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Abstract Background Among the 10% of pancreatic cancers that occur in a familial context, around a third carry a pathogenic variant in a cancer predisposition gene. Genetic studies of pancreatic cancer predisposition are limited by high mortality rates amongst index patients and other affected family members. The genetic risk for pancreatic cancer is often shared with breast cancer susceptibility genes, most notably BRCA2, PALB2, ATM and BRCA1. Therefore, we hypothesized that additional shared genetic etiologies might be uncovered by studying families presenting with both breast and pancreatic cancer. Methods Focusing on a multigene panel of 276 DNA Damage Repair (DDR) genes, we performed next-generation sequencing in a cohort of 41 families with at least three breast cancer cases and one pancreatic cancer. When the index patient with pancreatic cancer was deceased, close relatives (first or second-degree) affected with breast cancer were tested (39 families). Results We identified 27 variants of uncertain significance in DDR genes. A splice site variant (c.1605 + 2T > A) in the RAD17 gene stood out, as a likely loss of function variant. RAD17 is a checkpoint protein that recruits the MRN (MRE11-RAD50-NBS1) complex to initiate DNA signaling, leading to DNA double-strand break repair. Conclusion Within families with breast and pancreatic cancer, we identified RAD17 as a novel candidate predisposition gene. Further genetic studies are warranted to better understand the potential pathogenic effect of RAD17 variants and in other DDR genes.

IntroductionEndosymbiotic bacteria in the genus Wolbachia have evolved numerous strategies for manipulating host reproduction in order to promote their own transmission. This includes the feminization of males into functional females, a well-studied phenotype in the isopod Armadillidium vulgare. Despite an early description of this phenotype in isopods and the development of an evolutionary model of host sex determination in the presence of Wolbachia, the underlying genetic mechanisms remain elusive.MethodsHere we present the first complete genomes of the three feminizing Wolbachia (wVulC, wVulP, and wVulM) known to date in A. vulgare. These genomes, belonging to Wolbachia B supergroup, contain a large number of mobile elements such as WO prophages with eukaryotic association modules. Taking advantage of these data and those of another Wolbachia-derived feminizing factor integrated into the host genome (f element), we used a comparative genomics approach to identify putative feminizing factors.ResultsThis strategy has enabled us to identify three prophage-associated genes secreted by the Type IV Secretion System: one ankyrin repeat domain-containing protein, one helix-turn-helix transcriptional regulator and one hypothetical protein. In addition, a latrotoxin-related protein, associated with phage relic genes, was shared by all three genomes and the f element.ConclusionThese putative feminization-inducing proteins shared canonical interaction features with eukaryotic proteins. These results pave the way for further research into the underlying functional interactions.

Abstract Background Wolbachia are vertically transmitted bacteria known to be the most widespread endosymbiont in arthropods. They induce various alterations of the reproduction of their host, including feminization of genetic males in isopod crustaceans. In the pill bug Armadillidium vulgare, the presence of Wolbachia is also associated with detrimental effects on host fertility and lifespan. Deleterious effects have been demonstrated on hemocyte density, phenoloxidase activity, and natural hemolymph septicemia, suggesting that infected individuals could have defective immune capacities. Since nothing is known about the molecular mechanisms involved in Wolbachia-A. vulgare interactions and its secondary immunocompetence modulation, we developed a transcriptomics strategy and compared A. vulgare gene expression between Wolbachia-infected animals (i.e., “symbiotic” animals) and uninfected ones (i.e., “asymbiotic” animals) as well as between animals challenged or not challenged by a pathogenic bacteria. Results Since very little genetic data is available on A. vulgare, we produced several EST libraries and generated a total of 28 606 ESTs. Analyses of these ESTs revealed that immune processes were over-represented in most experimental conditions (responses to a symbiont and to a pathogen). Considering canonical crustacean immune pathways, these genes encode antimicrobial peptides or are involved in pathogen recognition, detoxification, and autophagy. By RT-qPCR, we demonstrated a general trend towards gene under-expression in symbiotic whole animals and ovaries whereas the same gene set tends to be over-expressed in symbiotic immune tissues. Conclusion This study allowed us to generate the first reference transcriptome ever obtained in the Isopoda group and to identify genes involved in the major known crustacean immune pathways encompassing cellular and humoral responses. Expression of immune-related genes revealed a modulation of host immunity when females are infected by Wolbachia, including in ovaries, the crucial tissue for the Wolbachia route of transmission.

Abstract Background The maternally inherited α-Proteobacteria Wolbachia pipientis is an obligate endosymbiont of nematodes and arthropods, in which they induce a variety of reproductive alterations, including Cytoplasmic Incompatibility (CI) and feminization. The genome of the feminizing wVulC Wolbachia strain harboured by the isopod Armadillidium vulgare has been sequenced and is now at the final assembly step. It contains an unusually high number of ankyrin motif-containing genes, two of which are homologous to the phage-related pk1 and pk2 genes thought to contribute to the CI phenotype in Culex pipiens. These genes encode putative bacterial effectors mediating Wolbachia-host protein-protein interactions via their ankyrin motifs. Results To test whether these Wolbachia homologs are potentially involved in altering terrestrial isopod reproduction, we determined the distribution and expression of both pk1 and pk2 genes in the 3 Wolbachia strains that induce CI and in 5 inducing feminization of their isopod hosts. Aside from the genes being highly conserved, we found a substantial copy number variation among strains, and that is linked to prophage diversity. Transcriptional analyses revealed expression of one pk2 allele (pk2b2) only in the feminizing Wolbachia strains of isopods. Conclusions These results reveal the need to investigate the functions of Wolbachia ankyrin gene products, in particular those of Pk2, and their host targets with respect to host sex manipulation.

