Clock gene per 2 daily rhythm: Correlation with the serum level of uncoupling protein 1 (UCP1) in goat and horse.

Recent studies evidenced that the circadian rhythm of Per2 is involved in adaptive thermogenesis by the modulating transcription of uncoupling protein 1 (UCP1). For this purpose, we investigated the linkage between the daily rhythm of Per2 and UCP1 in ruminant and non-ruminant mammalian species. Five clinically healthy, not pregnant, and not lactating Maltese female goats and five clinically healthy, not pregnant, and not lactating Italian Saddle horses were enrolled in the study. All animals were housed under natural photoperiod (sunrise 05:05, sunset 20:55) and environmental temperature and humidity. Goats were kept individually in 3.0 × 2.0 m box, horses were housed individually in 3.5 × 3.5 m box; all boxes were equipped with an opening window. On each subject, blood samples were collected every 4 h for a 48-h period. The Per2 gene expression was determined on blood samples collected in PAX gene Blood RNA Tube, whereas UCP1 concentration was assessed on serum. Per2 and UCP1 levels were statistically influenced by the species (p < 0.0001) and the time of data collection (p < 0.0001), but not by the day of monitoring. Per2 showed daily rhythmicity, statistically different in mesor and amplitude between the two species, diurnal in goats, nocturnal in horses; with the same robustness. UCP1 did not show daily rhythmicity. During the experimental period the two parameters showed a negative correlation in horses. According to the findings herein obtained, we can claim that the role of Per2 in the thermogenesis induced by the beige adipocytes throughout UCP1 activation did not reflect what found in other mammal species, but further studies are required to establish their correlation in equids.

Effect of different temperatures on survival and development of Aelurostrongylus abstrusus (Railliet, 1898) larvae.

Aim of the study was to get in-depth information on Aelurostrongylus abstrusus first-stage larvae (L1s) survival at different temperatures and to assess the capability of these larvae to develop into the third infective stage (L3s). Faeces of a naturally infected cat were split into two aliquots: the first was divided in subsamples assigned to four groups (F1-F4); from the second aliquot, L1s were extracted by Baermann technique, suspended in water and divided into four groups (W1-W4). Groups were stored at different temperatures (F1/W1 -20 ± 1°C; F2/W2 +4 ± 1°C; F3/W3 +14 ± 1°C; and F4/W4 +28 ± 1°C) and L1s vitality assessed every seven days. The capability of L1s stored in water to develop into L3s in snails was evaluated at the beginning and every 21 days. The L1s of W2 and F2 groups remained viable for a longer period (231 and 56 days, respectively) compared to those of other groups. The capability of L1s to moult into L3s in snails showed a decreasing trend; the group W2's L1s maintained the capability to moult into L3s for the longest time (day 189) compared to the other groups. The time of survival of A. abstrusus L1s is influenced by temperature. However, the species seems to be more resistant to temperature variations than other feline lungworms, and this may explain its wider distribution across Europe.

Infrared thermography as a rapid and non-invasive diagnostic tool to detect inflammatory foot diseases in dairy cows.

In this study the potential usefulness of infrared thermography (IRT) as a non-invasive tool to rapidly screen the most common non-infectious foot lesions in dairy cows was evaluated. Thirty-eight healthy cows and 38 cows affected by foot diseases were enrolled. Diseased cows showed the following disorders at lateral and medial claw in the hind foot: white line lesion, sole ulcer, sole haemorrhage, horizontal fissure, axial fissure. Thermography images of hind foot were collected for each animal using a digital infrared camera. Foot temperature was measured in four regions: central area of the hind foot (A1), interdigital area of the hind foot (A2), lateral (A3) and medial (A4) claw in the hind foot. Higher temperature values in the regions A1 and A2 compared to A3 and A4 were found in both healthy and diseased cows (p0.001). Cows affected by foot diseases showed higher foot temperature values compared to healthy cows (p0.05) in all considered regions. This study highlights the potential application of IRT as a reliable, practical tool for detection of hoof lesions in dairy cows. Multiple scanning images and comparisons between affected and healthy anatomical structures could be useful in defining the consistency of abnormality.

Different training schedules influence platelet aggregation in show jumping horses.

