Detection of Crimean-Congo haemorrhagic fever virus in Hyalomma marginatum ticks, southern France, May 2022 and April 2023.

Crimean-Congo haemorrhagic fever (CCHF), a potentially severe zoonotic viral disease causing fever and haemorrhagic manifestations in humans. As the Crimean-Congo haemorrhagic fever virus (CCHFV) has been detected in ticks in Spain and antibodies against the virus in ruminant sera in Corsica, it was necessary to know more about the situation in France. In 2022-2023, CCHFV was detected in 155 ticks collected from horses and cattle in southern France.

Multiplexed-Based Assessment of DNA Damage Response to Chemotherapies Using Cell Imaging Cytometry.

The current methods for measuring the DNA damage response (DDR) are relatively labor-intensive and usually based on Western blotting, flow cytometry, and/or confocal immunofluorescence analyses. They require many cells and are often limited to the assessment of a single or few proteins. Here, we used the Celigo® image cytometer to evaluate the cell response to DNA-damaging agents based on a panel of biomarkers associated with the main DDR signaling pathways. We investigated the cytostatic or/and the cytotoxic effects of these drugs using simultaneous propidium iodide and calcein-AM staining. We also describe new dedicated multiplexed protocols to investigate the qualitative (phosphorylation) or the quantitative changes of eleven DDR markers (H2AX, DNA-PKcs, ATR, ATM, CHK1, CHK2, 53BP1, NBS1, RAD51, P53, P21). The results of our study clearly show the advantage of using this methodology because the multiplexed-based evaluation of these markers can be performed in a single experiment using the standard 384-well plate format. The analyses of multiple DDR markers together with the cell cycle status provide valuable insights into the mechanism of action of investigational drugs that induce DNA damage in a time- and cost-effective manner due to the low amounts of antibodies and reagents required.

Carboxylate-functionalized foldamer inhibitors of HIV-1 integrase and Topoisomerase 1: artificial analogues of DNA mimic proteins.

Inspired by DNA mimic proteins, we have introduced aromatic foldamers bearing phosphonate groups as synthetic mimics of the charge surface of B-DNA and competitive inhibitors of some therapeutically relevant DNA-binding enzymes: the human DNA Topoisomerase 1 (Top1) and the human HIV-1 integrase (HIV-1 IN). We now report on variants of these anionic foldamers bearing carboxylates instead of phosphonates. Several new monomers have been synthesized with protecting groups suitable for solid phase synthesis (SPS). Six hexadecaamides have been prepared using SPS. Proof of their resemblance to B-DNA was brought by the first crystal structure of one of these DNA-mimic foldamers in its polyanionic form. While some of the foldamers were found to be as active as, or even more active than, the original phosphonate oligomers, others had no activity at all or could even stimulate enzyme activity in vitro. Some foldamers were found to have differential inhibitory effects on the two enzymes. These results demonstrate a strong dependence of inhibitory activity on foldamer structure and charge distribution. They open broad avenues for the development of new classes of derivatives that could inhibit the interaction of specific proteins with their DNA target thereby influencing the cellular pathways in which they are involved.

Safety and efficacy of temsirolimus as second line treatment for patients with recurrent bladder cancer.

