Cellular bases of hypofractionated radiotherapy protocols for lung cancer.

The extreme demand on health systems due to the COVID-19 pandemic has led to reconsider hypofractionation. Although the best clinical efficacy of these schemes is being demonstrated, the biological bases have not been established. Thus, after validating basic clinical parameters, through complementary in vitro models, we characterized the cellular and molecular mechanisms of hypofractionation protocols. Cell cultures of human lung cancer cell line A549 were irradiated with 0, 2, 4, 8, 12, 16 and 20 Gy. The clastogenic, cytotoxic, proliferative and clonogenic capacities and bystander effect were evaluated. In addition, we assessed survival and toxicity in a retrospective study of 49 patients with lung cancer. Our findings showed that the greater efficacy of ablative regimens should not only be attributed to events of direct cell death induced by genotoxic damage, but also to a lower cell repopulation and the indirect action of clastogenic factors secreted. These treatments were optimal in terms of 1- and 2-year overall survival (74 and 65%, respectively), and progression-free survival at 1 and 2 years (71 and 61%, respectively). The greater efficacy of high doses per fraction could be attributed to a multifactorial mechanism that goes beyond the 4Rs of conventional radiotherapy.

Ageing decreases the healing of wounds in the skin of alcohol-preferring rats.

OBJECTIVE: Alcohol consumption combined with ageing alters the healing process of the skin. We evaluated whether ageing decreases the healing of incisional wounds in the skin of Wistar rats of Universidade de Chile of variety B (UChB). METHOD: A total of 20 adult rats and 20 older UChB rats, divided into two groups which underwent surgical aggression in the anterior region of the abdomen, were used: G1, adult rats (100 days old, control) with water and 10% ethanol; G2, aged rats (540 days old, experimental) with water and 10% ethanol; evaluated at 4, 7, 14 and 21 days after surgery. RESULTS: Ageing did not alter the rupture force and collagen elasticity and resistance. There were increases in telomerase with the implementation of cellular senescence, in interleukin 1-alpha (IL-1α) at 14 days of healing, in epidermal growth factor (EGF) at 14 and 21 days of healing with delayed growth and development of keratinocytes, also an increase of IL-ß at 4 days, and decrease in tumour necrosis factor (TNFα) at 7 days, associated with chronic scarring. There was an increase in vascular endothelial growth factor (VEGF) at 4 and 7 days, responsible for the early vessels re-establishment. There was a decrease in transforming growth factor 2-beta (TGFß2) and ß3 at 4 and 7 days of healing respectively, and estradiol at 4 days. CONCLUSION: Ageing decreases the skin healing in incisional wounds in alcohol-preferring rats.

P-MAPA and Interleukin-12 Reduce Cell Migration/Invasion and Attenuate the Toll-Like Receptor-Mediated Inflammatory Response in Ovarian Cancer SKOV-3 Cells: A Preliminary Study.

Immunotherapies have emerged as promising complementary treatments for ovarian cancer (OC), but its effective and direct role on OC cells is unclear. This study examined the combinatory effects of the protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride, known as P-MAPA, and the human recombinant interleukin-12 (hrIL-12) on cell migration/invasion, apoptosis, toll-like receptor (TLR)-mediated inflammation, and cytokine/chemokine profile in human OC cell line SKOV-3. P-MAPA and IL-12 showed cancer cell toxicity under low doses after 48 h. Although apoptosis/necrosis and the cell cycle were unchanged by the treatments, P-MAPA enhanced the sensitivity to paclitaxel (PTX) and P-MAPA associated with IL-12 significantly reduced the migratory potential and invasion capacity of SKOV-3 cells. P-MAPA therapy reduced TLR2 immunostaining and the myeloid differentiation factor 88 (MyD88), but not the TLR4 levels. Moreover, the combination of P-MAPA with IL-12 attenuated the levels of MyD88, interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-kB p65). The IL-12 levels were increased and P-MAPA stimulated the secretion of cytokines IL-3, IL-9, IL-10, and chemokines MDC/CCL22 and, regulated on activation, normal T cells expressed and secreted (RANTES)/CCL5. Conversely, combination therapy reduced the levels of IL-3, IL-9, IL-10, MDC/CCL22, and RANTES/CCL5. Collectively, P-MAPA and IL-12 reduce cell dynamics and effectively target the TLR-related downstream molecules, eliciting a protective effect against chemoresistance. P-MAPA also stimulates the secretion of anti-inflammatory molecules, possibly having an immune response in the OC microenvironment.