Depending on the intensity, duration and type of physical exercise, equine metabolism has to adapt to nervous, cardiovascular, endocrine and respiratory system requirements. In horses, exercise and training are known to have considerable effects on the mechanisms of hemostatic system involving platelet activity. The aim of the present study was to evaluate the effect of different training schedules on platelet aggregation in 15 Italian Saddle jumping horses. Animals were divided into three equal groups: Group A was subjected to a high intensity-training program; group B to a light training program, group C included sedentary horses. From each animal, blood samples were collected by jugular venipuncture at rest on the 1st, 3rd and 5th days, and afterwards, once a week, for a total of 5 weeks data recording, in order to assess the maximum degree of platelet aggregation and the initial velocity of aggregation (slope) platelet aggregation. Two-way analysis of variance (ANOVA) showed a significant effect of the different training schedules on studied parameters. The results revealed a different degree of platelet aggregation and a different initial velocity of platelet aggregation that changes during the different training schedules in horses that could represent a different protective endothelial mechanism. These findings could have an important role for a clearer knowledge of the physiological reference values of platelet aggregation and for a better interpretation of these variations during the training.

Leukocyte modifications during the first month after foaling in mares and their newborn foals.

During early post-partum period both neonatal foals and peripartum mares are most susceptible to diseases. The aim of this study was to establish physiologic modifications of leukogram during the first month after foaling in mares and their newborn foals. To this end blood samples were collected from nine mares and nine foals (T0-T10), every three days from the 1st day until the 30th day after foaling. Samples were analysed for white blood cell (WBC) count and differential leucocyte counts. Two-way repeated measure analysis of variance (ANOVA) showed, in postpartum mares WBC showed significant higher values at T0 (9.02±0.76) in respect to other time points, and at T2 (8.08±0.53) and T3 (7.92±0.59) compared to T1 (6.98±0.43), whereas in foals lower WBC values at T0 (6.11±0.49) compared to other experimental periods except T1 (6.90±0.94), and at T1 compared to T8 (7.95±0.61) and T10 (7.90±0.36) were observed. The differential leucocyte counts showed significant modifications in the percentage of neutrophils (π<0.001) and lymphocytes (p<0.001) both in postpartum mares and in foals during the experimental period. Furthermore ANOVA showed significant differences between postpartum mares and foals (P<0.01) in all studied parameters, and between postpartum mares and control mares in WBC and neutrophils values. The obtained results provide suitable information about the influence of foaling on leukogram of periparturient mares and reveal WBC dynamics in newborn foals during the first month post-partum.

Metabolic profile of broodmares during late pregnancy and early post-partum.

Pregnancy represents a specific physiological status characterized by continuous adjustments that affect maternal metabolism of all nutrients. In the last trimester of pregnancy, mare's nutrient requirements greatly increase and most pregnancy-associated diseases are likely to occur. Therefore, we aimed to assess the metabolic profile of broodmares focusing on the last 3 months of pregnancy and the early post-partum. Fifteen pregnant mares (Group A) were monitored from 263 ± 3 days of pregnancy until 21 days after foaling. Seven non-pregnant mares (Group B) were used as the control group. Blood samples were collected weekly by jugular venipuncture throughout the experimental period, and additional blood samples were collected within 24 ± 12 h of foaling. Obtained sera were analyzed for urea (Ur), creatinine (Cre), total protein (TP), total, direct and indirect bilirubin (tB, dB, iB), triglycerides (TG), total cholesterol (tChol) and ß-hydroxybutyrate (BHB). During pregnancy, Ur (p = 0.015) and dB (p = 0.028) were higher in Group A than Group B. Serum Cre, tB, iB and TG (p < 0.001) decreased after foaling; furthermore, Group A exhibited lower tChol (p < 0.001) and higher BHB (p < 0.001) than Group B during the study. There were significant interactions of time × peripartum on Ur (p = 0.007), Cre (p < 0.001), tB (p = 0.018), TG (p < 0.001) and tChol (p < 0.001). These results can be used in the interpretation of biochemical studies in late gestation mares which develop systemic disease. The improved understanding of the metabolic profile during the peripartum might assist in monitoring the health status of the broodmares to promote foetal growth and well-being.

Effect of storage time on haematological parameters in mullet, Mugil cephalus.

