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This study explores the effects of dietary supplementation with passion fruit peel pectin (Passiflora edulis) and red yeast cell walls (Sporidiobolus pararoseus) on growth performance, immunity, intestinal morphology, gene expression, and gut microbiota of Nile tilapia (Oreochromis niloticus). Nile tilapia with an initial body weight of approximately 15 ± 0.06 g were fed four isonitrogenous (29.09-29.94%), isolipidic (3.01-4.28%), and isoenergetic (4119-4214 Cal/g) diets containing 0 g kg -1 pectin or red yeast cell walls (T1 - Control), 10 g kg -1 pectin (T2), 10 g kg -1 red yeast (T3), and a combination of 10 g kg -1 pectin and 10 g kg -1 red yeast (T4) for 8 weeks. Growth rates and immune responses were assessed at 4 and 8 weeks, while histology, relative immune and antioxidant gene expression, and gut microbiota analysis were conducted after 8 weeks of feeding. The results showed that the combined supplementation (T4) significantly enhanced growth performance metrics, including final weight, weight gain, specific growth rate, and feed conversion ratio, particularly by week 8, compared to T1, T2, and T3 (P < 0.05). Immunological assessments revealed increased lysozyme and peroxidase activities in both skin mucus and serum, with the T4 group showing the most pronounced improvements. Additionally, antioxidant and immune-related gene expression, including glutathione peroxidase (GPX), glutathione reductase (GSR), and interleukin-1 (IL1), were upregulated in the gut, while intestinal morphology exhibited improved villus height and width. Gut microbiota analysis indicated increased alpha and beta diversity, with a notable rise in beneficial phyla such as Actinobacteriota and Firmicutes in the supplemented groups. These findings suggest that the combined use of pectin and red yeast cell walls as prebiotics in aquaculture can enhance the health and growth of Nile tilapia, offering a promising alternative to traditional practices. Further research is needed to determine optimal dosages for maximizing these benefits., (© 2024. The Author(s).)

Chitosan (CH) shows great potential as an immunostimulatory feed additive in aquaculture. This study evaluates the effects of varying dietary CH levels on the growth, immunity, intestinal morphology, and antioxidant status of Nile tilapia ( Oreochromis niloticus ) reared in a biofloc system. Tilapia fingerlings (mean weight 13.54 ± 0.05 g) were fed diets supplemented with 0 (CH0), 5 (CH5), 10 (CH10), 20 (CH20), and 40 (CH40) mL·kg -1 of CH for 8 weeks. Parameters were assessed after 4 and 8 weeks. Their final weight was not affected by CH supplementation, but CH at 10 mL·kg -1 significantly improved weight gain (WG) and specific growth rate (SGR) compared to the control ( p < 0.05) at 8 weeks. Skin mucus lysozyme and peroxidase activities were lower in the chitosan-treated groups at weeks 4 and 8. Intestinal villi length and width were enhanced by 10 and 20 mL·kg -1 CH compared to the control. However, 40 mL·kg -1 CH caused detrimental impacts on the villi and muscular layer. CH supplementation, especially 5-10 mL·kg -1 , increased liver and intestinal expressions of interleukin 1 ( IL-1 ), interleukin 8 ( IL-8 ), LPS-binding protein ( LBP ), glutathione reductase ( GSR ), glutathione peroxidase ( GPX ), and glutathione S-transferase ( GST-α ) compared to the control group. Overall, dietary CH at 10 mL·kg -1 can effectively promote growth, intestinal morphology, innate immunity, and antioxidant capacity in Nile tilapia fingerlings reared in biofloc systems.

Colorectal cancer (CRC) is the second leading cause of cancer-related death, mostly due to metastatic disease and the fact that many patients already show signs of metastasis at the time of first diagnosis. Current CRC therapies negatively impact patients' quality of life and have little to no effect on combating the tumor once the dissemination has started. Danio rerio (zebrafish) is a popular animal model utilized in cancer research. One of its main advantages is the ease of xenograft transplantation due to the fact that zebrafish larvae lack the adaptative immune system, guaranteeing the impossibility of rejection. In this review, we have presented the many works that choose zebrafish xenograft as a tool for the study of CRC, highlighting the methods used as well as the promising new therapeutic molecules that have been identified due to this animal model., (© 2024. The Author(s).)

Aim: We evaluated the impact of inpatient and outpatient treatment provided by an infant nutrition foundation in Las Heras, Mendoza, Argentina and identified the factors that influenced nutritional recovery., Methods: This 2010-2018 retrospective study was based on 300 children up to 5 years of age with primary malnutrition, who were treated by an inpatient recovery centre, then an outpatient prevention centre. We analysed the children's height, weight, psychomotor development and living conditions when they were admitted, discharged and had received 1 year of outpatient treatment. There were full data on 241 children and just admission and discharge data for 59., Results: The children's mean age on admission and weight were 14.8 ± 12.4 months and 6.9 ± 2.3 kg and they stayed in hospital for a mean of 59.5 ± 49.7 days. We observed a significant increase in the weight-for-age, height-for-age and weight-for-height z-scores when all three time points were compared (p < 0.001). Psychomotor development improved considerably in all patients after treatment. The factors that negatively influenced nutritional recovery were higher age at admission, suboptimal breastfeeding practices, low birth weight, longer hospital stays, younger maternal age and overcrowded housing., Conclusion: Combining inpatient recovery and outpatient preventive treatment was effective for undernourished children in Argentina., (© 2023 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.)

