Using label-free proteomics to elucidate factors involved in the human response to orthohantavirus infection

Orthohantaviruses are a genus of Bunyaviruses carried by a range of rodent hosts. They are found in endemic foci across the globe and are associated with the zoonotic diseases haemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome. Orthohantaviruses are enzootic to south-eastern, central and northern parts of continental Europe. The virus has historically been considered absent from the British Isles, but the endemic presence of Seoul orthohantavirus (SEOV) was demonstrated in wild and pet rats in the UK in 2012. For the last six years several human cases of orthohantavirus-infections have been diagnosed in the UK with associated acute renal failure. Currently, treatment for haemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome is purely symptomatic. The mechanisms underlying the pathogenesis of orthohantavirus infections are not fully understood though several lines of enquiry have demonstrated the multifaceted nature of the disease, with direct virally mediated mechanisms, indirect immune mediate mechanism and genetic host factors all likely contributors to the clinical picture. Clinical proteomics is a field of study in which high-throughput analytical approaches are used to investigate proteins in clinical samples. A major advantage of the proteomic approach is that no a priori knowledge is required for proteome analysis, making it an excellent hypothesis generating experimental method. The high-throughput facets of proteomic methods also make the approach very useful for infections such as orthohantavirus, for which aspects of its pathology remains unclear, and the relative rarity of the associated diseases result in limited access to biological samples. The aims of this study were to use label-free proteomics to discover biomarkers of orthohantavirus infection in clinical human sera and to study the cellular interactome of recombinant SEOV Cherwell glycoproteins in a human kidney cell line, with the objective of gaining further insights into mechanism involved in the processing of viral proteins through host cells. A pipeline for investigating the human serum proteome during diseased and healthy conditions was established. Using label-free proteomics in association with immunological methods, afamin and galectin 3-binding protein were identified and evaluated as putative biomarkers of orthohantavirus infection. Both proteins showed promise as factors involved in the response to acute Seoul orthohantavirus infection. Their relevance as markers of disease were supported by their functional annotation, with afamin playing a possible role in the cellular response to oxidative stress, and galectin 3-binding protein being associated with pro-inflammatory and pro-coagulant activity. Further validation studies with larger sample cohorts are warranted. Initial investigations into the in vitro interactome of recombinant SEOV Cherwell glycoproteins identified 12 potential associations between host and viral proteins. While functional analysis of selected proteins revealed a potential for relevance in the viral life cycle, further follow-up of these findings will be required to establish whether the identified associations are firstly, true associations, and secondly, biologically relevant interactions

An Article that Changed the Course of History?

Yehuda Blum's article, ostensibly devoted to an examination of the lawfulness of a military order under the law of occupation, actually explored a preliminary question – whether Jordan had valid title to the West Bank (referred to as ‘Judea and Samaria’). Concluding that Jordan had no title, Blum concluded that the law of occupation did not apply. This reflection revisits Blum's thesis. It suggests that Blum's argument failed to elucidate the relevant legal questions and therefore his conclusion was hasty. It would be distressing to think that it was Blum's article that convinced Israeli decision-makers to deny the formal applicability of the law of occupation to the West Bank and Gaza.</jats:p

Mitochondrial Involvement in Vertebrate Speciation? The Case of Mito-nuclear Genetic Divergence in Chameleons

Compatibility between the nuclear (nDNA) and mitochondrial (mtDNA) genomes is important for organismal health. However, its significance for major evolutionary processes such as speciation is unclear, especially in vertebrates. We previously identified a sharp mtDNA-specific sequence divergence between morphologically indistinguishable chameleon populations (Chamaeleo chamaeleon recticrista) across an ancient Israeli marine barrier (Jezreel Valley). Because mtDNA introgression and gender-based dispersal were ruled out, we hypothesized that mtDNA spatial division was maintained by mito-nuclear functional compensation. Here, we studied RNA-seq generated from each of ten chameleons representing the north and south populations and identified candidate nonsynonymous substitutions (NSSs) matching the mtDNA spatial distribution. The most prominent NSS occurred in 14 nDNA-encoded mitochondrial proteins. Increased chameleon sample size (N = 70) confirmed the geographic differentiation in POLRMT, NDUFA5, ACO1, LYRM4, MARS2, and ACAD9. Structural and functionality evaluation of these NSSs revealed high functionality. Mathematical modeling suggested that this mito-nuclear spatial divergence is consistent with hybrid breakdown. We conclude that our presented evidence and mathematical model underline mito-nuclear interactions as a likely role player in incipient speciation in vertebrates