Abstract The order Holosporales (Alphaproteobacteria) encompasses obligate intracellular bacterial symbionts of diverse Eukaryotes. These bacteria have highly streamlined genomes and can have negative fitness effects on the host. Herein, we present a comparative analysis of the first genome sequences of ‘Ca. Hepatincola porcellionum’, a facultative symbiont occurring extracellularly in the midgut glands of terrestrial isopods. Using a combination of long-read and short-read sequencing, we obtained the complete circular genomes of two Hepatincola strains and an additional metagenome-assembled draft genome. Phylogenomic analysis validated its phylogenetic position as an early-branching family-level clade relative to all other established Holosporales families associated with protists. A 16S rRNA gene survey revealed that this new family encompasses diverse bacteria associated with both marine and terrestrial host species, which expands the host range of Holosporales bacteria from protists to several phyla of the Ecdysozoa (Arthropoda and Priapulida). Hepatincola has a highly streamlined genome with reduced metabolic and biosynthetic capacities as well as a large repertoire of transmembrane transporters. This suggests that this symbiont is rather a nutrient scavenger than a nutrient provider for the host, likely benefitting from a nutrient-rich environment to import all necessary metabolites and precursors. Hepatincola further possesses a different set of bacterial secretion systems compared to protist-associated Holosporales, suggesting different host-symbiont interactions depending on the host organism.

The logical problem of artificial intelligence—the question of whether the notion sometimes referred to as ‘strong’ AI is self-contradictory—is, essentially, the question of whether an artificial form of life is possible. This question has an immediately paradoxical character, which can be made explicit if we recast it (in terms that would ordinarily seem to be implied by it) as the question of whether an unnatural form of nature is possible. The present paper seeks to explain this paradoxical kind of possibility by arguing that machines can share the human form of life and thus acquire human mindedness, which is to say they can be intelligent, conscious, sentient, etc. in precisely the way that a human being typically is.

Abstract Immune-mediated inflammatory diseases are characterized by variability in disease presentation and severity but studying it is a challenging task. Defining the limits of a healthy immune system is therefore a prior step to capture variability in disease conditions. The goal of this study is to characterize the global immune cell composition along with their influencing factors. Blood samples were collected from 2 independent cohorts of respectively 389 (exploratory) and 208 (replication) healthy subjects. Twelve immune cells were measured in blood together with biological parameters. Three complementary clustering approaches were used to evaluate if variability related to the immune cells could be characterized as clusters or as a continuum. Large coefficients of variation confirmed the inter-individual variability of immune cells. Considering all subset variations in an overall analysis, it appeared that the immune makeup was organized as a continuum through the two cohorts. Some intrinsic and environmental factors affected the inter-individual variability of cells but without unveiling separable groups with similar features. This study provides a framework based on complementary clustering approach for analyzing inter-individual variability of immune cells. Our analyses support the absence of clusters in our two healthy cohorts. Also, our study reports some influence of age, gender, BMI, cortisol, season and CMV infection on immune variability.

People with metastatic breast cancer face many challenges and disparities in obtaining optimal cancer care. These challenges are accentuated in underserved patient populations across Europe, who are less likely to receive quality healthcare for reasons including socioeconomic inequalities, educational or cultural status, or geographic location. While there are many local and national initiatives targeted to address these challenges, there remains a need to reduce disparities and improve access to healthcare to improve outcomes, with a focus on multidisciplinary stakeholder engagement.In October 2019, a range of experts in metastatic breast cancer, including healthcare professionals, patient representatives, policymakers and politicians, met to discuss and prioritize the critical needs of underserved patient populations with metastatic breast cancer in Europe. Six key challenges faced by these communities were identified: the need for amplification of the metastatic breast cancer patient voice, better and wider implementation of high-quality guidelines for metastatic breast cancer, more collaboration between stakeholders, tailored support for patients from different cultural and ethnic backgrounds, improved data sharing, and work-related issues. The Expert Panel then conceived and discussed potential actionable goals to address each key challenge. Their conclusions present a set of interrelated approaches to address the different challenges and could serve as the basis for concerted improvement of the lives of patients with metastatic breast cancer in Europe.

Abstract Using the isopod Armadillidium vulgare as a case study, we review the significance of the "bacterial dosage model", which connects the expression of the extended phenotype to the rise of the Wolbachia load. In isopods, the Insulin-like Androgenic Gland hormone (IAG) induces male differentiation: Wolbachia feminizes males through insulin resistance, presumably through defunct insulin receptors. This should prevent an autocrine development of the androgenic glands so that females differentiate instead: feminization should translate as IAG silencing and increased Wolbachia load in the same developmental window. In line with the autocrine model, uninfected males expressed IAG from the first larval stage on, long before the androgenic gland primordia begin to differentiate, and exponentially throughout development. In contrast in infected males, expression fully stopped at stage 4 (juvenile), when male differentiation begins. This co-occurred with the only significant rise in the Wolbachia load throughout the life-stages. Concurrently, the raw expression of the bacterial Secretion Systems co-increased, but they were not over-expressed relative to the number of bacteria. The isopod model leads to formulate the "bacterial dosage model" throughout extended phenotypes as the conjunction between bacterial load as the mode of action, timing of multiplication (pre/post-zygotic), and site of action (soma vs. germen).