Interactions of ethanol and caffeine on apoptosis in the rat cerebellum (voluntary ethanol consumers).

Ethanol alters motricity, learning, cognition, and cellular metabolism in the cerebellum. The combination of ethanol with caffeine by consuming "energy drinks" is becoming increasingly popular among young people. We analyzed the use of ethanol and caffeine on apoptosis in the cerebellum of UChB rats. The adult rats were divided into three groups (n = 14/group): UChB group: rats fed with 1:10 (v/v) ethanol ad libitum (free choice for water or ethanol) drinking from >1.9 mL of ethanol/kg body weight/day, Control group and UChB/caffeine group (free choice for water or ethanol + caffeine 300 mg/L). The treatments occurred from Day 100 till Day 150, totalizing 50 days of ethanol/caffeine ingestion. Cerebellar sections were subjected to immunohistochemistry and gene expression for Real Time-PCR (RT-PCR) for Caspase-3, XIAP, and insulin-like growth factor 1-receptor (IGF-1R). The results showed a significant increase in the gene expression of Caspase-3 and XIAP in UChB group. On the other hand, the animals of the UChB/caffeine group showed similar results to the Controls. Regarding IGFR-1, there was greater expression in the UChB groups with strong labeling in Purkinje cells. Ethanol produces neuronal and glial neurodegeneration on the cerebellum of UChB rats. The simultaneous ingestion of ethanol and caffeine reversed the ethanol damages acting caffeine with a neuroprotective effect.

Melatonin Reduces Angiogenesis in Serous Papillary Ovarian Carcinoma of Ethanol-Preferring Rats.

Angiogenesis is a hallmark of ovarian cancer (OC); the ingrowth of blood vessels promotes rapid cell growth and the associated metastasis. Melatonin is a well-characterized indoleamine that possesses important anti-angiogenic properties in a set of aggressive solid tumors. Herein, we evaluated the role of melatonin therapy on the angiogenic signaling pathway in OC of an ethanol-preferring rat model that mimics the same pathophysiological conditions occurring in women. OC was chemically induced with a single injection of 7,12-dimethylbenz(a)anthracene (DMBA) under the ovarian bursa. After the rats developed serous papillary OC, half of the animals received intraperitoneal injections of melatonin (200 µg/100 g body weight/day) for 60 days. Melatonin-treated animals showed a significant reduction in OC size and microvessel density. Serum levels of melatonin were higher following therapy, and the expression of its receptor MT1 was significantly increased in OC-bearing rats, regardless of ethanol intake. TGFß1, a transforming growth factor-beta1, was reduced only after melatonin treatment. Importantly, vascular endothelial growth factor (VEGF) was severely reduced after melatonin therapy in animals given or not given ethanol. Conversely, the levels of VEGF receptor 1 (VEGFR1) was diminished after ethanol consumption, regardless of melatonin therapy, and VEGFR2 was only reduced following melatonin. Hypoxia-inducible factor (HIF)-1α was augmented with ethanol consumption, and, notably, melatonin significantly reduced their levels. Collectively, our results suggest that melatonin attenuates angiogenesis in OC in an animal model of ethanol consumption; this provides a possible complementary therapeutic opportunity for concurrent OC chemotherapy.

Quantitative Proteomic Profiling Reveals That Diverse Metabolic Pathways Are Influenced by Melatonin in an in Vivo Model of Ovarian Carcinoma.

To obtain more information into the molecular mechanisms underlying ovarian cancer (OC), we proposed a comparative proteomic analysis in animals receiving long-term melatonin as therapy or only vehicle using multidimensional protein identification combined with mass spectrometry. To induce tumor, a single dose of 100 µg 7,12-dimethylbenz(a)anthracene (DMBA) dissolved in 10 µL of sesame oil was injected under the left ovarian bursa of 20 Fischer 344 rats. The right ovaries were injected with sesame oil only. After tumors were developed, half of the animals received intraperitoneal administration of melatonin (200 µg/100g body weight/day) for 60 days. Melatonin therapy promoted down-regulation in numerous proteins involved in OC signaling pathways. The most significant portion of these proteins are involved in several metabolic processes, mainly those associated with mitochondrial systems, generation of metabolites and energy, hypoxia-inducible factor-1 signaling, antigen processing and presentation, endoplasmic reticulum stress-associated pathways, and cancer-related proteoglycans. A small number of proteins that were overexpressed by melatonin therapy included ATP synthase subunit ß, fatty acid-binding protein, and 10-kDa heat shock protein. Taken together, our findings suggest that melatonin therapy efficiently modulated important signaling pathways involved in OC, and these proteins might be further targets that should be explored in new therapeutic opportunities for OC.