A neuronal enhancer network upstream of MEF2C is compromised in patients with Rett-like characteristics

Mutations in myocyte enhancer factor 2C (MEF2C), an important transcription factor in neurodevelopment, are associated with a Rett-like syndrome. Structural variants (SVs) upstream of MEF2C, which do not disrupt the gene itself, have also been found in patients with a similar phenotype, suggesting that disruption of MEF2C regulatory elements can also cause a Rett-like phenotype. To characterize those elements that regulate MEF2C during neural development and that are affected by these SVs, we used genomic tools coupled with both in vitro and in vivo functional assays. Through circularized chromosome conformation capture sequencing (4C-seq) and the assay for transposase-accessible chromatin using sequencing (ATAC-seq), we revealed a complex interaction network in which the MEF2C promoter physically contacts several distal enhancers that are deleted or translocated by disease-associated SVs. A total of 16 selected candidate regulatory sequences were tested for enhancer activity in vitro, with 14 found to be functional enhancers. Further analyses of their in vivo activity in zebrafish showed that each of these enhancers has a distinct activity pattern during development, with eight enhancers displaying neuronal activity. In summary, our results disentangle a complex regulatory network governing neuronal MEF2C expression that involves multiple distal enhancers. In addition, the characterized neuronal enhancers pose as novel candidates to screen for mutations in neurodevelopmental disorders, such as Rett-like syndrome

Gene set of nuclear-encoded mitochondrial regulators is enriched for common inherited variation in obesity

There are hints of an altered mitochondrial function in obesity. Nuclear-encoded genes are relevant for mitochondrial function (3 gene sets of known relevant pathways: (1) 16 nuclear regulators of mitochondrial genes, (2) 91 genes for oxidative phosphorylation and (3) 966 nuclear-encoded mitochondrial genes). Gene set enrichment analysis (GSEA) showed no association with type 2 diabetes mellitus in these gene sets. Here we performed a GSEA for the same gene sets for obesity. Genome wide association study (GWAS) data from a case-control approach on 453 extremely obese children and adolescents and 435 lean adult controls were used for GSEA. For independent confirmation, we analyzed 705 obesity GWAS trios (extremely obese child and both biological parents) and a population-based GWAS sample (KORA F4, n = 1,743). A meta-analysis was performed on all three samples. In each sample, the distribution of significance levels between the respective gene set and those of all genes was compared using the leading-edge-fraction-comparison test (cut-offs between the 50(th) and 95(th) percentile of the set of all gene-wise corrected p-values) as implemented in the MAGENTA software. In the case-control sample, significant enrichment of associations with obesity was observed above the 50(th) percentile for the set of the 16 nuclear regulators of mitochondrial genes (p(GSEA,50) = 0.0103). This finding was not confirmed in the trios (p(GSEA,50) = 0.5991), but in KORA (p(GSEA,50) = 0.0398). The meta-analysis again indicated a trend for enrichment (p(MAGENTA,50) = 0.1052, p(MAGENTA,75) = 0.0251). The GSEA revealed that weak association signals for obesity might be enriched in the gene set of 16 nuclear regulators of mitochondrial genes

Comparative effectiveness of intensity modulated radiation therapy to 3-dimensional conformal radiation in locally advanced lung cancer: pathological and clinical outcomes.

