The deubiquitylase USP15 regulates topoisomerase II alpha to maintain genome integrity.

Ubiquitin-specific protease 15 (USP15) is a widely expressed deubiquitylase that has been implicated in diverse cellular processes in cancer. Here we identify topoisomerase II (TOP2A) as a novel protein that is regulated by USP15. TOP2A accumulates during G2 and functions to decatenate intertwined sister chromatids at prophase, ensuring the replicated genome can be accurately divided into daughter cells at anaphase. We show that USP15 is required for TOP2A accumulation, and that USP15 depletion leads to the formation of anaphase chromosome bridges. These bridges fail to decatenate, and at mitotic exit form micronuclei that are indicative of genome instability. We also describe the cell cycle-dependent behaviour for two major isoforms of USP15, which differ by a short serine-rich insertion that is retained in isoform-1 but not in isoform-2. Although USP15 is predominantly cytoplasmic in interphase, we show that both isoforms move into the nucleus at prophase, but that isoform-1 is phosphorylated on its unique S229 residue at mitotic entry. The micronuclei phenotype we observe on USP15 depletion can be rescued by either USP15 isoform and requires USP15 catalytic activity. Importantly, however, an S229D phospho-mimetic mutant of USP15 isoform-1 cannot rescue either the micronuclei phenotype, or accumulation of TOP2A. Thus, S229 phosphorylation selectively abrogates this role of USP15 in maintaining genome integrity in an isoform-specific manner. Finally, we show that USP15 isoform-1 is preferentially upregulated in a panel of non-small cell lung cancer cell lines, and propose that isoform imbalance may contribute to genome instability in cancer. Our data provide the first example of isoform-specific deubiquitylase phospho-regulation and reveal a novel role for USP15 in guarding genome integrity.

Epithelial-mesenchymal transition, IP3 receptors and ER-PM junctions: translocation of Ca2+ signalling complexes and regulation of migration.

Disconnection of a cell from its epithelial neighbours and the formation of a mesenchymal phenotype are associated with profound changes in the distribution of cellular components and the formation of new cellular polarity. We observed a dramatic redistribution of inositol trisphosphate receptors (IP3Rs) and stromal interaction molecule 1 (STIM1)-competent endoplasmic reticulum-plasma membrane junctions (ER-PM junctions) when pancreatic ductal adenocarcinoma (PDAC) cells disconnect from their neighbours and undergo individual migration. In cellular monolayers IP3Rs are juxtaposed with tight junctions. When individual cells migrate away from their neighbours IP3Rs preferentially accumulate at the leading edge where they surround focal adhesions. Uncaging of inositol trisphosphate (IP3) resulted in prominent accumulation of paxillin in focal adhesions, highlighting important functional implications of the observed novel structural relationships. ER-PM junctions and STIM1 proteins also migrate to the leading edge and position closely behind the IP3Rs, creating a stratified distribution of Ca(2+) signalling complexes in this region. Importantly, migration of PDAC cells was strongly suppressed by selective inhibition of IP3Rs and store-operated Ca(2+) entry (SOCE), indicating that these mechanisms are functionally required for migration.

The Reverse Superficial Sural Artery Flap Revisited for Complex Lower Extremity and Foot Reconstruction.

BACKGROUND: Soft-tissue defects of the distal lower extremity and foot present significant challenges to the reconstructive surgeon. The reverse superficial sural artery flap (RSSAF) is a popular option for many of these difficult wounds. Our initial experience with this flap at multiple institutions resulted in a 50% failure rate, mostly because of critical venous congestion. To overcome this, we have modified our operative technique, which has produced a more reliable flap. METHODS: All patients reconstructed with an RSSAF between May 2002 and September 2013 were retrospectively reviewed. In response to a high rate of venous congestion in an early group of patients, we adopted a uniform change in operative technique for a late group of patients. A key modification was an increase in pedicle width to at least 4 cm. Outcomes of interest included postoperative complications and limb salvage rate. RESULTS: Twenty-seven patients were reconstructed with an RSSAF (n = 12 for early group, n = 15 for late group). Salvage rate in the early group was 50% compared with 93% in the late group (P = 0.02). Postoperative complications (75% vs. 67%, P = 0.70) were similar between groups. Venous congestion that required leech therapy was 42% in the early group (n = 5) and 0% in the late group (P = 0.01). CONCLUSIONS: Venous congestion greatly impairs the survival of the RSSAF. A pedicle width of at least 4 cm is recommended to maintain venous drainage and preserve flap viability.