The aim of the present study was to assess the effect of storage time at +4 °C on red blood cell count (RBC), haematocrit (Hct), haemoglobin (Hb), white blood cell count (WBC), thrombocyte count (TC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) in mullet (Mugil cephalus) using an automatic method. After blood collection (T0 ), all samples were analyzed using both the manual and automatic method. To test the validation of the automatic method, a paired t-test was applied, and no statistical difference was observed. The samples were successively divided into four different aliquots and stored at +4 °C to assess the haematological parameters using the automatic method. The first aliquot was refrigerated for 6 h, the second one for 24 h, the third one for 48 h and the last one at for 72 h. One-way repeated-measures ANOVA showed a significant effect of storage time (P < 0.05) on Hb, WBC, TC, MCH and MCHC. These results suggest that haematological parameters can be assessed within 6 h from blood collection when samples are stored at +4 °C because long-term storage modifies the results of the analyses. Further studies on these parameters could be still needed in various fish species to validate an appropriate method for haematological analysis useful not only for the evaluation of the health status of animal living in captivity and in aquaculture but also to have reliability environmental haematological biomarkers.

Clock genes determination in whole blood in goats housed under a long light cycle.

An innate 24 h circadian clock drives various behavioral processes via expression of clock genes that regulate circadian rhythmicity and temporal signals. Elucidating the gene expression in goats may contribute to improving the knowledge of the regulation of circadian rhythms in this species. Five nonpregnant and nonlactating Maltese goats with no evidence of disease were kept in an indoor pen under the natural long photoperiod (05:05-20:56 h) and natural environmental temperature (23°C and 60% RH). They were fed an Alfalfa hay and concentrate mixture provided twice a day; water was available ad libitum. Blood samples were collected every 4 h over a 48 h period into PAX gene Blood RNA Tubes and stored at -80°C until processing. Clock genes (Clock; Cry1; Cry2; Per2; Per3) were determined using real-time quantitative polymerase chain reaction. During the experimental period, locomotor activity was monitored by an actigraphy-based data logger that records a digitally integrated measure of motor activity as a means to assess indices of discomfort during study and stability of the circadian rhythm. All of the tested genes showed daily rhythmicity in their expression in whole blood. Differences in their circadian parameters were observed. Mesor and amplitude were statistically different among the tested gene (Mesor: F(4.30) = 205.30; p < .0001; amplitude: F(4.30) = 104.80; p < .0001), with each gene showing its acrophase at a different time of day (F(4.30) = 81.17; p < .0001), and differences were observed between the two days of monitoring (F(1.30) = 10.25; p = .003). The application of two-way analysis of variance (ANOVA) on robustness of rhythm values did not show statistical differences among the tested genes (F(4.30) = 1.83; p = .14) and between the two days of monitoring (F(1.30) = 1.16; p = .28). Locomotor activity data recording were in accordance with the data reported in literature, indicating the absence of discomfort or alteration of circadian rhythms during the experimental period. Our results support the presence of a cyclic transcription of clock genes in whole blood of healthy goats housed under a long light natural photoperiod and natural environmental conditions.

Secreted Frizzled-related protein 4 (sFRP4) chemo-sensitizes cancer stem cells derived from human breast, prostate, and ovary tumor cell lines.

This study investigated molecular signals essential to sustain cancer stem cells (CSCs) and assessed their activity in the presence of secreted frizzled-related protein 4 (sFRP4) alone or in combination with chemotherapeutic drugs. SFRP4 is a known Wnt antagonist, and is also pro-apoptotic and anti-angiogenic. Additionally, sFRP4 has been demonstrated to confer chemo-sensitization and improve chemotherapeutic efficacy. CSCs were isolated from breast, prostate, and ovary tumor cell lines, and characterized using tumor-specific markers such as CD44+/CD24-/CD133+. The post-transcription data from CSCs that have undergone combinatorial treatment with sFRP4 and chemotherapeutic drugs suggest downregulation of stemness genes and upregulation of pro-apoptotic markers. The post-translational modification of CSCs demonstrated a chemo-sensitization effect of sFRP4 when used in combination with tumor-specific drugs. SFRP4 in combination with doxorubicin/cisplatin reduced the proliferative capacity of the CSC population in vitro. Wnt/ß-catenin signaling is important for proliferation and self-renewal of CSCs in association with human tumorigenesis. The silencing of this signaling pathway by the application of sFRP4 suggests potential for improved in vivo chemo-responses.

