Time interval between primary radiotherapy and salvage laryngectomy: a predictor of pharyngocutaneous fistula formation.

Salvage laryngectomy (SL) is associated with high levels of morbidity. Rates of pharyngocutaneous fistulae (PCF) are as high as 35 % in some series. Patients at highest risk of such complications may be candidates for altered surgical management in terms of additional tissue transfer, or delayed tracheoesophageal puncture. This study investigates the relationship between the time from primary radiotherapy (RT) to salvage surgery and the development of PCF. 26 consecutive patients who underwent SL between 2000 and 2010 were identified from our institutional database. Demographic, staging, treatment and complication data were collected. Subgroup analysis was performed using the Student's t test or Mann-Whitney U test for continuous variables and either Chi-squared test or Fisher's Exact test for categorical variables. 26 patients underwent SL between October 2003 and July 2010. Of these, 15 (58 %) developed a PCF. On analysis of the time between pre-operative RT and surgery, a significant difference was seen, with a mean time of 19.5 months in those who developed a PCF versus 47.0 months in those who did not (p = 0.02). Patient characteristics, treatment, and pathology results were comparable between the two groups. There was no significant difference in distribution of the other covariates between the PCF and non-PCF groups. We reported a high rate of PCF and identified an association between PCF and a short time from primary treatment to salvage surgery. Identifying factors associated with higher rates of post-operative morbidity allows surgeons to adapt surgical planning in an attempt to minimize rates of PCF.

The morphological applicability of a novel endovascular aneurysm sealing (EVAS) system (Nellix) in patients with abdominal aortic aneurysms.

OBJECTIVE: Endovascular aneurysm sealing (EVAS) using the Nellix system is a promising alternative to endovascular repair (EVR) and open surgery for abdominal aortic aneurysms (AAA). The aim of this study was to investigate the proportion of patients with AAA who are morphologically suitable for treatment with Nellix. METHODS: Patients presenting with AAA were investigated at two regionalised vascular units. Separate cohorts were identified, who had undergone infrarenal EVR, open aneurysm repair, fenestrated endovascular repair (FEVR) or non-operative management. Pre-operative morphology was quantified using three-dimensional computed tomography according to a validated protocol. Each aneurysm was assessed for compliance with the instructions for use (IFU) of Nellix RESULTS: 776 patients were identified with mean age 75 ± 9 years. 730/776 (94.1%) had undergone infrarenal EVR, 6/776 (0.8%) open repair, 27/776 (3.5%) FEVR and 13/776 (1.7%) had been managed non-operatively. 544/776 (70.1%) of all AAA were morphologically suitable for Nellix. 533/730 (73.0%) of patients who had undergone infrarenal EVR were compliant with Nellix IFU, compared with 497/730 (68.1%), 379/730 (51.9%) and 214/730 (29.3%) with the IFU for Medtronic Endurant (p = .04) or Cook Zenith (p < .01) and Gore C3 Excluder (p < .01) endografts respectively. CONCLUSIONS: Nellix technology appears widely applicable to contemporary infrarenal AAA practice, and may provide an option for patients that are outside current EVR device instructions for use. However, formal outcomes study is still required, and will ultimately dictate the clinical relevance of this feasibility study. The major limitation to anatomic suitability for Nellix is currently the maximum patent lumen diameter of large AAA.

Experience from the selection and nutritional preparation for Expedition ICE MAIDEN: the first successful all-female unassisted Antarctic traverse.