OBJECTIVE: Intensity-modulated radiotherapy (IMRT) has better normal-tissue sparing compared with 3-dimensional conformal radiation (3DCRT). We sought to assess the impact of radiation technique on pathological and clinical outcomes in locally advanced non-small cell lung cancer (LANSCLC) treated with a trimodality strategy. METHODS: Retrospective review of LANSCLC patients treated from August 2012 to August 2018 at Sheba Medical Center, Israel. The trimodality strategy consisted of concomitant chemoradiation to 60 Gray (Gy) followed by completion surgery. The planning target volume (PTV) was defined by co-registered PET/CT. Here we compare the pathological regression, surgical margin status, local control rates (LC), disease free (DFS) and overall survival (OS) between 3DCRT and IMRT. RESULTS: Our cohort consisted of 74 patients with mean age 62.9 years, male in 51/74 (69%), adenocarcinoma in 46/74 (62.1%), stage 3 in 59/74 (79.7%) and chemotherapy in 72/74 (97.3%). Radiation mean dose: 59.2 Gy (SD ± 3.8). Radiation technique : 3DCRT in 51/74 (68.9%), IMRT in 23/74 (31%). Other variables were similar between groups.Major pathological response (including pathological complete response or less than 10% residual tumor cells) was similar: 32/51 (62.7%) in 3DCRT and 15/23 (65.2%) in IMRT, p=0.83. Pathological complete response (pCR) rates were similar: 17/51 (33.3%) in 3DCRT and 8/23 (34.8%) in IMRT, p=0.9. Surgical margins were negative in 46/51 (90.1%) in 3DCRT vs. 17/19 (89.4%) in IMRT (p=1.0).The 2-year LC rates were 81.6% (95% CI 69-89.4%); DFS 58.3% (95% CI 45.5-69%) and 3-year OS 70% (95% CI57-80%). Comparing radiation techniques, there were no significant differences in LC (p=0.94), DFS (p=0.33) and OS (p=0.72). CONCLUSION: When used to treat LANSCLC in the neoadjuvant setting, both IMRT and 3DCRT produce comparable pathological and clinical outcomes. ADVANCES IN KNOWLEDGE: This study validates the real-world effectiveness of IMRT compared to 3DCRT

Revisiting Egyptian Foreign Policy towards Israel under Mubarak: From Cold Peace to Strategic Peace

This article is the first academic study of Egyptian foreign policy towards Israel under Hosni Mubarak (1981–2011). It challenges a deeply entrenched conventional wisdom that Egypt pursued a cold-peace foreign policy towards Israel throughout this period. We demonstrate that Egyptian foreign policy towards Israel was dynamic – comprising cold peace (1981–91), a hybrid foreign policy of cold peace and strategic peace (1991–2003), and a pure strategic peace posture (2003–11). We also use the case of Egyptian foreign policy towards Israel as a heuristic to develop a conception of a new type of peace, strategic peace, as an intermediary analytical category between cold and stable peace

Mitochondrial DNA Variation, but Not Nuclear DNA, Sharply Divides Morphologically Identical Chameleons along an Ancient Geographic Barrier

The Levant is an important migration bridge, harboring border-zones between Afrotropical and palearctic species. Accordingly, Chameleo chameleon, a common species throughout the Mediterranean basin, is morphologically divided in the southern Levant (Israel) into two subspecies, Chamaeleo chamaeleon recticrista (CCR) and C. c. musae (CCM). CCR mostly inhabits the Mediterranean climate (northern Israel), while CCM inhabits the sands of the north-western Negev Desert (southern Israel). AFLP analysis of 94 geographically well dispersed specimens indicated moderate genetic differentiation (PhiPT = 0.097), consistent with the classical division into the two subspecies, CCR and CCM. In contrast, sequence analysis of a 637 bp coding mitochondrial DNA (mtDNA) fragment revealed two distinct phylogenetic clusters which were not consistent with the morphological division: one mtDNA cluster consisted of CCR specimens collected in regions northern of the Jezreel Valley and another mtDNA cluster harboring specimens pertaining to both the CCR and CCM subspecies but collected southern of the Jezreel Valley. AMOVA indicated clear mtDNA differentiation between specimens collected northern and southern to the Jezreel Valley (PhiPT = 0.79), which was further supported by a very low coalescent-based estimate of effective migration rates. Whole chameleon mtDNA sequencing (∼17,400 bp) generated from 11 well dispersed geographic locations revealed 325 mutations sharply differentiating the two mtDNA clusters, suggesting a long allopatric history further supported by BEAST. This separation correlated temporally with the existence of an at least 1 million year old marine barrier at the Jezreel Valley exactly where the mtDNA clusters meet. We discuss possible involvement of gender-dependent life history differences in maintaining such mtDNA genetic differentiation and suggest that it reflects (ancient) local adaptation to mitochondrial-related traits