BACKGROUND: Bladder cancer is the 7th cause of death from cancer in men and 10th in women. Metastatic patients have a poor prognosis with a median overall survival of 14 months. Until recently, vinflunine was the only second-line chemotherapy available for patients who relapse. Deregulation of the PI3K/AKT/mTOR pathway was observed in more than 40% of bladder tumors and suggested the use of mTOR as a target for the treatment of urothelial cancers. METHODS: This trial assessed the efficacy of temsirolimus in a homogenous cohort of patients with recurrent or metastatic bladder cancer following first-line chemotherapy. Efficacy was measured in terms of non-progression at two months according to the RECIST v1.1 criteria. Based on a two-stage optimal Simon's design, 15 non-progressions out of 51 evaluable patients were required to claim efficacy. Patients were treated at a weekly dose of 25 mg IV until progression, unacceptable toxicities or withdrawal. RESULTS: Among the 54 patients enrolled in the study between November 2009 and July 2014, 45 were assessable for the primary efficacy endpoint. A total of 22 (48.9%) non-progressions were observed at 2 months with 3 partial responses and 19 stable diseases. Remarkably, 4 patients were treated for more than 30 weeks. Fifty patients experienced at least a related grade1/2 (94%) and twenty-eight patients (52.8%) a related grade 3/4 adverse event. Eleven patients had to stop treatment for toxicity. This led to recruitment being halted by an independent data monitoring committee with regard to the risk-benefit balance and the fact that the primary objective was already met. CONCLUSIONS: While the positivity of this trial indicates a potential benefit of temsirolimus for a subset of bladder cancer patients who are refractory to first line platinum-based chemotherapy, the risk of adverse events associated with the use of this mTOR inhibitor would need to be considered when such an option is envisaged in this frail population of patients. It also remains to identify patients who will benefit the most from this targeted therapy. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01827943 (trial registration date: October 29, 2012); Retrospectively registered.

Early objective response may not be a prognostic factor of survival for patients with metastatic urothelial carcinoma: from a retrospective analysis of a cohort of 113 patients.

BACKGROUND: This study aims to better define prognostic factors for patients with metastatic urothelial carcinoma (mUC), and to identify patients who will benefit from first-line cisplatin-based chemotherapy. We test the hypothesis that early objective response (EOR), defined as the occurrence of an objective response following 2 or 3 courses of chemotherapy, could be a prognostic factor for overall survival (OS) and thus be used to guide treatment decisions. Data from 113 patients with evaluable mUC receiving first-line cisplatin-based treatment between January 2004 and December 2006 was collected retrospectively from prospectively-maintained databases across seven French cancer centers. Clinical factors potentially associated with survival and EOR were analyzed in univariate and multivariate analysis. RESULTS: One hundred three patient records were complete and available for inclusion in the multivariate model. Four factors were independently associated with OS: Performance status 1 and 2 (HR 2.3 [95 % CI 1.3-3.9], p = 0.002; HR 3.4 [95 % CI 1.6-7.2], p = 0.001 respectively); presence of visceral metastases (HR 2.2 [95 % CI 1.3-3.9], p = 0.004); abnormal hemoglobin levels (HR 1.7 [95 % CI 1.01-2.8], p = 0.045); disease progression (HR 10.1 [95 % CI 4.2-24.1], p < 0.001). CONCLUSIONS: This study confirms the prognostic factors previously reported in first-line chemotherapy for mUC. However, we failed to demonstrate that EOR was an independent predictive factor of OS. Nevertheless, an early response evaluation is recommended since early progression is an important parameter that can be used to decide whether treatment should be interrupted and changed for alternative strategies integrating the concept of personalized medicine or new immune therapies.

Epidemiology, molecular virology and diagnostics of Schmallenberg virus, an emerging orthobunyavirus in Europe.

After the unexpected emergence of Bluetongue virus serotype 8 (BTV-8) in northern Europe in 2006, another arbovirus, Schmallenberg virus (SBV), emerged in Europe in 2011 causing a new economically important disease in ruminants. The virus, belonging to the Orthobunyavirus genus in the Bunyaviridae family, was first detected in Germany, in The Netherlands and in Belgium in 2011 and soon after in the United Kingdom, France, Italy, Luxembourg, Spain, Denmark and Switzerland. This review describes the current knowledge on the emergence, epidemiology, clinical signs, molecular virology and diagnosis of SBV infection.

Major efficacy of trabectedin in 2 metastatic osteosarcoma patients with wild-type Asp1104 ERCC5 tumor status.

