Gestational folate deficiency alters embryonic gene expression and cell function.

Folic acid is a nutrient essential for embryonic development. Folate deficiency can cause embryonic lethality or neural tube defects and orofacial anomalies. Folate receptor 1 (Folr1) is a folate binding protein that facilitates the cellular uptake of dietary folate. To better understand the biological processes affected by folate deficiency, gene expression profiles of gestational day 9.5 (gd9.5) Folr1-/- embryos were compared to those of gd9.5 Folr1+/+ embryos. The expression of 837 genes/ESTs was found to be differentially altered in Folr1-/- embryos, relative to those observed in wild-type embryos. The 837 differentially expressed genes were subjected to Ingenuity Pathway Analysis. Among the major biological functions affected in Folr1-/- mice were those related to 'digestive system development/function', 'cardiovascular system development/function', 'tissue development', 'cellular development', and 'cell growth and differentiation', while the major canonical pathways affected were those associated with blood coagulation, embryonic stem cell transcription and cardiomyocyte differentiation (via BMP receptors). Cellular proliferation, apoptosis and migration were all significantly affected in the Folr1-/- embryos. Cranial neural crest cells (NCCs) and neural tube explants, grown under folate-deficient conditions, exhibited marked reduction in directed migration that can be attributed, in part, to an altered cytoskeleton caused by perturbations in F-actin formation and/or assembly. The present study revealed that several developmentally relevant biological processes were compromised in Folr1-/- embryos.

Proposal of a pathway for enteric virus groups detection as indicators of faecal contamination to enhance the evaluation of microbiological quality in freshwater in Argentina.

An environmental survey was conducted in order to assess the frequency of detection of picobirnavirus (PBV), human adenovirus (HAdV) and infective enterovirus (iEV) as indicators of faecal contamination in freshwater, and to determine their potential as reporters of the presence of other enteric viruses, such as group A rotavirus (RVA). The study was carried out over a three-year period (2013-2015) in the San Roque Dam, Córdoba, Argentina. The overall frequency detection was 62.9% for PBV, 64.2% for HAdV and 70.4% for iEV. No significant differences were observed in the rates of detection for any of these viruses through the years studied, and a seasonal pattern was not present. Whenever there was RVA detection in the samples analyzed, there was also detection of iEV and/or HAdV and/or PBV. At least one of the viral groups analyzed was demonstrated in the 100% of the samples with faecal coliforms values within the guideline limits. In this setting, especially in those samples which reveal faecal indicator bacteria within the guideline limit, we propose to carry out a pathway, involving PBV, HAdV and iEV detection in order to enhance the evaluation of microbiological quality in freshwater in Argentina. The proposed methodological strategy could report faecal contamination in water, mainly of human origin, and the condition of the matrix to maintain viral viability. In addition, the viral groups selected could report the presence of RV.

Developmental toxicity of e-cigarette aerosols.

Maternal smoking during pregnancy represents a major public health concern increasing the risk for low birth weight, congenital anomalies, preterm birth, fetal mortality, and morbidity. In an effort to diminish adverse developmental effects of exposure to cigarette smoking, pregnant women, and women of reproductive age, are increasingly turning to electronic nicotine delivery systems (ENDS), such as e-cigarettes, as an alternative. Given that health risks associated with ENDS use during pregnancy are largely unknown, there is an acute need to determine risks vs. benefits of e-cigarette use by pregnant women. While the most recent Surgeon General's Report on the "Health Consequences of Smoking" states that "the evidence is sufficient to infer that nicotine adversely affects maternal and fetal health during pregnancy, contributing to multiple adverse outcomes," it remains unclear whether use of ENDS represents a "safer alternative" to tobacco smoking during pregnancy. This is due, in part, to the lack of sufficient and conclusive evidence concerning whether or not maternal e-cigarette use adversely affects embryonic/fetal development. While several recent developmental studies have challenged the safety of nicotine inhalation via ENDS, the true risks of smoking e-cigarettes during the first trimester of pregnancy-the period of organogenesis-are largely unknown. Moreover, evidence is emerging that even nicotine-free e-cigarette aerosols may harm the developing conceptus, suggesting that components of e-cigarette liquid, including flavorings, may be developmentally toxicity. Focused human epidemiological analyses, and carefully designed animal studies are critically needed to address the question of the safety of ENDS use during pregnancy.