Abstract Background Isopods have colonized all environments, partly thanks to their ability to decompose the organic matter. Their enzymatic repertoire, as well as the one of their associated microbiota, has contributed to their colonization success. Together, these holobionts have evolved several interesting life history traits to degrade the plant cell walls, mainly composed of lignocellulose. It has been shown that terrestrial isopods achieve lignocellulose degradation thanks to numerous and diverse CAZymes provided by both the host and its microbiota. Nevertheless, the strategies for lignocellulose degradation seem more diversified in isopods, in particular in aquatic species which are the least studied. Isopods could be an interesting source of valuable enzymes for biotechnological industries of biomass conversion. Results To provide new features on the lignocellulose degradation in isopod holobionts, shotgun sequencing of 36 metagenomes of digestive and non-digestive tissues was performed from several populations of four aquatic and terrestrial isopod species. Combined to the 15 metagenomes of an additional species from our previous study, as well as the host transcriptomes, this large dataset allowed us to identify the CAZymes in both the host and the associated microbial communities. Analyses revealed the dominance of Bacteroidetes and Proteobacteria in the five species, covering 36% and 56% of the total bacterial community, respectively. The identification of CAZymes and new enzymatic systems for lignocellulose degradation, such as PULs, cellulosomes and LPMOs, highlights the richness of the strategies used by the isopods and their associated microbiota. Conclusions Altogether, our results show that the isopod holobionts are promising models to study lignocellulose degradation. These models can provide new enzymes and relevant lignocellulose-degrading bacteria strains for the biotechnological industries of biomass conversion.

The combination of radiotherapy (RT) with immunotherapy represents a promising treatment modality for non-small cell lung cancer (NSCLC) patients. As only a minority of patients shows a persistent response today, a spacious optimization window remains to be explored. Previously we showed that fractionated RT can induce a local immunosuppressive profile. Based on the evolving concept of an immunomodulatory role for vagal nerve stimulation (VNS), we tested its therapeutic and immunological effects alone and in combination with fractionated RT in a preclinical-translational study. Lewis lung carcinoma-bearing C57Bl/6 mice were treated with VNS, fractionated RT or the combination while a patient cohort with locally advanced NSCLC receiving concurrent radiochemotherapy (ccRTCT) was enrolled in a clinical trial to receive either sham or effective VNS daily during their 6 weeks of ccRTCT treatment. Preclinically, VNS alone or with RT showed no therapeutic effect yet VNS alone significantly enhanced the activation profile of intratumoral CD8+ T cells by upregulating their IFN-γ and CD137 expression. In the periphery, VNS reduced the RT-mediated rise of splenic, but not blood-derived, regulatory T cells (Treg) and monocytes. In accordance, the serological levels of protumoral CXCL5 next to two Treg-attracting chemokines CCL1 and CCL22 were reduced upon VNS monotherapy. In line with our preclinical findings on the lack of immunological changes in blood circulating immune cells upon VNS, immune monitoring of the peripheral blood of VNS treated NSCLC patients (n=7) did not show any significant changes compared to ccRTCT alone. As our preclinical data do suggest that VNS intensifies the stimulatory profile of the tumor infiltrated CD8+ T cells, this favors further research into non-invasive VNS to optimize current response rates to RT-immunotherapy in lung cancer patients.

Abstract Background Isopods constitute a particular group of crustaceans that has successfully colonized all environments including marine, freshwater and terrestrial habitats. Their ability to use various food sources, especially plant biomass, might be one of the reasons of their successful spread. All isopods, which feed on plants and their by-products, must be capable of lignocellulose degradation. This complex composite is the main component of plants and is therefore an important nutrient source for many living organisms. Its degradation requires a large repertoire of highly specialized Carbohydrate-Active enZymes (called CAZymes) which are produced by the organism itself and in some cases, by its associated microbiota. The acquisition of highly diversified CAZymes could have helped isopods to adapt to their diet and to their environment, especially during land colonization. Results To test this hypothesis, isopod host CAZomes (i.e. the entire CAZyme repertoire) were characterized in marine, freshwater and terrestrial species through a transcriptomic approach. Many CAZymes were identified in 64 isopod transcriptomes, comprising 27 de novo datasets. Our results show that marine, freshwater and terrestrial isopods exhibit different CAZomes, illustrating different strategies for lignocellulose degradation. The analysis of variations of the size of CAZy families shows these are expanded in terrestrial isopods while they are contracted in aquatic isopods; this pattern is probably resulting from the evolution of the host CAZomes during the terrestrial adaptation of isopods. We show that CAZyme gene duplications and horizontal transfers can be involved in adaptive divergence between isopod CAZomes. Conclusions Our characterization of the CAZomes in 64 isopods species provides new insights into the evolutionary processes that enabled isopods to conquer various environments, especially terrestrial ones.

In this work, we highlight the influence of the material uncertainties (magnetic permeability, electric conductivity of a Mn-Zn ferrite core, and electric permittivity of wire insulation) on the RLC parameters of a wound inductor extracted from the finite element method. To that end, the finite element method is embedded in a Monte Carlo simulation. We show that considering mentioned different material properties as real random variables, leads to significant variations in the distributions of the RLC parameters.