INTRODUCTION: Expedition ICE MAIDEN (Ex IM) was the first all-female unsupported crossing of Antarctica. We describe the prerequisite selection and training, comparing those who formed the final team with other participants, and discuss how the expedition diet was established. METHODS: All women serving in the British Army were invited to participate. Following initial assessments, successful women completed three training/selection ski expeditions. Between expeditions 1 and 2, participants completed 6 months rigorous UK-based training. Weight was measured before and after the 6 months UK-based training, expeditions 2 and 3, and body composition by skinfold before and after expedition 2. Participant feedback, body composition and weight changes were applied to modify the expedition diet and provide weight gain targets prior to Ex IM. RESULTS: Following 250 applications, 50 women were assessed and 22, 12 and seven women attended training expeditions 1, 2 and 3, respectively. The final team of six women lost more weight than other participants during UK-based training (mean (SD) change -1.3 (1.5) kg vs -0.5 (1.6) kg, respectively, p=0.046) and during training expedition 2 (-2.8 (0.8) kg vs -1.7 (0.4) kg, respectively, p=0.048), when they also gained more lean mass (+2.1 (0.8) kg vs +0.4 (0.7) kg, respectively, p=0.004). The Ex IM diet provided 5000 kCal/day, comprising approximately 45% carbohydrate, 45% fat and 10% protein. Median (range) weight change between expedition 3 and Ex IM was +8.7 (-1.9 to +14.3) kg. CONCLUSIONS: The selected Ex IM team demonstrated favourable training-associated body composition changes. Training-associated weight loss informed the expeditionary diet design.

High-Efficiency Genome Editing of Streptomyces Species by an Engineered CRISPR/Cas System.

Next-generation sequencing technologies have rapidly expanded the genomic information of numerous organisms and revealed a rich reservoir of natural product gene clusters from microbial genomes, especially from Streptomyces, the largest genus of known actinobacteria at present. However, genetic engineering of these bacteria is often time consuming and labor intensive, if even possible. In this chapter, we describe the design and construction of pCRISPomyces, an engineered Type II CRISPR/Cas system, for targeted multiplex gene deletions in Streptomyces lividans, Streptomyces albus, and Streptomyces viridochromogenes with editing efficiency ranging from 70% to 100%. We demonstrate pCRISPomyces as a powerful tool for genome editing in Streptomyces.

Antibiotic-loaded bone void filler accelerates healing in a femoral condylar rat model.

AIMS: Demineralised bone matrix (DBM) is rarely used for the local delivery of prophylactic antibiotics. Our aim, in this study, was to show that a graft with a bioactive glass and DBM combination, which is currently available for clinical use, can be loaded with tobramycin and release levels of antibiotic greater than the minimum inhibitory concentration for Staphylococcus aureus without interfering with the bone healing properties of the graft, thus protecting the graft and surrounding tissues from infection. MATERIALS AND METHODS: Antibiotic was loaded into a graft and subsequently evaluated for drug elution kinetics and the inhibition of bacterial growth. A rat femoral condylar plug model was used to determine the effect of the graft, loaded with antibiotic, on bone healing. RESULTS: We found that tobramycin loaded into a graft composed of bioglass and DBM eluted antibiotic above the minimum inhibitory concentration for three days in vitro. It was also found that the antibiotic loaded into the graft produced no adverse effects on the bone healing properties of the DBM at a lower level of antibiotic. CONCLUSION: This antibiotic-loaded bone void filler may represent a promising option for the delivery of local antibiotics in orthopaedic surgery. Cite this article: Bone Joint J 2016;98-B:1126-31.

Inhibition of p130cas tyrosine phosphorylation by calyculin A.

P130cas is a dominant tyrosine phosphorylated protein in v-src-and v-crk-transformed cells. Tyrosine phosphorylation also occurs in response to integrin-mediated cell adhesion. P130cas has a unique structure with multiple SH2 and SH3 binding sites, which makes it a candidate docking protein that might be involved in several signal transduction pathways. Little is known about how p130cas itself is regulated. In this report we present evidence that tyrosine phosphorylated p130cas was rapidly dephosphorylated in several lymphatic cell lines after treatment with calyculin A, a serine/threonine phosphatase inhibitor. A similar result was obtained with okadaic acid, but higher concentrations and longer incubation times were required. Constitutive phosphorylation as well as receptor-cross linking-induced p130cas phosphorylation was inhibited. Furthermore, the p130cas-Crk association was disrupted by treatment of cells with calyculin A. However, the p130cas-Lyn association was not affected. These results suggest that calyculin A specifically affects SH2 domain-mediated protein-protein interactions and that Lyn does not bind to a susceptible SH2 domain. Furthermore, the data presented is consistent with the existence of a calyculin A-sensitive phosphatase or tyrosine kinase that may be a critical regulator of p130cas tyrosine phosphorylation.