Effects of 5-Aza-2'-deoxycytidine (decitabine) on gene expression.

5-Aza-2'-deoxycytidine (AzaD), also known as Decitabine, is a deoxycytidine analog that is typically used to activate methylated and silenced genes by promoter demethylation. However, a survey of the scientific literature indicates that promoter demethylation may not be the only (or, indeed, the major) mechanism by which AzaD affects gene expression. Regulation of gene expression by AzaD can occur in several ways, including some that are independent of DNA demethylation. Results from several studies indicate that the effect of AzaD on gene expression is highly context-dependent and can differ for the same gene under different environmental settings. This may, in part, be due to the nature of the silencing mechanism(s) involved - DNA methylation, repressive histone modifications, or a combination of both. The varied effects of AzaD on such context-dependent regulation of gene expression may underlie some of the diverse responses exhibited by patients undergoing AzaD therapy. In this review, we describe the salient properties of AzaD with particular emphasis on its diverse effects on gene expression, aspects that have barely been discussed in most reviews of this interesting drug.

Erratum to: 2016 WSES guidelines on acute calculous cholecystitis.

[This corrects the article DOI: 10.1186/s13017-016-0082-5.].

Developmental cigarette smoke exposure II: Hepatic proteome profiles in 6 month old adult offspring.

Utilizing a mouse model of 'active' developmental cigarette smoke exposure (CSE) [gestational day (GD) 1 through postnatal day (PD) 21] characterized by offspring low birth weight, the impact of developmental CSE on liver proteome profiles of adult offspring at 6 months of age was determined. Liver tissue was collected from Sham- and CSE-offspring for 2D-SDS-PAGE based proteome analysis with Partial Least Squares-Discriminant Analysis (PLS-DA). A similar study conducted at the cessation of exposure to cigarette smoke documented decreased gluconeogenesis coupled to oxidative stress in weanling offspring. In the current study, exposure throughout development to cigarette smoke resulted in impaired hepatic carbohydrate metabolism, decreased serum glucose levels, and increased gluconeogenic regulatory enzyme abundances during the fed-state coupled to decreased expression of SIRT1 as well as increased PEPCK and PGC1α expression. Together these findings indicate inappropriately timed gluconeogenesis that may reflect impaired insulin signaling in mature offspring exposed to 'active' developmental CSE.

2016 WSES guidelines on acute calculous cholecystitis.

Acute calculus cholecystitis is a very common disease with several area of uncertainty. The World Society of Emergency Surgery developed extensive guidelines in order to cover grey areas. The diagnostic criteria, the antimicrobial therapy, the evaluation of associated common bile duct stones, the identification of "high risk" patients, the surgical timing, the type of surgery, and the alternatives to surgery are discussed. Moreover the algorithm is proposed: as soon as diagnosis is made and after the evaluation of choledocholitiasis risk, laparoscopic cholecystectomy should be offered to all patients exception of those with high risk of morbidity or mortality. These Guidelines must be considered as an adjunctive tool for decision but they are not substitute of the clinical judgement for the individual patient.

Developmental cigarette smoke exposure II: Kidney proteome profile alterations in 6 month old adult offspring.

Cigarette smoke exposure (CSE) during gestation and early development suppresses the growth trajectory in offspring. In prior studies utilizing a mouse model of 'active' developmental CSE (GD1-PD21), low birth weight induced by CSE persisted throughout the neonatal period and was present at the cessation of exposure at weaning with proportionally smaller kidney mass that was accompanied by impairment of carbohydrate metabolism. In the present study, littermates of those characterized in the prior study were maintained until 6 months of age at which time the impact of developmental CSE on the abundance of proteins associated with cellular metabolism in the kidney was examined. Kidney protein abundances were examined by 2D-SDS-PAGE based proteome profiling with statistical analysis by Partial Least Squares-Discriminant Analysis. Key findings of this study include a persistence of impact of developmental CSE past the original exposure period on the nucleic acid and carbohydrate metabolism networks and oxidant scavenging pathways.

Developmental cigarette smoke exposure II: Hippocampus proteome and metabolome profiles in adult offspring.

