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Pressure ulcers (PrUs) remain a concern for clinicians, patients, caregivers, and researchers. Although data on prevalence and incidence are available, as well as evidence-based prevention and management intervention, PrU healing time is underreported. OBJECTIVE: The objective of this study was to evaluate the healing time of Stage II PrUs. METHODS: Secondary analysis of data collected from a multicenter randomized clinical trial was undertaken. Patients (a) with a Stage II PrU, (b) older than 18 years, and (c) who had given informed consent were included. The endpoints of the study were complete re-epithelialization of the PrU measured with the Pressure Ulcer Scale for Healing Tool 3.0 and the healing time. A network of 46 healthcare centers located in northern Italy participated in the study. RESULTS: Two hundred seventy patients with an average age of 83.9 years (95% confidence interval [CI], 82.71Y85.10) were recruited. Among 270 Stage II PrUs included, 153 lesions healed (56.7%), whereas 74 (27.4%) were still present after 10 weeks of follow-up. For 43 lesions (15.9%), the follow-up evaluation was interrupted because of patient death or transfer to units not included in the study. The PrUs healed on an average of 22.9 days (95% CI, 20.47Y25.37 days), with a median of 18 days. The average healing time for PrUs of less than 3.1 cm2 was significantly shorter (19.2 days; 95% CI, 16.6Y21.8) compared with those 3.1 cm2 or greater (31.0 days; 95% CI, 26.4Y35.6 days) (P = .000). CONCLUSIONS: To achieve complete re-epithelialization in Stage II PrUs, it takes approximately 23 days. This is quite a long time if we consider that pressures of only 60 to 70mmHg for between 30 and 240minutes are needed to cause tissue damage. On average, a small ulcer heals 12 days faster compared with those with a surface of 3.1 cm2 or greater. KEYWORDS: Stage II pressure ulcer, healing time, re-epithelialization

Antimicrobial peptides (AMPs) are secreted in the airway and contribute to initial defence against inhaled pathogens. Infections of the respiratory tract are a major cause of morbidity and mortality in preterm newborns and in patients with cystic fibrosis (CF). In this latter group, the state of chronic lung infection is due to the ability of bacteria to grow as mucoid biofilm, a condition characterised by overproduction and release of polysaccharides (PSs). In this study, we investigate the effect of PSs produced by lung pathogens such as Pseudomonas aeruginosa, Klebsiella pneumoniae andmembers of the Burkholderia cepacia complex on the antibacterial activity of structurally different peptides. The AMPs tested in this study include the cathelicidin LL-37 and the β-defensin hBD-3 from humans, both released at the alveolar level, as well as peptides from other mammals, i.e. SMAP-29, PG-1 and Bac7(1-35). Susceptibility assays, time killing and membrane permeabilization kinetics experiments were carried out to establish whether PSs produced by lung pathogens may be involved in the poor defence reaction of infected lungs and thus explain infection persistence. All the PSs investigated inhibited, albeit to a different extent, the antibacterial activity of the peptides tested, suggesting that their presence in the lungs of patients with CF may contribute to the decreased defence response of this district upon infection by PS-producing microorganisms. The results also show that inhibition of the antibacterial activity is not simply due to ionic interaction between the negatively charged PSs and the cationic AMPs, but it also involves other structural features of both interactors.

The aneuploidy-inducing activity of alachlor and dichlorvos, two pesticides representing an important source of human exposure to potential carcinogens, has been evaluated in a cytokinesis block micronucleus assay combined with anti-kinetochore (CREST) staining to detect chromosome loss and in situ hybridization with chromosome-specific centromeric probes for the analysis of non-disjunction. Cytofluorimetric analysis to assess potential interference of the chemicals with cell cycle progression and TUNEL assay to detect apoptosis were also performed. The results obtained show that both environmental compounds induced significant and doserelated increases of total micronuclei (MN) and CRESTpositive MN as compared with the concurrent solvent control. The chemicals were also capable of promoting chromosome non-disjunction. However, the two pesticides differed in their mode of action: alachlor induced both chromosomal aberrations and aneuploidy, while the genotoxic activity of dichlorvos was only related to aneuploidy induction. Cytofluorimetric analyses showed that dichlorvos caused a marked accumulation of cells in the G2/M phase of cell cycle and indicate a potential for this chemical to interfere with mitosis. Furthermore, dichlorvos induced CREST-positive MN at a concentration lower than the one producing apoptosis, suggesting that dichlorvos-induced aneuploid cells may persist in the growing cell population

