Factors affectingwater drainage long-time series in the salinized low-lying coastal area of Ravenna (Italy)

The low-lying coastal area of Ravenna (North-eastern Italy), like the majority of delta and coastal zones around the world, is affected by groundwater salinization due to natural processes (such as low topography, natural land subsidence, seawater encroachment along estuaries, etc.) and anthropogenic activities (i.e., increased anthropogenic subsidence rate, sea level rise, geofluids extraction, and drainage). Among all factors causing aquifer salinization, water drainage plays an important role in lowering the hydraulic head and favouring saltwater seepage in the Ravenna coastal aquifer. A network of drainage canals and water pumping stations first allowed for the reclamation of the low-lying territory and today are fundamental to keep land and infrastructures dry and maintain effective soil depth for agriculture practices. The aim of this work is to identify and assess factors affecting water drainage long-time series (1971-2017) of the most important mechanical drainage basin in this low-lying coastal area. Statistical analyses of drainage, climate, and land use change datasets help constrain the relative weight of each single factor potentially causing an increase of water drainage through time. The results show that, among these factors, subsidence rates and seepage processes are the most significant. The data trends also indicate that the climate, especially in terms of precipitation amount and extreme events, played no important role during the studied time interval. The process of infiltration soil capacity loss due to urbanization and consequent soil sealing probably has a small secondary effect. Moreover, an increase in pumping through time will exacerbate aquifer salinization and compromise freshwater availability in the coastal area

Cryopreservation of Ovarian Tissue in Pediatric Patients

Cancer treatments improve the survival rate of children and adolescents; however chemo- and radiotherapy result in gonadal damage leading to acute ovarian failure and sterility. Ovarian tissue cryopreservation allows long-term storage of primordial follicles and represents the only possibility of preserving the potential fertility in prepubertal girls. The aim of the present study is to describe our experience in ovarian tissue cryopreservation in 45 pediatric patients. The number of follicles per square millimeter of the overall section area and follicle quality were evaluated histologically. A strong negative correlation was found between age and follicular density in patients both prior to and after chemotherapy (P < 0.0001). Damage in follicular quality, that is, increased oocyte vacuolization and detachment of the oocyte from granulosa cells, was found after chemotherapy. Ovarian tissue cryopreservation, preferably performed before initiation of chemotherapy, should be offered to pediatric patients, including prepubertal girls, at risk of sterility

Long-term storage does not impact the quality of cryopreserved human ovarian tissue

Background: Ovarian tissue cryopreservation is an emerging technique, also addressed to very young cancer patients, for whom it is not possible to perform an ovarian stimulation for oocytes freezing, before gonadotoxic treatment. In this cases, ovarian tissue must be cryopreserved for a long period of time and it is very important to know if it maintains fertility function after a long period of storage. Here we aimed to assess the effect of long-term storage on preservation and viability of cryopreserved human ovarian tissue. Methods: Descriptive study of three cases of cancer patients whose cryopreserved ovarian tissue remained stored for 18 years. Long-term stored tissue was examined by histological and immunohistochemical analysis, transmission electron microscopy, TUNEL assay and LIVE/DEAD viability/citotoxicity test. Results: Ovarian tissue stored for 18 years showed a good morphology. Follicles presented negative staining for estrogen and progesterone receptors, positive staining for ki67 in granulosa cells and/or oocytes and for bcl2 in granulosa cells. Regarding stroma, patch/focal positive expression was found for estrogen receptor and ki67, diffusely positive expression for progesterone receptor and bcl2. After long-term storage, ultrastructural examination showed sub-cellular integrity of follicles and interstitial oedema foci. No apoptosis was observable by TUNEL assay. Stromal cell viability remained &gt;97 % during the culture period. Conclusion: The evaluation of different aspects o f the tissue provides evidence that the storage time does not impact on tissue quality and gives hope especially to cancer girls, whose tissues could remain cryopreserved for a very long time

Epigallocatechin-3-gallate inhibits doxorubicin-induced inflammation on human ovarian tissue

