A new LC-MS/MS method for simultaneous and quantitative detection of bisphenol-a and steroids in target tissues: A power tool to characterize the interference of bisphenol-a exposure on steroid levels

Bisphenol A (BPA), an endocrine disruptor, may affect in situ steroidogenesis and alter steroids levels. The present work proposes a liquid chromatography tandem mass spectrometry method to simultaneously quantify BPA, 17β-Estradiol and testosterone in two target tissues: testis and visceral fat mass. Analytes were isolated and lipophilic impurities removed by two serial steps: liquid-liquid and solid phase extraction. All compounds were separated in a single gradient run by Kinetex F5 column and detected via multiple reaction monitoring using a triple quadrupole with a TurboIon electrospray source in both negative and positive modes. The method is selective and very sensitive. In the investigated concentration range, the linearity of the detector response is verified in both tissues. The use of specific SPE cartridges for affinity chromatography purification allows obtaining high percentages of process efficiency (68.0-83.3% for testicular tissue; 63.7-70.7% for visceral fat mass). Good repeatability and reproducibility was observed. The validated method can be efficiently applied for direct biological monitoring in testis and visceral fat mass from mice exposed to BPA. The quantification of compounds in a single assay could be achieved without a loss of sensitivity

KDM4 Involvement in Breast Cancer and Possible Therapeutic Approaches

Breast cancer (BC) is the second leading cause of cancer death in women, although recent scientific and technological achievements have led to significant improvements in progression-free disease and overall survival of patients. Genetic mutations and epigenetic modifications play a critical role in deregulating gene expression, leading to uncontrolled cell proliferation and cancer progression. Aberrant histone modifications are one of the most frequent epigenetic mechanisms occurring in cancer. In particular, methylation and demethylation of specific lysine residues alter gene accessibility via histone lysine methyltransferases (KMTs) and histone lysine demethylases (KDMs). The KDM family includes more than 30 members, grouped into six subfamilies and two classes based on their sequency homology and catalytic mechanisms, respectively. Specifically, the KDM4 gene family comprises six members, KDM4A-F, which are associated with oncogene activation, tumor suppressor silencing, alteration of hormone receptor downstream signaling, and chromosomal instability. Blocking the activity of KDM4 enzymes renders them “druggable” targets with therapeutic effects. Several KDM4 inhibitors have already been identified as anticancer drugs in vitro in BC cells. However, no KDM4 inhibitors have as yet entered clinical trials due to a number of issues, including structural similarities between KDM4 members and conservation of the active domain, which makes the discovery of selective inhibitors challenging. Here, we summarize our current knowledge of the molecular functions of KDM4 members in BC, describe currently available KDM4 inhibitors, and discuss their potential use in BC therapy

The cannabinoid receptor cb1 stabilizes sperm chromatin condensation status during epididymal transit by promoting disulphide bond formation

The cannabinoid receptor CB1 regulates differentiation of spermatids. We recently characterized spermatozoa from caput epididymis of CB1-knock-out mice and identified a considerable number of sperm cells with chromatin abnormality such as elevated histone content and poorly condensed chromatin. In this paper, we extended our findings and studied the role of CB1 in the epididymal phase of chromatin condensation of spermatozoa by analysis of spermatozoa from caput and cauda epididymis of wild-type and CB1-knock-out mouse in both a homozygous or heterozygous condition. Furthermore, we studied the impact of CB1-gene deletion on histone displacement mechanism by taking into account the hyperacetylation of histone H4 and players of displacement such as Chromodomain Y Like protein (CDYL) and Bromodomain testis-specific protein (BRDT). Our results show that CB1, via local and/or endocrine cell-to-cell signaling, modulates chromatin remodeling mechanisms that orchestrate a nuclear condensation extent of mature spermatozoa. We show that CB1-gene deletion affects the epididymal phase of chromatin condensation by interfering with inter-/intra-protamine disulphide bridges formation, and deranges the efficiency of histone removal by reducing the hyper-acetylation of histone H4. This effect is independent by gene expression of Cdyl and Brdt mRNA. Our results reveal a novel and important role for CB1 in sperm chromatin condensation mechanisms

Nuclear size as estrogen-responsive chromatin quality parameter of mouse spermatozoa.