Soluble vascular cell adhesion molecule (VCAM)-Fc fusion protein induces leukotriene C4 secretion in platelet-activating factor-stimulated eosinophils.

Eosinophil adhesion to vascular cell adhesion molecule-1 (VCAM-1) is important for cellular recruitment into allergic inflammatory sites. To determine whether eosinophil adhesion to VCAM-1 affects cell function, leukotriene C4 (LTC4) was measured. Human eosinophils were incubated with platelet-activating factor (PAF) in the presence or absence of soluble VCAM-Fc fusion protein (sVCAM-Fc) or immobilized VCAM-Fc. sVCAM-Fc induced a concentration-dependent increase in LTC4 secretion, which was dependent on the presence of PAF and not blocked by cyclic peptides shown to inhibit alpha4beta1-dependent adhesion. Likewise, soluble ICAM-Fc induced a concentration-dependent LTC4 secretion. LTC4 secretion was induced by the calcium ionophore, A23187, and the combination of sVCAM-Fc and A23187 had synergistic properties. It is interesting to note that Mn2+ or anti-beta1 monoclonal antibody, TS2/16, inhibited LTC4 secretion induced by sVCAM-Fc and PAF. Eosinophil adhesion to VCAM-Fc or interleukin-1 beta-stimulated endothelial cells did not induce LTC4 secretion. These data suggest that sVCAM-Fc-induced LTC4 secretion depends on distinct signals from those of eosinophil adhesion.

Central ventilatory depression by oral propranolol.

Propranolol, 20 mg, was given orally four times daily for 5 days and placebo four times daily for 5 days in a randomized, double-blind fashion to nine healthy subjects. At the beginning of the study and on the last day of each medication pulse rate, blood pressure, airways resistance, maximum expiratory flow versus volume (using air and using 80% helium and 20% oxygen), spirometry before and after inhaled isoproterenol, and ventilatory and occlusion pressure responses to rebreathing carbon dioxide were measured. Results were compared by the Wilcoxon signed-rank test. Propranolol was associated with decreases in pulse rate (P = 0.002), systolic blood pressure (P = 0.024), and diastolic blood pressure (P = 0.027). There were no differences in airway resistance or the change of expiratory flow with helium-oxygen. Propranolol did not alter the preisoproterenol spirometry values but did reduce the response to isoproterenol. There were decreases in ventilatory responses (P = 0.004) and occlusion pressure responses (P = 0.006) at an end-tidal carbon dioxide of 60 mm Hg. Propranolol's beta-adrenergic blockade suppresses central ventilatory response to carbon dioxide.

Interleukin-1beta expression is induced by adherence and is enhanced by Fc-receptor binding to immune complex in THP-1 cells.

Adherence of monocytes to endothelial cells and subsequently to basement membrane represents the initial steps in monocyte migration from the vasculature to the interstitium. We investigated the role of adhesion to endothelial cells and basement membrane in the induction of the cytokine IL-1beta. We demonstrated that mRNA for IL-1beta is induced in adherent THP-1 cells, but not in a matrix-specific manner. Adherence to fibrinogen, however, causes an increase in mRNA for IL-1beta. A background level of IL-1beta mRNA induction was observed in cells adherent to all matrices, including the non-specific human serum albumin substrate, as compared to non-adherent cells cultured in teflon troughs. In addition, antibodies to CD11a, CD11b, beta1 integrin, VLA4, (alpha)v(beta)3 (VNR), and ICAM-1 did not induce significant IL-1beta mRNA when THP-1 cells were adherent to those immunoglobulins. THP-1 cells adherent to immune complexes of anti-CD11a, anti-CD11b, anti-VLA4, anti-VNR, and anti-ICAM-1 showed greater mRNA induction than cells adherent to primary antibodies alone. THP-1 cells adherent to non-specific immune complexes gave the highest level of mRNA induction. Secretion of IL-1beta protein, measured by ELISA at 24 h, was greatest when cells were adherent to immobilized immune complexes or to fibrinogen. Our results demonstrate that a general adherence-induced increase in IL-1beta gene expression is greatly enhanced by the presence of immune complex.

