8-Methoxypsoralen Plus Ultraviolet A Reduces the Psoriatic Response to Imiquimod in a Murine Model.

The effects of 8-Methoxypsoralen plus ultraviolet A (PUVA) or ultraviolet B (UVB) alone on imiquimod-induced psoriasis were examined in a mouse model. Mouse skin was treated with repetitive sub-phototoxic doses of PUVA or UVB before or during the induction of toll-like receptor 7/8 activation and psoriasis through the application of imiquimod. PUVA, to a greater degree than UVB, suppressed the established imiquimod-induced psoriatic phenotype, but pretreatment with PUVA prior to administration of imiquimod also reduced the susceptibility of murine skin to respond to imiquimod to a greater degree than did pretreatment with UVB. PUVA downregulated baseline levels of miRNA27a and 29a, as well as interferon-γ, interleukin-17 and -9, cytokines, which drive psoriatic inflammation. Microarray analysis showed enrichment of senescence pathway genes linked to upregulation of p16/p21 proteins after PUVA pretreatment. However, the anti-psoriatic effect of PUVA was lost when there was an interval of 7 days between final exposure to PUVA and the start of administration of imiquimod. This indicated that (UVB and) PUVA diminished imiquimod-induced established psoriatic inflammation, but also primed the skin in favour of a reduced responsiveness to toll-like receptor activation.

NorUrsodeoxycholic acid ameliorates cholemic nephropathy in bile duct ligated mice.

BACKGROUND & AIMS: Severe cholestasis may cause cholemic nephropathy that can be modeled in common bile duct ligated (CBDL) mice. We aimed to explore the therapeutic efficacy and mechanisms of norursodeoxycholic acid (norUDCA) in cholemic nephropathy. METHODS: In 8-week CBDL mice fed with norUDCA (prior or post CBDL) or chow we evaluated serum urea levels, urine cytology and urinary neutrophil gelatinase associated lipocalin (uNGAL), kidney and liver tissue quantification of fibrosis by hydroxyproline content and gene chip expression looking at key genes of inflammation and fibrosis. Moreover, we comprehensively analysed bile acid profiles in liver, kidney, serum and urine samples. RESULTS: NorUDCA-fed CBDL mice had significantly lower serum urea and uNGAL levels and less severe cholemic nephropathy as demonstrated by normal urine cytology, significantly reduced tubulointerstitial nephritis, and renal fibrosis as compared to controls. NorUDCA underwent extensive metabolism to produce even more hydrophilic compounds that were significantly enriched in kidneys. CONCLUSION: NorUDCA ameliorates cholemic nephropathy due to the formation of highly hydrophilic metabolites enriched in kidney. Consequently, norUDCA may represent a medical treatment for cholemic nephropathy. LAY SUMMARY: The term cholemic nephropathy describes renal dysfunction together with characteristic morphological alterations of the kidney in obstructive cholestasis that can be mimicked by ligation of the common bile duct in mice. Feeding the hydrophilic bile acid norUDCA to bile duct ligated mice leads to a significant amelioration of the renal phenotype due to the formation of highly hydrophilic metabolites enriched in the kidney and may therefore represent a medical treatment for cholemic nephropathy.

Mutations in KPTN cause macrocephaly, neurodevelopmental delay, and seizures.

The proper development of neuronal circuits during neuromorphogenesis and neuronal-network formation is critically dependent on a coordinated and intricate series of molecular and cellular cues and responses. Although the cortical actin cytoskeleton is known to play a key role in neuromorphogenesis, relatively little is known about the specific molecules important for this process. Using linkage analysis and whole-exome sequencing on samples from families from the Amish community of Ohio, we have demonstrated that mutations in KPTN, encoding kaptin, cause a syndrome typified by macrocephaly, neurodevelopmental delay, and seizures. Our immunofluorescence analyses in primary neuronal cell cultures showed that endogenous and GFP-tagged kaptin associates with dynamic actin cytoskeletal structures and that this association is lost upon introduction of the identified mutations. Taken together, our studies have identified kaptin alterations responsible for macrocephaly and neurodevelopmental delay and define kaptin as a molecule crucial for normal human neuromorphogenesis.