Endorepellin, the angiostatic module of perlecan, interacts with both the α2ß1 integrin and vascular endothelial growth factor receptor 2 (VEGFR2): a dual receptor antagonism.

Endorepellin, the C-terminal module of perlecan, negatively regulates angiogenesis counter to its proangiogenic parental molecule. Endorepellin (the C-terminal domain V of perlecan) binds the α2ß1 integrin on endothelial cells and triggers a signaling cascade that leads to disruption of the actin cytoskeleton. Here, we show that both perlecan and endorepellin bind directly and with high affinity to both VEGF receptors 1 and 2, in a region that differs from VEGFA-binding site. In both human and porcine endothelial cells, this interaction evokes a physical down-regulation of both the α2ß1 integrin and VEGFR2, with concurrent activation of the tyrosine phosphatase SHP-1 and downstream attenuation of VEGFA transcription. We demonstrate that endorepellin requires both the α2ß1 integrin and VEGFR2 for its angiostatic activity. Endothelial cells that express α2ß1 integrin but lack VEGFR2, do not respond to endorepellin treatment. Thus, we provide a new paradigm for the activity of an antiangiogenic protein and mechanistically explain the specificity of endorepellin for endothelial cells, the only cells that simultaneously express both receptors. We hypothesize that a mechanism such as dual receptor antagonism could operate for other angiostatic fragments.

Splinting after carpal tunnel release: current practice, scientific evidence, and trends.

BACKGROUND: Postoperative splinting is common after carpal tunnel release, despite a lack of scientific evidence supporting its value. The purpose of this study was to characterize postoperative splinting regimens among hand surgeons and to identify trends in splint use after this procedure. METHODS: Questionnaires were mailed to members of the American Society for Surgery of the Hand. Recipients were asked to record whether and for how long they use splints after carpal tunnel release. They were also asked to indicate their training (i.e., orthopedic, plastic, or general surgery). Results were compared with those of previously conducted surveys. RESULTS: One thousand ninety-one questionnaires were returned, for a response rate of 48 percent. Fifty-three percent of respondents use splinting postoperatively. Duration of splinting varied tremendously, from 1 to 42 days. Splinting patterns were similar across all training backgrounds. In comparison with related surveys conducted in 1987 and 1997, a trend is evident toward less use of splinting after carpal tunnel release. CONCLUSIONS: The use and duration of splinting after carpal tunnel release vary widely among hand surgeons. This divergence of practice implies that there is little therapeutic benefit to splinting after this procedure, a concept supported by substantial scientific evidence and by a trend away from splinting over the past 20 years.

The effect of magnetic fields on wound healing: experimental study and review of the literature.