Exposure to cigarette smoke during development is linked to neurodevelopmental delays and cognitive impairment including impulsivity, attention deficit disorder, and lower IQ. Utilizing a murine experimental model of "active" inhalation exposure to cigarette smoke spanning the entirety of gestation and through human third trimester equivalent hippocampal development [gestation day 1 (GD1) through postnatal day 21 (PD21)], we examined hippocampus proteome and metabolome alterations present at a time during which developmental cigarette smoke exposure (CSE)-induced behavioral and cognitive impairments are evident in adult animals from this model system. At six month of age, carbohydrate metabolism and lipid content in the hippocampus of adult offspring remained impacted by prior exposure to cigarette smoke during the critical period of hippocampal ontogenesis indicating limited glycolysis. These findings indicate developmental CSE-induced systemic glucose availability may limit both organism growth and developmental trajectory, including the capacity for learning and memory.

Cigarette smoke induces proteasomal-mediated degradation of DNA methyltransferases and methyl CpG-/CpG domain-binding proteins in embryonic orofacial cells.

Orofacial clefts, the most prevalent of developmental anomalies, occur with a frequency of 1 in 700 live births. Maternal cigarette smoking during pregnancy represents a risk factor for having a child with a cleft lip and/or cleft palate. Using primary cultures of first branchial arch-derived cells (1-BA cells), which contribute to the formation of the lip and palate, the present study addressed the hypothesis that components of cigarette smoke alter global DNA methylation, and/or expression of DNA methyltransferases (Dnmts) and various methyl CpG-binding proteins. Primary cultures of 1-BA cells, exposed to 80µg/mL cigarette smoke extract (CSE) for 24h, exhibited a >13% decline in global DNA methylation and triggered proteasomal-mediated degradation of Dnmts (DNMT-1 and -3a), methyl CpG binding protein 2 (MeCP2) and methyl-CpG binding domain protein 3 (MBD-3). Pretreatment of 1-BA cells with the proteasomal inhibitor MG-132 completely reversed such degradation. Collectively, these data allow the suggestion of a potential epigenetic mechanism underlying maternal cigarette smoke exposure-induced orofacial clefting.

An in vitro model of the tumor-lymphatic microenvironment with simultaneous transendothelial and luminal flows reveals mechanisms of flow enhanced invasion.

The most common cancers, including breast and skin, disseminate initially through the lymphatic system, yet the mechanisms by which tumor cells home towards, enter and interact with the lymphatic endothelium remain poorly understood. Transmural and luminal flows are important biophysical cues of the lymphatic microenvironment that can affect adhesion molecules, growth factors and chemokine expression as well as matrix remodeling, among others. Although microfluidic models are suitable for in vitro reconstruction of highly complex biological systems, the difficult assembly and operation of these systems often only allows a limited throughput. Here we present and characterize a novel flow chamber which recapitulates the lymphatic capillary microenvironment by coupling a standard Boyden chamber setup with a micro-channel and a controlled fluidic environment. The inclusion of luminal and transmural flow renders the model more biologically relevant, combining standard 3D culture techniques with advanced control of mechanical forces that are naturally present within the lymphatic microenvironment. The system can be monitored in real-time, allowing continuous quantification of different parameters of interest, such as cell intravasation and detachment from the endothelium, under varied biomechanical conditions. Moreover, the easy setup permits a medium-high throughput, thereby enabling downstream quantitative analyses. Using this model, we examined the kinetics of tumor cell (MDA-MB-231) invasion and transmigration dynamics across lymphatic endothelium under varying flow conditions. We found that luminal flow indirectly upregulates tumor cell transmigration rate via its effect on lymphatic endothelial cells. Moreover, we showed that the addition of transmural flow further increases intravasation, suggesting that distinct flow-mediated mechanisms regulate tumor cell invasion.

Grafting after sinus lift with anorganic bovine bone alone compared with 50:50 anorganic bovine bone and autologous bone: results of a pilot randomised trial at one year.