BACKGROUND: Treatment of osteosarcoma of the extremities consists of surgical resection preceded and followed by chemotherapy, including high-dose methotrexate or adriamycin-based protocols. When distant relapse occurs, therapeutic options are scarce. Trabectedin, a DNA-binding agent, is indicated for the treatment of patients with advanced soft tissue sarcomas after failure of anthracyclines and ifosfamide. In this indication, the 6-month progression-free survival is about 35-40%. Recent reports showed that some specific single nucleotide polymorphisms (SNPs) from DNA repair genes could be associated with sensitivity to trabectedin in soft tissue sarcomas. CASE REPORTS: We report our experience of 2 metastatic, heavily pre-treated osteosarcoma patients who were treated with trabectedin. Pyrosequencing analyses of tumors from both patients for several SNPs of the ERCC1, ERCC5 and BRAC1 genes were performed. Both patients showed major response to trabectedin, which was interestingly related with homozygoty of the common guanine allele of ERCC5 (G/G genotype; Asp/Asp) after pyrosenquencing analysis of tumors from both patients. This polymorphism was previously shown to be associated with better outcome in soft tissue sarcoma patients treated with trabectedin. CONCLUSION: Homozygoty for the wild-type Asp1104 SNP of the ERCC5 gene was found in 2 cases of relapsed osteosarcoma, who responded to trabectedin.

Innovation in Radionuclide Therapy for the Treatment of Prostate Cancers: Radiochemical Perspective and Recent Therapeutic Practices.

Prostate cancer represents the second cause of death by cancer in males in western countries. While early-stage diseases are accessible to surgery and/or external radiotherapy, advanced metastatic prostate cancers are primarily treated with androgen deprivation therapy, to which new generation androgen receptor antagonists or taxane-based chemotherapies are added in the case of tumor relapse. Nevertheless, patients become invariably resistant to castration with a median survival that rarely exceeds 3 years. This fostered the search for alternative strategies, independent of the androgen receptor signaling pathway. In this line, radionuclide therapies may represent an interesting option as they could target either the microenvironment of sclerotic bone metastases with the use of radiopharmaceuticals containing samarium-153, strontium-89 or radium-223 or tumor cells expressing the prostate-specific membrane antigen (PSMA), a protein found at the surface of prostate cancer cells. This review gives highlights the chemical properties of radioligands targeting prostate cancer cells and recapitulates the clinical trials evaluating the efficacy of radionuclide therapies, alone or in combination with other approved treatments, in patients with castration-resistant prostate tumors. It discusses some of the encouraging results obtained, especially the benefit on overall survival that was reported with [177Lu]-PSMA-617. It also addresses the specific requirements for the use of this particular class of drugs, both in terms of medical staff coordination and adapted infrastructures for efficient radioprotection.

Improving the response to oxaliplatin by targeting chemotherapy-induced CLDN1 in resistant metastatic colorectal cancer cells.

BACKGROUND: Tumor resistance is a frequent cause of therapy failure and remains a major challenge for the long-term management of colorectal cancer (CRC). The aim of this study was to determine the implication of the tight junctional protein claudin 1 (CLDN1) in the acquired resistance to chemotherapy. METHODS: Immunohistochemistry was used to determine CLDN1 expression in post-chemotherapy liver metastases from 58 CRC patients. The effects of oxaliplatin on membrane CLDN1 expression were evaluated by flow cytometry, immunofluorescence and western blotting experiments in vitro and in vivo. Phosphoproteome analyses, proximity ligation and luciferase reporter assays were used to unravel the mechanism of CLDN1 induction. RNAseq experiments were performed on oxaliplatin-resistant cell lines to investigate the role of CLDN1 in chemoresistance. The "one-two punch" sequential combination of oxaliplatin followed by an anti-CLDN1 antibody-drug conjugate (ADC) was tested in both CRC cell lines and murine models. RESULTS: We found a significant correlation between CLDN1 expression level and histologic response to chemotherapy, CLDN1 expression being the highest in resistant metastatic residual cells of patients showing minor responses. Moreover, in both murine xenograft model and CRC cell lines, CLDN1 expression was upregulated after exposure to conventional chemotherapies used in CRC treatment. CLDN1 overexpression was, at least in part, functionally related to the activation of the MAPKp38/GSK3ß/Wnt/ß-catenin pathway. Overexpression of CLDN1 was also observed in oxaliplatin-resistant CRC cell lines and was associated with resistance to apoptosis, suggesting an anti-apoptotic role for CLDN1. Finally, we demonstrated that the sequential treatment with oxaliplatin followed by an anti-CLDN1 ADC displayed a synergistic effect in vitro and in in vivo. CONCLUSION: Our study identifies CLDN1 as a new biomarker of acquired resistance to chemotherapy in CRC patients and suggests that a "one-two punch" approach targeting chemotherapy-induced CLDN1 expression may represent a therapeutic opportunity to circumvent resistance and to improve the outcome of patients with advanced CRC.