Patients with Amyotrophic Lateral Sclerosis Have High Interest in and Limited Access to Genetic Testing.

Although genetic testing for amyotrophic lateral sclerosis (ALS) is widely available, it is unknown what proportion of patients with ALS have access to genetic counseling and testing, and patient attitudes towards ALS genetic testing have not been studied. We conducted a national survey of ALS patients enrolled in the Agency for Toxic Substances and Disease Registry, which consisted of multiple choice questions and two 12 item Likert scale series assessing respondents' experience with and attitude toward genetic testing. The survey had an 8 % response rate, with 449 completed responses. Genetic testing was offered to 33.4 % and completed by 67.1 % of those offered. A minority of respondents (12.5 %) saw a genetic counselor, and were much more likely to be offered genetic testing (p = 0.0001). Respondents with a family history of ALS (8.4 %) were more likely to be offered testing (p = 0.0001) and complete testing (p = 0.05). Respondents with a family history of ALS were more likely to report a favorable attitude towards genetic testing (p = 0.0003), as were respondents who saw a genetic counselor (p = 0.02). The majority of respondents (82.7 %) felt that genetic testing should be offered to all patients with ALS. Our results indicate that ALS patients may have limited access to genetic testing, but perceive benefit from this service. Development of practice guidelines for genetic testing in ALS, to include the routine offer of genetic counseling, may result in broader and more consistent access to these services.

Zinc intake and status in Austria in the light of different reference values.

Zinc has been identified as a critical micronutrient also in high-income countries. There is still some uncertainty about the evaluation of zinc sufficiency due to divergent daily intake reference values. We wanted to exemplify this issue using data from the Austrian Study on Nutritional Status 2012. Plasma zinc concentrations were measured in a nationally representative sample of 872 persons aged 6-80 years (55.5 % female). Dietary zinc intake was estimated from two 24h dietary recalls. Additionally, parameters of the antioxidative status (plasma malondialdehyde (MDA), total antioxidative capacity) and activities of alkaline phosphatase (AP), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px)) were determined. Zinc status was marginal in schoolchildren (40 % of boys and 22 % of girls) and in elderly (28 % of men and 33 % of women). Dietary zinc intake was also unsatisfactory in these groups with 38 % of boys and 32 % of girls and 64.5 % of older men below the nationally recommended intake levels. However, the adequacy of zinc intake varied with different reference values. Adults were more likely to meet the D-A-CH reference values and those from the European Food Safety Authority than the recommendations of the International Zinc Nutrition Consultative Group (IZiNCG) and the Institute of Medicine, whereas children met the IZiNCG values best. Zinc status correlated weakly with AP activity (r = -0.298, p < 0.001) and some antioxidant status markers (CAT, MDA, GSH-PX, SOD), especially in the elderly (MDA: r = -0.527, p < 0.001, and SOD: r = -0.466, p = 0.002). Our results suggest a suboptimal zinc supply in Austria particularly among schoolchildren and older adults.

Monocytic cell differentiation from band-stage neutrophils under inflammatory conditions via MKK6 activation.