Following publication of the original article [1], the authors reported that there was an error in Figure 9, which contained a misplaced picture. The authors confirm that all of the published results and conclusions of the paper remain unchanged, as well as the figure legends. The authors apologize for any confusion caused. The corrected Figure 9 is shown as follows:

Reproductive parasites such as Wolbachia are able to manipulate the reproduction of their hosts by inducing parthenogenesis, male-killing, cytoplasmic incompatibility or feminization of genetic males. Despite extensive studies, no underlying molecular mechanism has been described to date. The goal of this study was to establish a system with a single Wolbachia strain that feminizes two different isopod species to enable comparative analyses aimed at elucidating the genetic basis of feminization. It was previously suggested that Wolbachia wVulC, which naturally induces feminization in Armadillidium vulgare, induces the development of female secondary sexual characters in transinfected Cylisticus convexus adult males. However, this does not demonstrate that wVulC induces feminization in C. convexus since feminization is the conversion of genetic males into functional females that occurs during development. Nevertheless, it suggests that C. convexus may represent a feminization model suitable for further development. Knowledge about C. convexus sexual differentiation is also essential for comparative analyses, as feminization is thought to take place just before or during sexual differentiation. Consequently, we first described gonad morphological differentiation of C. convexus and compared it with that of A. vulgare. Then, wVulC was injected into male and female C. convexus adult individuals. The feminizing effect was demonstrated by the combined appearance of female secondary sexual characters in transinfected adult males, as well as the presence of intersexes and female biases in progenies in which wVulC was vertically transmitted from transinfected mothers. The establishment of a new model of feminization of a Wolbachia strain in a heterologous host constitutes a useful tool towards the understanding of the molecular mechanism of feminization.

Epidermal growth factor receptor (EGFR) and c-MET receptors are expressed on many non-small cell lung cancer (NSCLC) cells. Current single agent therapeutic targeting of a mutant EGFR has a high efficacy in the clinic, but is not curative. Here, we investigated the combination of targeting EGFR and c-MET pathways in NSCLC cells resistant to receptor tyrosine kinase inhibitors (TKIs), using RNA interference and inhibition by TKIs. Different NSCLC cell lines with various genomic characteristics (H358, H1650 and H1975) were transfected with EGFR-specific-siRNA, T790M-specific-siRNA, c-MET siRNA or the combination. Subsequently EGFR TKIs (gefitinib, erlotinib or afatinib) or monoclonal antibody cetuximab were combined respectively with the c-MET-specific TKI su11274 in NSCLC cell lines. The cell proliferation, viability, caspase-3/7 activity and apoptotic morphology were monitored by spectrophotometry, fluorimetry and fluorescence microscopy. The combined effect of EGFR TKIs, or cetuximab and su11274, was evaluated using a combination index. The results showed that the cell lines that were relatively resistant to EGFR TKIs, especially the H1975 cell line containing the resistance T790M mutation, were found to be more sensitive to EGFR-specific-siRNA. The combination of EGFR siRNA plus c-MET siRNA enhanced cell growth inhibition, apoptosis induction and inhibition of downstream signaling in EGFR TKI resistant H358, H1650 and H1975 cells, despite the absence of activity of the c-MET siRNA alone. EGFR TKIs or cetuximab plus su11274 were also consistently superior to either agent alone. The strongest biological effect was observed when afatinib, an irreversible pan-HER blocker was combined with su11274, which achieved a synergistic effect in the T790M mutant H1975 cells. In a conclusion, our findings offer preclinical proof of principle for combined inhibition as a promising treatment strategy for NSCLC, especially for patients in whom current EGFR-targeted treatments fail due to the presence of the T790M-EGFR-mutation or high c-MET expression.

Aberrant expression of microRNA-146a (miR-146a) has been reported to be involved in the development and progression of various types of cancers. However, its role in non-small cell lung cancer (NSCLC) has not been elucidated. The aim of this study was to investigate the contribution of miR-146a to various aspects of the malignant phenotype of human NSCLCs. In functional experiments, miR-146a suppressed cell growth, induced cellular apoptosis and inhibited EGFR downstream signaling in five NSCLC cell lines (H358, H1650, H1975, HCC827 and H292). miR-146a also inhibited the migratory capacity of these NSCLC cells. On the other hand, miR-146a enhanced the inhibition of cell proliferation by drugs targeting EGFR, including both TKIs (gefitinib, erlotinib, and afatinib) and a monoclonal antibody (cetuximab). These effects were independent of the EGFR mutation status (wild type, sensitizing mutation or resistance mutation), but were less potent compared to the effects of siRNA targeting of EGFR. Our results suggest that these effects of miR-146a are due to its targeting of EGFR and NF-κB signaling. We also found, in clinical formalin fixed paraffin embedded (FFPE) lung cancer samples, that low expression of miR-146a was correlated with advanced clinical TNM stages and distant metastasis in NSCLC (P

Abstract Background The BRCA1 and BRCA2 mutation spectrum and mutation detection rates according to different family histories were investigated in 521 subjects from 322 unrelated Slovenian cancer families with breast and/or ovarian cancer. Methods The BRCA1 and BRCA2 genes were screened using DGGE, PTT, HRM, MLPA and direct sequencing. Results Eighteen different mutations were found in BRCA1 and 13 in BRCA2 gene. Mutations in one or other gene were found in 96 unrelated families. The mutation detection rates were the highest in the families with at least one breast and at least one ovarian cancer - 42% for BRCA1 and 8% for BRCA2. The mutation detection rate observed in the families with at least two breast cancers with disease onset before the age of 50 years and no ovarian cancer was 23% for BRCA1 and 13% for BRCA2. The mutation detection rate in the families with at least two breast cancers and only one with the disease onset before the age of 50 years was 11% for BRCA1 and 8% for BRCA2. In the families with at least two breast cancers, all of them with disease onset over the age of 50 years, the detection rate was 5% for BRCA2 and 0% for BRCA1. Conclusion Among the mutations detected in Slovenian population, 5 mutations in BRCA1 and 4 mutations in BRCA2 have not been described in other populations until now. The most frequent mutations in our population were c.181T > G, c.1687C > T, c.5266dupC and c.844_850dupTCATTAC in BRCA1 gene and c.7806-2A > G, c.5291C > G and c.3978insTGCT in BRCA2 gene (detected in 69% of BRCA1 and BRCA2 positive families).