Gene expression signature predicting high-grade prostate cancer responses to oxaliplatin.

Prostate cancer is one of the leading causes of cancer-related deaths among men. Several prognostic factors allow differentiation of low-grade tumors from high-grade tumors with high metastatic potential. High-grade tumors are currently treated with hormone therapy, to which taxanes are added when the tumors become resistant to castration. Clinical trials with other anticancer agents did not take into account the genetic backgrounds of the tumors, and most trials demonstrated low response rates. Here we used an in silico approach to screen for drug candidates that might be used as alternatives to taxanes, on the basis of a published expression signature involving 86 genes that could distinguish high-grade and low-grade tumors (Proc Natl Acad Sci USA 103:10991-10996, 2006). We explored the National Cancer Institute databases, which include data on the gene expression profiles of 60 human tumor cell lines and the in vitro sensitivities of the cell lines to anticancer drugs, and we identified several genes in the signature for which expression levels were correlated with chemosensitivity. As an example of the validation of this in silico approach, we identified a set of six genes for which expression levels could predict cell sensitivity to oxaliplatin but not cisplatin. This signature was validated in vitro through silencing of the genes in DU145, LNCaP, and C4-2B prostate cancer cells, which was accompanied by changes in oxaliplatin but not cisplatin cytotoxicity. These results demonstrate the relevance of our approach for the identification of both alternative treatments for high-grade prostate cancers and new biomarkers to predict clinical tumor responses.

The polyphenolic ellagitannin vescalagin acts as a preferential catalytic inhibitor of the α isoform of human DNA topoisomerase II.

Polyphenolic ellagitannins are natural compounds that are often associated with the therapeutic activity of plant extracts used in traditional medicine. They display cancer-preventing activity in animal models by a mechanism that remains unclear. Potential targets have been proposed, including DNA topoisomerases II (Top2). Top2α and Top2ß, the two isoforms of the human Top2, play a crucial role in the regulation of replication, transcription, and chromosome segregation. They are the target of anticancer agents used in the clinic such as anthracyclines (e.g., doxorubicin) or the epipodophyllotoxin etoposide. It was recently shown that the antitumor activity of etoposide was due primarily to the inhibition of Top2α, whereas inhibition of Top2ß was responsible for the development of secondary malignancies, pointing to the need for more selective Top2α inhibitors. Here, we show that the polyphenolic ellagitannin vescalagin preferentially inhibits the decatenation activity of Top2α in vitro, by a redox-independent mechanism. In CEM cells, we also show that transient small interfering RNA-mediated down-regulation of Top2α but not of Top2ß conferred a resistance to vescalagin, indicating that the α isoform is a preferential target. We further confirmed that Top2α inhibition was due to a catalytic inhibition of the enzyme because it did not induce DNA double-strand breaks in CEM-treated cells but prevented the formation of Top2α- rather than Top2ß-DNA covalent complexes induced by etoposide. To our knowledge, vescalagin is the first example of a catalytic inhibitor for which cytotoxicity is due, at least in part, to the preferential inhibition of Top2α.

Deciphering the role of the ERCC2 gene polymorphism on anticancer drug sensitivity.