Post-translational modifications of core histones give a major contribution in driving changes in chromatin conformation and compaction. Here we investigated the role of histone deacetylation on the mitotic process by inhibiting histone deacetylases shortly before mitosis in human primary fibroblasts. Cells entering mitosis with hyperacetylated histones displayed altered chromatin conformation associated with decreased reactivity to the anti Ser 10 phospho H3 antibody, increased recruitment of protein phosphatase 1-ƒÔ on mitotic chromosomes, and depletion of heterochromatin protein 1 from the centromeric heterochromatin. Inhibition of histone deacetylation prior to mitosis produced defective chromosome condensation and impaired mitotic progression in living cells, suggesting that improper chromosome condensation may induce mitotic checkpoint activation. In situ hybridization analysis on anaphase cells demonstrated the presence of chromatin bridges, which were caused by persisting cohesion along sister chromatid arms after centromere separation. Thus, the presence of hyperacetylated chromatin during mitosis impairs proper chromosome condensation during the pre-anaphase stages, resulting in poor sister chromatid resolution. Lagging chromosomes consisting of single or paired sisters were also induced by the presence of hyperacetylated histones, indicating that the less constrained centromeric organization associated with heterochromatin protein 1 depletion may promote the attachment of kinetochores to microtubules coming from both poles.

Because in experimental hepatocarcinogenesis apoptosis increases from normal to preneoplastic to carcinoma tissue, proapoptotic factors, such as activin-A, may represent useful markers for hepatocellular carcinoma (HCC). In this study, serum activin-A was measured in 99 cirrhotic patients, of whom 55 had HCC. Activin-A concentrations were higher in HCC patients (median, 2.33 ng/ml; range, 0.41-8.12) than in patients with nonmalignant cirrhosis (1.28 ng/ml; range, 0.35-6.25) (P.05). All 12 patients with activin-A greater than 3 ng/ml and serum alpha-fetoprotein greater than 30 ng/ml had HCC, in comparison to 32 of 41 patients who had only one and to 11 of 46 patients who had both markers below these cutoffs (P.0001). No correlation was found between activin-A and alpha-fetoprotein in the two groups, whereas in patients with HCC, activin-A was strictly correlated with serum aspartate aminotransferase (P.001). Activin-A mRNA for inhibin betaA subunit was expressed both in tumor and nontumor liver tissues in a case of HCC superimposed on cirrhosis and was not expressed in a case of HCC without cirrhosis. In conclusion, cirrhotic patients with HCC have high serum activin-A, to the production of which both the cirrhotic liver and the liver tumor are likely to contribute.

Faithful chromosome segregation is mandatory for cell and organismal viability. Kinetochores, large protein assemblies embedded in centromeric chromatin, establish a mechanical link between chromosomes and spindle microtubules. The KMN network, a conserved 10-subunit kinetochore complex, harbors the microtubule-binding interface. RWD domains in the KMN subunits Spc24 and Spc25 mediate kinetochore targeting of the microtubule-binding subunits by interacting with the Mis12 complex, a KMN subcomplex that tethers directly onto the underlying chromatin layer. Here, we show that Knl1, a KMN subunit involved in mitotic checkpoint signaling, also contains RWD domains that bind the Mis12 complex and that mediate kinetochore targeting of Knl1. By reporting the first 3D electron microscopy structure of the KMN network, we provide a comprehensive framework to interpret how interactions of RWD-containing proteins with the Mis12 complex shape KMN network topology. Our observations unveil a regular pattern in the construction of the outer kinetochore.

Oligopeptidase B (OpdB) is a serine peptidase widespread among bacteria and protozoa that has emerged as a virulence factor despite its function has not yet been precisely established. By using an OpdB-overexpressing Escherichia coli strain, we found that the overexpressed peptidase makes the bacterial cells specifically less susceptible to several proline-rich antimicrobial peptides known to penetrate into the bacterial cytosol, and that its level of activity directly correlates with the degree of resistance. We established that E. coli OpdB can efficiently hydrolyze in vitro cationic antimicrobial peptides up to 30 residues in length, even though they contained several prolines, shortening them to inactive fragments. Two consecutive basic residues are a preferred cleavage site for the peptidase. In the case of a single basic residue, there is no cleavage if proline residues are present in the P1 and P2 positions. These results also indicate that cytosolic peptidases may cause resistance to antimicrobial peptides that have an intracellular mechanism of action, such as the proline-rich peptides, and may contribute to define the substrate specificity of the E. coli OpdB.