Chemotherapy protocol can destroy the reproductive potential of young cancer patients. Doxorubicin (DOX) is a potent anthracycline commonly used in the treatment of numerous malignancies. The purpose of the study was to evaluate the ovarian toxicity of DOX via inflammation and the possible protective effect of the green tea polyphenol epigallocatechin-3-gallate (EGCG). Ovarian tissue of three patients was cultured with 1 \ub5g/ml DOX and/or 10 \ub5g/ml EGCG for 24 and 48 h. Levels of inflammatory factors were determined by quantitative Real-Time PCR, western blot, zimography, and multiplex bead-based immunoassay. Morphological evaluation, damaged follicle count and TUNEL assay were also performed. DOX influenced inflammatory responses by inducing a significant increase in the expression of pro-inflammatory cytokines, such as tumor necrosis factor-\u3b1 (TNF-\u3b1) and cyclooxigenase-2 (COX-2), of inflammatory interleukins (IL), such as interleukin-6 (IL-6) and interleukin-8 (IL-8), and the inflammatory proteins mediators metalloproteinase-2 and metalloproteinase-9 (MMP2 and MMP9). IL-8 secretion in the culture supernatants and MMP9 activity also significantly raised after DOX treatment. Moreover, a histological evaluation of the ovarian tissue showed morphological damage to follicles and stroma after DOX exposure. EGCG significantly reduced DOX-induced inflammatory responses and improved the preservation of follicles. DOX-induced inflammation could be responsible for the ovarian function impairment of chemotherapy. EGCG could have a protective role in reducing DOX-mediated inflammatory responses in human ovarian tissue

Diagnostic and cost utility of whole exome sequencing in peripheral neuropathy.

OBJECTIVE: To explore the diagnostic utility and cost effectiveness of whole exome sequencing (WES) in a cohort of individuals with peripheral neuropathy. METHODS: Singleton WES was performed in individuals recruited though one pediatric and one adult tertiary center between February 2014 and December 2015. Initial analysis was restricted to a virtual panel of 55 genes associated with peripheral neuropathies. Patients with uninformative results underwent expanded analysis of the WES data. Data on the cost of prior investigations and assessments performed for diagnostic purposes in each patient was collected. RESULTS: Fifty patients with a peripheral neuropathy were recruited (median age 18 years; range 2-68 years). The median time from initial presentation to study enrollment was 6 years 9 months (range 2 months-62 years), and the average cost of prior investigations and assessments for diagnostic purposes AU$4013 per patient. Eleven individuals received a diagnosis from the virtual panel. Eight individuals received a diagnosis following expanded analysis of the WES data, increasing the overall diagnostic yield to 38%. Two additional individuals were diagnosed with pathogenic copy number variants through SNP microarray. CONCLUSIONS: This study provides evidence that WES has a high diagnostic utility and is cost effective in patients with a peripheral neuropathy. Expanded analysis of WES data significantly improves the diagnostic yield in patients in whom a diagnosis is not found on the initial targeted analysis. This is primarily due to diagnosis of conditions caused by newly discovered genes and the resolution of complex and atypical phenotypes

Expanding the clinical and genetic spectrum of ALPK3 variants: Phenotypes identified in pediatric cardiomyopathy patients and adults with heterozygous variants

Introduction: Biallelic damaging variants in ALPK3, encoding alpha-protein kinase 3, cause pediatric-onset cardiomyopathy with manifestations that are incompletely defined. Methods and Results: We analyzed clinical manifestations of damaging biallelic ALPK3 variants in 19 pediatric patients, including nine previously published cases. Among these, 11 loss-of-function (LoF) variants, seven compound LoF and deleterious missense variants, and one homozygous deleterious missense variant were identified. Among 18 live-born patients, 8 exhibited neonatal dilated cardiomyopathy (44.4%; 95% CI: 21.5%-69.2%) that subsequently transitioned into ventricular hypertrophy. The majority of patients had extracardiac phenotypes, including contractures, scoliosis, cleft palate, and facial dysmorphisms. We observed no association between variant type or location, disease severity, and/or extracardiac manifestations. Myocardial histopathology showed focal cardiomyocyte hypertrophy, subendocardial fibroelastosis in patients under 4 years of age, and myofibrillar disarray in adults. Rare heterozygous ALPK3 variants were also assessed in adult-onset cardiomyopathy patients. Among 1548 Dutch patients referred for initial genetic analyses, we identified 39 individuals with rare heterozygous ALPK3 variants (2.5%; 95% CI: 1.8%-3.4%), including 26 missense and 10 LoF variants. Among 149 U.S. patients without pathogenic variants in 83 cardiomyopathy-related genes, we identified six missense and nine LoF ALPK3 variants (10.1%; 95% CI: 5.7%-16.1%). LoF ALPK3 variants were increased in comparison to matched controls (Dutch cohort, P = 1.6×10−5; U.S. cohort, P = 2.2×10−13). Conclusion: Biallelic damaging ALPK3 variants cause pediatric cardiomyopathy manifested by DCM transitioning to hypertrophy, often with poor contractile function. Additional extracardiac features occur in most patients, including musculoskeletal abnormalities and cleft palate. Heterozygous LoF ALPK3 variants are enriched in adults with cardiomyopathy and may contribute to their cardiomyopathy. Adults with ALPK3 LoF variants therefore warrant evaluations for cardiomyopathy