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BACKGROUND: Phelan-McDermid syndrome (PMS) is a rare neurodevelopmental disorder caused by haploinsufficiency of the SHANK3 gene and characterized by global developmental delays, deficits in speech and motor function, and autism spectrum disorder (ASD). Monogenic causes of ASD such as PMS are well suited to investigations with novel therapeutics, as interventions can be targeted based on established genetic etiology. While preclinical studies have demonstrated that the neuropeptide oxytocin can reverse electrophysiological, attentional, and social recognition memory deficits in Shank3-deficient rats, there have been no trials in individuals with PMS. The purpose of this study is to assess the efficacy and safety of intranasal oxytocin as a treatment for the core symptoms of ASD in a cohort of children with PMS. METHODS: Eighteen children aged 5-17 with PMS were enrolled. Participants were randomized to receive intranasal oxytocin or placebo (intranasal saline) and underwent treatment during a 12-week double-blind, parallel group phase, followed by a 12-week open-label extension phase during which all participants received oxytocin. Efficacy was assessed using the primary outcome of the Aberrant Behavior Checklist-Social Withdrawal (ABC-SW) subscale as well as a number of secondary outcome measures related to the core symptoms of ASD. Safety was monitored throughout the study period. RESULTS: There was no statistically significant improvement with oxytocin as compared to placebo on the ABC-SW (Mann-Whitney U = 50, p = 0.055), or on any secondary outcome measures, during either the double-blind or open-label phases. Oxytocin was generally well tolerated, and there were no serious adverse events. LIMITATIONS: The small sample size, potential challenges with drug administration, and expectancy bias due to relying on parent reported outcome measures may all contribute to limitations in interpreting results. CONCLUSION: Our results suggest that intranasal oxytocin is not efficacious in improving the core symptoms of ASD in children with PMS. Trial registration NCT02710084.

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Ultraviolet (UV) radiation is an accepted etiological factor in cutaneous melanoma (CM), however its role in uveal melanoma (UM) is controversial. Partly as a consequence, CM and UM are often considered to be separate conditions, and advances in the treatment of CM have not led to joint clinical trials or parallel improvements in survival of UM. This study hypothesized that a subset of UM tumors displays evidence of genetic changes consistent with UV-related damage similar to that shown in CM. Analysis of the Broad Institute's Firebrowse depository of 80 UM samples and 343 CM samples, together with the Sanger Institute's Catalogue of Somatic Mutations in Cancer depository of 995 UM and 12,447 CM samples was undertaken to identify the most frequently mutated genes, mutation types, and specific nucleotide variants (SNVs) in each condition. Somatic mutation data were cross-correlated and shared mutations assessed against known effects of UV radiation. The proportion of samples with C>T substitutions (a classic genetic marker of UV-related damage) was higher in UM than CM on both DNA strands (17.0% vs 13.1%, p=0.038). The most frequently encountered cross-correlated mutated genes between UM and CM were, in order, BRAF, NRAS, TP53, CDKN2A, TERT, PTEN, ARID2, and KMT2C, with multiple common BRAF point mutations. Each cross-correlated mutation, and each common point mutation in BRAF, was associated with UV-related mechanistic changes. These findings support the hypothesis that the etiology of a substantial minority of UMs may be more UV dependent than previously recognized.

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Intravitreal injection of a therapeutic substance is the most common procedure performed in ophthalmology. It has a low incidence of serious complications but is associated with a small chance of endophthalmitis. Although the rate of endophthalmitis is between 0.019% and 0.09%, the associated visual morbidity is often devastating. Procedural changes have evolved over the years to improve patient comfort and reduce injection-related injury and infection. Despite the availability of published evidence, there remains considerable variations and lack of consensus in practical clinical settings. In addition, emerging literature concerning the use of speculums, the use of prophylactic topical antibiotics, and the setting of injections continues to impact the ophthalmologist's injection practice. This article provides an up to date assessment of various aspects of the procedure such as the setting, ventilation, type of anaesthetic, and control of sterility during the procedure; including discussions on performing bilateral eye same-day injections and the use of antibiotics.

