstration that xenotropic MuLV-related viruses can produce an authentic human infection, and strongly implicate RNase L activity in the prevention or clearance of infection in vivo. These findings also raise questions about the possible relationship between exogenous infection and cancer development in genetically susceptible individuals

Synchronizing chromosome segregation by flux-dependent force equalization at kinetochores

The synchronous movement of chromosomes during anaphase ensures their correct inheritance in every cell division. This reflects the uniformity of spindle forces acting on chromosomes and their simultaneous entry into anaphase. Although anaphase onset is controlled by the spindle assembly checkpoint, it remains unknown how spindle forces are uniformly distributed among different chromosomes. In this paper, we show that tension uniformity at metaphase kinetochores and subsequent anaphase synchrony in Drosophila S2 cells are promoted by spindle microtubule flux. These results can be explained by a mechanical model of the spindle where microtubule poleward translocation events associated with flux reflect relaxation of the kinetochore–microtubule interface, which accounts for the redistribution and convergence of kinetochore tensions in a timescale comparable to typical metaphase duration. As predicted by the model, experimental acceleration of mitosis precludes tension equalization and anaphase synchrony. We propose that flux-dependent equalization of kinetochore tensions ensures a timely and uniform maturation of kinetochore–microtubule interfaces necessary for error-free and coordinated segregation of chromosomes in anaphase