Highly Expressed in Cancer protein 1 (Hec1) is a subunit of the Ndc80 complex, a constituent of the mitotic kinetochore. HEC1 has been shown to be overexpressed in many cancers, suggesting that HEC1 upregulation is involved in the generation and/or maintenance of the tumour phenotype. However, the regulation of Hec1 expression in normal and tumour cells and the molecular alterations promoting accumulation of this protein in cancer cells are still unknown. Here we show that elevated Hec1 protein levels are characteristic of transformed cell lines of different origins and that kinetochore recruitment of this protein is also increased in cancer cell lines in comparison with normal human cells. Using different cell synchronization strategies, Hec1 expression was found to be tightly regulated during the cell cycle in both normal and cancer cells. A limited proteasome-dependent degradation of Hec1 cellular content was observed at mitotic exit, with no evident differences between normal and cancer cells. Interestingly, increased expression of HEC1 mRNA and Hec1 protein was observed after transient silencing of the retinoblastoma gene by siRNA or following microRNA-mediated permanent depletion of the retinoblastoma protein in HCT116 cells. Our data provide evidence for a functional link between Hec1 expression and the pRb pathway. These observations suggest that disruption of pRb function may lead to chromosome segregation errors and mitotic defects through Hec1 overexpression. This may importantly contribute to aneuploidy and chromosomal instability in RB-defective cancer cells.

In contrast to many antimicrobial peptides, members of the proline-rich group of antimicrobial peptides inactivate Gram-negative bacteria by a non-lytic mechanism. Several lines of evidence indicate that they are internalized into bacteria and their activity mediated by interaction with unknown cellular components. With the aim of identifying such interactors, we selected mutagenized Escherichia coli clones resistant to the proline-rich Bac7(1-35) peptide and analysed genes responsible for conferring resistance, whose products may thus be involved in the peptide's mode of action. We isolated a number of genomic regions bearing such genes, and one in particular coding for SbmA, an inner membrane protein predicted to be part of an ABC transporter. An E. coli strain carrying a point mutation in sbmA, as well as other sbmA-null mutants, in fact showed resistance to several proline-rich peptides but not to representative membranolytic peptides. Use of fluorescently labelled Bac7(1-35) confirmed that resistance correlated with a decreased ability to internalize the peptide, suggesting that a bacterial protein, SbmA, is necessary for the transport of, and for susceptibility to, proline-rich antimicrobial peptides of eukaryotic origin.

Proline-rich peptides are a unique group of antimicrobial peptides that exert their activity selectively against Gram-negative bacteria through an apparently non-membranolytic mode of action that is not yet well understood. We have investigated the mechanism underlying the antibacterial activity of the proline-rich cathelicidin Bac7 against Salmonella enterica and Escherichia coli. The killing and membrane permeabilization kinetics as well as the cellular localization were assessed for the fully active N-terminal fragment Bac7(1-35), its all-D enantiomer and for differentially active shortened fragments. At sub-micromolar concentrations, Bac7(1-35) rapidly killed bacteria by a non-lytic, energy-dependent mechanism, whereas its D-enantiomer was inactive. Furthermore, while the L-enantiomer was rapidly internalized into bacterial cells, the D-enantiomer was virtually excluded. At higher concentrations (>or=64 microM), both L- and D-Bac7(1-35) were instead able to kill bacteria also via a lytic mechanism. Overall, these results suggest that Bac7 may inactivate bacteria via two different modes of action depending on its concentration: (i) at near-MIC concentrations via a mechanism based on a stereospecificity-dependent uptake that is likely followed by its binding to an intracellular target, and (ii) at concentrations several times the MIC value, via a non-stereoselective, membranolytic mechanism.

The interaction of two cathelicidin antimicrobial peptides, LL-37 and SMAP-29, with three bacterial polysaccharides, respectively, produced by Pseudomonas aeruginosa , Burkholderia cepacia and Klebsiella pneumoniae , was investigated to identify possible mechanisms adopted by lung pathogens to escape the action of innate immunity effectors. In vitro assays indicated that the antibacterial activity of both peptides was inhibited to a variable extent by the three polysaccharides. Circular dichroism experiments showed that these induced an α-helical conformation in the two peptides, with the polysaccharides from K. pneumoniae and B. cepacia showing, respectively, the highest and the lowest effect. Fluorescence measurements also indicated the presence of peptide–polysaccharide interactions. A model is proposed in which the binding of peptides to the polysaccharide molecules induces, at low polysaccharide to peptide ratios, a higher order of aggregation, due to peptide–peptide interactions. Overall, these results suggest that binding of the peptides by the polysaccharides produced by lung pathogens can contribute to the impairment of peptide-based innate defenses of airway surface.