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Switchgrass (Panicum virgatum L.) is a native perennial grass with potential as a biofuel crop. The smut fungus, Tilletia maclaganii (Berk.) Clint., is associated with significant biomass reduction in switchgrass in the Midwest (4), but has not been reported in the northeast United States in more than 60 years (New York in 1890 and Pennsylvania in 1946) (2,3). From 2007 to 2010, smutted panicles were observed on the majority of plants in stands of several switchgrass cultivars at the USDA-NRCS Plant Materials Center in Big Flats (Chemung County), NY; in production fields of several switchgrass cultivars near Meadville (Crawford County), PA; and in an ornamental bed of switchgrass in Ithaca (Tompkins County), NY. Smutted panicles emerged 3 to 4 weeks prior to healthy panicles, had a compact, club-shaped appearance, and enlarged florets with swollen ovaries that readily released a powdery mass of odorless, rusty orange-to-dark brown teliospores when pinched. The entire caryopsis of every floret within a panicle was smutted and the infected plants appeared stunted, indicative of systemic infection. The fungus from each location was identified as T. maclaganii based on host, habit, and teliospore morphology (3). Teliospores were pale yellowish brown to reddish brown, varied from globose to slightly irregular in shape, and averaged 21 µm (18 to 25 µm) in diameter. The exospore was thick (2 to 3 µm), finely verrucose, and no sheath was present. True sterile cells, pale yellow and 10 to 18 µm in diameter, were sparsely present. Teliospores germinated and formed large (40 to 60 × 3 to 6 µm), nonconjugating basidiospores within 20 h on 2% water agar (WA). Occasionally, we also found the floret-infecting species T. pulcherrima (1) on switchgrass at very low incidence in Big Flats, NY, but it was easily distinguished from T. maclaganii. Stratified seeds (3 g) of 'Shelter', washed and found to be free of teliospores, were dusted with 0.04 g of teliospores of T. maclaganii isolate Tm001NY09 (Cornell Plant Pathology Herbarium Accession CUP-67931) harvested from infected 'Shelter' in Big Flats, NY in 2009. Inoculated and noninoculated seeds were sown in seedling trays, transplanted, and evaluated at panicle emergence. There were no symptoms on plants from noninoculated seeds. Symptoms on inoculated plants were consistent with field observations and teliospores were reisolated from infected panicles and cultured on 2% WA. Teliospores harvested from a single panicle infected with Tm001NY09 were used for culturing and DNA extraction. The fully annotated sequence of the rDNA internal transcribed spacer and 5.8S regions of this isolate were deposited in GenBank (Accession No. JF745116). Smut outbreaks in New York and Pennsylvania suggest that T. maclaganii must be managed effectively if switchgrass production is to be sustainable in the Northeast. References: (1) L. M. Carris et al. Plant Dis. 92:1707, 2008. (2) R. Durán and G. W. Fischer. The Genus Tilletia. Washington State University, Pullman, WA, 1961. (3) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , May 3, 2011 (4) P. M. Thomsen et al. Online publication. doi:10.1094/PHP-2008-0317-01-RS. Plant Health Progress, 2008.

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BACKGROUND: Diabetes inhibits dark adaptation and both processes alter the electroretinogram (ERG) in similar ways. This study aimed to investigate the relationship between oscillatory potentials (OPs) and the b-wave during dark adaptation and to determine if this relationship changes during the development of diabetes. METHODS: Twenty-one rats were assigned to adaptation, control and diabetic groups. Rats were dark adapted for periods between 20 minutes and 4 hours, and ERGs recorded. Diabetes was induced with streptozotocin, and ERGs measured after 3, 6, 9 and 12 weeks after injection. RESULTS: Increasing periods of dark adaptation led to a logarithmic increase in the amplitude of the b-wave and the OPs. This was accompanied by a decrease in the peak times of the OPs and b-wave. Total OP amplitude and b-wave amplitude were linearly related, allowing an empirical OP constant to be developed to describe the relationship between the two parameters. Diabetes led to a progressive decrease in the amplitude and increase in the peak time of all waves. The OP constant decreased in a linear fashion with increasing duration of diabetes. CONCLUSIONS: It is argued that OP masking of the b-wave could explain previous inconsistencies in reported ERG changes in diabetes and that a slowing of dark adaptation does not account for these ERG changes. The report concludes that the OPs and b-wave amplitudes and latencies are intimately related in the normal retina and that this correlation is lost predictably during the development of diabetes.

