Coordinated NADPH oxidase/hydrogen peroxide functions regulate cutaneous sensory axon de- and regeneration.

Tissue wounding induces cutaneous sensory axon regeneration via hydrogen peroxide (H2O2) that is produced by the epithelial NADPH oxidase, Duox1. Sciatic nerve injury instead induces axon regeneration through neuronal uptake of the NADPH oxidase, Nox2, from macrophages. We therefore reasoned that the tissue environment in which axons are damaged stimulates distinct regenerative mechanisms. Here, we show that cutaneous axon regeneration induced by tissue wounding depends on both neuronal and keratinocyte-specific mechanisms involving H2O2 signaling. Genetic depletion of H2O2 in sensory neurons abolishes axon regeneration, whereas keratinocyte-specific H2O2 depletion promotes axonal repulsion, a phenotype mirrored in duox1 mutants. Intriguingly, cyba mutants, deficient in the essential Nox subunit, p22Phox, retain limited axon regenerative capacity but display delayed Wallerian degeneration and axonal fusion, observed so far only in invertebrates. We further show that keratinocyte-specific oxidation of the epidermal growth factor receptor (EGFR) at a conserved cysteine thiol (C797) serves as an attractive cue for regenerating axons, leading to EGFR-dependent localized epidermal matrix remodeling via the matrix-metalloproteinase, MMP-13. Therefore, wound-induced cutaneous axon de- and regeneration depend on the coordinated functions of NADPH oxidases mediating distinct processes following injury.

Nutrition in the prevention and treatment of endometriosis: A review.

Endometriosis is characterized by the presence of endometrial tissues outside the uterine lining, typically on the external surface of the uterus, the ovaries, fallopian tubes, abdominal wall, or intestines. The prevalence of endometriosis in North America, Australia, and Europe is ~1-5% in women of reproductive age. Treatment options for endometriosis are limited. While over-the-counter medications may be used to reduce acute pain, hormonal treatments are common and may interfere with fertility. In more severe cases, laparoscopic excision procedures and even hysterectomies are used to treat the pain associated with endometriosis. Nutritional interventions may be helpful in the prevention and treatment of endometriosis and associated pain. Reducing dietary fat and increasing dietary fiber have been shown to reduce circulating estrogen concentrations, suggesting a potential benefit for individuals with endometriosis, as it is an estrogen-dependent disease. Meat consumption is associated with greater risk of developing endometriosis. Anti-inflammatory properties of plant-based diets may benefit women with endometriosis. Additionally, seaweed holds estrogen-modulating properties that have benefitted postmenopausal women and offers potential to reduce estradiol concentrations in pre-menopausal women. Furthermore, consumption of vitamin D has been shown to reduce endometrial pain via increased antioxidant capacity and supplementation with vitamins C and E significantly reduced endometriosis symptoms, compared with placebo. More randomized clinical trials are needed to elucidate the role of diet in endometriosis.

Dietary advanced glycation end-products and postmenopausal hot flashes: A post-hoc analysis of a 12-week randomized clinical trial.

OBJECTIVE: Postmenopausal hot flashes are associated with an increased risk of cardiovascular disease and diabetes. Because dietary advanced glycation end-products (AGEs) may act as endocrine disruptors, this study examined the potential association of modifications to the intake of dietary AGEs with the frequency and severity of postmenopausal hot flashes. METHODS: Postmenopausal women (n = 84) reporting ≥2 moderate-to-severe hot flashes daily were randomly assigned to either the intervention group or the control group. The former were asked to follow a low-fat, vegan diet, including cooked soybeans (1/2 cup [86 g]/day) for 12 weeks, and the latter continued their usual diets for 12 weeks. Frequency and severity of hot flashes were recorded with a mobile application. Three-day diet records were analyzed using the Nutrition Data System for Research software and dietary AGEs were estimated, using a database. Seventy-one participants completed the whole study and 63 provided complete hot flash and dietary data for the AGEs analysis (n = 31 in the intervention and n = 24 in the control group). Pearson correlations were used to assess the association between changes in hot flashes and dietary AGEs. RESULTS: Dietary AGEs decreased in the intervention group by 73 %, that is by 5509 ku/day on average (95 % -7009 to -4009; p < 0.001), compared with the control group (+458; 95 % CI -835 to +1751; p = 0.47; treatment effect -5968 ku/day [95 % CI -7945 to -3991]; Gxt, p < 0.001). Severe hot flashes decreased by 92 % (p < 0.001) and moderate-to-severe hot flashes decreased by 88 % in the intervention group (p < 0.001). Changes in dietary AGEs correlated with changes in severe (r = +0.39; p = 0.002) and moderate hot flashes (r = +0.34; p = 0.009) and remained significant after adjustment for changes in energy intake (r = +0.45; p < 0.001; and r = +0.37; p = 0.004, respectively) and changes in body mass index (r = +0.37; p = 0.004; and r = +0.27; p = 0.04, respectively). The reduction in dietary AGEs required to achieve a predicted reduction in hot flashes by 1/day was 6933 ku/day for severe and 4366 ku/day for moderate-to-severe hot flashes. CONCLUSIONS: The reduction in dietary AGEs with a low-fat plant-based diet was associated with a significant reduction in the frequency of severe and moderate-to-severe postmenopausal hot flashes, independent of changes in energy intake and weight loss. Plant-based diets could be used not only to alleviate vasomotor symptoms in postmenopausal women, but also to reduce other health risks associated with AGEs. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04587154.