Our aim was to compare the outcome of implants inserted in maxillary sinuses augmented with anorganic bovine bone grafts compared with those augmented with mixed 50:50 bovine and autologous bone grafts. Twenty sinuses with 1-4mm of residual crestal height below the maxillary sinuses were randomised into two groups according to a parallel group design (n=10 in each). Sinuses were grafted using a lateral approach. In one group the grafts were 50:50 anorganic bovine bone and autologous bone and in the other anorganic bovine bone alone. After 7 months, 32 implants had been inserted. Outcome measures were survival of implants, complications, marginal changes in the height of the bone, and soft tissue variables (pocket probing depth and bleeding on probing). Probabilities of less than 0.05 were accepted as significant. No patient failed to complete the trial and no implant had failed at 1 year. There were some minor complications. After 12 months, the mean (SD) marginal bone loss (mm) was 1.06 (0.61) in the 50:50 group and 1.19 (0.53) in the anorganic bovine group. The mean (SD) values for pocket probing depth (mm) and bleeding on probing (score) were 2.49 (0.38) and 1.59 (0.82) in the 50:50 group and 2.31 (0.64) and 1.36 (0.87) in the anorganic bovine group (neither difference was significant). The present data are consistent with the hypothesis that the outcome of implants inserted in sinuses grafted with either material is comparable.

Pharmacokinetics of concomitant cisplatin and paclitaxel administered by hyperthermic intraperitoneal chemotherapy to patients with peritoneal carcinomatosis from epithelial ovarian cancer.

BACKGROUND: Hyperthermic intraperitoneal chemotherapy (HIPEC) is advised as a treatment option for epithelial ovarian cancer (EOC) with peritoneal carcinomatosis. This study was designed to define the pharmacokinetics of cisplatin (CDDP) and paclitaxel (PTX) administered together during HIPEC. METHODS: Thirteen women with EOC underwent cytoreductive surgery (CRS) and HIPEC, with CDDP and PTX. Blood, peritoneal perfusate and tissue samples were harvested to determine drug exposure by high-performance liquid chromatography and matrix-assisted laser desorption ionization imaging mass spectrometry (IMS). RESULTS: The mean maximum concentrations of CDDP and PTX in perfusate were, respectively, 24.8±10.4 µg ml(-1) and 69.8±14.3 µg ml(-1); in plasma were 1.87±0.4 µg ml(-1) and 0.055±0.009 µg ml(-1). The mean concentrations of CDDP and PTX in peritoneum at the end of HIPEC were 23.3±8.0 µg g(-1) and 30.1±18.3 µg(-1)g(-1), respectively. The penetration of PTX into the peritoneal wall, determined by IMS, was about 0.5 mm. Grade 3-4 surgical complications were recorded in four patients, five patients presented grade 3 and two patients presented grade 4 hematological complications. CONCLUSIONS: HIPEC with CDDP and PTX after CRS is feasible with acceptable morbidity and has a favorable pharmacokinetic profile: high drug concentrations are achieved in peritoneal tissue with low systemic exposure. Larger studies are needed to demonstrate its efficacy in patients with microscopic postsurgical residual tumours in the peritoneal cavity.

Developmental cigarette smoke exposure: hippocampus proteome and metabolome profiles in low birth weight pups.

Exposure to cigarette smoke during development is linked to neurodevelopmental delays and cognitive impairment including impulsivity, attention deficit disorder, and lower IQ. However, brain region specific biomolecular alterations induced by developmental cigarette smoke exposure (CSE) remain largely unexplored. In the current molecular phenotyping study, a mouse model of 'active' developmental CSE (serum cotinine > 50 ng/mL) spanning pre-implantation through third trimester-equivalent brain development (gestational day (GD) 1 through postnatal day (PD) 21) was utilized. Hippocampus tissue collected at the time of cessation of exposure was processed for gel-based proteomic and non-targeted metabolomic profiling with partial least squares-discriminant analysis (PLS-DA) for selection of features of interest. Ingenuity pathway analysis was utilized to identify candidate molecular and metabolic pathways impacted within the hippocampus. CSE impacted glycolysis, oxidative phosphorylation, fatty acid metabolism, and neurodevelopment pathways within the developing hippocampus.

Computer-assisted implant surgery and immediate loading in edentulous ridges with dental fresh extraction sockets. Two years results of a prospective case series study.