Recently, we have investigated the endocannabinoid involvement in chromatin remodeling events occurring in male spermatids. Indeed, we have demonstrated that genetic inactivation of the cannabinoid receptor type 1 (Cnr1) negatively influences chromatin remodeling mechanisms, by reducing histone displacement and indices of sperm chromatin quality (chromatin condensation and DNA integrity). Conversely, Cnr1 knock-out (Cnr1-/-) male mice, treated with estrogens, replaced histones and rescued chromatin condensation as well as DNA integrity. In the present study, by exploiting Cnr1+/+, Cnr+/- and Cnr1-/- epididymal sperm samples, we show that histone retention directly correlates with low values of sperm chromatin quality indices determining sperm nuclear size elongation. Moreover, we demonstrate that estrogens, by promoting histone displacement and chromatin condensation rescue, are able to efficiently reduce the greater nuclear length observed in Cnr1-/- sperm. As a consequence of our results, we suggest that nucleus length may be used as a morphological parameter useful to screen out spermatozoa with low chromatin quality

Laparoscopic approach to Meckel's diverticulum

Aim.To retrospective review the laparoscopic management of Meckel Diverticulum (MD) in two Italian Pediatric Surgery Centers. METHODS: Between January 2002 and December 2012, 19 trans-umbilical laparoscopic-assisted (TULA) procedures were performed for suspected MD. The children were hospitalized for gastrointestinal bleeding and/or recurrent abdominal pain. Median age at diagnosis was 5.4 years (range 6 mo-15 years). The study included 15 boys and 4 girls. All patients underwent clinical examination, routine laboratory tests, abdominal ultrasound and technetium-99m pertechnetate scan, and patients with bleeding underwent gastrointestinal endoscopy. The abdominal exploration was performed with a 10 mm operative laparoscope. Pneumoperitoneum was established based on the body weight. Systematic overview of the peritoneal cavity allowed the ileum to be grasped with an atraumatic instrument. The complete exploration and surgical treatment of MD were performed extracorporeally, after intestinal exteriorization through the umbilicus. All patients' demographics, main clinical features, diagnostic investigations, operative time, histopathology reports, conversion rate, hospital stay and complications were registered and analyzed. RESULTS: MD was identified in 17 patients, while 1 had an ileal duplication and 1 a jejunal hemangioma. Fifteen patients had painless intestinal bleeding, while 4 had recurrent abdominal pain and exhibited cyst like structures in an ultrasound study. Eleven patients had a positive technetium-99m pertechnetate scan. In the patients with bleeding, gastrointestinal endoscopy did not name the source of hemorrhage. All patients were subjected to a TULA surgical procedure. An intestinal resection/anastomosis was performed in 14 patients, while 4 had a wedge resection of the diverticulum and 1 underwent stapling diverticulectomy. All surgical procedures were performed without conversion to open laparotomy. Mean operative time was 75 min (range 40-115 min). No major surgical complications were recorded. The median hospital stay was 5-7 d (range 4-13 d). All patients are asymptomatic at a median follow up of 4, 5 years (range 10 mo-10 years). CONCLUSION: Trans-umbilical laparoscopic-assisted Meckel's diverticulectomy is safe and effective in the treatment of MD, with excellent results. KEYWORDS: Gastrointestinal bleeding; Ileal duplication; Jejunal hemangioma; Laparoscopy; Meckel’s diverticulum; Minimal invasive surgery; One trocar surger

Fetal-Perinatal Exposure to Bisphenol-A Affects Quality of Spermatozoa in Adulthood Mouse

Bisphenol-A (BPA) is considered an endocrine disruptor with estrogenic activity. It is described as an environment-polluting industrial chemical whose adverse effects on the male reproductive system depend on the period of exposure (i.e., fetal, prepubertal, or adult life). We exposed male mice to BPA during the fetal-perinatal period (from 10 days post coitum to 31 days post partum) and investigated the impact of this early-life exposure on gamete health in adulthood animals at 78 days of age. Both in control and BPA-exposed mice, viability and motility of spermatozoa, as well as sperm motility acquisition and chromatin condensation of spermatozoa, have been evaluated. Results reveal harmful effect of BPA on viability and motility of sperm cells as well as on chromatin condensation status during epididymal maturation of spermatozoa. In particular, BPA exposure interferes with biochemical mechanism useful to stabilize sperm chromatin condensation, as it interferes with oxidation of thiol groups associated to chromatin