A dietary intervention for postmenopausal hot flashes: A potential role of gut microbiome. An exploratory analysis.

OBJECTIVE: This study examined the role of gut microbiome changes in mediating the effects of a dietary intervention on the frequency and severity of postmenopausal vasomotor symptoms METHODS: Postmenopausal women (n = 84) reporting ≥2 moderate-to-severe hot flashes daily were randomly assigned, in 2 successive cohorts, to an intervention including a low-fat, vegan diet and cooked soybeans (½ cup [86 g] daily) or to stay on their usual diet. Over a 12-week period, frequency and severity of hot flashes were recorded with a mobile application. In a subset of 11 women, gut microbiome was analyzed at baseline and after 12 weeks of the dietary intervention (low-fat vegan diet with soybeans), using deep shotgun metagenomic sequencing. Differences in the microbiome between baseline and 12 weeks were assessed by comparing alpha diversity with Wilcoxon signed rank tests, beta diversity with permanovaFL, and taxon abundance with Wilcoxon signed rank tests. Pearson correlations were used to assess the association between changes in hot flashes and gut bacteria. RESULTS: In the subset for which microbiome testing was done, total hot flashes decreased by 95 % during the dietary intervention (p = 0.007); severe hot flashes disappeared (from 0.6 to 0.0/day; p = 0.06); and moderate-to-severe hot flashes decreased by 96 % (p = 0.01). Daytime and nighttime hot flashes were reduced by 96 % (p = 0.01) and 94 % (p = 0.004), respectively. Alpha and beta diversity did not significantly differ in the intervention group between baseline and 12 weeks. Two families (Enterobacteriaceae and Veillonellaceae), 5 genera (Erysipelatoclostridium, Fusicatenibacter, Holdemanella, Intestinimonas, and Porphyromonas), and 6 species (Clostridium asparagiforme, Clostridium innocuum, Bacteroides thetaiotaomicron, Fusicatenibacter saccharivorans, Intestinimonas butyriciproducens, Prevotella corporis, and Streptococcus sp.) were differentially abundant, but after correction for multiple comparisons, these differences were no longer significant. Changes in the relative abundance of Porphyromonas and Prevotella corporis were associated with the reduction in severe day hot flashes both unadjusted (r = 0.61; p = 0.047; and r = 0.69; p = 0.02), respectively), and after adjustment for changes in body mass index (r = 0.63; p = 0.049; and r = 0.73; p = 0.02), respectively). Changes in relative abundance of Clostridium asparagiforme were associated with the reduction in total severe hot flashes (r = 0.69; p = 0.019) and severe night hot flashes (r = 0.82; p = 0.002) and the latter association remained significant after adjustment for changes in body mass index (r = 0.75; p = 0.01). CONCLUSIONS: This exploratory analysis revealed potential associations between changes in vasomotor symptoms in response to a diet change and changes in the gut microbiome. Larger randomized clinical trials are needed to investigate these findings.

Distinct molecular mechanisms of acquired resistance to temozolomide in glioblastoma cells.