ERCC2 [Xeroderma pigmentosum (XP) group D] belongs to the nucleotide excision repair pathway. It is also part of the TFIIH transcription complex and is required for the association of the cyclin-dependent kinase (CDK)-activating kinase (CAK) subcomplex with TFIIH. Using the NCI-60 panel of human tumor cell lines, we had shown that the ERCC2 gene variant Gln(751) was significantly associated to increased taxanes sensitivity and decreased ERCC2 gene expression. Since TFIIH is involved in both DNA repair and cell cycle progression, we hypothesized that quantitative or qualitative ERCC2 alterations might cause CAK liberation, allowing its activation of the G(2)/M transition. Enhancing mitosis entry would lead to hypersensitivity to spindle poisons, explaining the effect of ERCC2 polymorphisms on taxane sensitivity. Starting from ERCC2-deficient XP6BE, we generated several isogenic clones differing only by the Lys751Gln variation. Wild-type and variant ERCC2-expressing clones recovered ultraviolet radiation and cisplatin resistance but presented similar sensitivity to paclitaxel, demonstrating that the amino acid change was not involved in paclitaxel differential sensitivity in the NCI-60 panel. Using small interfering RNA approach, we knocked down ERCC2 expression and observed a block in the G(2)/M phase, with a consistent increase in paclitaxel sensitivity and no change in cisplatin sensitivity. We observed in addition an increase in CDK1 activity, as evaluated by histone H1 phosphorylation. We evaluated messenger RNA (mRNA) half-life in the isogenic lines and observed a more rapid degradation in cells bearing the variant construct. We concluded that the increased paclitaxel sensitivity of ERCC2 variant cell lines is a consequence of lower gene expression, likely due to decreased stability of the variant ERCC2 mRNA.

[Molecular signatures in castration-resistant prostate cancers: An update]. / Signatures moléculaires dans les cancers de la prostate résistants à la castration : état des lieux.

Castration resistant prostate cancers are highly heterogenous at the molecular level. With the use of new generation sequencing technologies, a series of alterations were identified, opening the way for potential alternative treatments that could be more efficient for specific molecular subtypes. In this brief update, we summarize the new targetable pathways that are currently investigated. In this era of liquid biopsy and of precision medicine, it seems pertinent to be able to screen for these alterations on a more systematic basis before initiating any treatment.

Seroprevalence of Crimean-Congo Hemorrhagic Fever Among Small Ruminants from Southern Romania.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease that can be contracted by direct contact with viremic animals or humans. Domestic animals are accidental hosts and contribute to the spread and amplification of the virus. The main objective of this study was to provide updated information related to CCHF virus (CCHFV) infection in Southern Romania by assessing the seroprevalence of CCHF in small ruminants (sheep and goats) using a double-antigen sandwich enzyme-linked immunosorbent assay and by detection of CCHFV in engorged ticks and serum samples using real-time RT-PCR. The overall seroprevalence of CCHF in small ruminants was 37.7% (95% CI 31.7 to 43.7). No statistical seroprevalence difference was observed between the two species of ruminants (p = 0.76), but a significant difference was established between the locations (p < 0.01). No CCHFV RNA was detected in tick pools and small ruminant's sera tested by real-time RT-PCR, although the high seroprevalence to CCHFV among ruminants indicates that CCHV or a closely related virus circulates in Southern Romania.

New Topoisomerase I mutations are associated with resistance to camptothecin.

BACKGROUND: Topoisomerase I (TOP1) is a nuclear enzyme that catalyzes the relaxation of supercoiled DNA during DNA replication and transcription. TOP1 is the molecular target of camptothecin and related drugs such as irinotecan and SN38 (irinotecan's active metabolite). Irinotecan is widely used as an anti-cancer agent in the treatment of metastatic colon cancer. However, its efficacy is often limited by the development of resistance. METHODS: We previously established several SN38 resistant HCT116-derived clones to study the mechanisms underlying resistance to SN38. Here, we investigated whether resistance to SN38 in these cell lines could be linked to the presence of TOP1 mutations and changes in its expression and activity. Functional analyses were performed on these cell lines challenged with SN38 and we specifically monitored the double strands breaks with γH2AX staining and replication activity with molecular combing. RESULTS: In SN38 resistant HCT116 clones we identified three new TOP1 mutations, which are located in the core subdomain III (p.R621H and p.L617I) and in the linker domain (p.E710G) and are packed together at the interface between these two domains. The presence of these TOP1 mutations in SN38 resistant HCT116 cells did not modify TOP1 expression or intrinsic activity. Conversely, following challenge with SN38, we observed a decrease of TOP1-DNA cleavage complexes and a reduction in double-stranded break formation). In addition, we showed that SN38 resistant HCT116 cells present a strong decrease in the SN38-dependent asymmetry of replication forks that is characteristic of SN38 sensitive HCT116 cells. CONCLUSIONS: These results indicate that the TOP1 mutations are involved in the development of SN38 resistance. We hypothesize that p.L617, p.R621 and p.E710 TOP1 residues are important for the functionality of the linker and that mutation of one of these residues is sufficient to alter or modulate its flexibility. Consequently, linker fluctuations could have an impact on SN38 binding by reducing the enzyme affinity for the drug.

