STUDY QUESTION Can high resolution array-CGH analysis on a cohort of women showing a primary ovarian insufficiency (POI) phenotype in young age identify copy number variants (CNVs) with a deleterious effect on ovarian function? SUMMARY ANSWER This approach has proved effective to clarify the role of CNVs in POI pathogenesis and to better unveil both novel candidate genes and pathogenic mechanisms. WHAT IS KNOWN ALREADY POI describes the progression toward the cessation of ovarian function before the age of 40 years. Genetic causes are highly heterogeneous and despite several genes being associated with ovarian failure, most of genetic basis of POI still needs to be elucidated. STUDY DESIGN, SIZE, DURATION The current study included 67 46,XX patients with early onset POI (<19 years) and 134 control females recruited between 2012 and 2016 at the Medical Cytogenetics and Molecular Genetics Lab, IRCCS Istituto Auxologico Italiano. PARTICIPANTS/MATERIALS, SETTING, METHODS High resolution array-CGH analysis was carried out on POI patients’ DNA. Results of patients and female controls were analyzed to search for rare CNVs. All variants were validated and subjected to a gene content analysis and disease gene prioritization based on the present literature to find out new ovary candidate genes. Case-control study with statistical analysis was carried out to validate our approach and evaluate any ovary CNVs/gene enrichment. Characterization of particular CNVs with molecular and functional studies was performed to assess their pathogenic involvement in POI. MAIN RESULTS AND THE ROLE OF CHANCE We identified 37 ovary-related CNVs involving 44 genes with a role in ovary in 32 patients. All except one of the selected CNVs were not observed in the control group. Possible involvement of the CNVs in POI pathogenesis was further corroborated by a case-control analysis that showed a significant enrichment of ovary-related CNVs/genes in patients ( P = 0.0132; P = 0.0126). Disease gene prioritization identified both previously reported POI genes (e.g. BMP15 , DIAPH2 , CPEB1 , BNC1 ) and new candidates supported by transcript and functional studies, such as TP63 with a role in oocyte genomic integrity and VLDLR which is involved in steroidogenesis. LARGE SCALE DATA ClinVar database ( ); accession numbers SCV000787656 to SCV000787743. LIMITATIONS, REASONS FOR CAUTION This is a descriptive analysis for almost all of the CNVs identified. Inheritance studies of CNVs in some non-familial sporadic cases was not performed as the parents’ DNA samples were not available. Addionally, RT-qP...

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Nuclear expression of the calcium-binding protein S100A4 is a biomarker of increased invasiveness in cholangiocarcinoma (CCA), a primary liver cancer with scarce treatment opportunities and dismal prognosis. In this study, we provide evidence that targeting S100A4 nuclear import by low dose paclitaxel (PTX), a microtubule stabilizing agent, inhibits CCA invasiveness and metastatic spread. Administration of low dose PTX to established (EGI-1) and primary (CCA-TV3) CCA cell lines expressing nuclear S100A4 triggered a marked reduction in nuclear expression of S100A4 without modifying its cytoplasmic levels, an effect associated with a significant decrease in cell migration and invasiveness. While low dose PTX did not affect cellular proliferation, apoptosis or cytoskeletal integrity, it significantly reduced SUMOylation of S100A4, a critical posttranslational modification that directs its trafficking to the nucleus. This effect of lose dose PTX was reproduced by ginkolic acid, a specific SUMOylation inhibitor. Downregulation of nuclear S100A4 by low dose PTX was associated with a strong reduction in RhoA and Cdc42 GTPase activity, MT1-MMP expression and MMP-9 secretion. In a SCID mouse xenograft model, low dose metronomic PTX treatment decreased lung dissemination of EGI-1 cells without significantly affecting their local tumor growth. In the tumor mass, nuclear S100A4 expression by CCA cells was significantly reduced, whereas rates of proliferation and apoptosis were unchanged. Overall, our findings highlight nuclear S100A4 as a candidate therapeutic target in CCA and establish a mechanistic rationale for the use of low dose PTX in blocking metastatic progression of cholangiocarcinoma.