Melatonin attenuates the TLR4-mediated inflammatory response through MyD88- and TRIF-dependent signaling pathways in an in vivo model of ovarian cancer.

BACKGROUND: Toll-like receptors (TLRs) are effector molecules expressed on the surface of ovarian cancer (OC) cells, but the functions of the TLR2/TLR4 signaling pathways in these cells remain unclear. Melatonin (mel) acts as an anti-inflammatory factor and has been reported to modulate TLRs in some aggressive tumor cell types. Therefore, we investigated OC and the effect of long-term mel therapy on the signaling pathways mediated by TLR2 and TLR4 via myeloid differentiation factor 88 (MyD88) and toll-like receptor-associated activator of interferon (TRIF) in an ethanol-preferring rat model. METHODS: To induce OC, the left ovary of animals either consuming 10% (v/v) ethanol or not was injected directly under the bursa with a single dose of 100 µg of 7,12-dimethylbenz(a)anthracene (DMBA) dissolved in 10 µL of sesame oil. The right ovaries were used as sham-surgery controls. After developing OC, half of the animals received i.p. injections of mel (200 µg/100 g b.w./day) for 60 days. RESULTS: Although mel therapy was unable to reduce TLR2 levels, it was able to suppress the OC-associated increase in the levels of the following proteins: TLR4, MyD88, nuclear factor kappa B (NFkB p65), inhibitor of NFkB alpha (IkBα), IkB kinase alpha (IKK-α), TNF receptor-associated factor 6 (TRAF6), TRIF, interferon regulatory factor 3 (IRF3), interferon ß (IFN-ß), tumor necrosis factor alpha (TNF-α), and interleukin (IL)-6. In addition, mel significantly attenuated the expression of IkBα, NFkB p65, TRIF and IRF-3, which are involved in TLR4-mediated signaling in OC during ethanol intake. CONCLUSION: Collectively, our results suggest that mel attenuates the TLR4-induced MyD88- and TRIF-dependent signaling pathways in ethanol-preferring rats with OC.

Maternal separation on the ethanol-preferring adult rat liver structure.

UNLABELLED: Background and rationale for the study. We designed to test whether there is interaction of maternal separation (MS) on the ethanol-preferring rats liver structure. The UCh rat pups were separated daily from their mothers during the stress hyporesponsive period (SHRP), between four and 14 days-old, always at the same time for four hours in a cage containing eight subdivisions, one for each pup. Subsequently, rats that presented the highest (UChB) and the lowest (UChA) ethanol (EtOH) consumption were selected to the study. Both UChB and UChA rats received 10% (v/v) EtOH and distilled water ad libitum until the end of the experiment (120 days-old). The liver was collected to histological routine for morphometric and stereological analyses, and immunohistochemistry. RESULTS: There was an interaction of MS and EtOH on the liver: increased liver mass, peritubular vessels, stellate cell numbers, steatosis and cell death, decreased necrosis, sinusoidal capillary diameters and cell proliferation. While there was a decrease in FSH, testosterone and 5α-di-hidrotestosterone, and increasing corticosterone and cholesterol. CONCLUSIONS: There is interaction of MS and EtOH on the liver structure, dependent on the amount of EtOH intake. Furthermore, the interaction of stress and drugs can increase or decrease their effects on the liver or indirectly via hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes.

Apoptosis of Purkinje and granular cells of the cerebellum following chronic ethanol intake.