ERCC5/XPG, ERCC1, and BRCA1 gene status and clinical benefit of trabectedin in patients with soft tissue sarcoma.

BACKGROUND: The objective of this study was to determine whether specific single nucleotide polymorphisms (SNPs) from nucleotide excision repair (NER) and homologous recombination (HR) DNA repair pathways are associated with sensitivity to trabectedin in patients with soft tissue sarcoma (STS). METHODS: The authors analyzed excision repair cross-complementation group 5/xeroderma pigmentosum group G (ERCC5/XPG) (NER), excision repair cross-complementation group 1 (ERCC1) (NER), and breast cancer 1 (BRCA1) (HR) SNPs and messenger RNA expression levels in tumor specimens from 113 patients with advanced STS who were enrolled in previously published phase 2 trials or in a compassionate-use program. The 6-month progression-free rate (PFR), progression-free survival (PFS), and overall survival (OS) were analyzed according to ERCC5, ERCC1, and BRCA1 status using log-rank tests. RESULTS: High expression of the common allele (aspartic acid at codon 1104) of ERCC5, high expression of ERCC1, and BRCA1 haplotype were associated significantly with improved PFR, PFS, and OS. The ERCC1 thymine-to-cytosine (T→C) SNP at codon 19007 and BRCA1 expression were not associated with outcome. On univariate analysis, tumor histology, favorable NER status (high expression of common ERCC5 and/or high ERCC1 expression status), and favorable BRCA1 haplotype (at least 1 triple-adenine plus guanine [AAAG] allele) were the sole variables associated significantly with PFS and OS. CONCLUSIONS: In the current study, ERCC5, ERCC1, and BRCA1 status represented a potential DNA repair signature that could be used for the prediction of clinical response to trabectedin in patients with STS.

In vivo biotinylated recombinant influenza A virus hemagglutinin for use in subtype-specific serodiagnostic assays.

There is an urgent need for robust subtype-specific serological tests to diagnose influenza A virus infections in poultry and mammals, including humans. Such assays require reliable subtype-specific sources of soluble and authentically folded seroreactive hemagglutinin (HA), one of the integral membrane proteins that determine the serological subtype of influenza viruses. To this purpose, a bigenic pFastBacDual baculovirus transfer vector allowing efficient invivo biotinylation of soluble HA homo-oligomers expressed via the secretory pathway was developed. An Avi-Tag allowed site-specific biotinylation by a coexpressed genetically modified BirA biotin ligase retained in the endoplasmic reticulum (ER). Highly seroreactive mono-biotinylated HA of recent H5 and H7 influenza A subtypes was secreted from recombinant baculovirus infected High-Five insect cells at levels sufficient to directly load streptavidin-coated enzyme-linked immunosorbent assay (ELISA) matrices, thereby avoiding any purification steps. The recombinant antigens retained authentic antigenicity, including conformation-dependent epitopes involved in hemagglutination inhibition as detected by monoclonal antibodies. This is the first bigenic invivo biotinylation system established for use in insect cells with secretable recombinant membrane proteins biotinylated by an ER-retained variant of BirA biotin ligase. The proposed technique is expected to significantly increase flexibility in the design of subtype-specific assays, thereby expanding the power of influenzaA virus serodiagnosis.

Internalization of Foldamer-Based DNA Mimics through a Site-Specific Antibody Conjugate to Target HER2-Positive Cancer Cells.