Background: AMBRA1 is an intrinsically disordered protein, working as a scaffold molecule to coordinate, by protein-protein interaction, many cellular processes, including autophagy, mitophagy, apoptosis and cell cycle progression. The zebrafish genome contains two ambra1 paralogous genes (a and b), both involved in development and expressed at high levels in the gonads. Characterization of the zebrafish paralogous genes mutant lines generated by CRISPR/Cas9 approach showed that ambra1b knockout leads to an all-male population., Results: We demonstrated that the silencing of the ambra1b gene determines a reduction of primordial germ cells (PGCs), a condition that, in the zebrafish, leads to the development of all-male progeny. PGC reduction was confirmed by knockdown experiments and rescued by injection of ambra1b and human AMBRA1 mRNAs, but not ambra1a mRNA. Moreover, PGC loss was not rescued by injection with human AMBRA1 mRNA mutated in the CUL4-DDB1 binding region, thus suggesting that interaction with this complex is involved in PGC protection from loss. Results from zebrafish embryos injected with murine Stat3 mRNA and stat3 morpholino suggest that Ambra1b could indirectly regulate this protein through CUL4-DDB1 interaction. According to this, Ambra1 +/- mice showed a reduced Stat3 expression in the ovary together with a low number of antral follicles and an increase of atretic follicles, indicating a function of Ambra1 in the ovary of mammals as well. Moreover, in agreement with the high expression of these genes in the testis and ovary, we found significant impairment of the reproductive process and pathological alterations, including tumors, mainly limited to the gonads., Conclusions: By exploiting ambra1a and ambra1b knockout zebrafish lines, we prove the sub-functionalization between the two paralogous zebrafish genes and uncover a novel function of Ambra1 in the protection from excessive PGC loss, which seems to require binding with the CUL4-DDB1 complex. Both genes seem to play a role in the regulation of reproductive physiology., (© 2023. The Author(s).)

Objectives: Prostate cancer (PCa) is the most common adenocarcinoma in men >50 y of age. It has a long latency period, which provides time for preventive strategies like incorporating healthy eating habits. Yerba mate (YM) intake has been associated with numerous health benefits. Since YM is one of the most popular infusions in Argentina, the of this study was to examine the influence of YM on PCa development., Methods: We carried out an in vivo model of PCa through subcutaneous inoculation of transgenic adenocarcinoma of the mouse prostate-C1 cells in C57BL/6 mice. Subsequently, the animals were divided into two groups: mate (25 mg/mL of YM in drinking water, n = 15), and control (only drinking water, n = 15). We also developed an in vitro model to study the direct effects of YM on three human PCa cell lines: lymph node carcinoma of the prostate (LNCaP), PC-3, and DU-145., Results: Our in vivo model showed that YM intake slightly reduced body weight, increased the latency of tumor appearance (P <0.01), and diminished the tumor volume (P <0.05) compared with the control group. In agreement, the expression of proliferating cell nuclear antigen, and nuclear estrogen receptor α were lower in the tumors of the mate animals (P <0.05). In vitro, YM decreased the viability, proliferation, and adhesion of the three tumor cell lines (P < 0.001) and retarded the migration of LNCaP (P <0.05) and DU-145 (P <0.005), without modifying the migration of PC-3 cells., Conclusions: YM showed anticancer effects in vitro and in vivo and were more effective on the androgen-sensitive cell line (LNCaP)., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Dopamine dyshomeostasis has been acknowledged among the determinants of nigrostriatal neuron degeneration in Parkinson's disease (PD). Several studies in experimental models and postmortem PD patients underlined increasing levels of the dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is highly reactive towards proteins. DOPAL has been shown to covalently modify the presynaptic protein αSynuclein (αSyn), whose misfolding and aggregation represent a major trait of PD pathology, triggering αSyn oligomerization in dopaminergic neurons. Here, we demonstrated that DOPAL elicits αSyn accumulation and hampers αSyn clearance in primary neurons. DOPAL-induced αSyn buildup lessens neuronal resilience, compromises synaptic integrity, and overwhelms protein quality control pathways in neurites. The progressive decline of neuronal homeostasis further leads to dopaminergic neuron loss and motor impairment, as showed in in vivo models. Finally, we developed a specific antibody which detected increased DOPAL-modified αSyn in human striatal tissues from idiopathic PD patients, corroborating the translational relevance of αSyn-DOPAL interplay in PD neurodegeneration., (© 2023. The Author(s).)

An intriguing hypothesis to explain the ubiquity of numerical abilities is that all vertebrates are born with hardwired neuronal networks for processing numbers. To date, only studies on human foetuses have clearly supported this hypothesis. Zebrafish hatch 48-72 h after fertilisation with an embryonic nervous system, providing a unique opportunity for investigating this hypothesis. Here, we demonstrated that zebrafish larvae exposed to vertical bars at birth acquired an attraction for bar stimuli and we developed a numerical discrimination task based on this preference. When tested with a series of discriminations of increasing difficulty (1vs.4, 1vs.3, 1vs.2, and 2vs.4 bars), zebrafish larvae reliably selected the greater numerosity. The preference was significant when stimuli were matched for surface area, luminance, density, and convex hull, thereby suggesting a true capacity to process numerical information. Converging results from two phylogenetically distant species suggests that numerical abilities might be a hallmark feature of vertebrates' brains., (© 2023. The Author(s).)

Colon cancer is one of the leading causes of death worldwide. In recent years, cannabinoids have been extensively studied for their potential anticancer effects and symptom management. Several in vitro studies reported anandamide's (AEA) ability to block cancer cell proliferation and migration, but evidence from in vivo studies is still lacking. Thus, in this study, the effects of AEA exposure in zebrafish embryos transplanted with HCT116 cells were evaluated. Totally, 48 hpf xenografts were exposed to 10 nM AEA, 10 nM AM251, one of the cannabinoid 1 receptor (CB1) antagonist/inverse agonists, and to AEA + AM251, to verify the specific effect of AEA treatment. AEA efficacy was evaluated by confocal microscopy, which demonstrated that these xenografts presented a smaller tumor size, reduced tumor angiogenesis, and lacked micrometastasis formation. To gain deeper evidence into AEA action, microscopic observations were completed by molecular analyses. RNA seq performed on zebrafish transcriptome reported the downregulation of genes involved in cell proliferation, angiogenesis, and the immune system. Conversely, HCT116 cell transcripts resulted not affected by AEA treatment. In vitro HCT116 culture, in fact, confirmed that AEA exposure did not affect cell proliferation and viability, thus suggesting that the reduced tumor size mainly depends on direct effects on the fish rather than on the transplanted cancer cells. AEA reduced cell proliferation and tumor angiogenesis, as suggested by socs3 and pcnp mRNAs and Vegfc protein levels, and exerted anti-inflammatory activity, as indicated by the reduction of il-11a, mhc1uba, and csf3b mRNA. Of note, are the results obtained in groups exposed to AM251, which presence nullifies AEA's beneficial effects. In conclusion, this study promotes the efficacy of AEA in personalized cancer therapy, as suggested by its ability to drive tumor growth and metastasis, and strongly supports the use of zebrafish xenograft as an emerging model platform for cancer studies., (© 2022. The Author(s).)