Ethanol alters motricity, learning, cognition, and cellular metabolism in the cerebellum. We evaluated the effect of ethanol on apoptosis in Golgi, Purkinje, and granule cells of the cerebellum in adult rats. There were two groups of 20 rats: a control group that did not consume ethanol and an experimental group of UChA rats that consumed ethanol at 10% (<2 g ethanol/kg body weight/day). At 120 days old, rats were anesthetized and decapitated, and their cerebella were collected and fixed. Cerebellar sections were subjected to immunohistochemistry for terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL), caspase-3, X-linked inhibitor of apoptosis protein (XIAP), and insulin-like growth factor 1-receptor (IGF-1R); real-time PCR (RT-PCR) to determine caspase-3, XIAP, and IGF-1R gene expression; and transmission electron microscopy (TEM). We identified fragmentation of DNA and an increase in caspase-3 protein and XIAP in Purkinje cells, whereas granule cells exhibited increased caspase-3 and XIAP. IGF-1R expression was unchanged. There was no significant difference in gene expression of caspase-3, XIAP, and IGF-1R. There were an increase in lipid droplets, a reduction in the cellular cytoplasm in electron-dense nuclei, and changes in the myelin sheath in the cerebellar cortex. In conclusion, our data demonstrated that ethanol induced apoptosis in the Purkinje and granule cells of the cerebellum of adult UChA rats.

Chronic ethanol consumption alters all-trans-retinoic acid concentration and expression of their receptors on the prostate: a possible link between alcoholism and prostate damage.

BACKGROUND: Ethanol (EtOH) alters the all-trans-retinoic acid (ATRA) levels in some tissues. Retinol and ATRA are essential for cell proliferation, differentiation, and maintenance of prostate homeostasis. It has been suggested that disturbances in retinol/ATRA concentration as well as in the expression of retinoic acid receptors (RARs) contribute to benign prostate hyperplasia and prostate cancer. This study aimed to evaluate whether EtOH consumption is able to alter retinol and ATRA levels in the plasma and prostate tissue as well as the expression of RARs, cell proliferation, and apoptosis index. METHODS: All animals were divided into 4 groups (n = 10/group). UChA: rats fed 10% (v/v) EtOH ad libitum; UChACo: EtOH-naïve rats without access to EtOH; UChB: rats fed 10% (v/v) EtOH ad libitum; UChBCo: EtOH-naïve rats without access to EtOH. Animals were euthanized by decapitation after 60 days of EtOH consumption for high-performance liquid chromatography and light microscopy analysis. RESULTS: EtOH reduced plasma retinol concentration in both UChA and UChB groups, while the retinol concentration was not significantly different in prostate tissue. Conversely, plasma and prostate ATRA levels increased in UChB group compared with controls, beyond the up-regulation of RARß and -γ in dorsal prostate lobe. Additionally, no alteration was found in cell proliferation and apoptosis index involving dorsal and lateral prostate lobe. CONCLUSIONS: We conclude that EtOH alters the plasma retinol concentrations proportionally to the amount of EtOH consumed. Moreover, high EtOH consumption increases the concentration of ATRA in plasma/prostate tissue and especially induces the RARß and RARγ in the dorsal prostate lobe. EtOH consumption and increased ATRA levels were not associated with cell proliferation and apoptosis in the prostate.

Pregnancy outcome in women with prior placenta accreta spectrum disorders treated with conservative-reconstructive surgery: analysis of 202 cases.

AIM: To report the outcome of pregnant women with a prior pregnancy complicated by placenta accreta spectrum (PAS) disorders treated with resective-conservative surgery at the time of cesarean section. MATERIALS AND METHODS: Retrospective analysis of pregnant women treated with conservative surgery in the prior pregnancy complicated by PAS disorders. The primary outcome was spontaneous preterm birth with intact membranes or following a preterm labor rupture of the membranes before 37 weeks of gestation. Secondary outcomes were uterine rupture, need for hysterectomy due to severe ante or intrapartum maternal hemorrhage, myometrial thinning at the time of cesarean section, 5 min Apgar score, birth weight centile, and the occurrence of small for gestational age newborns. All these outcomes were observed in women with prior PAS treated with conservative resective surgery divided according to the topographical surgical classification. RESULT: Pregnancies included: 89.6% (181/202) related to PAS type 1; 7.9% (16/202) related to PAS type 2, and 2.5% (5/202) related to PAS type 3. 90% of cases (162/179) (95 CI: 90.3-90.6) completed the pregnancy at term (greater than 37 weeks). The average intergenesic period was 15 months for PAS type 1 and 2 (SD 4,76) (Q1:12; Q3:19), and 18 months for PAS 3 (SD 6,56) (Q1:14; Q3:19). A few mothers presented some complications PPROM 1; premature labor 4; hypertension 2; atony 1; overweight 1; and gestational diabetes 2. The mean age was 30 years (T1), 31 years (T2), and 36 years (T3·). The uterine segment was thicker than usual except for one case of partial uterine dehiscence (twins). There were no placenta previa or PAS, a uterine atony case, and there was one case of hysterectomy by patient request. CONCLUSIONS: Subsequent pregnancies after use of resective-reconstructive for PAS has demonstrated to have similar maternal and neonatal outcomes to typical gestation and cesarean delivery.