Abstract Background Both recurrent and population specific mutations have been found in different areas of the world and more specifically in ethnically defined or isolated populations. The population of Slovenia has over several centuries undergone limited mixing with surrounding populations. The current study was aimed at establishing the mutation spectrum of BRCA1/2 in the Slovenian breast/ovarian cancer families taking advantage of a complete cancer registration database. A second objective was to determine the cancer phenotype of these families. Methods The original population database was composed of cancer patients from the Institute of Oncology Ljubljana in Slovenia which also includes current follow-up status on these patients. The inclusion criteria for the BRCA1/2 screening were: (i) probands with at least two first degree relatives with breast and ovarian cancer; (ii) probands with only two first degree relatives of breast cancer where one must be diagnosed less than 50 years of age; and (iii) individual patients with breast and ovarian cancer, bilateral breast cancer, breast cancer diagnosed before the age of 40 and male breast cancer without any other cancer in the family. Results Probands from 150 different families met the inclusion criteria for mutation analysis of which 145 consented to testing. A BRCA1/2 mutation was found in 56 (39%). Two novel large deletions covering consecutive exons of BRCA1 were found. Five highly recurrent specific mutations were identified (1806C>T, 300T>G, 300T>A, 5382insC in the BRCA1 gene and IVS16-2A>G in the BRCA2 gene). The IVS16-2A>G in the BRCA2 gene appears to be a unique founder mutation in the Slovenian population. A practical implication is that only 4 PCR fragments can be used in a first screen and reveal the cancer predisposing mutation in 67% of the BRCA1/2 positive families. We also observed an exceptionally high frequency of 4 different pathogenic missense mutations, all affecting one of the cryptic cysteine residues of the BRCA1 Ring Finger domain. Conclusion A high mutation detection rate and the frequent occurrence of a limited array of recurring mutations facilitate BRCA1/2 mutation screening in Slovenian families.

Abstract Non-small cell lung cancer (NSCLC) patients harboring activating mutations in epidermal growth factor receptor (EGFR) are sensitive to therapy with EGFR tyrosine kinase inhibitors (TKI). Despite remarkable clinical responses using EGFR TKI, surviving drug tolerant cells serve as a reservoir from which drug resistant tumors may emerge. This study addresses the need for improved efficacy of EGFR TKI by identifying targets involved in functional drug tolerance against them. To this aim, a high-throughput siRNA kinome screen was performed using two EGFR TKI-sensitive EGFR-mutant NSCLC cell lines in the presence/absence of the second-generation EGFR TKI afatinib. From the screen, Serine/Threonine/Tyrosine Kinase 1 (STYK1) was identified as a target that when downregulated potentiates the effects of EGFR inhibition in vitro. We found that chemical inhibition of EGFR combined with the siRNA-mediated knockdown of STYK1 led to a significant decrease in cancer cell viability and anchorage-independent cell growth. Further, we show that STYK1 selectively interacts with mutant EGFR and that the interaction is disrupted upon EGFR inhibition. Finally, we identified fibroblast growth factor 1 (FGF1) as a downstream effector of STYK1 in NSCLC cells. Accordingly, downregulation of STYK1 counteracted the afatinib-induced upregulation of FGF1. Altogether, we unveil STYK1 as a valuable target to repress the pool of surviving drug tolerant cells arising upon EGFR inhibition. Co-targeting of EGFR and STYK1 could lead to a better overall outcome for NSCLC patients.

Abstract Background We examined 15 years of key performance indicators (KPIs) of the population-based mammography screening programme (PMSP) in Flanders, Belgium. Methods Individual screening data were linked to the national cancer registry to obtain oncological follow-up. We benchmarked crude KPI results against KPI-targets set by the European guidelines and KPI results of other national screening programmes. Temporal trends were examined by plotting age-standardised KPIs against the year of screening and estimating the Average Annual Percentage Change (AAPC). Results PMSP coverage increased significantly over the period of 15 years (+ 7.5% AAPC), but the increase fell to + 1.6% after invitation coverage was maximised. In 2016, PMSP coverage was at 50.0% and opportunistic coverage was at 14.1%, resulting in a total coverage by screening of 64.2%. The response to the invitations was 49.8% in 2016, without a trend. Recall rate decreased significantly (AAPC -1.5% & -5.0% in initial and subsequent regular screenings respectively) while cancer detection remained stable (AAPC 0.0%). The result was an increased positive predictive value (AAPC + 3.8%). Overall programme sensitivity was stable and was at 65.1% in 2014. In initial screens of 2015, the proportion of DCIS, tumours stage II+, and node negative invasive cancers was 18.2, 31.2, and 61.6% respectively. In subsequent regular screens of 2015, those proportions were 14.0, 24.8, and 65.4% respectively. Trends were not significant. Conclusion Besides a suboptimal attendance rate, most KPIs in the Flemish PMSP meet EU benchmark targets. Nonetheless, there are several priorities for further investigation such as a critical evaluation of strategies to increase screening participation, organising a biennial radiological review of interval cancers, analysing the effect that preceding opportunistic screening has on the KPI for initial screenings, and efforts to estimate the impact on breast cancer mortality.