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BACKGROUND: The transglutaminase (TG) family consists of eight distinct isoforms. TG types 1, 3 and 5 play a major role in normal skin development, with TG2 also being elevated during dermal wounding. TG1, 3 and 5 are responsible for the cross-linking of keratin precursors and formation of the cornified envelope during keratinocyte differentiation. TG2 may play a role in keratinocyte basement membrane cross-linking. Abnormal TG expression has been demonstrated in Darier disease, Netherton syndrome, psoriasis and lamellar ichthyosis. During a recent investigation of skin contraction in tissue-engineered skin, transglutaminase inhibitors were found to produce hyperproliferation and parakeratosis. OBJECTIVES: Accordingly, this study was designed to study the effect of pan-transglutaminase inhibition on morphology of tissue-engineered skin and expression of keratinocyte differentiation and proliferation-associated antigens. METHODS: We used a tissue-engineered model of human skin, based on de-epidermized acellular human dermis, seeded with normal keratinocytes and dermal fibroblasts and cultured at an air-liquid interface. The pan-transglutaminase inhibitors putrescine, NTU283 (1-dimethyl,2-[(oxopropyl)thio]imidazolium) and NTU285 (N-benzyloxycarbonyl-l-glutaminyl-6-dimethylsulfonium-5-oxo-l-norleucine) were added to the culture medium. After 28 days, histology and immunohistochemistry for collagen IV, involucrin and cytokeratins 6, 10 and 16 were performed. RESULTS: Keratinocyte hyperproliferation and parakeratosis were seen in response to transglutaminase inhibition. Inhibition of transglutaminase also resulted in loss of basement membrane collagen IV. Involucrin and cytokeratins 6 and 16 were confined to the basal layers in control composites but expressed throughout the epidermis in response to transglutaminase inhibition. A distinct band of expression of cytokeratin 10 was seen in the upper stratum granulosum of control composites but only patchy expression was seen after transglutaminase expression. CONCLUSIONS: Pan-transglutaminase inhibition inhibits terminal differentiation of keratinocytes, leading to a hyperproliferative epidermis with parakeratosis and enhanced expression of involucrin and cytokeratins 6 and 16. Expression of the differentiation-associated cytokeratin, cytokeratin 10, is reduced. Basement membrane integrity is also lost as a result of transglutaminase inhibition.

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The aim of this work was to investigate the interrelated effects of glucose, nitric oxide (NO) and erythropoietin on neuronal survival in retinal cultures, thereby exploring the mechanism of neuronal death in the diabetic retina. Rat retinal cells were cultured in low (5 mM) or high (15 mM) glucose concentrations. After 9 days, cell viability was assessed by (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and NO production was determined by the Griess reaction. Immunohistochemistry was used to quantify GABA-labelled neurones and cells staining for DNA breakdown. High or low glucose concentrations had no effect on basal NO production or the survival of neurones in culture, but treatment with N-nitro-L-arginine methyl ester reduced extracellular levels of NO and increased neuronal survival at both concentrations of glucose. Erythropoietin decreased cell death and NO levels, but only in cultures grown in low concentrations of glucose. It is concluded that erythropoietin's neurotrophic function in the retina is attenuated at glucose concentrations similar to those which occur in diabetes.

