Understanding signaling cascades in melanoma

Understanding regulatory pathways involved in melanoma development and progression has advanced significantly in recent years. It is now appreciated that melanoma is the result of complex changes in multiple signaling pathways that affect growth control, metabolism, motility and the ability to escape cell death programs. Here we review the major signaling pathways currently known to be deregulated in melanoma with an implication to its development and progression. Among these pathways are Ras, B-Raf, MEK, PTEN, phosphatidylinositol-3 kinase (PI3Ks) and Akt which are constitutively activated in a significant number of melanoma tumors, in most cases due to genomic change. Other pathways discussed in this review include the [Janus kinase/signal transducer and activator of transcription (JAK/STAT), transforming growth factor-beta pathways which are also activated in melanoma, although the underlying mechanism is not yet clear. As a paradigm for remodeled signaling pathways, melanoma also offers a unique opportunity for targeted drug development.Fil: Lopez Bergami, Pablo Roberto. Sanford-burnham Medical Research Institute; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Fitchmann, B. Sanford-burnham Medical Research Institute; Estados UnidosFil: Ronai, Ze´ev. Sanford-burnham Medical Research Institute; Estados Unido

Is metal theft committed by organized crime groups, and why does it matter?

Using the example of metal theft in the United Kingdom, this study used mixed methods to evaluate the accuracy of police estimates of the involvement of organised crime groups (OCGs) in crime. Police estimate that 20-30% of metal theft is committed by OCGs, but this study found that only 0.5% of metal thieves had previous convictions for offences related to OCGs, that only 1.3% were linked to OCGs by intelligence information, that metal thieves typically offended close to their homes and that almost no metal thefts involved sophisticated offence methods. It appears that police may over-estimate the involvement of OCGs in some types of crime. The reasons for and consequences of this over-estimation are discussed

The use of focus group discussion methodology: Insights from two decades of application in conservation

This is the final version of the article. Available from Wiley via the DOI in this recordFocus group discussion is frequently used as a qualitative approach to gain an in-depth understanding of social issues. The method aims to obtain data from a purposely selected group of individuals rather than from a statistically representative sample of a broader population. Even though the application of this method in conservation research has been extensive, there are no critical assessment of the application of the technique. In addition, there are no readily available guidelines for conservation researchers. Here, we reviewed the applications of focus group discussion within biodiversity and conservation research between 1996 and April 2017. We begin with a brief explanation of the technique for first-time users. We then discuss in detail the empirical applications of this technique in conservation based on a structured literature review (using Scopus). The screening process resulted in 170 articles, the majority of which (67%, n = 114,) were published between 2011 and 2017. Rarely was the method used as a stand-alone technique. The number of participants per focus group (where reported) ranged from 3 to 21 participants with a median of 10 participants. There were seven (median) focus group meetings per study. Focus group discussion sessions lasted for 90 (median) minutes. Four main themes emerged from the review: understanding of people's perspectives regarding conservation (32%), followed by the assessment of conservation and livelihoods practices (21%), examination of challenges and impacts of resource management interventions (19%) and documenting the value of indigenous knowledge systems (16%). Most of the studies were in Africa (n = 76), followed by Asia (n = 44), and Europe (n = 30). We noted serious gaps in the reporting of the methodological details in the reviewed papers. More than half of the studies (n = 101) did not report the sample size and group size (n = 93), whereas 54 studies did not mention the number of focus group discussion sessions while reporting results. Rarely have the studies provided any information on the rationale for choosing the technique. We have provided guidelines to improve the standard of reporting and future application of the technique for conservation.N.T.O. was funded by Cambridge Overseas Trusts, The Wildlife Conservation Society, Wildlife Conservation Network and WildiZe Foundation. NM was funded by the NERC grant (NE/R006946/1), Fondation Wiener Anspach and the Scriven post doctoral fellowships. K.W. was sup-ported by the Australian Research Council Centre of Excellence for Environmental Decisions (CE11001000104) and Future Fellowship (FT100100413) programs and funded by the Australian Government

Binding of Soluble Yeast β-Glucan to Human Neutrophils and Monocytes is Complement-Dependent