Abomasa of 185 chamois shot during 5 consecutive hunting seasons were collected as part of a health monitoring program in an alpine area of Italy and examined for nematodes. The data were obtained during both the preceding period and that following a severe die-off caused by a pneumonia outbreak. Prevalence, mean abundance, mean intensity, and Thul Importance index were consistently high, in particular for Haemonchus contortus, having a low host specificity and high pathogenic potential. Species typical of cervids were also consistently detected. The abomasal nematode community showed an isolationist structure, suggesting its composition was primarily determined by external factors such as interspecific interaction among host species and environmental conditions. The effect of different factors (host sex, sampling site, and time) on nematode counts and aggregation were analyzed and discussed considering the peculiarities of the study site and the chamois population crash. In the light of parallel results for health monitoring, abomasal parasitism could represent a predisposing factor for the observed die-off.It has been shown that macroparasites play a role in regulating wildlife populations (see Tompkins et al., 2002 for review). However, the effect of parasites on free-ranging ruminant populations remains difficult to evaluate under field conditions due to the wide range of factors that can influence the establishment and abundance of parasite species. Host-parasite interactions may not always be clearly evident, thus leading to contradictory interpretations. Furthermore, the use of macroparasites as an index of population health, in particular, counts of gastrointestinal helminths, has often been criticized (Demarais et al., 1983;Waid et al., 1985;Rossi et al., 1997; Pérez et al., 2003). Nevertheless, different studies have demonstrated that parasites can have an impact on health status in natural ruminant populations (e.g., Gulland, 1992;Stien et al., 2002), and further data, both from an ecological perspective and a management point of view, are needed.If the effect of parasites is considered as the result of various interactions between host, parasite, and environment, studies should ideally focus on (1) the ecological correlates determining the composition and distribution of parasite communities (e.g., climatic conditions and distribution of hosts [Calvete et al., 2003]), (2) the epidemiology and host specificity of the species most likely to exert an impact on hosts (e.g., selected generalist nematodes ), and (3) the pathogenic potential of different parasite species and the contributing factors (e.g., food shortage [Gulland, 1992]). Experimental studies are generally unfeasible in wild populations for ethical and logistical reasons. However, evaluation of the parasite community within a host during particular events such as mortality outbreaks or under extreme conditions may provide an opportunity to correlate parasite populations with predisposing factors.The present study is part of a 5-yr ...

The aim of the study was to perform an investigation on the concentration of 19 minerals and cortisol in red deer (Cervus elaphus) hair, a matrix that is easy to collect with non-invasive and painless sampling, able to represent an integrative values of long-term substance concentrations, and able to give useful information, also when performed on dead animals, given its extreme stability over time. In the study thirty-five animals were included, coming from two different sides of a valley in the Stelvio National Park, where official water analysis had pointed out elevated concentrations of As in one of the two orographic sides. Hair cortisol concentrations were measured using a RIA(Radio Immuno Assay), while minerals were detected using ICP-MS (Inductively Coupled Plasma- Mass Spectrometry). Results showed a negative relationship between cortisol and some mineral concentrations (Li, Co, As, Cd, Cr and Tl) and significant differences in some mineral concentrations between park areas (Al, Co, Cu, Cd and Ni). As, Cr and cortisol differences approached statistical significance. This preliminary study represents a step forward in the study of wildlife allostatic load and a valid method for applications in wildlife management programs, in environmental studies and in public health programs.