During inflammation, neutrophils are rapidly mobilized from the bone marrow storage pool into peripheral blood (PB) to enter lesional sites, where most rapidly undergo apoptosis. Monocytes constitute a second wave of inflammatory immigrates, giving rise to long-lived macrophages and dendritic cell subsets. According to descriptive immunophenotypic and cell culture studies, neutrophils may directly "transdifferentiate" into monocytes/macrophages. We provide mechanistic data in human and murine models supporting the existence of this cellular pathway. First, the inflammatory signal-induced MKK6-p38MAPK cascade activates a monocyte differentiation program in human granulocyte colony-stimulating factor-dependent neutrophils. Second, adoptively transferred neutrophils isolated from G-CSF-pretreated mice rapidly acquired monocyte characteristics in response to inflammatory signals in vivo. Consistently, inflammatory signals led to the recruitment of osteoclast progenitor cell potential from ex vivo-isolated G-CSF-mobilized human blood neutrophils. Monocytic cell differentiation potential was retained in left-shifted band-stage neutrophils but lost in neutrophils from steady-state PB. MKK6-p38MAPK signaling in HL60 model cells led to diminishment of the transcription factor C/EBPα, which enabled the induction of a monocytic cell differentiation program. Gene profiling confirmed lineage conversion from band-stage neutrophils to monocytic cells. Therefore, inflammatory signals relayed by the MKK6-p38MAPK cascade induce monocytic cell differentiation from band-stage neutrophils.

Synergistic effects of NOD1 or NOD2 and TLR4 activation on mouse sickness behavior in relation to immune and brain activity markers.

Toll-like receptors (TLRs) and nuclear-binding domain (NOD)-like receptors (NLRs) are sensors of bacterial cell wall components to trigger an immune response. The TLR4 agonist lipopolysaccharide (LPS) is a strong immune activator leading to sickness and depressed mood. NOD agonists are less active but can prime immune cells to augment LPS-induced cytokine production. Since the impact of NOD and TLR co-activation in vivo has been little studied, the effects of the NOD1 agonist FK565 and the NOD2 agonist muramyl dipeptide (MDP), alone and in combination with LPS, on immune activation, brain function and sickness behavior were investigated in male C57BL/6N mice. Intraperitoneal injection of FK565 (0.001 or 0.003mg/kg) or MDP (1 or 3mg/kg) 4h before LPS (0.1 or 0.83mg/kg) significantly aggravated and prolonged the LPS-evoked sickness behavior as deduced from a decrease in locomotion, exploration, food intake and temperature. When given alone, FK565 and MDP had only minor effects. The exacerbation of sickness behavior induced by FK565 or MDP in combination with LPS was paralleled by enhanced plasma protein and cerebral mRNA levels of proinflammatory cytokines (IFN-γ, IL-1ß, IL-6, TNF-α) as well as enhanced plasma levels of kynurenine. Immunohistochemical visualization of c-Fos in the brain revealed that NOD2 synergism with TLR4 resulted in increased activation of cerebral nuclei relevant to sickness. These data show that NOD1 or NOD2 synergizes with TLR4 in exacerbating the immune, sickness and brain responses to peripheral immune stimulation. Our findings demonstrate that the known interactions of NLRs and TLRs at the immune cell level extend to interactions affecting brain function and behavior.

Use of whole genome expression analysis in the toxicity screening of nanoparticles.

The use of nanoparticles (NPs) offers exciting new options in technical and medical applications provided they do not cause adverse cellular effects. Cellular effects of NPs depend on particle parameters and exposure conditions. In this study, whole genome expression arrays were employed to identify the influence of particle size, cytotoxicity, protein coating, and surface functionalization of polystyrene particles as model particles and for short carbon nanotubes (CNTs) as particles with potential interest in medical treatment. Another aim of the study was to find out whether screening by microarray would identify other or additional targets than commonly used cell-based assays for NP action. Whole genome expression analysis and assays for cell viability, interleukin secretion, oxidative stress, and apoptosis were employed. Similar to conventional assays, microarray data identified inflammation, oxidative stress, and apoptosis as affected by NP treatment. Application of lower particle doses and presence of protein decreased the total number of regulated genes but did not markedly influence the top regulated genes. Cellular effects of CNTs were small; only carboxyl-functionalized single-walled CNTs caused appreciable regulation of genes. It can be concluded that regulated functions correlated well with results in cell-based assays. Presence of protein mitigated cytotoxicity but did not cause a different pattern of regulated processes.

