Solar Radiation Investigations; a Foundation Study

Skin cancer is a global epidemic that is increasing annually. However, our knowledge of the mechanisms involved in skin carcinogenesis is relatively poor. Investigative studies to date have predominantly employed fluorescent UV A and I or UVB lamps. The information gained from such studies has pioneered this area of research effectively, however, the typical unimodal Gaussian distribution of such irradiators do not reflect that of solar radiation nor do they account for potential waveband interactions. Advancing technologies in solar simulation have opened up this field to more environmentally and biologically relevant exposures, not only in terms of distribution but also irradiance. To begin, this study addressed issues regarding the biological relevance of four different irradiators with respect to solar radiation. During this investigation the different exposure media employed (cell culture medium and PBS) were found to elicit significantly different results in terms of cell survival which were in direct conflict with the transmittance properties of the exposure media. The differential effects of the media were further investigated using endpoints that assessed the role of reactive oxygen species, mechanistic processes ( caspase-3 activity, mitochondrial membrane potential) and genomic perturbations (mitotic index, comet assay) in response to solar simulated irradiation. These results prompted further investigations into the effects of solar simulated radiation on cell culture medium. Medium transfer experiments showed that cell culture medium irradiated in the absence of cells was cytotoxic to unirradiated cells. Solar simulated radiation induced bystander effects were also investigated to determine if the presence of cells during irradiation had an effect on the cytotoxicity of the irradiated medium. Thus, this study assessed the two most fundamental parameters in non-ionising radiation in vitro investigations in order to form solid foundations upon which more detailed investigations into the mechanisms of skin carcinogenesis can confidently be performed

Revolutions, coups, and clashes: Using implicit motivations to predict the severity of intranational political unrest

Research has found that war is likely to break out in times when leaders are high in power motives and low in affiliation, however research has been limited to conflicts between Western countries. We examine 4 revolutionary movements in the Philippines to examine whether this pattern applies to political violence across cultures and conflict types (i.e., within-country vs. between-country). We also explore the role of achievement motives in intranational political unrest. We gathered speeches during 4 times of civil unrest in the Philippines to study implicit motives at various levels of threat. All 4 occurred in the same country, city, and street in the Philippines, with some of the same actors. We scored speeches for power, affiliation, and achievement motives. The highest power and lowest affiliation motives occurred during the most violent conflict. In addition, we found that higher violence was associated with lower achievement motive

Medium Mediated Effects Increase cell Killing in a Human Keratinocyte Cell Line Exposed to Solar Simulated Radiation

Purpose: The objective of this study is to investigate whether cell culture medium is 40 a biologically relevant exposure medium that can be employed in non-ionising photobiological investigations. 42 Methods: The effect of solar simulated irradiation on cell culture medium and its ability to elicit cell death was studied. The role of reactive oxygen species (ROS), cell 44 secreted factors, and the contribution of individual components of the medium were investigated. 46 Results: Cell death was found to be primarily mediated through the formation of ROS via riboflavin photosensitisation and degradation in the cell culture medium. Phenol 48 red was found to significantly reduce the cell killing ability of riboflavin. Exposures in riboflavin free medium resulted in significantly increased cell survival compared to 50 identical exposures in riboflavin containing medium. Conclusions: This study has shown that solar radiation toxicity is augmented by cell 52 culture medium due to the presence of riboflavin. Results suggest that exposures performed in phenol red free medium may serve to increase phototoxic effects if 54 riboflavin is present. Riboflavin free media is recommended for solar radiation investigations to eliminate concerns regarding riboflavin photosensitisation and nutrient 56 deprivation

Solar Simulated Radiation Induced Cell Death Depends on Spectral Distribution and Irradiance But Not Output Delivery

Photo biological investigations are dependent on calibration and characterisation to determine the relevance of an artificial irradiator to the study at hand. The importance of this has been voiced in the literature. However, the importance of output delivery is relatively unknown. The biological relevance of a high energy, rapidly pulsing solar simulator was investigated using the clonogenic assay and was found to be reciprocity law compliant despite an exaggerated UV irradiance in excess of 1600 Wm-2 delivered per pulse. In fact, it was found to be the least cytotoxic irradiator compared to a second solar simulator and a UVB fluorescent lamp with continuous UV irradiances of 55 Wm-2 and 6.4 Wm-2 respectively. The reduced survival observed with the continuous irradiators is attributed to differences in spectral irradiance and distribution, particularly in the UVB, which in the absence of thorough calibration and characterisation may have resulted in erroneous conclusions

Assessment of 2-Year Storage Conditions on Protein, RNA, and DNA in Unstained Human Tissue Sections, Including a Novel Multiplex Digital Gene Expression Profiling Method with Implications for Biobanking.