The immunomodulatory properties of yeast β-1,3/1,6 glucans are mediated through their ability to be recognized by human innate immune cells. While several studies have investigated binding of opsonized and unopsonized particulate β-glucans to human immune cells mainly via complement receptor 3 (CR3) or Dectin-1, few have focused on understanding the binding characteristics of soluble β-glucans. Using a well-characterized, pharmaceutical grade, soluble yeast β-glucan, this study evaluated and characterized the binding of soluble β-glucan to human neutrophils and monocytes. The results demonstrated that soluble β-glucan bound to both human neutrophils and monocytes in a concentration-dependent and receptor-specific manner. Antibodies blocking the CD11b and CD18 chains of CR3 significantly inhibited binding to both cell types, establishing CR3 as the key receptor recognizing the soluble β-glucan in these cells. Binding of soluble β-glucan to human neutrophils and monocytes required serum and was also dependent on incubation time and temperature, strongly suggesting that binding was complement-mediated. Indeed, binding was reduced in heat-inactivated serum, or in serum treated with methylamine or in serum reacted with the C3-specific inhibitor compstatin. Opsonization of soluble β-glucan was demonstrated by detection of iC3b, the complement opsonin on β-glucan-bound cells, as well as by the direct binding of iC3b to β-glucan in the absence of cells. Binding of β-glucan to cells was partially inhibited by blockade of the alternative pathway of complement, suggesting that the C3 activation amplification step mediated by this pathway also contributed to binding

Preponderance of the oncogenic V599E and V599K mutations in B-raf kinase domain is enhanced in melanoma cutaneous/subcutaneous metastases

BACKGROUND: Downstream of Ras, the serine/threonine kinase B-raf has been reported to be mutated, among other carcinomas, in a substantial subset of primary melanomas with a preponderance of mutations within the kinase domain including the activating V599E and V599K transitions. METHODS: We here investigated a representative series of 60 resection specimens of cutaneous and subcutaneous melanoma metastases for the presence of mutations within the activation segment (exon 15) of the B-raf kinase domain by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) gel electrophoresis. RESULTS: Sequencing of cloned PCR-SSCP amplicons resulted in 24 (40%) samples harbouring somatic mutations which is not exceeding the mutation frequency in recently investigated primary melanomas. The activating mutation T1796A was present in 24/60 (40%) resection specimens, followed in frequency by the oncogenic g1795A mutation in 8/60 (13%) cases. As to the B-raf protein sequence, the acidic amino acid transitions V599E and V599K were predicted in 19/60 (32%) and 6/60 (10%) cases, resepectively, but were not associated with enhanced risk for subsequent metastasis in patients' follow up. In comparison to the primary melanomas that we recently investigated, the spectrum of predicted B-raf protein mutations narrowed significantly in the cutaneous/subcutaneous metastases. Unexpectedly, V599 and V599E mutations were absent in cutaneous/subcutaneous metastases derived from acrolentiginous melanomas as preceding primary tumours. CONCLUSION: During transition from primary melanomas towards cutaneous/subcutaneous metastases, the spectrum of predicted B-raf mutations narrows significantly. Focusing on the V599E and V599K, these oncogenic mutations are likely to affect melanocyte-specific pathways controlling proliferation and differentiation

Toll-Like Receptor Agonists Synergize with CD40L to Induce Either Proliferation or Plasma Cell Differentiation of Mouse B Cells

In a classical dogma, pathogens are sensed (via recognition of Pathogen Associated Molecular Patterns (PAMPs)) by innate immune cells that in turn activate adaptive immune cells. However, recent data showed that TLRs (Toll Like Receptors), the most characterized class of Pattern Recognition Receptors, are also expressed by adaptive immune B cells. B cells play an important role in protective immunity essentially by differentiating into antibody-secreting cells (ASC). This differentiation requires at least two signals: the recognition of an antigen by the B cell specific receptor (BCR) and a T cell co-stimulatory signal provided mainly by CD154/CD40L acting on CD40. In order to better understand interactions of innate and adaptive B cell stimulatory signals, we evaluated the outcome of combinations of TLRs, BCR and/or CD40 stimulation. For this purpose, mouse spleen B cells were activated with synthetic TLR agonists, recombinant mouse CD40L and agonist anti-BCR antibodies. As expected, TLR agonists induced mouse B cell proliferation and activation or differentiation into ASC. Interestingly, addition of CD40 signal to TLR agonists stimulated either B cell proliferation and activation (TLR3, TLR4, and TLR9) or differentiation into ASC (TLR1/2, TLR2/6, TLR4 and TLR7). Addition of a BCR signal to CD40L and either TLR3 or TLR9 agonists did not induce differentiation into ASC, which could be interpreted as an entrance into the memory pathway. In conclusion, our results suggest that PAMPs synergize with signals from adaptive immunity to regulate B lymphocyte fate during humoral immune response

Intra- and Inter-Tumor Heterogeneity of BRAFV600EMutations in Primary and Metastatic Melanoma