Mutations in B4GALNT1 (GM2 synthase) underlie a new disorder of ganglioside biosynthesis.

Glycosphingolipids are ubiquitous constituents of eukaryotic plasma membranes, and their sialylated derivatives, gangliosides, are the major class of glycoconjugates expressed by neurons. Deficiencies in their catabolic pathways give rise to a large and well-studied group of inherited disorders, the lysosomal storage diseases. Although many glycosphingolipid catabolic defects have been defined, only one proven inherited disease arising from a defect in ganglioside biosynthesis is known. This disease, because of defects in the first step of ganglioside biosynthesis (GM3 synthase), results in a severe epileptic disorder found at high frequency amongst the Old Order Amish. Here we investigated an unusual neurodegenerative phenotype, most commonly classified as a complex form of hereditary spastic paraplegia, present in families from Kuwait, Italy and the Old Order Amish. Our genetic studies identified mutations in B4GALNT1 (GM2 synthase), encoding the enzyme that catalyzes the second step in complex ganglioside biosynthesis, as the cause of this neurodegenerative phenotype. Biochemical profiling of glycosphingolipid biosynthesis confirmed a lack of GM2 in affected subjects in association with a predictable increase in levels of its precursor, GM3, a finding that will greatly facilitate diagnosis of this condition. With the description of two neurological human diseases involving defects in two sequentially acting enzymes in ganglioside biosynthesis, there is the real possibility that a previously unidentified family of ganglioside deficiency diseases exist. The study of patients and animal models of these disorders will pave the way for a greater understanding of the role gangliosides play in neuronal structure and function and provide insights into the development of effective treatment therapies.

Mutation of HERC2 causes developmental delay with Angelman-like features.

BACKGROUND: Deregulation of the activity of the ubiquitin ligase E6AP (UBE3A) is well recognised to contribute to the development of Angelman syndrome (AS). The ubiquitin ligase HERC2, encoded by the HERC2 gene is thought to be a key regulator of E6AP. METHODS AND RESULTS: Using a combination of autozygosity mapping and linkage analysis, we studied an autosomal-recessive neurodevelopmental disorder with some phenotypic similarities to AS, found among the Old Order Amish. Our molecular investigation identified a mutation in HERC2 associated with the disease phenotype. We establish that the encoded mutant HERC2 protein has a reduced half-life compared with its wild-type counterpart, which is associated with a significant reduction in HERC2 levels in affected individuals. CONCLUSIONS: Our data implicate a model in which disruption of HERC2 function relates to a reduction in E6AP activity resulting in neurodevelopmental delay, suggesting a previously unrecognised role of HERC2 in the pathogenesis of AS.

Dual functionality of diacylglycerols in water-in-oil emulsion gel systems.

Distearin (DS) can be used as an emulsifier, due to its surface activity derived from the amphiphilic nature of the molecule, moreover, it can also crystallize and form a 3D crystal network that can induce oil gelation. The current research aimed to examine the ability to combine both emulsifying and oil gelation properties to structure and stabilize water-in-oil emulsion gel system. Different water contents and DS concentrations produce emulsion gels with different textural attributes while incorporating up to 30% of water in a 15% wt. DS-based oleogel resulted in stable white gels. Microscopy imaging confirmed the formation of a water-in-oleogel type emulsion gel characterized by DS crystallization in the continuous phase and at the interface through Pickering mechanism. A positive relation was observed between the G' and hardness values and water content, suggesting gel strengthening resulted from interactions between the DS crystals at the interface and the continuous phase, as suggested by the active filler theory. Thermal analysis revealed two broad melting events at the temperature range of 42.2-44.9 °C and 55.9-58.6 °C for emulsion gels with 10-30% water content, suggesting initial melting of ß' polymorph and transition to ß during melting, which was confirmed by XRD. The results showed that homogenization significantly improved the oil retention of the gels due to increased crystal surface area, while water addition slightly reduced it. Compared with traditional emulsions or oleogels, this water-in-oil gel system demonstrated prolonged stability and enhanced mechanical properties due to the dual functionality of DS at the water/oil interface and bulk.