Abstract Background In the majority of familial breast cancer (BC) families, the etiology of the disease remains unresolved. To identify missing BC heritability resulting from relatively rare variants (minor allele frequency ≤ 1%), we have performed whole exome sequencing followed by variant analysis in a virtual panel of 492 cancer-associated genes on BC patients from BRCA1 and BRCA2 negative families with elevated BC risk. Methods BC patients from 54 BRCA1 and BRCA2-negative families with elevated BC risk and 120 matched controls were considered for germline DNA whole exome sequencing. Rare variants identified in the exome and in a virtual panel of cancer-associated genes [492 genes associated with different types of (hereditary) cancer] were compared between BC patients and controls. Nonsense, frame-shift indels and splice-site variants (strong protein-damaging variants, called PDAVs later on) observed in BC patients within the genes of the panel, which we estimated to possess the highest probability to predispose to BC, were further validated using an alternative sequencing procedure. Results Exome- and cancer-associated gene panel-wide variant analysis show that there is no significant difference in the average number of rare variants found in BC patients compared to controls. However, the genes in the cancer-associated gene panel with nonsense variants were more than two-fold over-represented in women with BC and commonly involved in the DNA double-strand break repair process. Approximately 44% (24 of 54) of BC patients harbored 31 PDAVs, of which 11 were novel. These variants were found in genes associated with known or suspected BC predisposition (PALB2, BARD1, CHEK2, RAD51C and FANCA) or in predisposing genes linked to other cancer types but not well-studied in the context of familial BC (EXO1, RECQL4, CCNH, MUS81, TDP1, DCLRE1A, DCLRE1C, PDE11A and RINT1) and genes associated with different hereditary syndromes but not yet clearly associated with familial cancer syndromes (ABCC11, BBS10, CD96, CYP1A1, DHCR7, DNAH11, ESCO2, FLT4, HPS6, MYH8, NME8 and TTC8). Exome-wide, only a few genes appeared to be enriched for PDAVs in the familial BC patients compared to controls. Conclusions We have identified a series of novel candidate BC predisposition variants/genes. These variants/genes should be further investigated in larger cohorts/case-control studies. Other studies including co-segregation analyses in affected families, locus-specific loss of heterozygosity and functional studies should shed further light on their relevance for BC risk.

Emerging anticancer agents such as the pan-FGFR Inhibitor have achieved remarkable improvements in the survival of patients with metastatic malignancies. Nevertheless they are still associated with specific ophthalmic toxicities. Understanding their pathophysiology can lead us to better clinical practice of life-threatening and vision-threatening circumstances. To investigate choroidal alterations as a potential pathophysiological mechanism of a serous detachment in bilateral pan-FGFR Inhibitor-Associated Retinopathy (FGFRAR), the morphology of the choroid and choriocapillaris were assessed. The choroidal thickness (ChT) and choriocapillaris flow void were measured by macular optical coherence tomography (OCT) and angiography (OCT-A), respectively. Data were collected at the baseline, then at one-month and two-months follow-ups after starting erdafitinib, in a single case of pulmonary angiosarcoma. Choroidal and choriocapillaris morphology showed stable ChT and choriocapillaris flow void at FGFRAR onset and relapse. To the best of our knowledge, this is the first analyzed case reported with flow-void OCT-angiography. Considering these results, FGFRAR in this patient does not seem to match the pachychoroid spectrum disorder definition; rather, an intracellular mechanism based on intracellular transduction pathways may be at work.

Abstract Objective Our aim was to produce a mono-biotinylated single domain antibody (‘nanobody’) specific for the epidermal growth factor receptor (EGFR), which is overexpressed in many cancer cells. The binding of the nanobody and its function are tested in cancer cells. The construct could be used to carry variable therapeutic or diagnostic load using biotin-streptavidin bridging. Results The EGFR-specific 7D12 nanobody was genetically fused to an IgA hinge linker and to a C-terminal biotin ligase acceptor sequence, allowing mono-biotinylation in E. coli. Expression was in strain BL21-DE3 from a T7 RNA polymerase driven pET22b vector. The biotinylated nanobody, isolated from the periplasm, was purified using streptavidin-mutein affinity chromatography. Final yields were up to 5 mg/l of cell culture. We showed that the construct could bind to EGFR expressing A431 epidermoid carcinoma cells, and to transiently transformed EGFR overexpressing HEK293T cells and not to EGFR negative control cells. The specificity for the EGFR was further demonstrated by immunoprecipitation. To test the functionality, PC9 non-small cell lung cancer cells were treated with mono-biotinylated nanobody or with streptavidin-coupled tetravalent nanobodies. Both were able to block mutant EGFR phosphorylation and slow down growth of PC9 cells. Tetravalent nanobodies were able to downregulate AKT phosphorylation.