The rationale for using small molecule inhibitors of oncogenic proteins as cancer therapies depends, at least in part, on the assumption that metastatic tumors are primarily clonal with respect to mutant oncogene. With the emergence of BRAFV600E as a therapeutic target, we investigated intra- and inter-tumor heterogeneity in melanoma using detection of the BRAFV600E mutation as a marker of clonality. BRAF mutant-specific PCR (MS-PCR) and conventional sequencing were performed on 112 tumors from 73 patients, including patients with matched primary and metastatic specimens (n = 18). Nineteen patients had tissues available from multiple metastatic sites. Mutations were detected in 36/112 (32%) melanomas using conventional sequencing, and 85/112 (76%) using MS-PCR. The better sensitivity of the MS-PCR to detect the mutant BRAFV600E allele was not due to the presence of contaminating normal tissue, suggesting that the tumor was comprised of subclones of differing BRAF genotypes. To determine if tumor subclones were present in individual primary melanomas, we performed laser microdissection and mutation detection via sequencing and BRAFV600E-specific SNaPshot analysis in 9 cases. Six of these cases demonstrated differing proportions of BRAFV600Eand BRAFwild-type cells in distinct microdissected regions within individual tumors. Additional analyses of multiple metastatic samples from individual patients using the highly sensitive MS-PCR without microdissection revealed that 5/19 (26%) patients had metastases that were discordant for the BRAFV600E mutation. In conclusion, we used highly sensitive BRAF mutation detection methods and observed substantial evidence for heterogeneity of the BRAFV600E mutation within individual melanoma tumor specimens, and among multiple specimens from individual patients. Given the varied clinical responses of patients to BRAF inhibitor therapy, these data suggest that additional studies to determine possible associations between clinical outcomes and intra- and inter-tumor heterogeneity could prove fruitful

A Requirement of TolC and MDR Efflux Pumps for Acid Adaptation and GadAB Induction in Escherichia coli

BACKGROUND: The TolC outer membrane channel is a key component of several multidrug resistance (MDR) efflux pumps driven by H(+) transport in Escherichia coli. While tolC expression is under the regulation of the EvgA-Gad acid resistance regulon, the role of TolC in growth at low pH and extreme-acid survival is unknown. METHODS AND PRINCIPAL FINDINGS: TolC was required for extreme-acid survival (pH 2) of strain W3110 grown aerobically to stationary phase. A tolC deletion decreased extreme-acid survival (acid resistance) of aerated pH 7.0-grown cells by 10(5)-fold and of pH 5.5-grown cells by 10-fold. The requirement was specific for acid resistance since a tolC defect had no effect on aerobic survival in extreme base (pH 10). TolC was required for expression of glutamate decarboxylase (GadA, GadB), a key component of glutamate-dependent acid resistance (Gad). TolC was also required for maximal exponential growth of E. coli K-12 W3110, in LBK medium buffered at pH 4.5-6.0, but not at pH 6.5-8.5. The TolC growth requirement in moderate acid was independent of Gad. TolC-associated pump components EmrB and MdtB contributed to survival in extreme acid (pH 2), but were not required for growth at pH 5. A mutant lacking the known TolC-associated efflux pumps (acrB, acrD, emrB, emrY, macB, mdtC, mdtF, acrEF) showed no growth defect at acidic pH and a relatively small decrease in extreme-acid survival when pre-grown at pH 5.5. CONCLUSIONS: TolC and proton-driven MDR efflux pump components EmrB and MdtB contribute to E. coli survival in extreme acid and TolC is required for maximal growth rates below pH 6.5. The TolC enhancement of extreme-acid survival includes Gad induction, but TolC-dependent growth rates below pH 6.5 do not involve Gad. That MDR resistance can enhance growth and survival in acid is an important consideration for enteric organisms passing through the acidic stomach

Activation of the MAPK pathway is a common event in uveal melanomas although it rarely occurs through mutation of BRAF or RAS

In contrast to cutaneous melanoma, there is no evidence that BRAF mutations are involved in the activation of the mitogen-activated protein kinase (MAPK) pathway in uveal melanoma, although there is increasing evidence that this pathway is activated frequently in the latter tumours. In this study, we performed mutation analysis of the RAS and BRAF genes in a panel of 11 uveal melanoma cell lines and 19 primary uveal melanoma tumours. In addition, Western blot and immunohistochemical analyses were performed on downstream members of the MAPK pathway in order to assess the contribution of each of these components. No mutations were found in any of the three RAS gene family members and only one cell line carried a BRAF mutation (V599E). Despite this, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), ERK and ELK were constitutively activated in all samples. These data suggest that activation of the MAPK pathway is commonly involved in the development of uveal melanoma, but occurs through a mechanism different to that of cutaneous melanoma