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BACKGROUND: Experimental studies have yielded a wealth of information related to the mechanism of ganglion cell death following injury either to the myelinated ganglion cell axon or to the ganglion cell body. However, no suitable animal models exist where injury can be directed to the optic nerve head region, particularly the unmyelinated ganglion cell axons. The process of relating the data from the various animal models to many different types of optic neuropathies in man must, therefore, be cautious. RESULTS: Extensive studies on the isolated optic nerve have yielded valuable information on the way white matter is affected by ischaemia and how certain types of compounds can attenuate the process. Moreover, there are now persuasive data on how ganglion cell survival is affected when the ocular blood flow is reduced in various animal models. As a consequence, the molecular mechanisms involved in ganglion cell death are fairly well understood and various pharmacological agents have been shown to blunt the process when delivered before or shortly after the insult. CONCLUSIONS: A battery of agents now exist that can blunt animal ganglion cell death irrespective of whether the insult was to the ganglion cell body or the myelinated axon. Whether this information can be applied for use in patients remains a matter of debate, and major obstacles need to be overcome before the laboratory studies may be applied clinically. These include the delivery of the pharmacological agents to the site of ganglion cell injury and side effects to the patients. Moreover, it is necessary to establish whether effective neuroprotection is only possible when the drug is administered at a defined time after injury to the ganglion cells. This information is essential in order to pursue the idea that a neuroprotective strategy can be applied to a disease like glaucoma, where ganglion cell death appears to occur at different times during the lifetime of the patient.

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OBJECTIVE: To develop tissue-engineered buccal mucosa (TEBM) for use in substitution urethroplasty, as urethral reconstruction is limited by the amount and type of tissue that is available for grafting, and BM has become the favoured tissue for use as a urethral substitute in the last decade. MATERIALS AND METHODS: After enzymatic treatment of a small (0.5 cm) BM biopsy the epidermis and dermis were mechanically separated. Oral keratinocytes were isolated from the epidermis and oral fibroblasts from the dermis. These cells were expanded and applied to sterilized de-epidermized dermis (DED) to obtain a full-thickness TE oral mucosa. Horizontal migration of keratinocytes on the DED was assessed using a tetrazolium-blue (MTT) assay. The TEBM was assessed histologically after mechanical stressing in vitro using catheterization and meshing. RESULTS: Histologically the TEBM closely resembled the native oral mucosa after culturing at an air-liquid interface for 2 weeks. The MTT assay showed good horizontal migration of keratinocytes on the DED. Serial histology revealed a gradually increasing thickness of the epidermis and remodelling of the dermis by the fibroblasts from day 1 to day 14. Despite subjecting the TEBM to mechanical stress the integrity of the epidermal-dermal junction was maintained. CONCLUSIONS: We report the successful culture of full-thickness TEBM for substitution urethroplasty, which is robust and suitable for clinical use.

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Alpha-melanocyte stimulating hormone (alpha-MSH) is known to have pleiotrophic functions including pigmentary, anti-inflammatory, antipyretic and immunoregulatory roles in the mammalian body. It is also reported to influence melanoma invasion with levels of alpha-, beta- and gamma-MSH correlated clinically with malignant melanoma development, but other studies suggest alpha-MSH acts to retard invasion. In the present study, we investigated the action of alpha-MSH on three human melanoma cell lines (HBL, A375-SM and C8161) differing in metastatic potential. alpha-melanocyte-simulating hormone reduced invasion through fibronectin and also through a human reconstructed skin composite model for the HBL line, and inhibited proinflammatory cytokine-stimulated activation of the NF-kappaB transcription factor. However, A375-SM and C8161 cells did not respond to alpha-MSH. Immunofluorescent microscopy and Western blotting identified melanocortin-1 receptor (MC-1R) expression for all three lines and MC-2R on HBL and A375-SM lines. Receptor binding identified a similar affinity for alpha-MSH for all three lines with the highest number of binding sites on HBL cells. Only the HBL melanoma line demonstrated a detectable cyclic adenosine monophosphate (cAMP) response to alpha-MSH, although all three lines responded to acute alpha-MSH addition (+(-)-N(6)-(2-phenylisopropyl)-adenosine (PIA)) with an elevation in intracellular calcium. The nonresponsive lines displayed MC-1R polymorphisms (C8161, Arg (wt) 151/Cys 151; A375-SM, homozygous Cys 151), whereas the HBL line was wild type. Stable transfection of the C8161 line with wild-type MC-1R produced cells whose invasion was significantly inhibited by alpha-MSH. From this data, we conclude that alpha-MSH can reduce melanoma cell invasion and protect cells against proinflammatory cytokine attack in cells with the wild-type receptor (HBL).