OBJECTIVE: Magnets are purported to aid wound healing despite a paucity of scientific evidence. The purpose of this study was to evaluate the effect of static magnetic fields on cutaneous wound healing in an animal model. The literature was reviewed to explore the historical and scientific basis of magnet therapy and to define its current role in the evidence-based practice of plastic surgery. METHODS: Standardized wounds were created on the backs of 33 Sprague-Dawley rats, which were divided into 3 groups with either a 23 gauss magnet (group 1), a sham magnet (group 2), or nothing (group 3) positioned over the wound. The rate of wound closure by secondary intention was compared between the groups. Literature review was conducted through searches of PubMed and Ovid databases for articles pertinent to magnets and wound healing. RESULTS: Wounds in the magnet group healed in an average of 15.3 days, significantly faster than those in either the sham group (20.9 days, P = .006) or control group (20.3 days, P < .0001). There was no statistically significant difference between the sham and control groups (P = .45). CONCLUSIONS: An externally applied, low-power, static magnetic field increases the rate of secondary healing. Review of the literature reveals conflicting evidence regarding the use of magnetic energy to aid the healing of bone, tendon, and skin. Level I studies are lacking and difficult to execute but are needed to define conclusively the role of magnets in clinical practice.

The effect of herbal medicines on platelet function: an in vivo experiment and review of the literature.

BACKGROUND: Herbal medicines are used by a considerable number of surgical patients. An increased risk of bleeding, substantiated by anecdotal reports, has been attributed to the use of certain herbs, and numerous in vitro experiments have identified some herbal extracts as platelet inhibitors. The purpose of this investigation was to determine whether standard commercial preparations of commonly used herbal medicines have an effect on platelet function in vivo and, by extension, to provide clinical scientific evidence of the safety of their use in the perioperative period. METHODS: Five commercially available herbal agents were investigated, including Ginkgo biloba, garlic, Asian ginseng, St. John's wort, and saw palmetto. In a blinded fashion, one of the agents was administered to 10 adult volunteers at the manufacturer's recommended dose for 2 weeks. At the end of the 2-week period, in vivo platelet function was quantified using the PFA-100 assay. After a 2-week "washout" period, the protocol was repeated using a different agent. This 4-week cycle was repeated for each of the five herbal agents, as well as the control agent aspirin. RESULTS: In vivo platelet function was not affected by the administration of any herbal agent and was markedly inhibited with the administration of aspirin. CONCLUSIONS: The herbal medicines investigated in this study do not affect platelet function in vivo. Neither this experiment nor a review of the literature supports the concern of perioperative bleeding in users of these herbal medicines.

The inhibitory effect of minocycline on hypertrophic scarring.

BACKGROUND: Matrix metalloproteinases are enzymes that serve to degrade the extracellular matrix, giving them a central role in the inflammatory and wound-healing processes; they have been implicated in the pathophysiology of hypertrophic scarring. The purpose of this study was to examine the effect of minocycline, a matrix metalloproteinase inhibitor, on hypertrophic scarring. METHODS: Standardized wounds were created on the ears of eight New Zealand White rabbits. Half of the rabbits received daily injections of minocycline, whereas the other half received daily injections of saline (control). After 4 weeks, the resulting ear scars were harvested. Histologic slides were prepared from the thickest cross-sections of the scars, and from these slides the cross-sectional area of each scar was measured. A hypertrophic index was calculated by comparing the area of the scar to the baseline value of unwounded skin. Statistical analysis was performed using the SAS/STAT NESTED Procedure for hierarchical data. RESULTS: Among the rabbits treated with minocycline, the mean hypertrophic index was 1.08 +/- 0.01, compared with 1.54 +/- 0.03 in the control group (p = 0.03), representing an 85 percent reduction in hypertrophic area. CONCLUSIONS: Systemically administered minocycline significantly reduces the severity of hypertrophic scarring in a rabbit model. Although not directly examined in this study, matrix metalloproteinase inhibition is hypothesized to be responsible for this effect.

The effect of tirofiban on microvascular thrombosis: crush model.