Temozolomide (TMZ) is an alkylating chemotherapeutic agent that prolongs the survival of patients with glioblastoma. Clinical benefit is more prominent in patients with methylation of the O(6) -methyl-guanine DNA methyltransferase (MGMT) promoter. However, all patients eventually suffer from tumor progression because their tumors become resistant to TMZ. Here, we modeled acquired TMZ resistance in glioma cells in vitro to identify underlying molecular mechanisms. To this end, the glioma cell lines LNT-229, LN-308, and LN-18 were exposed repetitively to increasing concentrations of TMZ to induce a stable resistant phenotype (R) defined by clonogenic survival assays. The molecular mechanisms mediating acquired resistance were assessed by immunoblot, PCR, and flow cytometry. Rescue experiments were performed with siRNA-mediated candidate gene silencing. We found in LN-18 cells constitutively expressing MGMT a strong up-regulation of MGMT levels in TMZ-resistant cells. TMZ resistance in the MGMT-negative cell lines LNT-229 and LN-308 was not associated with de novo expression of MGMT. Instead, we found a down-regulation of several DNA mismatch-repair proteins in resistant LNT-229 cells. A TMZ-resistant phenotype was also achieved by silencing selected DNA mismatch repair proteins in parental LNT-229 cells. No obvious mechanism of resistance was identified in the third cell line, LN-308, except for reduced methylation of LINE-1 repetitive elements. In conclusion, we demonstrate that different molecular mechanisms may contribute to the development of acquired TMZ resistance in glioma cells, indicating the need to develop distinct strategies to overcome resistance.

Impact of genetically modified organism requirements on gene therapy development in the EU, Japan, and the US.

Advanced therapies are emerging as an important class of medicinal products; among these, gene therapies are advancing at an exceptional rate. However, one of the major challenges for gene therapies relates to the additional regulatory requirements for genetically modified organisms. In this paper, we provide an overview of the regulatory requirements for genetically modified organisms in the European Union, Japan, and the United States. We share our experience in managing these requirements and their impact on the adeno-associated virus gene therapies that are under development at Pfizer. Specifically, we discuss the relative complexity of the approval process and the impact of risk assessment expectations on the clinical development of genetically modified organisms. We also compare the regulatory processes and timelines of various regions based on our experience with adeno-associated viral vectors. Finally, we propose that genetically modified organisms, for which pathogenicity and replication competency are well controlled, should be regulated solely under medicinal product regulations and be exempt from additional requirements for genetically modified organisms. Even if an exemption is not implemented, it should still be possible to significantly reduce the sponsor and agency burden by simplifying and harmonizing documentation and data requirements as well as timelines for applications for genetically modified organisms.

SOCS3 promoter methylation is mutually exclusive to EGFR amplification in gliomas and promotes glioma cell invasion through STAT3 and FAK activation.

The suppressor of cytokine signaling 3 (SOCS3) gene is one of eight structurally related genes of the SOCS family and has been suggested to function as a tumor suppressor by inhibition of the JAK/STAT signaling pathway. We investigated 60 human gliomas of different histological types for SOCS3 alterations and found frequent SOCS3 promoter hypermethylation and transcriptional downregulation. However, SOCS3 promoter hypermethylation was virtually absent in primary glioblastomas, which are characterized by frequent epidermal growth factor receptor (EGFR) amplification and overexpression. Assessment of the relationship between SOCS3 and EGFR aberrations revealed that SOCS3 promoter hypermethylation was inversely related to both the EGFR gene dosage as well as the EGFR protein expression, thus suggesting SOCS3 inactivation as a mechanism substituting for EGFR activation in a subset of gliomas. In support of this hypothesis, stable shRNA-mediated SOCS3 knock-down in U251 glioblastoma cells resulted in an activation of EGFR-related signaling pathways, i.e. an increase in the activation levels of STAT3, FAK and to a lesser extent MAPK, while the AKT phosphorylation levels remained unaffected. Functionally, SOCS3-depletion caused strongly increased tumor cell invasion with no obvious effect on tumor cell proliferation. In summary, our findings suggest that SOCS3 inactivation by promoter hypermethylation is mutually exclusive to EGFR activation in gliomas and preferentially promotes glioma cell invasion through STAT3 and FAK activation.

Investigating the role of interleukin 10 on Eimeria intestinal pathogenesis in broiler chickens.