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The invasion of melanoma is complex and multi-staged and involves changes in both cell/extracellular matrix (ECM) and cell/cell interactions. Female steroids and alpha-MSH have also been reported to influence metastatic melanoma progression, but their mechanisms of action are unknown. Accordingly, our aim was to establish in vitro models to examine (a) the influence of sex steroids and alpha-melanocyte-stimulating hormone (alpha-MSH) on tumour invasion and the influence of (b) ECM proteins and (c) adjacent cells on melanoma invasion. In the first model, melanoma cell invasion through fibronectin over 20 hr under serum-free conditions was used to investigate the effects of 17beta-oestradiol and oestrone on the invasion of human melanoma cell lines, A375-SM and HBL. A375-SM, but not HBL cells, proved very susceptible to inhibition by female steroids. However, invasion of the HBL line was inhibited by alpha-MSH. Using the second model of reconstructed human skin based on de-epidermised acellular dermis, we found that the HBL cells on their own failed to invade into the dermis (irrespective of the presence or absence of the basement membrane). However, there was a significant synergistic interaction between keratinocytes, fibroblasts and HBL cells, such that a modest invasion of HBLs into the dermis was seen within 2 weeks when other skin cells were present. In contrast, A375-SM cells showed a significant ability to invade the dermis in the absence of other cells, with less invasion when other skin cells were present. In summary, these models have provided new information on the extent to which melanoma cell invasion is sensitive to oestrogenic steroids and to alpha-MSH and to interaction, not only with adjacent skin cells but also to the presence of basement membrane antigens.

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AIM: To compare the immediate and late outcomes of patients treated by a policy of routine stent implantation with routine balloon angioplasty and the use of stents only when an ideal result has not been obtained. METHODS: A nine centre, multinational, randomised study of 300 patients with coronary artery disease thought suitable for treatment of a single lesion by balloon angioplasty or stent implantation. Only new lesions in patients who had not undergone previous bypass surgery were included, and totally occluded vessels were excluded. RESULTS: The initial procedure was considered successful in 96% of patients. There was more complete angiographic restoration of luminal diameter in patients treated by elective stent (minimum lumen diameter (MLD) 2.68 mm for stent v 2.27 mm for balloon; p < 0.007), but analysis of the subgroup of balloon angioplasty patients who crossed over to stenting showed that they achieved similar results to the elective stent group. Late luminal loss was greater in stented patients than in those undergoing balloon angioplasty only, and by six months the angiographic benefit of stenting had disappeared (MLD 1.90 mm for stent group v 2.00 mm for balloon angioplasty). Angiographic and clinical results in the balloon angioplasty group were assisted by the high crossover rate (30.1%). Both groups had similar symptom relief, with 58.9% of patients improving by two or more angina grades. The need for further revascularisation was also similar in the two groups at one year (18.2% in the stented group v 17.1% in the balloon angioplasty group). Haemorrhagic complications at the local arterial entry site were more common than expected and were distributed equally between the patients receiving full anticoagulation and those receiving antiplatelet treatment only. The results of both Wiktor stent placement and balloon angioplasty were similar to the findings in the stent group in previous randomised studies (Benestent II, STRESS). CONCLUSIONS: Provisional stenting appears to offer the same longer term outcome as elective stenting in this selected group of patients. Improvement in the results of conventional balloon angioplasty in the past 10 years means that a policy of obtaining an ideal result without the use of stents appears to be practicable in many of these patients, with consequent cost savings.

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The relation between length of training and Dan rank was significant for 110 male black-belts, although the complication of tied ranks was noted.