A benzothiophene-carboxamide is a potent inhibitor of IL-1beta induced VCAM-1 gene expression in human endothelial cells.

Vascular endothelial cells respond to cytokines such as IL-1 beta or TNF-alpha by undergoing a number of functional alterations. Among these alterations is the induction of cell surface adhesion molecules, including VCAM-1. In this report, we investigated the effects of a 3-alkoxybenzo[beta]thiophene-2-carboxamide (BZT) on the cytokine induction of VCAM-1 expression and activation of the transcription factor NF-kappa B in human endothelial cells. BZT blocked the IL-1 beta induced cell surface expression of VCAM-1 in human endothelial cells but did not prevent nuclear translocation of NF-kappa B. This study demonstrates that BZT is a potent inhibitor of VCAM-1 expression in human endothelial cells.

Mapping of MCP-1 functional domains by peptide analysis and site-directed mutagenesis.

Monocyte chemoattractant protein-1 (MCP-1) is a member of the beta chemokine family which acts through specific seven transmembrane receptors to recruit monocytes, basophils, and T lymphocytes to sites of inflammation. To identify regions of the human MCP-1 protein which are important for its biological activity, we have synthesized domain-specific peptides and tested their ability to antagonize MCP-1 binding and chemotaxis in THP-1 cells. We have found that an intercysteine first loop peptide encompassing amino acids 13-35 inhibits MCP-1 binding and chemotactic activity, while peptides representing the amino-terminus (amino acids 1-10), second loop (amino acids 37-51), and carboxy-terminus (amino acids 56-71) of MCP-1 have no effect. In addition, we have found that cyclization of the first loop peptide by disulfide linkage and blocking the C-terminus of the peptide by amidation increases the activity of this peptide to block MCP-1 binding and chemotaxis. In order to specifically identify amino acid residues within the first loop that are crucial for MCP-1 functional activity, we have substituted alanine for tyrosine (Y13A) or arginine (R18A) in MCP-1 recombinant proteins. While baculovirus produced wild type and R18A MCP-1 proteins are indistinguishable in their ability to induce THP-1 chemotaxis and show modest effects in binding activity compared to commercially available recombinant MCP-1 protein, the Y13A point mutation causes a dramatic loss in function. The identification of functional domains of MCP-1 will assist in the design of MCP-1 receptor antagonists which may be clinically beneficial in a number of inflammatory diseases.

Familial band heterotopias simulating tuberous sclerosis.

We report the clinical and neuroimaging findings of a mother and daughter with seizure disorders and band heterotopias seen on magnetic resonance imaging studies. These clinicoradiologic findings simulate those for a diagnosis of tuberous sclerosis complex. Clinicians should be aware of this migrational anomaly and its neuroimaging characteristics, as well as the potential for this specific migrational anomaly to be genetically transmitted.

Adaptations of forearm circulation to varied intensities and sets of heavy exercise.