BACKGROUND: Microvascular anastomotic thrombosis is a significant clinical problem, particularly in crush and avulsion injuries. Platelet deposition plays a particularly important role in the initiation and propagation of microvascular thrombosis, whereas thrombin has little effect in the acute phase of thrombus formation. Nevertheless, heparin (a specific thrombin inhibitor) remains the most widely used microvascular irrigant. The purpose of this study was to evaluate tirofiban HCl (Aggrastat), a glycoprotein IIb/IIIa inhibitor, and its role in preventing postoperative thrombosis in a crush anastomosis model. METHODS: A crush injury model using the rat femoral artery was used. End-to-end microvascular repairs were performed. One milliliter of irrigant was used within the vessel lumen before placement of the last suture. The irrigant used was randomized into one of four groups: lactated Ringer's as a control, tirofiban (50 microg/ml), heparin (100 U/ml), and a combination of heparin (100 U/ml) and tirofiban (50 microg/ml). The vessels were reexamined 24 hours postoperatively and patency was assessed. A total of 62 vessels were used for the study. RESULTS: The patency rate was two of 20 (10 percent) for the control group, 13 of 22 (59 percent) for the tirofiban group, one of 10 (20 percent) for the heparin group, and eight of 10 (80 percent) for the heparin plus tirofiban group. This study demonstrates a statistically significant improvement in patency with tirofiban irrigation in a crush anastomosis rat model when compared with saline or heparin alone. CONCLUSIONS: Clinically, tirofiban may have utility as a potent anticoagulant and is potentially useful in microvascular injuries that have a significant crush/avulsion component.

Analysis of freshwater pathogens: a guide to rational empiric antibiotic coverage.

Fresh water injuries are often contaminated with bacteria that are not typically encountered in other wounds. Their treatment should include empiric administration of appropriate antibiotics. This study identifies the most common pathogens found in the Lake of the Ozarks and their antibiotic sensitivity. Eleven of the twelve lake water samples (92%) had a positive culture result. Sixty-seven percent of lake water samples contained at least two strains of bacteria. Five different bacterial species of gram negative rods were isolated. All isolates were sensitive to Cefotetan, Ceftazidime, Ceftriaxone, Imipenem, Levofloxacin, Tobramycin, and Trimetheprim/sulfa. Antibiotic coverage after traumatic wounds required gram positive coverage. Our study suggests the addition of gram negative coverage for penetrating trauma contaminated by fresh water.

Analysis of lipocyte viability after liposuction.

Free fat grafts from liposuction aspirate can be used as donor material for soft-tissue augmentation. The purpose of this study was to attempt to identify a subpopulation of adipose cells within liposuction aspirate with the greatest viability and, it is hoped, a greater chance for increased survival after transplantation. Liposuction samples were obtained from 20 individuals (16 women, four men; age range, 27 to 49 years). These samples were then centrifuged at 50 g. At 2-minute intervals, specimens from three different areas (superficial, middle, deep) were obtained from each specimen. After collagenase degradation, the specimens were stained with trypan blue, and the number of viable cells were counted. The bottom (deepest) layer consistently contained the highest number of viable cells after centrifugation: 250 percent more viable cells when compared with the top layer (p < 0.0001) and 140 percent more viable cells when compared with the middle layer (p < 0.0002). Centrifugation beyond 2 minutes did not increase the number or proportion of viable adipocytes. When using aspirated fat from liposuction for soft-tissue augmentation, centrifugation for 2 minutes at 50 g will stratify the adipocytes, with more viable cells being found at the deepest layer. Using only this bottom portion of the fat layer for transplantation will yield a fat graft with a greater number of viable adipocytes, potentially improving fat graft survival and decreased fat graft resorption.

Fungal infections of the perionychium.

There are two types of fungi (yeasts and molds) both of which can cause superficial infections of the perionychium. Yeasts (such as Candida albicans) grow as single cells and reproduce by asexual budding. In contrast, molds grow in long filaments, called hyphae. There are approximately 100,000 species of fungi that have been characterized. Most of these are ubiquitous. Fortunately only about 200 are human pathogens, and only a handful are commonly found to be associated with human disease. This article discusses causes, symptoms, diagnosis, and treatment of the most common fungal infections of the perionychium, including superficial dermatophytosis, onychomycosis, and chronic paronychia.