Renewable Energy Communities consist in an emerging decentralized market mechanism which allows local energy exchanges between end-users, bypassing the traditional wholesale/retail market structure. In that configuration, local consumers and prosumers gather in communities and can either cooperate or compete towards a common objective, such as the minimization of the electricity costs and/or the minimization of greenhouse gas emissions for instance. This paper proposes data analytics modules which aim at helping the community members to schedule the usage of their resources (generation and consumption) in order to minimize their electricity bill. A day-ahead local wind power forecasting algorithm, which relies on state-of-the-art Machine Learning techniques currently used in worldwide forecasting contests, is in that way proposed. We develop furthermore an original method to improve the performance of neural network forecasting models in presence of abnormal wind power data. A technique for computing representative profiles of the community members electricity consumption is also presented. The proposed techniques are tested and deployed operationally on a pilot Renewable Energy Community established on an Medium Voltage network in Belgium, involving 2.25MW of wind and 18 Small and Medium Enterprises who had the possibility to freely access the results of the developed data modules by connecting to a dedicated web platform. We first show that our method for dealing with abnormal wind power data improves the forecasting accuracy by 10% in terms of Root Mean Square Error. The impact of the developed data modules on the consumption behaviour of the community members is then quantified, by analyzing the evolution of their monthly self-consumption and self-sufficiency during the pilot. No significant changes in the members behaviour, in relation with the information provided by the models, were observed in the recorded data. The pilot was however perturbed by the COVID-19 crisis which had a significant impact on the economic activity of the involved companies. We conclude by providing recommendations for the future set up of similar communities.

This paper addresses the voltage control problem in medium-voltage distribution networks. The objective is to cost-efficiently maintain the voltage profile within a safe range, in presence of uncertainties in both the future working conditions, as well as the physical parameters of the system. Indeed, the voltage profile depends not only on the fluctuating renewable-based power generation and load demand, but also on the physical parameters of the system components. In reality, the characteristics of loads, lines and transformers are subject to complex and dynamic dependencies, which are difficult to model. In such a context, the quality of the control strategy depends on the accuracy of the power flow representation, which requires to capture the non-linear behavior of the power network. Relying on the detailed analytical models (which are still subject to uncertainties) introduces a high computational power that does not comply with the real-time constraint of the voltage control task. To address this issue, while avoiding arbitrary modeling approximations, we leverage a deep reinforcement learning model to ensure an autonomous grid operational control. Outcomes show that the proposed model-free approach offers a promising alternative to find a compromise between calculation time, conservativeness and economic performance.

Sebastian Grève interviews Felix Mühlhölzer on his work on the philosophy of mathematics.

INTRODUCTION:Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibition is the preferred first-line treatment of advanced adenocarcinoma of the lung that harbors EGFR activating tyrosine kinase domain mutations. Most data available pertain to Asian populations in which such mutations are more prevalent. We report on the long-term results of first-line treatment with erlotinib in Caucasian patients with advanced adenocarcinoma of the lung that have a somatic EGFR mutation in their tumor. METHODS:Multicenter academic prospective phase II study with erlotinib in patients with an activating EGFR tyrosine kinase (TK) domain somatic mutation (any exon encoding the kinase domain) in the tumor and no prior treatment for their advanced disease. RESULTS:Phenotypic preselecting of 229 patients led to a high EGFR mutation detection rate of 24% of which 46 patients were included in the phase II study. With a progression free survival (PFS) of 81% at three months the study met its primary endpoint for presumed superiority over chemotherapy. With an overall median PFS of 11 months and a median overall survival (OS) of 23 months, the results compare favorably with results obtained in randomized studies using TKI in first line in EGFR mutation positive adenocarcinoma of the lung. CONCLUSION:The present study reinforces the use of EGFR tyrosine kinase inhibition (TKI) as a first line treatment of choice for advanced adenocarcinoma of the lung carrying an activating EGFR mutation. The mutation rate in preselected Caucasian patients is higher than previously reported. Issues relevant for clinical practice are discussed. TRIAL REGISTRATION:ClinicalTrials.gov NCT00339586.

Wolbachia bacteria are obligate intracellular alpha-Proteobacteria of arthropods and nematodes. Although widespread among isopod crustaceans, they have seldom been found in non-isopod crustacean species. Here, we report Wolbachia infection in fourteen new crustacean species. Our results extend the range of Wolbachia infections in terrestrial isopods and amphipods (class Malacostraca). We report the occurrence of two different Wolbachia strains in two host species (a terrestrial isopod and an amphipod). Moreover, the discovery of Wolbachia in the goose barnacle Lepas anatifera (subclass Thecostraca) establishes Wolbachia infection in class Maxillopoda. The new bacterial strains are closely related to B-supergroup Wolbachia strains previously reported from crustacean hosts. Our results suggest that Wolbachia infection may be much more widespread in crustaceans than previously thought. The presence of related Wolbachia strains in highly divergent crustacean hosts suggests that Wolbachia endosymbionts can naturally adapt to a wide range of crustacean hosts. Given the ability of isopod Wolbachia strains to induce feminization of genetic males or cytoplasmic incompatibility, we speculate that manipulation of crustacean-borne Wolbachia bacteria might represent potential tools for controlling crustacean species of commercial interest and crustacean or insect disease vectors.