Nine male and five female college students were tested on four occasions to determine the effects of various exhaustive weightlifting stimuli on forearm blood flow as measured by strain-gauge plethysmography. Maximum voluntary dynamic wrist curl strength was determined in the first session, which was followed by random treatments of exercise with 70, 80, and 90% of the weight subjects could curl only one time (1 RM) on three separate days. During each treatment, after a 15-min rest, subjects performed three sets of exercise movements at a pace of 0.5 Hz until voluntary exhaustion. Each set of continuous repetitions was followed by a 166-s recovery period. Blood flows (ml flow X 100 ml tissue-1 X min-1) were determined at 10, 36, 62, 88, 114, 140, and 166 s of recovery. Data were analyzed using ANOVA tests for a three (intensities) by three (sets) complete factorial design with repeated measures on all factors. Generally, 90% flows were significantly (P less than 0.05) lower than both 70 and 80% flows. Also, 70% flows were statistically equal to 80% flows. There were significant (P less than 0.05) set effects at 10, 36, 114, and 166 s of recovery. Blood flows for set 3 exceeded those for set 1 at 10, 36, 114, and 166 s of recovery, and blood flows for set 2 exceeded those for set 1 at 166 s of recovery.

Effects of color and pattern on implicit and explicit picture memory.

The degree to which repetition priming is perceptually specific is informative about the mechanisms of implicit memory as well as of perceptual processing. In 2 sets of experiments with pictures as stimuli, we tested the effects of color and pattern manipulations between study and test on implicit memory (i.e., naming facilitation) and explicit memory (i.e., 2 forms of recognition). These manipulations did not affect priming. However, participants were able to explicitly detect stimulus changes at above-chance levels. Changes in color also produced small decrements in participants' ability to judge that repeated stimuli were old on a recognition test. Experiment 2 showed diminished priming with changes in the stimulus exemplar (i.e., a different picture of the same named object) from study to test, which demonstrated that the picture-naming paradigm is sensitive to changes in physical attributes. The results suggest that physical attributes that are not essential to the formation of a shape representation do not influence repetition priming in a basic identification paradigm. Suggestions for how priming may be mediated are discussed.

Biochemical and molecular analysis of spontaneous and induced mutations at the mouse Mod-1 locus.

We have analyzed five Mod-1 (malic enzyme) mutants at the molecular and biochemical level. Four of these mutants, three electrophoretic variants and one null mutant, were induced by ethylnitrosourea (ENU). Another null mutant was the result of a spontaneous mutation. All of these mutations were heritable in a Mendelian fashion and viable in the homozygous condition. Restriction endonuclease and Southern blot analysis revealed that the spontaneous null mutant possessed an altered restriction fragment banding pattern. All of the ENU-induced mutants possessed normal restriction fragment banding patterns. All 5 mutants produced normal levels of Mod-1-specific mRNA. Only the spontaneous null mutant produced mRNA with altered size, which was consistent with the altered DNA-banding pattern. MOD-1 enzyme activity levels were normal in the three ENU-induced mutants with altered electrophoretic mobility. Enzyme activity was significantly lower than normal in tissues from animals homozygous for the null alleles, however, using Western blot analysis, low but significant levels of MOD-1 protein in Mod-1 null homozygotes were detected.

Molecular analysis of the TK locus in L5178Y large and small colony mouse lymphoma cell mutants induced by hycanthone methanesulfonate.

Mouse lymphoma cells of the L5178Y TK + /-3.7.2C line were exposed to varying concentrations of the anti-schistosomal drug hycanthone methanesulfonate. The trifluorothymidine (TFT)-resistant cells fell into two classes based on colony size. Southern blot analyses were performed using NcoI-digested DNA from a number of large and small mutant colonies from each treatment group. Two different restriction fragment banding patterns were identified in these analyses, those colonies that contained the 6.4 kb NcoI restriction fragment and those that did not. A total of 471 mutant colonies were analyzed and 84.5% (398) of these colonies did not exhibit the 6.4 kb fragment. There did not appear to be a hycanthone methanesulfonate dose response effect in the number of colonies that did not contain the 6.4 kb fragment among the treated groups. In addition, 82% (154 out of 188) of spontaneous mutants did not contain the 6.4 kb fragment. The results imply that greater than 80% of all spontaneous mutations found in the mouse lymphoma assay regardless of colony size do not contain the 6.4 kb fragment and each may be considered to be a large scale mutation. In addition, greater than 80% of the hycanthone induced mutations in the mouse lymphoma assay do not contain the 6.4 kb fragment and thus may be considered to be a large scale mutation.