INTRODUCTION: The two-stage surgical approach for implant placement first documented in 1977 by Brånemark, represents today the most used protocol for placing implants. AIM: Aim of this prospective case series study was to compare the clinical and radiological performance of 12 edentulous jaws treated with of a modified prosthetic and surgical protocol for 3D software planning, guided surgery, immediate loading of implants inserted in edentulous jaws and extraction sockets and restored with Cad-Cam Zirconia and titanium full arch frameworks. PATIENTS AND METHODS: This work was designed as a prospective case series study. Twelve patients have been consecutively rehabilitated with an immediately loaded implant supported fixed full prosthesis. A total of 72 implants, Nobel Replace Tapered Groovy; Nobel Biocare AB, Goteborg, Sweden) 26 of which were inserted in fresh extraction sockets, were inserted. Outcome measures were implants survival, radiographic marginal bone-levels and bone remodeling, soft tissue parameters and complications. RESULTS: All patients reached 24 months follow-up, and no patients dropped out from the study. The cumulative survival rate was 100%; after 24 months mean marginal bone remodelling value was: 1.35 ± 0.25, mean PPD value was 2.75 ± 0.40 mm and mean BOP value was 3.8% ± 1.8%. Only minor prosthetic complications were recorded. CONCLUSIONS: These data seem to validate this surgical and prosthetic protocol with valid results when applied in selected cases.

Release of targeted p53 from the mitochondrion as an early signal during mitochondrial dysfunction.

Increased accumulation of p53 tumor suppressor protein is an early response to low-level stressors. To investigate the fate of mitochondrial-sequestered p53, mouse embryonic fibroblast cells (MEFs) on a p53-deficient genetic background were transfected with p53-EGFP fusion protein led by a sense (m53-EGFP) or antisense (c53-EGFP) mitochondrial import signal. Rotenone exposure (100nM, 1h) triggered the translocation of m53-EGFP from the mitochondrion to the nucleus, thus shifting the transfected cells from a mitochondrial p53 to a nuclear p53 state. Antibodies for p53 serine phosphorylation or lysine acetylation indicated a different post-translational status of recombinant p53 in the nucleus and mitochondrion, respectively. These data suggest that cycling of p53 through the mitochondria may establish a direct pathway for p53 signaling from the mitochondria to the nucleus during mitochondrial dysfunction. PK11195, a pharmacological ligand of mitochondrial TSPO (formerly known as the peripheral-type benzodiazepine receptor), partially suppressed the release of mitochondria-sequestered p53. These findings support the notion that p53 function mediates a direct signaling pathway from the mitochondria to nucleus during mitochondrial dysfunction.

Neurobehavioral phenotype of C57BL/6J mice prenatally and neonatally exposed to cigarette smoke.

Although maternal cigarette smoking during pregnancy is a well-documented risk factor for a variety of adverse pregnancy outcomes, how prenatal cigarette smoke exposure affects postnatal neurobehavioral/cognitive development remains poorly defined. In order to investigate the cause of an altered behavioral phenotype, mice developmentally exposed to a paradigm of 'active' maternal cigarette smoke is needed. Accordingly, cigarette smoke exposed (CSE) and air-exposed C57BL/6J mice were treated for 6h per day in paired inhalation chambers throughout gestation and lactation and were tested for neurobehavioral effects while controlling for litter effects. CSE mice exhibited less than normal anxiety in the elevated zero maze, transient hypoactivity during a 1h locomotor activity test, had longer latencies on the last day of cued Morris water maze testing, impaired hidden platform learning in the Morris water maze during acquisition, reversal, and shift trials, and impaired retention for platform location on probe trials after reversal but not after acquisition or shift. CSE mice also showed a sexually dimorphic response in central zone locomotion to a methamphetamine challenge (males under-responded and females over-responded), and showed reduced anxiety in the light-dark test by spending more time on the light side. No differences on tests of marble burying, acoustic startle response with prepulse inhibition, Cincinnati water maze, matching-to-sample Morris water maze, conditioned fear, forced swim, or MK-801-induced locomotor activation were found. Collectively, the data indicate that developmental cigarette smoke exposure induces subnormal anxiety in a novel environment, impairs spatial learning and reference memory while sparing other behaviors (route-based learning, fear conditioning, and forced swim immobility). The findings add support to mounting evidence that developmental cigarette smoke exposure has long-term adverse effects on brain function.