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The purpose of this study was to compare the invasive properties of normal human cutaneous melanocytes and of a cutaneous melanoma cell line (HBL) in a three-dimensional model of reconstructed human skin. Specifically, we asked to what extent the pigmentary and invasive behaviour of both cells is influenced by their interaction with adjacent skin cells (keratinocytes and fibroblasts) and the basement membrane (BM). In the presence of a BM, normal human melanocytes within this model remained within the basal layer of keratinocytes and did not pigment spontaneously. When the BM was removed, melanocytes were found suprabasally and pigmented extensively. No significant invasion of melanocytes into the dermis was detected in the presence or absence of the BM. HBL melanoma cells showed no significant ability to invade into the dermis in the absence of other cells, irrespective of the presence or absence of the BM. However, when added to keratinocytes and fibroblasts, HBL cells showed a capacity to invade into the dermis, both in the presence and absence of the BM. Associated with HBL invasion into the dermis, we noted significant keratinocyte entry into the dermis. On their own, keratinocytes entered the dermis in the absence of the BM but showed no significant penetration into the dermis when the BM was present. In summary, this model demonstrates clear differences between melanocytes and a melanoma cell line with respect to their invasive properties. It also allows demonstration of interactions between cells, and between cells and the BM. The study also provides evidence for a synergistic interaction between this melanoma cell line and keratinocytes in penetrating the BM.

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The State-Trait Anxiety Inventory was administered to 17 female karateka immediately prior to Shotokan karate practice. As no difference between mean Trait and State Anxiety scores was found, it was suggested that this may be seen as a reflection of the changing times.

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The aim of this study was to identify a sterilization technique for the preparation of human allodermis which could be used as a dermal component in wound healing and as the dermal base for production of dermal-epidermal composites for one-stage grafting in patients. We report that it is possible to produce dermal-epidermal composites which perform well in vitro and in vivo using a standard ethylene oxide sterilization methodology. Prevention of ethylene oxide-induced damage to the dermis was achieved using gentle dehydration of the skin prior to ethylene oxide sterilization. The issue of whether viable fibroblasts are required for composite production was examined in comparative studies using glycerol vs. ethylene oxide sterilized dermis. Where good collagen IV retention was achieved following preparation of acellular de-epidermized dermis there was no advantage to having fibroblasts present in vitro or in vivo; however, where collagen IV retention was poor or where keratinocytes were initially expanded in culture then there was a significant advantage to introducing fibroblasts to the composites during their preparative 10-day period in vitro. The requirement for fibroblasts became less evident when composites were grafted on to nude mice. In conclusion, we report a protocol for the successful sterilization of human allodermis to achieve an acellular dermis with good retention of collagen IV. This acellular dermis would be appropriate for clinical use as a dermal replacement material. It can also be used for the production of dermal-epidermal composites using autologous keratinocytes (with or without fibroblasts).

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11 experienced black-belt subjects were individually timed on each of the five Heian kata and then timed again when performing as part of a group. The pull of the group had a significant effect upon timing on two of the kata.

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Animal bites are seen almost daily in the emergency department, and the majority heal without complication. Pasteurella multocida is frequently the causative organism of localized wound infections and cellulitis in this patient population. P. multocida infection is usually associated with close contact with pets, such as dogs and cats, that harbor this organism as normal oral flora. Meningitis and septic arthritis are very rare sequelae of P. multocida infection. This case report presents a patient with P. multocida bacteremia, meningitis, and septic arthritis developing together as a complication of a cat bite.

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The importance of a dermal element when providing permanent wound cover for skin loss has become evident as the shortcomings of pure epidermal grafts are recognized. We are developing a skin composite formed from sterilized human de-epidermized acellular dermis, keratinocytes and fibroblasts with the ultimate aim of using this composite to cover full-thickness excised burn wounds. These composites can be prepared with or without basement membrane (BM) antigens initially present on the dermis. This study investigates the importance of retaining BM antigens on the dermis to the production and appearance of these composites in vitro. Skin composites prepared from dermis with BM antigens either present or absent initially were studied throughout 3 weeks. Composites with BM antigens present initially were significantly better than those initially lacking BM antigens in: (i) the degree of epithelial cell attachment to the underlying dermis (hemidesmosomes were seen only in the former); (ii) the morphology of the epithelial layer; (iii) the consistent presence of collagen IV and laminin and the increasing expression of tenascin; and (iv) the amount of soluble collagen IV and fibronectin detected in the conditioned media. We conclude that an initial BM antigen template is vital in this skin composite model for the attachment and differentiation of the epithelial layer and for the subsequent remodelling of the BM in vitro.

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