Most of crustacean immune responses are well described for the aquatic forms whereas almost nothing is known for the isopods that evolved a terrestrial lifestyle. The latter are also infected at a high prevalence with Wolbachia, an endosymbiotic bacterium which affects the host immune system, possibly to improve its transmission. In contrast with insect models, the isopod Armadillidium vulgare is known to harbor Wolbachia inside the haemocytes.In A. vulgare we characterized three haemocyte types (TEM, flow cytometry): the hyaline and semi-granular haemocytes were phagocytes, while semi-granular and granular haemocytes performed encapsulation. They were produced in the haematopoietic organs, from central stem cells, maturing as they moved toward the edge (TEM). In infected individuals, live Wolbachia (FISH) colonized 38% of the haemocytes but with low, variable densities (6.45±0.46 Wolbachia on average). So far they were not found in hyaline haemocytes (TEM). The haematopoietic organs contained 7.6±0.7×10(3)Wolbachia, both in stem cells and differentiating cells (FISH). While infected and uninfected one-year-old individuals had the same haemocyte density, in infected animals the proportion of granular haemocytes in particular decreased by one third (flow cytometry, Pearson's test = 12 822.98, df = 2, p

Abstract Background The epidermal growth factor receptor (EGFR) is a validated therapeutic target in non-small cell lung cancer (NSCLC). However, current single agent receptor targeting does not achieve a maximal therapeutic effect, and some mutations confer resistance to current available agents. In the current study we have examined, in different NSCLC cell lines, the combined effect of RNA interference targeting the EGFR mRNA, and inactivation of EGFR signaling using different receptor tyrosine kinase inhibitors (TKIs) or a monoclonal antibody cetuximab. Methods NSCLC cells (cell lines HCC827, H292, H358, H1650, and H1975) were transfected with EGFR siRNA and/or treated with the TKIs gefitinib, erlotinib, and afatinib, and/or with the monoclonal antibody cetuximab. The reduction of EGFR mRNA expression was measured by real-time quantitative RT-PCR. The down-regulation of EGFR protein expression was measured by western blot, and the proliferation, viability, caspase3/7 activity, and apoptotic morphology were monitored by spectrophotometry, fluorimetry, and fluorescence microscopy. The combined effect of EGFR siRNA and different drugs was evaluated using a combination index. Results EGFR-specific siRNA strongly inhibited EGFR protein expression almost equally in all cell lines and inhibited cell growth and induced cell apoptosis in all NSCLC cell lines studied, albeit with a different magnitude. The effects on growth obtained with siRNA was strikingly different from the effects obtained with TKIs. The effects of siRNA probably correlate with the overall oncogenic significance of the receptor, which is only partly inhibited by the TKIs. The cells which showed weak response to TKIs, such as the H1975 cell line containing the T790M resistance mutation, were found to be responsive to siRNA knockdown of EGFR, as were cell lines with downstream TKI resistance mutations. The cell line HCC827, harboring an exon 19 deletion mutation, was more than 10-fold more sensitive to TKI proliferation inhibition and apoptosis induction than any of the other cell lines. Cetuximab alone had no relevant in vitro activity at concentrations obtainable in the clinic. The addition of EGFR siRNA to either TKIs or cetuximab additively enhanced growth inhibition and induction of apoptosis in all five cell lines, independent of the EGFR mutation status (wild-type or sensitizing mutation or resistant mutation). The strongest biological effect was observed when afatinib was combined with an EGFR-specific siRNA. Conclusions EGFR knockdown by siRNA further decreases the cell growth of lung cancer cells that are treated with TKIs or cetuximab alone, confirming that single agent drug targeting does not achieve a maximal biological effect. The siRNA inhibits EGFR oncogenic activity that bypasses downstream "resistance" mutations such as KRAS and PTEN. The combined treatment of siRNA and EGFR inhibitory agents is additive. The combination of a potent, irreversible kinase inhibitor such as afatinib, with EGFR-specific siRNAs should be further investigated as a new strategy in the treatment of lung cancer and other EGFR dependent cancers, including those with downstream resistance mutations.

Abstract Background Real-time quantitative RT-PCR (RT-qPCR) is a "gold" standard for measuring steady state mRNA levels in RNA interference assays. The knockdown of the epidermal growth factor receptor (EGFR) gene with eight individual EGFR small interfering RNAs (siRNAs) was estimated by RT-qPCR using three different RT-qPCR primer sets. Results Our results indicate that accurate measurement of siRNA efficacy by RT-qPCR requires careful attention for the selection of the primers used to amplify the target EGFR mRNA. Conclusions We conclude that when assessing siRNA efficacy with RT-qPCR, more than one primer set targeting different regions of the mRNA should be evaluated and at least one of these primer sets should amplify a region encompassing the siRNA recognition sequence.

Abstract Background The status of the EGFR and HER2-neu genes has not been fully defined in ovarian cancer. An integrated analysis of both genes could help define the proportion of patients that would potentially benefit from targeted therapies. Methods We determined the tumour mutation status of the entire tyrosine kinase (TK) domain of the EGFR and HER2-neu genes in a cohort of 52 patients with invasive epithelial ovarian cancer as well as the gene copy number and protein expression of both genes in 31 of these patients by DGGE and direct sequecing, immunohistochemistry and Fluorescent in Situ Hybridisation (FISH). Results The EGFR was expressed in 59% of the cases, with a 2+/3+ staining intensity in 38%. HER2-neu expression was found in 35%, with a 2/3+ staining in 18%. No mutations were found in exons 18–24 of the TK domains of EGFR and HER2-neu. High polysomy of the EGFR gene was observed in 13% of the invasive epthelial cancers and amplification of the HER2-neu gene was found in 10% and correlated with a high expression level by immunohistochemistry. Mutations within the tyrosine kinase domain were not found in the entire TK domain of both genes, but have been found in very rare cases by others. Conclusion Genomic alteration of the HER2-neu and EGFR genes is frequent (25%) in ovarian cancer. EGFR/HER2-neu targeted therapies should be investigated prospectively and specifically in that subset of patients.