Tumor cells can interact with neighboring adipose cells and adipocyte dedifferentiation appears to be an important aspect of tumorigenesis. We evaluated the size of adipocytes in human adipose explants from normal (hRAN) and kidney cancer (hRAT); changes in the expression of WAT and BAT/beige markers in hRAN and hRAT; the expression of epithelial-mesenchymal transition (EMT) cell markers in human kidney tumor (786-O, ACHN and Caki-1); and non-tumor (HK-2) epithelial cell lines incubated with the conditioned media (CMs) of hRAN and hRAT. We observed that hRAT adipocytes showed a significantly minor size compared to hRAN adipocytes. Also, we observed that both Prdm16 and Tbx1 mRNA and the expression of UCP1, TBX1, PPARγ, PCG1α, c/EBPα LAP and c/EBPα LIP was significantly higher in hRAT than hRAN. Finally, we found an increase in vimentin and N-cadherin expression in HK-2 cells incubated for 24 h with hRAT-CMs compared to hRAN- and control-CMs. Furthermore, desmin and N-cadherin expression also increased significantly in 786-O when these cells were incubated with hRAT-CMs compared to the value observed with hRAN- and control-CMs. We observed a significant decrease in E-cadherin expression in the ACHN cell line incubated with hRAT-CMs versus hRAN- and control-CMs. However, we did not observe changes in E-cadherin expression in HK-2, 786-O or Caki-1. The results obtained, together with the results previously published by our group, allow us to conclude that perirenal white adipose tissue browning contributes to tumor development in kidney cancer. In addition, hRAT-CMs increases the expression of mesenchymal markers in renal epithelial cells, which could indicate a regulation of EMT due to this adipose tissue., (© 2022. The Author(s).)

Background: Dysregulation of the autophagic flux is linked to a wide array of human diseases, and recent findings highlighted the central role of autophagy in reproduction, as well as an association between impairment of autophagy and behavioural disorders. Here we deepened on the possible multilevel link between impairment of the autophagic processes and reproduction at both the physiological and the behavioural level in a zebrafish mutant model., Methods: Using a KO epg5 zebrafish line we analysed male breeding success, fertility rate, offspring survival, ejaculate quality, sperm and testes morphology, and courtship behaviour. To this aim physiological, histological, ultrastructural and behavioural analyses on epg5 +/+ and mutant epg5 -/- males coupled to WT females were applied., Results: We observed an impairment of male reproductive performance in mutant epg5 -/- males that showed a lower breeding success with a reduced mean number of eggs spawned by their WT female partners. The spermatogenesis and the ability to produce fertilising ejaculates were not drastically impaired in our mutant males, whereas we observed a reduction of their courtship behaviour that might contribute to explain their lower overall reproductive success., Conclusion: Collectively our findings corroborate the hypothesis of a multilevel link between the autophagic process and reproduction. Moreover, by giving a first glimpse on behavioural disorders associated to epg5 KO in model zebrafish, our results open the way to more extensive behavioural analyses, also beyond the reproductive events, that might serve as new tools for the molecular screening of autophagy-related multisystemic and neurodegenerative diseases., Competing Interests: Conflicts of interest Authors declare that they have no competing interests., (Copyright © 2021 Chang Gung University. Published by Elsevier B.V. All rights reserved.)

Glucocorticoids mainly exert their biological functions through their cognate receptor, encoded by the nr3c1 gene. Here, we analysed the glucocorticoids mechanism of action taking advantage of the availability of different zebrafish mutant lines for their receptor. The differences in gene expression patterns between the zebrafish gr knock-out and the gr s357 mutant line, in which a point mutation prevents binding of the receptor to the hormone-responsive elements, reveal an intricate network of GC-dependent transcription. Particularly, we show that Stat3 transcriptional activity mainly relies on glucocorticoid receptor GR tethering activity: several Stat3 target genes are induced upon glucocorticoid GC exposure both in wild type and in gr s357/s357 larvae, but not in gr knock-out zebrafish. To understand the interplay between GC, their receptor, and the mineralocorticoid receptor, which is evolutionarily and structurally related to the GR, we generated an mr knock-out line and observed that several GC-target genes also need a functional mineralocorticoid receptor MR to be correctly transcribed. All in all, zebrafish mutants and transgenic models allow in vivo analysis of GR transcriptional activities and interactions with other transcription factors such as MR and Stat3 in an in-depth and rapid way.

Maternal milk consumption can cause changes in the mammary epithelium of the offspring that result in the expression of molecules involved in the induction of differentiation, reducing the risk of developing mammary cancer later in life. We previously showed that animals that maintained a higher intake of maternal milk had a lower incidence of mammary cancer. In the present study, we evaluated one of the possible mechanisms by which the consumption of maternal milk could modify the susceptibility to mammary carcinogenesis. We used Sprague Dawley rats reared in litters of 3 (L3), 8 (L8), or 12 (L12) pups per mother in order to generate a differential consumption of milk. Whole mounts of mammary glands were performed to analyze the changes in morphology. Using real-time polymerase chain reaction (PCR), we analyzed the expression of mammary Pinc, Tbx3, Stat6, and Gata3 genes. We use the real-time methylation-specific polymerase chain reaction method to assess the methylation status of Stat6 and Gata3 CpG sites. Our findings show an increase in the size of the epithelial tree and a smaller number of ducts called terminal end buds in L3 vs. L12. We observed an increased expression of mRNA of Stat6, Gata3, Tbx3, and a lower expression of Pinc in L3 with respect to L12. Stat6 and Gata3 are more methylated in the CpG islands of the promoter analyzed in L12 vs. L3. In conclusion, the increased consumption of maternal milk during the postnatal stage generates epigenetic and morphological changes associated with the differentiation of the mammary gland.