Evaluation of formalin-free tissue fixation for RNA and microRNA studies.

FineFix, RCL-2 and HOPE, three formalin-free fixatives, were compared to the common used formalin fixed tissue samples of lung cancer and were evaluated for their effects on quality, quantity and integrity of RNA and microRNA. Two commercially available RNA extraction Kits (RNeasy FFPE by Qiagen and RecoverAll™ Nucleic Acid Isolation by Ambion) were tested and optimized in order to determine an extraction protocol for RNA as well as miRNA independent of the fixative. Two selected miRNAs were quantified via TaqMan MicroRNA assays. The optimized RNA extraction protocol for Qiagen's Kit leads to similar results for RNA quality and integrity for all fixatives. Highest RNA yield was obtained for formalin and the highest average miRNA ratio was found for FineFix. RNA fragments smaller than 500 bases were detected in FineFix, formalin and RCL2 fixed tissues; HOPE was the only fixative showing long fragments in one third of the samples. Our findings demonstrate that formalin-free fixatives are in general not superior for RNA studies. With our optimized RNA extraction protocol, there is no difficulty in extracting great amounts of RNA with high quality. According to the quality obtained, quantitative real-time PCR analysis can be performed without any negative impact. Similar results can be achieved for the tested fixatives and therefore no fixative seems to represent a new "gold-standard" for tissue fixation.

Transcriptional activation of the tad type IVb pilus operon by PypB in Yersinia enterocolitica.

Type IV pili are virulence factors in various bacteria and mediate, among other functions, the colonization of diverse surfaces. Various subclasses of type IV pili have been identified, but information on pilus expression, biogenesis, and the associated phenotypes is sparse for the genus Yersinia. We recently described the identification of PypB as a transcriptional regulator in Yersinia enterocolitica. Here we show that the pypB gene is associated with the tad locus, a genomic island that is widespread among bacterial and archaeal species. The genetic linkage of pypB with the tad locus is conserved throughout the yersiniae but is not found among other bacteria carrying the tad locus. We show that the genes of the tad locus form an operon in Y. enterocolitica that is controlled by PypB and that pypB is part of this operon. The tad genes encode functions necessary for the biogenesis of the Flp subfamily of type IVb pili initially described for Aggregatibacter actinomycetemcomitans to mediate a tight-adherence phenotype. In Y. enterocolitica, the Flp pilin protein shows some peculiarities in its amino acid sequence that imply similarities as well as differences compared to typical motifs found in the Flp subtype of type IVb pili. Flp is expressed and processed after PypB overproduction, resulting in microcolony formation but not in increased adherence to biotic or abiotic surfaces. Our data describe the transcriptional regulation of the tad type IVb pilus operon by PypB in Y. enterocolitica but fail to show most previously described phenotypes associated with this type of pilus in other bacteria.

A regulatory network controls expression of the in vivo-expressed HreP protease of Yersinia enterocolitica.

The human enteropathogen Yersinia enterocolitica survives and replicates in the lymphoid tissues of its host. Previous in vivo analyses of gene expression revealed that various chromosomal genes are expressed at this stage of infection, but not in vitro. One of these, termed hreP, encodes a protease that is necessary for full virulence of Y. enterocolitica. Using transposon mutagenesis, we identified three genes, pypA, pypB, and pypC, as positive regulators of hreP transcription. PypA is an inner membrane protein with no significant similarity to any known proteins; PypB is a ToxR-like transmembrane transcriptional regulator; and PypC is a cytoplasmic transcriptional regulator with an OmpR-like winged helix-turn-helix DNA binding motif. We show that all Pyp proteins are able to activate hreP independently of each other and that PypB and PypC interact directly with the hreP promoter region. Furthermore, pypB and pypC are autoregulated and regulate each other. Additional data indicate that transcription of hreP is repressed by the histone-like nucleoid-structuring protein H-NS in a temperature-dependent manner. Our data reveal a new regulatory network that might have implications for the controlled expression of further virulence-associated functions in Yersinia.