Placenta accreta spectrum: a hysterectomy can be prevented in almost 80% of cases using a resective-reconstructive technique.

OBJECTIVE: To describe the use of surgical repair (One-step resective-conservative surgery) in all cases of placenta accreta spectrum. STUDY DESIGN: Multicentre retrospective case series from tertiary referral hospitals in Argentina. A total of 452 patients were accepted from 12 hospitals presenting suspicion of invasive placenta by auxiliary methods (ultrasound, Doppler and MRI). At the time of the surgery, placenta accreta spectrum was classified according to invasion topography (specific blood supply) and local features (proximity to other structures, adhesion process, and multiple anastomotic blood vessels). Type 1: upper posterior bladder; type 2: parametrial; type 3: low posterior bladder; and type 4: low posterior bladder and fibrosis. After the ligature of newly formed vessels between the uterus and pelvic organs, the fetus was delivered through an upper segmental hysterotomy. Hemostasis was achieved by selective ligature of vesical-uterine and colpo-uterine vessels. Then, the invaded myometrium and the entire placenta were removed totally in bloc and until detected healthy tissue in both edges, to guarantee the most physiological hysterotomy in the uterine segment. The uterus was closed with a polyglactin suture, double-layer technique. The main outcome measurements were the uterine conservation, the blood loss and other complications classified according to intrasurgical classification. RESULTS: From 452 accepted patients, 326 patients had a confirmed diagnosis of placenta accreta spectrum by histology analysis and surgical-clinical findings. In 126 cases, placenta accreta spectrum was excluded used the same diagnostic criteria (Type 0 or false positive PAS). They were identified 248 cases as type 1, 44 as type 2, 23 as type 3 and 11 as type 4. Uterine conservation was possible in the 81% of type 1 invasion with 500 mL of blood loss (interquartile range, IQR = Q3 - Q1). The modified Pfannenstiel was the most commonly used incision, while midline incision was chosen in all emergencies or in patients with a previous midline incision. Hysterotomy made in the upper part of the uterine segment presented normally attached placentas and not accreta. Selective vessel ligature, also named custom-made hemostasis method (CMHM) was effective at stopping or preventing bleeding associated with PAS. The entire placenta and the invaded area are removed in block, to guarantee to perform the uterine repair with healthy tissue and to avoid a recurrence in the subsequent cesarean. The uterine-ovary artery axis is never occluded or obliterate to guarantee the uterine-endometrial and ovary blood supply as before surgery. No significant differences existed according to the population; however, the presence of total occlusive placenta previa was more frequent in types 3 and 4, which were also associated with older mothers and age-related collagen changes. Lateral and lower segment invasions (types 2 and 3) were most commonly associated with previous terminations of pregnancy, curettage, and manual removal of the placenta. Blood loss and technical difficulty were clearly associated to the invaded area, while invasion degree was a poor marker to predicting bleeding or complications in all locations Uterine conservation was possible in 202/248 (81.5%) of type 1, 21/44 (47.7%) of type 2, 5/23 (21.8%) of type 3 and 0/11 (0%) of type four cases. Type 0 (false positive) were excluded of statistical analysis, and the uterus was preserved in 100% of cases. In a separate report, we will describe the maternal and fetal outcomes as well as 204 subsequent pregnancies after the use of one-step resective reconstructive technique. CONCLUSIONS: Using the resective-reconstructive approach (one-step conservative surgery) to the management of invasive placenta, the uterus can be preserved with minimal morbidity and reduced blood loss in almost 80% of cases. Précis preventing hysterectomy in 80% of placenta accreta spectrum.

Neonatal outcome after conservative-reconstructive surgery for placenta accreta spectrum disorders.