Glucocorticoids (GCs) are steroid hormones that contribute to the regulation of many physiological processes, such as inflammation, metabolism and stress response, mainly through binding to their cognate receptor, GR, which works as a ligand-activated transcription factor. Due to their pleiotropy and the common medical use of these steroids to treat patients affected by different pathologies, the investigation of their mechanisms of action is extremely important in biology and clinical research. The evolutionary conservation of GC physiological function, biosynthesis pathways, as well as the sequence and structure of the GC nuclear receptors has stimulated, in the last 20 years, the use of zebrafish (a teleost of Cyprinidae family) as a reliable model organism to investigate this topic. In this review, we wanted to collect many of the most significant findings obtained by the the scientific community using zebrafish to study GCs and their receptors. The paper begins by describing the experiments with transient knockdown of zebrafish gr to gain insights, mainly during development, and continues with the discoveries provided by the generation of transgenic reporter lines. Finally, we discuss how the generation of mutant lines for either gr or the enzymes involved in GC synthesis has significantly advanced our knowledge on GC biology.

The pleiotropic effects of glucocorticoids in metabolic, developmental, immune and stress response processes have been extensively investigated; conversely, their roles in reproduction are still less documented. It is well known that stress or long-lasting therapies can cause a strong increase in these hormones, negatively affecting reproduction. Moreover, the need of glucocorticoid (GC) homeostatic levels is highlighted by the reduced fertility reported in the zebrafish glucocorticoid receptor mutant ( nr3c1 ia30/ia30 ) line (hereafter named gr -/- ). Starting from such evidence, in this study, we have investigated the role of glucocorticoid receptor (Gr) in the reproduction of female zebrafish. Key signals orchestrating the reproductive process at the brain, liver, and ovarian levels were analyzed using a multidisciplinary approach. An impairment of the kiss-GnRH system was observed at the central level in ( gr -/- ) mutants as compared to wild-type ( wt ) females while, in the liver, vitellogenin (vtg) mRNA transcription was not affected. Changes were instead observed in the ovary, particularly in maturing and fully grown follicles (classes III and IV), as documented by the mRNA levels of signals involved in oocyte maturation and ovulation. Follicles isolated from gr -/- females displayed a decreased level of signals involved in the acquisition of competence and maturation, causing a reduction in ovulation with respect to wt females. Fourier transform infrared imaging (FTIRI) analysis of gr -/- follicle cytoplasm showed major changes in macromolecule abundance and distribution with a clear alteration of oocyte composition. Finally, differences in the molecular structure of the zona radiata layer of gr -/- follicles are likely to contribute to the reduced fertilization rate observed in mutants.

Objective: Hemoxigenase 1 (HO-1) is an enzyme that has anti-apoptotic and proliferative effects on tumor cells. However, there is little epidemiological and clinical evidence on the role of HO-1 in urologic tumors., Objective: To determine if there is correlation between the expression of HO-1 and the histological characteristics, evolution, Disease Free Survival (DFS) and cancer mortality in Clear Cell Renal Cell Carcinoma (cRCC)., Materials and Methods: A retrospective study including 34 patients (9 women and 25 men) with cRCC from the "Servicio de Urología del Policlínico Neuquén" (Argentina) throughout 2003-2008. The expression of HO-1 by Immunohistochemistry (IHC) was determined. The statistical analysis was performed using the Student'sT test and Pearson correlation coefficient (p≤0.05). RESULTS: HO-1 was expressed in the epithelial cells of the tubules from normal kidney tissue and in the cytoplasmof cRCC tumor cells. There were no differences in the HO-1 expression related to the gender, age, tumorsize, stage of disease and 5 years DFS. High FuhrmancRCC had a greater expression of HO-1 compared with low Fuhrman cRCC (p≤0.05). The score of immunostaining for HO-1 was greater in those tumors located in the mesorrenal area, which coincidentally presented a more advanced stage of the disease. CONCLUSIONS: Over expression of HO-1 in tumors located in the interpolar zone and with high Furhman grade suggest that HO-1 could be a good adjunctive marker for the aggressiveness of the cRCC.

Recognition memory is the capacity to recognize previously encountered objects, events or places. This ability is crucial for many fitness-related activities, and it appears very early in the development of several species. In the laboratory, recognition memory is most often investigated using the novel object recognition test (NORt), which exploits the tendency of most vertebrates to explore novel objects over familiar ones. Despite that the use of larval zebrafish is rapidly increasing in research on brain, cognition and neuropathologies, it is unknown whether larvae possess recognition memory and whether the NORt can be used to assess it. Here, we tested a NOR procedure in zebrafish larvae of 7-, 14- and 21-days post-fertilization (dpf) to investigate when recognition memory first appears during ontogeny. Overall, we found that larvae explored a novel stimulus longer than a familiar one. This response was fully significant only for 14-dpf larvae. A control experiment evidenced that larvae become neophobic at 21-dpf, which may explain the poor performance at this age. The preference for the novel stimulus was also affected by the type of stimulus, being significant with tri-dimensional objects varying in shape and bi-dimensional geometrical figures but not with objects differing in colour. Further analyses suggest that lack of effect for objects with different colours was due to spontaneous preference for one colour. This study highlights the presence of recognition memory in zebrafish larvae but also revealed non-cognitive factors that may hinder the application of NORt paradigms in the early developmental stages of zebrafish., Competing Interests: The authors declare there are no competing interests., (©2020 Bruzzone et al.)

In zebrafish, two paralogous genes, activating molecule in beclin-1 (BECN1)-regulated autophagy ambra1a and ambra1b , both required for the autophagic process and during development, encode the protein AMBRA1, a positive regulator of early steps of autophagosome formation. As transcripts for both genes are expressed during embryogenesis in the heart region, in this work, we investigated the effects of ambra1a and ambra1b knockdown on heart development by means of morpholino oligonucleotides (MOs). Silencing of the two proteins by MOs directed against the ATG translation initiation codon affects cardiac morphogenesis, resulting in a small, string-like heart with pericardial edema, whereas treatment with splice-blocking MOs does not lead to overt cardiac phenotypes, thus revealing the relevance of maternally supplied ambra1 transcripts for heart development. Co-injection of both ATG-MOs determines a more severe cardiac phenotype, with prominent pericardial edema. Whole-mount in situ hybridization (WMISH) for myosin light chain 7 ( myl7 ), as well as ambra1 ATG-MO microinjection in zebrafish transgenic line expressing green fluorescent protein in the heart, revealed defects with the heart jogging process followed by imperfect cardiac looping. Moreover, WMISH of homeodomain transcription factor 2 isoform c ( pitx2c ) transcripts showed both bilateral and reversed pitx2c expression in morphants. The morphants' cardiac phenotypes were effectively rescued by co-injection of MOs with human AMBRA1 ( hAMBRA1 ) messenger RNA (mRNA), pointing at the conservation of Ambra1 functions during evolution. Co-injections of ambra1 ATG-MOs with a hAMBRA1 mRNA mutated in the protein phosphatase 2a (PP2A) binding sites ( hAMBRA1 PXP ) were not able to rescue the cardiac phenotypes, at the difference from wild-type hAMBRA1 mRNA, and treatment of zebrafish embryos with the specific PP2A inhibitor cantharidin resulted in similar developmental cardiac defects. These results suggest a critical role for AMBRA1 in vertebrate heart development, likely involving the binding site for the PP2A phosphatase.