Skin Microbiome Modulates the Effect of Ultraviolet Radiation on Cellular Response and Immune Function.

The skin is colonized by a diverse microbiome intricately involved in various molecular and cellular processes within the skin and beyond. UV radiation is known to induce profound changes in the skin and modulate the immune response. However, the role of the microbiome in UV-induced immune suppression has been overlooked. By employing the standard model of contact hypersensitivity (using germ-free mice) we found diminished UV-induced systemic immune suppression in the presence of microbiome. Upon UV exposure, we found enhanced epidermal hyperplasia and neutrophilic infiltration in the presence and enhanced numbers of mast cells and monocyte or macrophages in the absence of microbiome. Transcriptome analysis revealed a predominant expression of cytokine genes related to pro-inflammatory milieu in the presence versus immunosuppressive milieu (with increased interleukin-10) in the absence of microbiome. Collectively, microbiome abrogates the immunosuppressive response to UV by modulating gene expression and cellular microenvironment of the skin.

Cbl-b deficiency provides protection against UVB-induced skin damage by modulating inflammatory gene signature.

Exposure of skin to ultraviolet (UV) radiation induces DNA damage, inflammation, and immune suppression that ultimately lead to skin cancer. However, some of the pathways that regulate these events are poorly understood. We exposed mice to UVB to study its early effects in the absence of Cbl-b, a known suppressor of antitumor immune response in the skin. Cbl-b-/- mice were protected from UV-induced cell damage as shown by the lower number of cyclobutane pyrimidine dimers and sunburn cells in exposed skin compared to wild-type mice. Microarray data revealed that deficiency of Cbl-b resulted in differential expression of genes involved in apoptosis evasion, tumor suppression and cell survival in UV-exposed skin. After UVB, Cbl-b-/- mice upregulated gene expression pattern associated with regulation of epidermal cell proliferation linked to Wnt signaling mediators and enzymes that relate to cell removal and tissue remodeling like MMP12. Additionally, the skin of Cbl-b-/- mice was protected from chronic inflammatory responses and epidermal hyperplasia in a 4-weeks UVB treatment protocol. Overall, our results suggest a novel role for Cbl-b in regulating inflammation and physiologic clearance of damaged cells in response to UVB by modulating inflammatory gene signature.

Patients with sporadic and familial amyotrophic lateral sclerosis found value in genetic testing.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is increasingly recognized as a genetic disease. There is no consensus, however, as to the role of genetic testing in the care of the ALS patient. METHODS: We conducted a survey to study patient access, attitudes, and experience with ALS genetic testing among patients enrolled in a US ALS registry. RESULTS: Among 449 survey respondents, 156 (34.7%) were offered testing and 105 of 156 (67.3%) completed testing. The majority of respondents with familial ALS (fALS) (31/45, 68.9%) were offered testing, while a minority of respondents with sporadic ALS (sALS) (111/404, 27.5%) were offered testing (p = .00001). Comparison of mean test experience scores between groups revealed that respondents with fALS were no more likely to report a favorable experience with genetic testing than those with sALS (p = .51). Respondents who saw a genetic counselor did not have significantly different test experience scores, compared to those who did not (p = .14). In addition, no differences in test experience scores were observed between those who received positive or negative genetic test results (p = .98). CONCLUSION: These data indicate that patients with ALS found value in clinical genetic testing.

Bacterial culture results from liver, gallbladder, or bile in 248 dogs and cats evaluated for hepatobiliary disease: 1998-2003.