AIM: To report the neonatal outcome after conservative-reconstructive surgery for placenta accreta spectrum (PAS) disorders. MATERIALS AND METHODS: Inclusion criteria were women undergoing conservative-reconstructive surgery for PAS. The outcomes explored were: 5 min Apgar score, birth weight, and need for ventilatory support (RS1 supplementary oxygen, RS2 nasal positive pressure ventilation, or RS3 mechanical ventilatory assistance). Descriptive statistics (means and standard deviations for quantitative and percentage and interquartile range for quantitative variables) were sued to report the data. RESULTS: 84% of women with PAS type 1 were delivered between 35 and 37 weeks of gestation. There was only one case of small for gestational age (SGA) newborn 81% of the newborns required admission to the NICU and 11% respiratory support of those pregnancies complicated by PAS type 2, 59% were delivered between 35 and 36.6 weeks. Neonatal birth weight was consistent with gestational age at birth for all the included cases, and there was no SGA newborn in this group. 84% of the newborns required admission to the NICU, while 21% respiratory support. All women with PAS type 3 were delivered between 30 and 33 weeks of gestation. Although all newborns were admitted to NICU and 73% required ventilatory support, there was no SGA case. Pregnancies complicated by PAS type 4 completed their pregnancy between weeks 35 and 37. There was no case affected by SGA; although all newborns were admitted to NICU, none required ventilatory support. CONCLUSIONS: Conservative surgery in pregnancies complicated by PAS does not seem to increase the risk of adverse neonatal outcomes. Early gestational age at birth and invasion in the inferior third of the lower uterine segment is associated with an increased incidence of neonatal complications, likely due to the earlier gestational age at delivery for these pregnancies.

Variations in maternal care alter corticosterone and 17beta-estradiol levels, estrous cycle and folliculogenesis and stimulate the expression of estrogen receptors alpha and beta in the ovaries of UCh rats.

BACKGROUND: Variations in maternal care are associated with neonatal stress, hormonal disturbances and reproductive injuries during adulthood. However, the effects of these variations on sex hormones and steroid receptors during ovary development remain undetermined. This study aimed to investigate whether variations in maternal care are able to influence the hormonal profile, follicular dynamics and expression of AR, ER-alpha and ER-beta in the ovaries of UCh rat offspring. METHODS: Twenty-four adult UCh rats, aged 120 days, were randomly divided into two groups (UChA and UChB) and mated. Maternal care was assessed from birth (day 0) to the 10th postnatal day (PND). In adulthood, twenty adult female rats (UChA and UChB offspring; n = 10/group), aged 120 days, were euthanized by decapitation during the morning estrus. RESULTS: UChA females (providing high maternal care) more frequently displayed the behaviors of carrying pups, as well as licking/grooming and arched back nursing cares. Also, mothers providing high care had elevated corticosterone levels. Additionally, offspring receiving low maternal care showed the highest estrous cycle duration, increased corticosterone and 17beta-estradiol levels, overexpression of receptors ER-alpha and ER-beta, increased numbers of primordial, antral and mature follicles and accentuated granulosa cell proliferation. CONCLUSIONS: Our study suggests that low maternal care alters corticosterone and 17beta-estradiol levels, disrupting the estrous cycle and folliculogenesis and differentially regulating the expression of ER-alpha and ER-beta in the ovaries of adult rats.

Melatonin reduces LH, 17 beta-estradiol and induces differential regulation of sex steroid receptors in reproductive tissues during rat ovulation.

BACKGROUND: Melatonin is associated with direct or indirect actions upon female reproductive function. However, its effects on sex hormones and steroid receptors during ovulation are not clearly defined. This study aimed to verify whether exposure to long-term melatonin is able to cause reproductive hormonal disturbances as well as their role on sex steroid receptors in the rat ovary, oviduct and uterus during ovulation. METHODS: Twenty-four adult Wistar rats, 60 days old (+/-250 g) were randomly divided into two groups. Control group (Co): received 0.9% NaCl 0.3 mL+95% ethanol 0.04 mL as vehicle; Melatonin-treated group (MEL): received vehicle+melatonin [100 µg/100 g BW/day] both intraperitoneally during 60 days. All animals were euthanized by decapitation during the morning estrus at 4 a.m. RESULTS: Melatonin significantly reduced the plasma levels of LH and 17 beta-estradiol, while urinary 6-sulfatoximelatonin (STM) was increased at the morning estrus. In addition, melatonin promoted differential regulation of the estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR) and melatonin receptor (MTR) along the reproductive tissues. In ovary, melatonin induced a down-regulation of ER-alpha and PRB levels. Conversely, it was observed that PRA and MT1R were up-regulated. In oviduct, AR and ER-alpha levels were down-regulated, in contrast to high expression of both PRA and PRB. Finally, the ER-beta and PRB levels were down-regulated in uterus tissue and only MT1R was up-regulated. CONCLUSIONS: We suggest that melatonin partially suppress the hypothalamus-pituitary-ovarian axis, in addition, it induces differential regulation of sex steroid receptors in the ovary, oviduct and uterus during ovulation.