Developmental cigarette smoke exposure: kidney proteome profile alterations in low birth weight pups.

The Brenner hypothesis states that a congenital reduction in nephron number predisposes to adult-onset hypertension and renal failure. The reduction in nephron number induced by proportionally smaller kidney mass may predispose offspring to glomerular hyperfiltration with maturity onset obesity. Developmental cigarette smoke exposure (CSE) results in intrauterine growth retardation with a predisposition to obesity and cardiovascular disease at maturity. Utilizing a mouse model of 'active' developmental CSE (gestational day [GD] 1-postnatal day [PD] 21; cotinine>50 ng/mL) characterized by persistently smaller offspring with proportionally decreased kidney mass, the present study examined the impact of developmental CSE on the abundance of proteins associated with cellular metabolism in the kidney. Following cessation of CSE on PD21, kidney tissue was collected from CSE and Sham exposed pups for 2D-SDS-PAGE based proteome profiling with statistical analysis by partial least squares-discriminant analysis (PLS-DA) with affected molecular pathways identified by ingenuity pathway analysis. Proteins whose expression in the kidney were affected by developmental CSE belonged to the inflammatory disease, cell to cell signaling/interaction, lipid metabolism, small molecule biochemistry, cell cycle, respiratory disease, nucleic acid and carbohydrate metabolism networks. The present findings indicate that developmental CSE alters the kidney proteome. The companion paper details the liver proteome alterations in the same offspring.

Developmental cigarette smoke exposure: liver proteome profile alterations in low birth weight pups.

Cigarette smoke is composed of over 4000 chemicals many of which are strong oxidizing agents and chemical carcinogens. Chronic cigarette smoke exposure (CSE) induces mild alterations in liver histology indicative of toxicity though the molecular pathways underlying these alterations remain to be explored. Utilizing a mouse model of 'active' developmental CSE (gestational day (GD) 1 through postnatal day (PD) 21; cotinine >50ng/mL) characterized by low birth weight offspring, the impact of developmental CSE on liver protein abundances was determined. On PD21, liver tissue was collected from pups for 2D SDS-PAGE based proteome analysis with statistical analysis by Partial Least Squares-Discriminant Analysis (PLS-DA). Protein spots of interest were identified by ESI-MS/MS with impacted molecular pathways identified by Ingenuity Pathway Analysis. Developmental CSE decreased the abundance of proteins associated with the small molecule biochemistry (includes glucose metabolism), lipid metabolism, amino acid metabolism, and inflammatory response pathways. Decreased gluconeogenic enzyme activity and lysophosphatidylcholine availability following developmental CSE were found and supports the impact of CSE on these pathways. Proteins with increased abundance belonged to the cell death and drug metabolism networks. Liver antioxidant enzyme abundances [glutathione-S-transferase (GST) and peroxiredoxins] were also altered by CSE, but GST enzymatic activity was unchanged. In summary, cigarette smoke exposure spanning pre- and post-natal development resulted in persistent decreased offspring weights, decreased abundances of liver metabolic proteins, decreased gluconeogenic activity, and altered lipid metabolism. The companion paper details the kidney proteome alterations in the same offspring.

Chromatin immunoprecipitation-promoter microarray identification of genes regulated by PRDM16 in murine embryonic palate mesenchymal cells.

The transcription factor PRDM16 regulates differentiation of brown adipocyte tissue in mice. Recently, however, it has been demonstrated that genetic knockout of Prdm16 in mice leads to a complete cleft of the secondary palate in offspring. To identify genes whose promoters bind PRDM16 in mouse embryonic palate/maxillary mesenchymal cells, we have conducted a chromatin immunoprecipitation-promoter microarray analysis (ChIP-Chip). One hundred and twenty-two gene promoters were identified as capable of binding PRDM16. These could be functionally grouped to include those on genes linked to muscle development, chondrogenesis and osteogenesis, in addition to many transcription factors. These results suggest that PRDM16 may play a role in differentiation of mesenchymal cells in the embryonic secondary palate that contribute to the anterior, bony palate and posterior, muscular palate.