Classical and molecular genetic analyses of his-3 mutants of Neurospora crassa. II. Southern blot analyses and molecular mechanisms of mutagenicity.

Previous studies (Overton et al., Mutation Res., 1989) on specific revertibility of 81 his-3 mutants have shown a correlation between complementation pattern and presumed genetic alteration similar to that shown by ad-3B mutants. In the present study, restriction enzyme analyses were used to further characterize the genetic alterations in individual his-3 mutants. The restriction fragment banding patterns of the majority of mutants were identical with that shown by wild-type 74-OR23-1A and were consistent with expectations based on previous data suggesting that they resulted from single base-pair alterations (Overton et al., Mutation Res., 1989). His-3 mutants with altered banding patterns were only found among those with polarized complementation patterns or noncomplementing mutants. One of the mutants with a polarized complementation pattern, 1-189-83, and another noncomplementing mutant, 1-189-85, are associated with genetic alterations proximal to the his-3 locus. In one other mutant, 1-226-565 (with a polarized complementation pattern), an insertion of approx. 2 kb has occurred in the proximal region of the his-3 locus. Two other mutants, 1-155-270 and 1-155-276 (both noncomplementing), contained a large insertion of approx. 12.8 kb in the proximal region of the his-3 locus.

Preliminary molecular analysis of the TK locus in L5178Y large- and small-colony mouse lymphoma cell mutants.

Mouse lymphoma cells of the L5178Y TK+/- -3.7.2C line were exposed to sidestream and mainstream cigarette smoke condensates (CSC). Cells which survived the trifluorothymidine (TFT) challenge fell in 2 classes: large- and small-colony formers. Southern blot analysis of NcoI-digested DNA from mutant colonies yielded 2 distinct restriction fragment banding patterns when probed with the thymidine kinase (TK) cDNA clone pMtk4. One such pattern was composed of 4 bands at 6.4, 5.5, 4.7 and 2.9 kilobase pairs (kb) and was identical to that of TK+/- controls. A second pattern differed from the first only in the absence of the 6.4-kb band. The majority (83/95) of both large and small colonies derived from cells exposed to CSC exhibited restriction fragment banding patterns lacking the 6.4-kb band. The data from the present study suggest that there is no association between mutant colony size and the presence of the 6.4-kb NcoI restriction fragment at the TK locus in the mouse lymphoma mutants analyzed.

Ethylene dibromide: negative results with the mouse dominant lethal assay and the electrophoretic specific-locus test.

Ethylene dibromide (1,2-dibromoethane; EDB) was tested for the induction of dominant lethal and electrophoretically-detectable specific-locus mutations in the germ cells of DBA/2J male mice. Males were treated with a single intraperitoneal injection of 100 mg/kg EDB and mated to two C57BL/6J females. In the dominant lethal assay, matings were carried out to measure the effect of EDB on meiotic and postmeiotic stages; germ cells representing spermatogonial stem cells were analyzed in the electrophoretic specific-locus test. Neither of these germ cell tests produced any evidence that EDB is a germ cell mutagen. It appears from these data and those reported in the literature that EDB, a genotoxic carcinogen that affects male fertility in some mammalian species, is not mutagenic in the germ cells of the male mouse.

A clinical comparison of the effect on the gingiva of the Prophy-Jet and the rubber cup and paste techniques.

This clinical trial compared the effect on the gingiva of the Prophy-Jet and the rubber cup and paste techniques of stain and supragingival plaque removal. Twenty-one human subjects with healthy gingiva or slight gingivitis participated. A split mouth design was used. The Prophy-Jet caused a statistically significant increase (P less than 0.05) in gingival irritation immediately posttreatment, but the differences were not deemed clinically significant. There were no statistically significant (P less than 0.05) or clinically significant differences in the effect on the gingiva between the two techniques at 7 and 21 days posttreatment. There was no lasting difference in gingival trauma between the two methods of stain and supragingival plaque removal in subjects with healthy gingiva or slight gingivitis.