BACKGROUND: Information is lacking on the prevalence and susceptibility patterns of bacterial isolates in dogs and cats with suspected hepatobiliary disease. OBJECTIVES: To characterize the prevalence, identity, and antimicrobial susceptibility of common hepatobiliary isolates from such patients. ANIMALS: Dogs and cats presented to the University of Wisconsin-Madison Veterinary Medical Teaching Hospital for which samples of bile, gallbladder, or liver were submitted for culture from 1998 to 2003, including 190 dogs (192 culture episodes) and 58 cats (61 culture episodes). METHODS: Cases were identified from the microbiology laboratory database. Data from patient medical records were extracted, including the history of antimicrobial administration, the presence of fever, the results of CBC and serum biochemistry, the presence of biliary obstruction or hepatobiliary inflammation, and the results of aerobic and anaerobic bacterial cultures and aerobic antimicrobial susceptibilities. RESULTS: Biliary cultures yielded a significantly higher percentage of positive results overall (30% [18 of 60]) than did hepatic cultures (7% [15 of 215]). In patients with cholecystitis, 62% (8 of 13) had positive biliary cultures. In patients with hepatic inflammation, 23% (7 of 30) had positive bile cultures, whereas only 6% (6 of 103) had positive hepatic cultures. Escherichia coli, Enterococcus spp., Bacteroides spp., Streptococcus spp., and Clostridium spp. were the most common true-positive isolates. More than 80% of Enterobacteriaceae were susceptible to ciprofloxacin or aminoglycosides, with only 30-67% susceptible to first-generation aminopenicillins and cephalosporins. Liver samples obtained by surgery or laparoscopy were more likely to yield positive cultures than those obtained by percutaneous needle biopsy.

MUG-Mel2, a novel highly pigmented and well characterized NRAS mutated human melanoma cell line.

NRAS mutation in melanoma has been associated with aggressive tumor biology and poor prognosis. Although targeted therapy has been tested for NRAS mutated melanoma, response rates still appear much weaker, than in BRAF mutated melanoma. While plenty of cell lines exist, however, only few melanogenic cell lines retain their in vivo characteristics. In this work we present an intensively pigmented and well-characterized cell line derived from a highly aggressive NRAS mutated cutaneous melanoma, named MUG-Mel2. We present the clinical course, unique morphology, angiogenic properties, growth characteristics using in vivo experiments and 3D cell culture, and results of the exome gene sequencing of an intensively pigmented melanogenic cell line MUG-Mel2, derived from a cutaneous metastasis of an aggressive NRAS p. Q61R mutated melanoma. Amongst several genetic alterations, mutations in GRIN2A, CREBP, PIK3C2G, ATM, and ATR were present. These mutations, known to reinforce DNA repair problems in melanoma, might serve as potential treatment targets. The aggressive and fast growing behavior in animal models and the obtained phenotype in 3D culture reveal a perfect model for research in the field of NRAS mutated melanoma.

Adverse effects of EMLA (lidocaine/prilocaine) cream and efficacy for the placement of jugular catheters in hospitalized cats.

EMLA is a lidocaine/prilocaine cream used for topical analgesia in human pediatric patients. The purpose of this study was to establish the safety of EMLA in clinically ill cats, to measure systemic absorption and to determine whether EMLA reduced the need for sedation for the placement of jugular catheters. Thirty-one cats were randomized to either a placebo or EMLA cream group. Cream was applied to a 10 cm(2) area over the jugular vein, with 1h of occlusive dressing. Neither anesthetic was systemically absorbed in any cat, and no adverse clinical signs were observed. Struggling during catheter placement was less in the EMLA-treated cats compared to placebo, but did not reach significance (P = 0.06). Jugular catheters were successfully placed in 60% of EMLA-treated cats and 38% of placebo cats; this difference was not statistically significant and may not justify the added steps of EMLA cream administration for this purpose. However, EMLA does appear to be safe in clinically ill cats, and may be useful for other applications such as for skin mass removal or repeated venepuncture.