STUDY QUESTION Can a targeted whole exome sequencing (WES) on a cohort of women showing a primary ovarian insufficiency (POI) phenotype at a young age, combined with a study of copy number variations, identify variants in candidate genes confirming their deleterious effect on ovarian function? SUMMARY ANSWER This integrated approach has proved effective in identifying novel candidate genes unveiling mechanisms involved in POI pathogenesis. WHAT IS KNOWN ALREADY POI, a condition occurring in 1% of women under 40 years of age, affects women’s fertility leading to a premature loss of ovarian reserve. The genetic causes of POI are highly heterogeneous and several determinants contributing to its prominent oligogenic inheritance pattern still need to be elucidated. STUDY DESIGN, SIZE, DURATION WES screening for pathogenic variants of 41 Italian women with non-syndromic primary and early secondary amenorrhoea occurring before age 25 was replicated on another 60 POI patients, including 35 French and 25 American women, to reveal statistically significant shared variants. PARTICIPANTS/MATERIALS, SETTING, METHODS The Italian POI patients’ DNA were processed by targeted WES including 542 RefSeq genes expressed or functioning during distinct reproductive or ovarian processes (e.g. DNA repair, meiosis, oocyte maturation, folliculogenesis and menopause). Extremely rare variants were filtered and selected by means of a Fisher Exact test using several publicly available datasets. A case-control Burden test was applied to highlight the most significant genes using two ad-hoc control female cohorts. To support the obtained data, the identified genes were screened on a novel cohort of 60 Caucasian POI patients and the same case-control analysis was carried out. Comparative analysis of the human identified genes was performed on mouse and Drosophila melanogaster by analysing the orthologous genes in their ovarian phenotype, and two of the selected genes were fruit fly modelled to explore their role in fertility. MAIN RESULTS AND THE ROLE OF CHANCE The filtering steps applied to search for extremely rare pathogenic variants in the Italian cohort revealed 64 validated single-nucleotide variants/Indels in 59 genes in 30 out of 41 screened women. Burden test analysis highlighted 13 ovarian genes as being the most enriched and significant. To validate these findings, filtering steps and Burden analysis on the second cohort of Caucasian patients yielded 11 significantly enriched genes. Among them, AFP, DMRT3, MOV10, FYN and MYC were significant in both patient cohorts and hence were considered strong candidates for POI. Mouse and Drosophila comparative analysis evaluated a conserved role through the evolution of several candidates, and functional studies using a Drosophila model, when applicable, supported the conserved role of the MOV10 armitage and DMRT3 dmrt93B orthologues in female fertility. LARGE SCALE DATA The datasets for the Italian cohort generated during the current study are publicly available at ClinVar database (http://www.ncbi.nlm.nih.gov/clinvar/): accession numbers SCV001364312 to SCV001364375. LIMITATIONS, REASONS FOR CAUTION This is a targeted WES analysis hunting variants in candidate genes previously identified by different genomic approaches. For most of the investigated sporadic cases, we could not track the parental inheritance, due to unavailability of the parents’ DNA samples; in addition, we might have overlooked additional rare variants in novel candidate POI genes extracted from the exome data. On the contrary, we might have considered some inherited variants whose clinical significance is uncertain and might not be causative for the patients’ phenotype. Additionally, as regards the Drosophila model, it will be extremely important in the future to have more mutants or RNAi strains available for each candidate gene in order to validate their role in POI pathogenesis. WIDER IMPLICATIONS OF THE FINDINGS The genomic, statistical, comparative and functional approaches integrated in our study convincingly support the extremely heterogeneous oligogenic nature of POI, and confirm the maintenance across the evolution of some key genes safeguarding fertility and successful reproduction. Two principal classes of genes were identified: (i) genes primarily involved in meiosis, namely in synaptonemal complex formation, asymmetric division and oocyte maturation and (ii) genes safeguarding cell maintenance (piRNA and DNA repair pathways). STUDY FUNDING/COMPETING INTEREST(S) This work was supported by Italian Ministry of Health grants ‘Ricerca Corrente’ (08C621_2016 and 08C924_2019) provided to IRCCS Istituto Auxologico Italiano, and by ‘Piano Sostegno alla Ricerca’ (PSR2020_FINELLI_LINEA_B) provided by the University of Milan; M.P.B. was supported by Telethon-Italy (grant number GG14181). There are no conflicts of interest.

<p>Supplementary Figure 5. Low dose PTX did not affect cytoskeletal integrity of EGI-1 cells.</p>

<p>Supplementary Figure 1. Schedule of treatment with low dose metronomic PTX and assessment by bioluminescence analysis in SCID mice xenografted with EGI-1 cells.</p>