Background: Formalin-fixed, paraffin-embedded (FFPE) tissues are a valuable resource for clinical and basic science research. Paraffin blocks and the resulting unstained sections (USS) are often stored for years before being used. Previous studies have evaluated the effects of time, temperature, humidity, and inert gases on preservation of USS; however, no study has examined all four variables together. Methods: In the current work, we prospectively and blindly assessed time points from 0 to 24 months, room versus refrigerated temperature, and presence of a desiccant and/or nitrogen atmosphere on a variety of benign and malignant tissues from North America and Africa. End points included immunohistochemistry (IHC), in situ hybridization (ISH), extracted RNA and DNA quantity and quality, and messenger RNA performance in a novel, multiplexed digital gene expression profiling assay of both housekeeping and tumor-specific genes. Results: We found that using current methods of antigen retrieval, staining, and extraction, the end points of IHC, ISH, RNA, and DNA were well preserved under the various conditions tested, with implications that pre-embedding factors contribute to variability in subsequent tissue integrity. We also document that spectrophotometric estimations of nucleic acid concentrations were in general estimated to be higher than with fluorimetric methods, which may be pertinent to end assay development. We further describe a new multiplex assay, the PlexSet digital gene expression assay, suitable for evaluating RNA quality in FFPE tissues. Conclusion: Altogether, these results may provide helpful guidance with regard to approaches for long-term storage conditions for USS

Clinical Impact of Immune Checkpoint Inhibitor (ICI) Response, DNA Damage Repair (DDR) Gene Mutations and Immune-Cell Infiltration in Metastatic Melanoma Subtypes

Molecular and histopathological analysis of melanoma subtypes has revealed distinct epidemiological, genetic, and clinical features. However, immunotherapy for advanced metastatic melanoma patients does not differ based on subtype. Response to immune checkpoint inhibitors (ICI) has been shown to vary, therefore, predictive biomarkers are needed in the design of precision treatments. Targeted sequencing and histopathological analysis (CD8 and CD20 immunohistochemistry) were performed on subtypes of metastatic melanoma (cutaneous melanoma (CM, n = 10); head and neck melanoma (HNM, n = 7); uveal melanoma (UM, n = 4); acral lentiginous melanoma (AM, n = 1) and mucosal melanoma (MM, n = 1) treated with ICI). Progression-free survival (PFS) was significantly associated with high CD8 expression (p = 0.025) and mutations in DNA damage repair (DDR) pathway genes (p = 0.012) in all subtypes but not with CD20 expression. Our study identified that immune cell infiltration and DDR gene mutations may have an impact in response to ICI treatment in metastatic melanoma but differs among subtypes. Therefore, a comprehensive understanding of the immune infiltration cells’ role and DDR gene mutations in metastatic melanoma may identify prognostic biomarkers

EBV-positive HIV-associated diffuse large B cell lymphomas are characterized by JAK/STAT (STAT3) pathway mutations and unique clinicopathologic features

Even in the era of highly active combination antiretroviral therapy (cART), patients with HIV have a disproportionate risk of developing aggressive lymphomas that are frequently Epstein-Barr virus (EBV)-related. Here, we investigate HIV-associated diffuse large B-cell lymphoma (HIV-DLBCL) and compare EBV-positive and EBV-negative cases. HIV-DLBCL were identified from two academic medical centres and characterised by immunohistochemistry, EBV status, fluorescence in situ hybridisation, cell of origin determination by gene expression profiling, and targeted deep sequencing using a custom mutation panel of 334 genes. We also applied the Lymphgen tool to determine the genetic subtype of each case. Thirty HIV-DLBCL were identified, with a median patient age of 46 years and male predominance (5:1). Thirteen cases (48%) were EBV-positive and 14 (52%) EBV-negative. Nine of the 16 tested cases (56%) had MYC rearrangement, three (19%) had BCL6 (two of which were double hit MYC/BCL6) and none had BCL2 rearrangements. Using the Lymphgen tool, half of the cases (15) were classified as other. All HIV-DLBCL showed mutational abnormalities, the most frequent being TP53 (37%), MYC (30%), STAT3 (27%), HIST1H1E (23%), EP300 (20%), TET2 (20%), SOCS1 (17%) and SGK1 (17%). EBV-negative cases were mostly of germinal centre B-cell (GCB) origin (62%), showed more frequent mutations per case (a median of 13·5/case) and significant enrichment of TP53 (57% vs. 15%; P = 0·046), SGK1 (36% vs. 0%; P = 0·04), EP300 (43% vs. 0%; P = 0·02) and histone-modifying gene (e.g. HIST1H1E, HIST1H1D, 79% vs. 31%; P = 0·02) mutations. EBV-positive cases were mostly of non-GCB origin (70%), with fewer mutations per case (median 8/case; P = 0·007), and these tumours were enriched for STAT3 mutations (P = 0·10). EBV-positive cases had a higher frequency of MYC mutations but the difference was not significant (36% vs. 15%; P = 0·38). EBV-association was more frequent in HIV-DLBCLs, arising in patients with lower CD4 counts at diagnosis (median 46·5 vs. 101, P = 0·018). In the era of cART, approximately half of HIV-DLBCL are EBV-related. HIV-DLBCL are enriched for MYC rearrangements, MYC mutations and generally lack BCL2 rearrangements, regardless of EBV status. Among HIV-DLBCL, tumours that are EBV-negative and EBV-positive appear to have important differences, the latter arising in context of lower CD4 count, showing frequent non-GCB origin, lower mutation burden and recurrent STAT3 mutations