The EPG5 protein is a RAB7A effector involved in fusion specificity between autophagosomes and late endosomes or lysosomes during macroautophagy/autophagy. Mutations in the human EPG5 gene cause a rare and severe multisystem disorder called Vici syndrome. In this work, we show that zebrafish epg5 -/- mutants from both heterozygous and incrossed homozygous matings are viable and can develop to the age of sexual maturity without conspicuous defects in external appearance. In agreement with the dysfunctional autophagy of Vici syndrome, western blot revealed higher levels of the Lc3-II autophagy marker in epg5 - /- mutants with respect to wild type controls. Moreover, starvation elicited higher accumulation of Lc3-II in epg5 - /- than in wild type larvae, together with a significant reduction of skeletal muscle birefringence. Accordingly, muscle ultrastructural analysis revealed accumulation of degradation-defective autolysosomes in starved epg5 - /- mutants. By aging, epg5 - /- mutants showed impaired motility and muscle thinning, together with accumulation of non-degradative autophagic vacuoles. Furthermore, epg5 - /- adults displayed morphological alterations in gonads and heart. These findings point at the zebrafish epg5 mutant as a valuable model for EPG5-related disorders, thus providing a new tool for dissecting the contribution of EPG5 on the onset and progression of Vici syndrome as well as for the screening of autophagy-stimulating drugs. Abbreviations: ATG: autophagy related; cDNA: complementary DNA; DIG: digoxigenin; dpf: days post-fertilization; EGFP: enhanced green fluorescent protein; EPG: ectopic P granules; GFP: green fluorescent protein; hpf: hours post-fertilization; IL1B: interleukin 1 beta; Lc3-II: lipidated Lc3; mpf: months post-fertilization; mRNA: messenger RNA; NMD: nonsense-mediated mRNA decay; PCR: polymerase chain reaction; qPCR: real time-polymerase chain reaction; RAB7A/RAB7: RAB7a, member RAS oncogene family; RACE: rapid amplification of cDNA ends; RFP: red fluorescent protein; RT-PCR: reverse transcriptase-polymerase chain reaction; SEM: standard error of the mean; sgRNA: guide RNA; UTR: untranslated region; WMISH: whole mount in situ hybridization; WT: wild type.

Environmental factors during perinatal life can lead to changes in the mammary gland, making it susceptible to cancer in adulthood. Breastfeeding has a special importance since it takes place at a critical period of growth and development of the newborn. We aimed to analyze if an appropriate lactation protects the offspring against mammary carcinogenesis during adult life and explore the mechanisms involved in the protective effect. One-day-old Sprague-Dawley female rats were randomly distributed in litters of three (L3), eight (L8) or 12 (L12) pups per dam, to induce a differential consumption of breast milk. At 55 days of age, the animals were treated with a single dose of dimethylbenzanthracene to study tumor latency, incidence and progression. Histological, immunohistochemical and Western blot studies were performed. We observed lower incidence and higher latency in L3 compared to the other groups. The mitotic index and expression of proliferating cell nuclear antigen (PCNA) was significantly augmented in tumors of L12 rats compared to L3 and L8, while the apoptotic index was augmented in tumors of L3 v. L12. Cleaved caspase 8 was significantly higher in tumors from L3 compared to L12. Tumors developed in L3 have a greater number of apoptotic bodies and a greater expression of caspase 8. These results demonstrate that the animals that maintained a higher intake of maternal milk (L3) presented lower incidence and greater tumor latency. Lower consumption of breast milk (L12) would increase tumor mitosis and the expression of PCNA, explaining the higher tumor incidence observed in this group.

To characterize the influence of hypothyroidism on the endocrine activity of mesenteric and omental adipose tissue (MOAT) and the peripheral regulation of energy balance (EB) in rats, we analyzed food intake (FI); basal metabolic rate (BMR); locomotor activity; body weight (BW); serum hormone concentrations and the expression of their receptors in MOAT. We evaluated the morphology and differentiation of adipocytes. Hypothyroidism decreased FI, BMR and BW. The percentage of visceral white adipose tissue (WAT) depots and the morphology of adipocytes were similar to euthyroid rats. Serum leptin and adiponectin expression in MOAT were altered by hypothyroidism. The expression of Perilipin 1, HSL, UCP1 and PRDM16 was significantly lower in MOAT of hypothyroid animals. Hypothyroidism in rats leads to a compensated EB by inducing a white adipocyte dysfunction and a decrease in BW, BMR, FI and adipokine secretions without changing the percentage of WAT depots and the morphology of the MOAT., (Copyright © 2019. Published by Elsevier B.V.)

Epidemiological studies describe estrogens as protectors in the development of colon cancer in postmenopausal women treated with hormone replacement therapy. However, the role of progesterone in colon cancer has been minimally studied and the results are controversial. For the above, the objective of this work was to determine the hormonal regulation exerted by natural ovarian steroids on proliferation and apoptosis in an experimental model of colon cancer in ovariectomized rats treated with 17-beta estradiol and progesterone. Sprague-Dawley rats were exposed to the carcinogen 1,2-dimethylhydrazine to induce colon tumors. Thirty days later, the rats were ovariectomized and treated with estradiol (60 μg/kg), progesterone (10 mg/kg), estradiol plus progesterone (60 μg/kg and 10 mg/kg) or vehicle. We observed no significant differences in colon cancer incidence and tumor multiplicity between the groups. Nevertheless, we observed a decrease in PCNA expression and a greater number of apoptotic index, higher expression of caspase 3, cleaved PARP and cleaved caspase 8 in tumors, confirming the activation of the extrinsic pathway of apoptosis by the combined treatment. In addition, we observed a higher expression of estrogen receptor beta in these tumors. We conclude that the action of both hormones, estradiol and progesterone, is necessary to reduce proliferation and increase apoptosis in colon tumors, probably through estrogen receptor beta activation.