Inhibition of protein-DNA interactions represents an attractive strategy to modulate essential cellular functions. We reported the synthesis of unique oligoamide-based foldamers that adopt single helical conformations and mimic the negatively charged phosphate moieties of B-DNA. These mimics alter the activity of DNA interacting enzymes used as targets for cancer treatment, such as DNA topoisomerase I, and they are cytotoxic only in the presence of a transfection agent. The aim of our study was to improve internalization and selective delivery of these highly charged molecules to cancer cells. For this purpose, we synthesized an antibody-drug conjugate (ADC) using a DNA mimic as a payload to specifically target cancer cells overexpressing HER2. We report the bioconjugation of a 16-mer DNA mimic with trastuzumab and its functional validation in breast and ovarian cancer cells expressing various levels of HER2. Binding of the ADC to HER2 increased with the expression of the receptor. The ADC was internalized into cells and was more efficient than trastuzumab at inhibiting their growth in vitro. These results provide proof of concept that it is possible to site-specifically graft high molecular weight payloads such as DNA mimics onto monoclonal antibodies to improve their selective internalization and delivery in cancer cells.

SARS-CoV-2 antibodies seroprevalence in dogs from France using ELISA and an automated western blotting assay.

Dogs are occasionally susceptible to SARS-CoV-2, developing few or no clinical signs. Epidemiological surveillance of SARS-CoV-2 in dogs requires testing to distinguish it from other canine coronaviruses. In the last year, significant advances have been made in the diagnosis of SARS-CoV-2, allowing its surveillance in both human and animal populations. Here, using ELISA and automated western blotting (AWB) assays, we performed a longitudinal study on 809 apparently healthy dogs from different regions of France to investigate anti-SARS-CoV-2 antibodies. There were three main groups: (i) 356 dogs sampled once before the pandemic, (ii) 235 dogs sampled once during the pandemic, and (iii) 218 dogs, including 82 dogs sampled twice (before and during the pandemic), 125 dogs sampled twice during the pandemic and 11 dogs sampled three times (once before and twice during the pandemic). Using ELISA, seroprevalence was significantly higher during the pandemic [5.5% (25/453)] than during the pre-pandemic period [1.1% (5/449)]. Among the 218 dogs sampled twice, at least 8 ELISA-seroconversions were observed. ELISA positive pre-pandemic sera were not confirmed in serial tests by AWB, indicating possible ELISA cross-reactivity, probably with other canine coronaviruses. A significant difference was observed between these two serological tests (Q = 88, p = 0.008). A clear correlation was observed between SARS-CoV-2 seroprevalence in dogs and the incidence of SARS-CoV-2 infection in human population from the same area. AWB could be used as a second line assay to confirm the doubtful and discrepant ELISA results in dogs. Our results confirm the previous experimental models regarding the susceptibility of dogs to SARS-CoV-2, suggesting that viral transmission from and between dogs is weak or absent. However, the new variants with multiple mutations could adapt to dogs; this hypothesis cannot be ruled out in the absence of genomic data on SARS-CoV-2 from dogs.

PXR Modulates the Prostate Cancer Cell Response to Afatinib by Regulating the Expression of the Monocarboxylate Transporter SLC16A1.

Resistance to castration is a crucial issue in the treatment of metastatic prostate cancer. Kinase inhibitors (KIs) have been tested as potential alternatives, but none of them are approved yet. KIs are subject of extensive metabolism at both the hepatic and the tumor level. Here, we studied the role of PXR (Pregnane X Receptor), a master regulator of metabolism, in the resistance to KIs in a prostate cancer setting. We confirmed that PXR is expressed in prostate tumors and is more frequently detected in advanced forms of the disease. We showed that stable expression of PXR in 22Rv1 prostate cancer cells conferred a resistance to dasatinib and a higher sensitivity to erlotinib, dabrafenib, and afatinib. Higher sensitivity to afatinib was due to a ~ 2-fold increase in its intracellular accumulation and involved the SLC16A1 transporter as its pharmacological inhibition by BAY-8002 suppressed sensitization of 22Rv1 cells to afatinib and was accompanied with reduced intracellular concentration of the drug. We found that PXR could bind to the SLC16A1 promoter and induced its transcription in the presence of PXR agonists. Together, our results suggest that PXR could be a biomarker of response to kinase inhibitors in castration-resistant prostate cancers.