Long-term exogenous melatonin treatment modulates overall feed efficiency and protects ovarian tissue against injuries caused by ethanol-induced oxidative stress in adult UChB rats.

BACKGROUND: Chronic ethanol intake leads to reproductive damage including reactive oxygen species formation, which accelerates the oxidative process. Melatonin is known to regulate the reproductive cycle, food/liquid intake, and it may also act as a potent antioxidant indoleamine. The aim of this study was to verify the effects of alcoholism and melatonin treatment on overall feed efficiency and to analyze its protective role against the oxidative stress in the ovarian tissue of UChB rats (submitted to 10% [v/v] voluntary ethanol consumption). METHODS: Forty adult female rats (n = 10/group) were finally selected for this study: UChB Co: drinking water only; and UChB EtOH: drinking ethanol at 2 to 6 ml/100 g/d + water, both receiving 0.9% NaCl + 95% ethanol 0.04 ml as vehicle. Concomitantly, UChB Co + M and UChB EtOH + M groups were infused with vehicle + melatonin (100 µg/100 g body weight/d) intraperitoneally over 60 days. All animals were euthanized by decapitation during the morning estrus (4 am). RESULTS: Body weight gain was reduced with ethanol plus melatonin after 40 days of treatment. In both melatonin-treated groups, it was observed a reduction in food-derived calories and liquid intake toward the end of treatment. The amount of consumed ethanol dropped during the treatment. Estrous cycle was longer in rats that received both ethanol and melatonin, with prolonged diestrus. Following to oxidative status, lipid hydroperoxide levels were higher in the ovaries of ethanol-preferring rats and decreased after melatonin treatment. Additionally, antioxidant activities of superoxide dismutase, glutathione peroxidase activity, and glutathione reductase activity were increased in melatonin-treated groups. CONCLUSIONS: We suggest that melatonin is able to affect feed efficiency and, conversely, it protects the ovaries against the oxidative stress arising from ethanol consumption.

Caffeine consumption attenuates ethanol-induced inflammation through the regulation of adenosinergic receptors in the UChB rats cerebellum.

Caffeine consumption is able to interfere in cellular processes related to inflammatory mechanisms by acting through the adenosinergic system. This study aimed to recognize alterations related to adenosinergic system and inflammatory process in the cerebellum of University of Chile Bibulous (UChB) rats after the consumption of ethanol and caffeine. UChB and Wistar rats, males at 5 months old, were divided into the groups (n = 15/group): (i) Control (Wistar rats receiving water); (ii) Ethanol group (UChB rats receiving ethanol solution at 10%) and (iii) Ethanol+caffeine group (UChB rats receiving ethanol solution at 10% added of 3 g/L of caffeine). The cerebellar tissue was collected and processed for immunohistochemistry, Reverse transcription polymerase chain reaction (RT-PCR) and western blotting techniques for the adenosinergic receptors A1 and A2a and inflammatory markers, including Nuclear factor kappa B (NFkB), TLR4, TLR2, MyD88, TNF-α, COX-2, iNOS and microglial marker Iba-1. Results showed ethanol and caffeine consumption differentially altering the immunolocalization of adenosinergic receptors and inflammatory markers in the cerebellar tissue. The A2a receptor was overexpressed in the Ethanol group and was evident in the glial cells. The Ethanol group had increased protein levels for NFκB and TLR4, expressively in Bergmann glia and Purkinje cells. Caffeine reduced the expression of these markers to levels similar to those found in the Control group. The A1 gene was upregulated the Ethanol group, but not its protein levels, suggesting post-transcriptional interference. In conclusion, caffeine seems to attenuate ethanol-induced inflammation in the cerebellum of UChB rats through the A1 and A2a modulation, playing a neuroprotective role in the chronic context of ethanol consumption.