We evaluated the effects of conditioned media (CMs) of human adipose tissue from renal cell carcinoma located near the tumor (hRATnT) or farther away from the tumor (hRATfT), on proliferation, adhesion and migration of tumor (786-O and ACHN) and non-tumor (HK-2) human renal epithelial cell lines. Human adipose tissues were obtained from patients with renal cell carcinoma (RCC) and CMs from hRATnT and hRATfT incubation. Proliferation, adhesion and migration were quantified in 786-O, ACHN and HK-2 cell lines incubated with hRATnT-, hRATfT- or control-CMs. We evaluated versican, adiponectin and leptin expression in CMs from hRATnT and hRATfT. We evaluated AdipoR1/2, ObR, pERK, pAkt y pPI3K expression on cell lines incubated with CMs. No differences in proliferation of cell lines was found after 24 h of treatment with CMs. All cell lines showed a significant decrease in cell adhesion and increase in cell migration after incubation with hRATnT-CMs vs. hRATfT- or control-CMs. hRATnT-CMs showed increased levels of versican and leptin, compared to hRATfT-CMs. AdipoR2 in 786-O and ACHN cells decreased significantly after incubation with hRATfT- and hRATnT-CMs vs. control-CMs. We observed a decrease in the expression of pAkt in HK-2, 786-O and ACHN incubated with hRATnT-CMs. This result could partially explain the observed changes in migration and cell adhesion. We conclude that hRATnT released factors, such as leptin and versican, could enhance the invasive potential of renal epithelial cell lines and could modulate the progression of the disease., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest.

Experimental hypothyroidism retards mammary carcinogenesis promoting apoptosis of tumor cells. β-catenin plays a critical role in cell adhesion and intracellular signaling pathways conditioning the prognosis of breast cancer. However, the mechanistic connections associated with the expression of β-catenin in thyroid status and breast cancer are not known. Therefore, we studied the relationship between the expression and localization of β-catenin and apoptosis in mammary tumors induced by 7,12-dimethylbenz(a)anthracene (DMBA) in hypothyroid (Hypot) and euthyroid (EUT) rats. Female Sprague Dawley rats were treated with a dose of DMBA (15 mg/rat) at 55 days of age and were then divided into two groups: HypoT (0.01% 6-N-propyl-2-thiouracil in drinking water, n = 54) and EUT (untreated control, n = 43). Latency, incidence and progression of tumors were determined. At sacrifice, tumors were obtained for immunohistological studies and Western Blot. The latency was longer (p < 0.05), the incidence was lower (p < 0.0001) and tumor growth was slower (p < 0.01) in HypoT rats compared to EUT. The expression of Bax, cleaved caspase-9 and caspase-3 was significantly higher in tumors of HypoT than in EUT (p < 0.05) indicating the activation of the intrinsic pathway. In this group, β-catenin was expressed in the plasma membrane and with less intensity, while its expression was nuclear and with greater intensity in the EUT (p < 0.05). Moreover, the expression of survivin was reduced in tumors of HypoT rats (p < 0.05). In conclusion, decreased expression of β-catenin and its normal location in membrane of mammary tumors are associated with augmented apoptosis via activation of the intrinsic pathway in HypoT rats.

Background: Adipose microenvironment is involved in signaling pathways that influence breast cancer. We aim to characterize factors that are modified: 1) in tumor and non tumor human breast epithelial cell lines when incubated with conditioned media (CMs) from human breast cancer adipose tissue explants (hATT) or normal breast adipose tissue explants (hATN); 2) in hATN-CMs vs hATT-CMs; 3) in the tumor associated adipocytes vs. non tumor associated adipocytes., Methods: We used hATN or hATT- CMs on tumor and non-tumor breast cancer cell lines. We evaluated changes in versican, CD44, ADAMTS1 and Adipo R1 expression on cell lines or in the different CMs. In addition we evaluated changes in the morphology and expression of these factors in slices of the different adipose tissues. The statistical significance between different experimental conditions was evaluated by one-way ANOVA. Tukey's post-hoc tests were performed within each individual treatment., Results: hATT-CMs increase versican, CD44, ADAMTS1 and Adipo R1 expression in breast cancer epithelial cells. Furthermore, hATT-CMs present higher levels of versican expression compared to hATN-CMs. In addition, we observed a loss of effect in cellular migration when we pre-incubated hATT-CMs with chondroitinase ABC, which cleaves GAGs chains bound to the versican core protein, thus losing the ability to bind to CD44. Adipocytes associated with the invasive front are reduced in size compared to adipocytes that are farther away. Also, hATT adipocytes express significantly higher amounts of versican, CD44 and Adipo R1, and significantly lower amounts of adiponectin and perilipin, unlike hATN adipocytes., Conclusions: We conclude that hATT secrete a different set of proteins compared to hATN. Furthermore, versican, a proteoglycan that is overexpressed in hATT-CMs compared to hATN-CMs, might be involved in the tumorogenic behavior observed in both cell lines employed. In addition, we may conclude that adipocytes from the tumor microenvironment show a less differentiated state than adipocytes from normal microenvironment. This would indicate a loss of normal functions in mature adipocytes (such as energy storage), in support of others that might favor tumor growth.