Eimeria species are intestinal protozoan parasites that cause lack of production, malabsorption and mortality in floor raised chickens. Administering an oral antibody to interleukin 10 (aIL-10) reduces the symptoms of coccidiosis in broilers, indicating interleukin 10 (IL-10) is key to Eimeria pathology. IL-10 is an anti-inflammatory cytokine and acts as a stand down signal to reduce inflammation and host pathology during disease. Related protozoan parasites exploit IL-10 to reduce pathogen-damaging host inflammatory responses. We hypothesize that IL-10 is increased during Eimeria infection through an unknown host-pathogen interaction, and by feeding aIL-10 to neutralize excess IL-10 the bird is allowed to mount an effective immune response to Eimeria. To determine the effects of aIL-10 during the intestinal immune response, intestinal pathology and the relationship between IL-10, interferon gamma (IFNγ) and Eimeria infection were evaluated in this study. In both experiments, broilers were administered either a 10x dose of Advent® Eimeria vaccine or saline. Duodenum, jejunum and cecum samples were collected, processed, stained and examined under a microscope. Evaluation of intestinal histomorphology during aIL-10 administration showed minimal differences in birds fed aIL-10 during infection compared to animals fed a control antibody during Eimeria infection. To further evaluate aIL-10's positive effect during infection, immunofluorescent histochemistry was performed on chicken intestines days 3-7 post Eimeria infection for IL-10 and IFNγ presence in intestinal mucosa in control and infected birds, in regions with and without visible Eimeria burden. IL-10 and IFNγ had significant changes between days 4.5-7 post-infection in birds fed aIL-10 compared to animals fed a control antibody. Overall we found that the duodenum had increased IL-10 presence and increased IFNγ presence, and the jejunum and cecum had decreased IL-10 presence and decreased IFNγ presence. These differences in spatial regulation of IL-10 and IFNγ may indicate Eimeria species induce slightly different cytokine responses.

Biodegradation of pharmaceuticals and endocrine disruptors with oxygen, nitrate, manganese (IV), iron (III) and sulfate as electron acceptors.

Biodegradation of pharmaceuticals and endocrine disrupting compounds was examined in long term batch experiments for a period of two and a half years to obtain more insight into the effects of redox conditions. A mix including lipid lowering agents (e.g. clofibric acid, gemfibrozil), analgesics (e.g. diclofenac, naproxen), beta blockers (e.g. atenolol, propranolol), X-ray contrast media (e.g. diatrizoic acid, iomeprol) as well as the antiepileptic carbamazepine and endocrine disruptors (e.g. bisphenol A, 17α-ethinylestradiol) was analyzed in batch tests in the presence of oxygen, nitrate, manganese (IV), iron (III), and sulfate. Out of the 23 selected substances, 14 showed a degradation of >50% of their initial concentrations under aerobic conditions. The beta blockers propranolol and atenolol and the analgesics pentoxifylline and naproxen showed a removal of >50% under anaerobic conditions. In particular naproxen proved to be degradable with oxygen and under most anaerobic conditions, i.e. with manganese (IV), iron (III), or sulfate. The natural estrogens estriol, estrone and 17ß-estradiol showed complete biodegradation under aerobic and nitrate-reducing conditions, with a temporary increase of estrone during transformation of estriol and 17ß-estradiol. Transformation of 17ß-estradiol under Fe(III)-reducing conditions resulted in an increase of estriol as well. Concentrations of clofibric acid, carbamazepine, iopamidol and diatrizoic acid, known for their recalcitrance in the environment, remained unchanged.

Excisions of severe cervical dysplasia: Are there mandatory diameters of the cone that need to be considered?

OBJECTIVE: To achieve optimal depth for negative margin cones after loop electrosurgical excision procedures (LEEP) for cervical dysplasia. MATERIAL AND METHODS: Retrospective cohort analysis of LEEP cones of 201 patients with cervical dysplasia during a four-year period. Analysed cones were divided into two different groups: cones with negative margins without dysplasia, and cones with margins positive for dysplasia. In order to determine the cut-off value of the depth of the resected cones, receiver operating characteristic (ROC) analysis was performed. RESULTS: Negative margins were found in 71.0% (n=49) of all cones, whereas positive margins were reported in 29.0% (n=20). Negative margin cones were achieved in 100% with a cone depth of ≥20 mm. A resection depth between 10-19.9 mm led to 73.0% negative margin cones. Calculation of cone volume shows for 2.0 cm3, a sensitivity of 79% and a specificity of 64%. Statistical analysis using an ROC model showed p=0.002. CONCLUSION: Forth greatest safety of patients, cone depths from LEEPs for cervical dysplasia should be ≥20 mm to achieve negative margins.