Stabilization and application of spray-dried tannase from Aspergillus fumigatus CAS21 in the presence of different carriers.

The Aspergillus fumigatus CAS21 tannase was spray dried with ß-cyclodextrin, Capsul® starch, soybean meal, lactose, and maltodextrin as adjuvants. The moisture content and water activity of the products ranged from 5.6 to 11.5% and from 0.249 to 0.448, respectively. The maximal tannase activity was achieved at 40-60 ºC and pH 5.0-6.0 for the powders containing ß-cyclodextrin and Capsul® starch, which was stable at 40 ºC and 40-60 ºC for 120 min, respectively. For all the dried products, tannase retained its activity of over 80% for 120 min at pH 5.0 and 6.0. Salts and solvents influenced the activity of the spray-dried tannase. The activity of the spray-dried tannase was maintained when preserved for 1 year at 4 ºC and 28 ºC. Spray-dried tannase reduced the content of tannins and polyphenolic compounds of leather effluent and sorghum flour and catalyzed the transesterification reaction. The spray drying process stabilized the tannase activity, highlighting the potential of dried products for biotechnological applications.

P-MAPA and IL-12 Differentially Regulate Proteins Associated with Ovarian Cancer Progression: A Proteomic Study.

To investigate the potential role of immunotherapies in the cellular and molecular mechanisms associated with ovarian cancer (OC), we applied a comparative proteomic toll using protein identification combined with mass spectrometry. Herein, the effects of the protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride, known as P-MAPA, and the human recombinant interleukin-12 (hrIL-12) were tested alone or in combination in human SKOV-3 cells. The doses and period were defined based on a previous study, which showed that 25 µg/mL P-MAPA and 1 ng/mL IL-12 are sufficient to reduce cell metabolism after 48 h. Indeed, among 2,881 proteins modulated by the treatments, 532 of them were strictly concordant and common. P-MAPA therapy upregulated proteins involved in tight junction, focal adhesion, ribosome constitution, GTP hydrolysis, semaphorin interactions, and expression of SLIT and ROBO, whereas it downregulated ERBB4 signaling, toll-like receptor signaling, regulation of NOTCH 4, and the ubiquitin proteasome pathway. In addition, IL-12 therapy led to upregulation of leukocyte migration, tight junction, and cell signaling, while cell communication, cell metabolism, and Wnt signaling were significantly downregulated in OC cells. A clear majority of proteins that were overexpressed by the combination of P-MAPA with IL-12 are involved in tight junction, focal adhesion, DNA methylation, metabolism of RNA, and ribosomal function; only a small number of downregulated proteins were involved in cell signaling, energy and mitochondrial processes, cell oxidation and senescence, and Wnt signaling. These findings suggest that P-MAPA and IL-12 efficiently regulated important proteins associated with OC progression; these altered proteins may represent potential targets for OC treatment in addition to its immunoadjuvant effects.

SWEET Transporters for the Nourishment of Embryonic Tissues during Maize Germination.

In maize seed germination, the endosperm and the scutellum nourish the embryo axis. Here, we examined the mRNA relative amount of the SWEET protein family, which could be involved in sugar transport during germination since high [14-C]-glucose and mainly [14-C]-sucrose diffusional uptake were found in embryo tissues. We identified high levels of transcripts for SWEETs in the three phases of the germination process: ZmSWEET4c, ZmSWEET6b, ZmSWEET11, ZmSWEET13a, ZmSWEET13b, ZmSWEET14b and ZmSWEET15a, except at 0 h of imbibition where the abundance of each ZmSWEET was low. Despite the major sucrose (Suc) biosynthesis capacity of the scutellum and the high level of transcripts of the Suc symporter SUT1, Suc was not found to be accumulated; furthermore, in the embryo axis, Suc did not decrease but hexoses increased, suggesting an efficient Suc efflux from the scutellum to nourish the embryo axis. The influx of Glc into the scutellum could be mediated by SWEET4c to take up the large amount of transported sugars due to the late hydrolysis of starch. In addition, sugars regulated the mRNA amount of SWEETs at the embryo axis. These results suggest an important role for SWEETs in transporting Suc and hexoses between the scutellum and the embryo axis, and differences in SWEET transcripts between both tissues might occur because of the different sugar requirements and metabolism.