Prolactin (PRL) is a key player in the development of mammary cancer. We studied the effects of parity or hyperprolactinemia on mammary carcinogenesis in OFA hr/hr treated with 7,12-dimethylbenzanthracene. They were divided into three groups: nulliparous (Null), primiparous (PL, after pregnancy and lactation), and hyperprolactinemic rats (I, implanted in the arcuate nucleus with 17β-estradiol). The tumor incidence was similar in the three groups. However, a higher percentage of regressing tumors was evident in the PL group. Serum PRL, mammary development, and mammary β-casein content were higher in I rats compared to Null. The expression of hormone receptors was similar in the different groups. However, mammary tissue from PL rats bearing tumors had increased expression of PRL and estrogen alpha receptors compared to rats free of tumors. Our results suggest that serum PRL levels do not have relevance on the incidence of tumors, probably because the low levels of PRL in OFA rats are not further decreased by PL like in other strains. However, supraphysiological levels of PRL affect carcinogenesis. PL induces regression of the tumors due to the differentiation produced on the mammary cells. Alterations in the expression of hormonal receptors may be involved in progression and regression of tumors.

Epidemiological and in vitro data have not provided conclusive evidence concerning the involvement of thyroid hormones (THs) on mammary carcinogenesis. We used an in vivo model to assess the relationship between THs, adipose tissue and breast cancer development. Female Sprague‑Dawley rats were treated with a dose of 7,12-dimethylbenz(a)anthracene (15 mg/rat) at 55 days of age and were then divided into four experimental groups: hypothyroid rats (HypoT, 0.01% 6-N-propyl-2-thiouracil in drinking water), untreated control (EUT); hyperthyroid rats (HyperT, 0.25 mg/kg/day T4 s.c.) and vehicle-treated control rats. The latency of tumor appearance and the incidence and progression of tumors were determined. At sacrifice, blood samples were collected for hormone determinations and samples of tumor and mammary glands were obtained for immunohistological studies. HypoT rats had retarded growth and an increase in mammary fat. The latency was longer (p<0.0001), the incidence rate was lower (p<0.05) and tumor growth was slower in HypoT rats compared to EUT and HyperT rats. Mitotic index and PCNA immunostaining were similar in all groups. HypoT rats showed increased apoptosis (p<0.05) as evaluated by the apoptotic index and TUNEL staining. No differences in serum prolactin and progesterone were observed. However, circulating estradiol (E2) was significantly lower in HypoT and HyperT rats. Serum leptin levels were reduced in HypoT rats even though the abdominal fat mass was similar in all groups. To note, the leptin level was higher in HypoT rats that developed mammary tumors than the level in non-tumoral HypoT rats. In conclusion, hypothyroidism altered animal growth, breast morphology, body composition, leptin secretion and serum E2 enhancing apoptosis and, consequently, retarding mammary carcinogenesis in rats.

Lactogenesis is a very complex process highly dependent on hormonal regulation. In the present study the time-course of the inhibitory actions of progesterone on prolactin secretion, mammary gland morphology and lactogenesis from mid- to late gestation in rodents was investigated. Groups of pregnant rats were luteectomised or administered with mifepristone on Day 10, 13, 15 or 17 of gestation and decapitated 28 or 48h later. Whole-blood samples and the inguinal mammary glands were taken for determinations of hormone levels and for measurement of mammary content of casein and lactose and for tissue morphology analyses, respectively. Luteectomy or mifepristone evoked prolactin increases only after Day 17 of gestation. Mammary content of casein was increased by both treatments regardless of timing or duration. Mifepristone was less effective than luteectomy in inducing lactose production and the effect was only observed after Day 15 of gestation. Analysis of mammary gland morphology confirmed the observed effect of progesterone on lactogenesis. Both treatments triggered remarkable secretory activity in the mammary gland, even without a parallel epithelial proliferation, demonstrating that the mammary epithelium is able to synthesise milk compounds long before its full lobulo-alveolar development is achieved, provided that progesterone action is abolished. Thus, the present study demonstrates that progesterone is a potent hormonal switch for the prolactin and prolactin-like effects on mammary gland development and its milk-synthesising capacity during pregnancy, and that its inhibitory action is already evident by mid-pregnancy in rodents.

Recent studies indicate that adipose tissue and adipocytokines might affect the development of prostate cancer (PCa). Leptin would have a stimulating effect on prostate cancer cells by inducing promotion and progression, whereas adiponectin would have a protective effect. The aim of this study was to determine the relation between body composition, leptin, and adiponectin levels with the prevalence and aggressiveness of PCa in men of Mendoza, Argentina. Seventy volunteers between 50 and 80 years (35 healthy men as control group and 35 with PCa) were selected. The PCa group was subclassified according to the Gleason Score (GS). Digital rectal examination, transrectal ultrasound, and prostatic biopsy were performed; PSA, testosterone, leptin, and adiponectin levels were determined; and a nutritional interview including anthropometric measurements and a food frequency questionnaire was carried out. Statistical analysis was performed by Student t test, ANOVA I, and Bonferroni (p < 0.05). Body mass index and percentage of body fat mass were not statistically different between PCa and control groups. However, body fat mass was higher in subjects with more aggressive tumors (p = 0.032). No differences were observed regarding leptin levels between the groups. Nevertheless, leptin levels were higher in subjects with high GS (p < 0.001). Adiponectin levels showed no statistical differences regarding the presence and aggressiveness of the tumor (p = 0.131). Finally, consumption and nutrient intake did not differ in the studied groups. In conclusion, body composition and leptin are related to the PCa aggressiveness but not with its prevalence.

Mifepristone (MIF) administration to cycling rats at proestrus induces hypersecretion of prolactin (PRL) at the following estrus. We aimed to assess whether this effect is due to the antiprogesterone or antiglucocorticoid action of MIF and to help underscore the nature of the circulating hormone(s) regulating PRL secretion at estrus. Female cycling rats in proestrus were treated with vehicle; the progesterone (Pg) and glucocorticoid receptor antagonists, MIF (5 mg/kg) or ORG-33628 (5 mg/kg); the glucocorticoid agonist dexamethasone (DEX; 27 mg/kg)±MIF; or the inhibitor of steroid synthesis aminoglutethimide (AG; 150 mg/kg)±MIF. The animals' blood was sampled the same day at 1800 h and at 1800 h of the following day to assess for circulating PRL and Pg levels. To distinguish antiglucocorticoid from antiprogesterone effects of MIF, we administered a highly specific neutralizing antibody against Pg. None of the antagonists modified serum PRL values at proestrus but increased PRL levels at estrus. DEX decreased the secretion of PRL at proestrus, yet the effect was entirely blocked by MIF. Furthermore, DEX decreased PRL at estrus in a MIF-reversible manner, suggesting that adrenal corticoids during proestrous may regulate PRL secretion at estrus. AG increased PRL secretion at estrus, whereas its association with MIF produced an even higher response. PRL concentration at estrus was not modified by the antiprogesterone antibody, suggesting that the effect of MIF is a consequence of its antiglucocorticoid effect and not due to its antiprogesterone properties. In conclusion, PRL secretion in the afternoon of the estrus is most likely regulated by glucocorticoids through an inhibitory action.