Facial attractiveness judgements reflect learning of parental age characteristics.

Mate preferences are shaped by infant experience of parental characteristics in a wide variety of species. Similar processes in humans may lead to physical similarity between parents and mates, yet this possibility has received little attention. The age of parents is one salient physical characteristic that offspring may attend to. The current study used computer-graphic faces to examine how preferences for age in faces were influenced by parental age. We found that women born to 'old' parents (over 30) were less impressed by youth, and more attracted to age cues in male faces than women with 'young' parents (under 30). For men, preferences for female faces were influenced by their mother's age and not their father's age, but only for long-term relationships. These data indicate that judgements of facial attractiveness in humans reflect the learning of parental characteristics.

Sources and processes affecting the spatio-temporal distribution of pharmaceuticals and X-ray contrast media in the water resources of the Lower Jordan Valley, Jordan.

The closed basin of the Lower Jordan Valley with the Dead Sea as final sink features high evapotranspiration rates and almost complete reuse of treated wastewater for irrigation farming. This study focuses on the water transfer schemes and the presence, spreading, and potential accumulation of pharmaceutical residues in the local water resources based on findings of a five-year monitoring program. Overall 16 pharmaceuticals and 9 iodinated X-ray contrast media were monitored in groundwater, surface water, and treated wastewater. A total of 95 samples were taken to cover all geographical settings and flow paths from origin (wastewater) to target (groundwater). Nine substances were detected in groundwater, with concentrations ranging between 11 ng/L and 33,000 ng/L. Sometimes, detection rates were higher than in comparable studies: Diatrizoic acid 75%, iopamidol 42%, iopromide 19%, iomeprol 11%, carbamazepine and iohexol 8%, ibuprofen 6%, and fenofibrate and iothalamic acid 3%. Concentrations in groundwater generally increase from north to south depending on the application of treated wastewater for irrigation. Almost all substances occurred most frequently and with highest concentrations in treated wastewater, followed by surface water and groundwater. As exception, diatrizoic acid was found more frequently in groundwater than in treated wastewater, with concentrations being similar. This indicates the persistence of diatrizoic acid with long residence times in local groundwater systems, but may also reflect changing prescription patterns, which would be in accordance with increasing iopamidol findings and surveys at local hospitals. Trend analyses confirm this finding and indicate a high probability of increasing iopamidol concentrations, while other substances did not reveal any trends. However, no proof of evaporative enrichment could be found. The high spatial and temporal variability of the concentrations measured calls for further systematic studies to assess the long-term evolution of organic trace substances in this reuse setting.

DNA hypermethylation and histone modifications downregulate the candidate tumor suppressor gene RRP22 on 22q12 in human gliomas.

RRP22 (Ras-related protein on chromosome 22) has been suggested as a candidate tumor suppressor in human cancers. Investigating a panel of 70 human gliomas, we found a frequent decrease in the RRP22 mRNA expression levels (67%), preferentially in high-grade gliomas [World Health Organization (WHO) grades III and IV] as compared with low-grade gliomas (WHO grade II). Moreover, reduced RRP22 mRNA expression was associated with shorter overall survival in 180 glioblastoma patients included in the National Institutes of Health Repository for Molecular Brain Neoplasia Data (NIH REMBRANDT) database. Decreased RRP22 expression levels were in part explained by 5'-CpG island hypermethylation and increased by the treatment with the demethylating agent 5-aza-2'-deoxycytidine in glioblastoma cell lines. In addition, the in vitro treatment with the histone deacetylase inhibitor trichostatin A alone resulted in RRP22 reexpression as well as a significant increase in the levels of RRP22 promoter DNA bound to pan-acetylated histone H3 and H4. Moreover, in primary human glioblastomas, we observed an increase of H3K9me3-bound and a decrease of pan-Ac-H3-bound RRP22 in comparison with non-neoplastic brain tissue, consistent with a heterochromatinization of the RRP22 promoter. Taken together, our findings demonstrate that both 5'-CpG island hypermethylation and histone modifications contribute to the frequent and prognostically unfavorable transcriptional downregulation of RRP22 in malignant gliomas.