Unlabelled: Several studies have demonstrated an inverse relation between serum selenium levels (Se) and advanced prostate cancer (PCa)., Objective: To determine and compare selenium plasma levels in patients with different prostatic pathologies., Material and Methods: It is a transversal, descriptive and comparative study. A sample of 64 men between 50 and 80 years old were selected for the study between 2007 and 2009. All volunteers underwent a digital rectal examination, prostate specific antigen level, ultrasound and transrectal prostate biopsy (12-14 chips). Prostate cancer was subclassified according to Gleason Score. Selenium was determined indirectly by serum Glutathione peroxidase (Kit Ransel, Randox SRL, Crumlin, UK). Statistical analysis was performed using ANOVA I (p<0.05)., Results: Glutathione Peroxidase level was 33.75+/-2.36 mg/ml in control patients. A decrease of 31.6% was observed in patients with BPH (23.08+/-1.57 mg/ml) and of (63.6%) in subjects with prostate cancer (12.28+/-1.03 mg/ml) (p<0,0001). There was no correlation with the Gleason Score., Conclusion: Serum Seleniun is lower in patients with prostatic pathologies being even more important in cancer patients regardless the Gleason Score.

Inadequate dietary patterns and sedentary lifestyles are believed to be important factors in predisposing people to obesity. This study analyzed the potential interaction between habitual physical activity and the carbohydrate (CHO)-fat distribution in 2 hypocaloric diets and the impact of such interplay on body composition changes. Forty healthy obese women, 20-50 years old, were randomly assigned to a high- or low-CHO energy-restricted diet, which was low or high in fat, respectively, during 10 weeks. Baseline and final measurements were performed to assess dietary habits, resting metabolic rate, and body composition changes. Physical activity was measured with a triaxial accelerometer and with a questionnaire. There were no significant differences in anthropometric and metabolic variables between both dietary groups at baseline. However, there was a positive correlation between total free-living physical activity and arm muscle preservation after 10 weeks (r = 0.371; p = 0.024). Interestingly, an interaction between macronutrient (CHO-fat distribution) intake and physical activity was found, since less-active subjects with a high-CHO-low-fat diet showed a greater fat loss than those more active with a lower-CHO-high-fat diet, whereas more-active subjects with a high-CHO-low-fat diet showed a smaller fat loss than those receiving a low-CHO-high-fat diet. Physical activity and the macronutrient content of energy-restricted diets, when designed to promote body fat mass reduction, should be considered together to better predict the outcome.

Introduction: Prostate cancer (CaP) is one of the most important causes of morbidity and mortality in the world. There is evidence that obesity and inadequate eating habits may promote CaP development., Objective: To analyze and compare the body mass index (BMI) and the food intake, especially fats and antioxidants, among subjects with CaP and those free of disease as a control group., Material and Methods: A sample of 40 men between 50 and 80 years old were selected for the study: 20 with CaP and 20 healthy men as control group. All volunteers underwent a digital rectal examination, prostate specific antigen level, ultrasound and transrectal prostate biopsy, and a nutritional interview where a dietary history and different anthropometric measurements were made. Statistical analysis was performed using the Student T test for independent samples (p < 0.05)., Results: BMI in the subjects with CaP was higher than in controls (29.8 kg/m2 vs. 27.96 kg/m2, p = 0.13) but not statistically significant. However, there was a direct correlation between BMI and tumor aggressiveness (r = 0.79, P < 0.001). Total, saturated, monounsaturated and polyunsaturated fat intake was significantly higher in subjects with CaP; while omega3 fatty acids, vitamin C and lycopene intake was significantly lower than in controls (p < 0.05)., Conclusions: A healthy weight and a diet low in total fat, saturated, monounsaturated and polyunsaturated fat and rich in n3 fatty acids, vitamin C and lycopene is associated with a lower risk of CaP.

Background: Many studies have investigated the association between obesity, adipose tissue-derived factors (leptin and adiponectin) and prostate cancer (CaP) but the results are still inconsistent., Methods: The aim of this study was to carry out a comprehensive review of the existing evidence about the role of leptin and adiponectin in prostate carcinogenesis and to provide an overview of it., Results: Recent evidence suggests that leptin may play a rol in prostate cancer progression, while adiponectin may act as an "antiprostatic cancer" adipokine., Conclusions: Obesity may promote the progression of established prostate cancer and and adipokines may provide a molecular mechanism whereby obesity exerts its effects on prostate tumour biology.

The aims of this study were to describe a female obese population according to their food intake and physical activity, and to compare two methods to estimate physical activity (PA). The study included a nutritional interview where a detailed dietary history was done, an initial clinical day in which measurements of body composition and basal metabolic rate (BMR) were carried out and an estimation of PA by means of a triaxial accelerometer and a PA questionnaire. The group of volunteers showed a mean BMI of 37.15 kg/m2, a waist/hip ratio of 0.82 and a mean body fat mass of 43.34%. The average of BMR was 1720 Kcal/d and the CRNP of 0.79. The total caloric intake was 3344 Kcal/d with a energy-distribution of 43.1% Carbohydrates, 16.4% Proteins, 40.3% Lipids. Weight and BMI showed a positive correlation (p < 0.05) with the sedentary index (SI). Also, weight presented a positive correlation with the heart rate (p < 0.05). The estimates of PA derived from the questionnaire showed a positive correlation with the triaxial accelerometer (p < 0.01); and this one, revealed a negative correlation with the SI (p < 0.01). Anthropometric, metabolic and food intake variables are comparable to the results found in obese women from similar socioeconomic background. The estimates of PA according to the questionnaire were significantly correlated to the results of the triaxial accelerometer, thus confirming the validity of the questionnaire to assess PA in an obese population.