Water temperature influences growth and gonad differentiation in European sea bass (Dicentrarchus labrax, L. 1758).

The effect of rearing temperature on gonad differentiation and growth was evaluated in sea bass (Dicentrarchus labrax). One control group (CG, n = 60) and two experimental groups (EG1, n = 30; EG2, n = 30) were selected. CG was reared at 15.0 °C during 10 days post-hatch (dph) and at 19.0 °C throughout the remaining larval and post-larval development. EG1 was exposed to 14.5 °C from 1 to 50 dph, followed by an increase to 20 °C until sampling (176 dph). EG2 was exposed to 14.5 °C from 1 to 37 dph, followed by an increase to 20 °C until sampling (226 dph); 30 fish from CG were randomly sampled at 176 dph (CG1, control for EG1) and 30 fish were sampled at 226 dph (CG2, control for EG2). Weight, total and fork length measurement, and gonad collection were performed. All biometric indices were higher in EG1 and EG2 than in CG1 and CG2, respectively. Histologic analysis reported 100% of undifferentiated gonads in CG1 and; EG1 reported 80% of undifferentiated gonads and 20% of gonads at early differentiation toward the male line. CG2 reported 34% of undifferentiated gonads, 10% of gonads at early differentiation toward the female line, 23% of gonads at early differentiation toward the male line, and 33% of gonads with intratesticular oocytes. EG2 reported 47% of gonads differentiated in testis, 33% gonads differentiated in ovary, and 20% of gonads with intratesticular oocytes. These results suggest that water temperature plays an important role in the process of gonad differentiation and growth of sea bass.

Wnt signaling in triple-negative breast cancer.

Wnt signaling regulates a variety of cellular processes, including cell fate, differentiation, proliferation and stem cell pluripotency. Aberrant Wnt signaling is a hallmark of many cancers. An aggressive subtype of breast cancer, known as triple-negative breast cancer (TNBC), demonstrates dysregulation in canonical and non-canonical Wnt signaling. In this review, we summarize regulators of canonical and non-canonical Wnt signaling, as well as Wnt signaling dysfunction that mediates the progression of TNBC. We review the complex molecular nature of TNBC and the emerging therapies that are currently under investigation for the treatment of this disease.

Serum levels of mitochondrial uncoupling protein 1, leptin, and lipids during late pregnancy and the early postpartum period in mares.

The aim of this study was to investigate the changes of serum mitochondrial uncoupling protein 1 (UCP1) and leptin levels as well as of lipid and lipoprotein profiles in mares during the peripartum period. Ten pregnant mares (group A) were monitored from 15 ± 3 days of pregnancy until 15 days after foaling, and 10 nonpregnant nonlactating mares constituted the control (group B). In group A, blood sampling was performed on Days 15 ± 3 and 7 ± 3 before foaling, on the day of foaling, and on Days 7 and 15 after foaling. In group B, blood sampling was performed on the same days as in group A. Serum levels were determined for UCP1, leptin, total lipids, phospholipids, triglycerides, total cholesterol (Total-Chol), high-density lipoproteins, low-density lipoproteins (LDLs), and very low-density lipoproteins (VLDLs). Two-way repeated-measures ANOVA was applied to evaluate the effects of peripartum period and group membership. All studied parameters except phospholipid levels (P > 0.05) showed significant changes in group A over the peripartum period (P < 0.0001). A significant effect of pregnancy was found on all studied parameters (P < 0.001), which showed lower levels in group A than in group B for most of the time points considered. Significant negative correlations were found between UCP1 and total lipids, triglycerides, VLDLs, Total-Chol, and LDL values. Positive correlations were found between leptin and total lipids, triglycerides, VLDLs, Total-Chol, and LDLs. These changes observed in mares during the peripartum period could represent a response to hormonal and metabolic adaptations occurring during specific physiological conditions such as late pregnancy and early postpartum. These changes should compensate for the energy loss occurring during these particular life phases and ensure a good body condition to protect mares against negative energy balance.

Dynamic modulation of platelet aggregation, albumin and nonesterified fatty acids during physical exercise in Thoroughbred horses.

The effect of exercise on platelet aggregation, albumin and nonesterified fatty acids (NEFAs) values and the correlation among these parameters were evaluated in ten clinically healthy and regularly trained Thoroughbred horses. All horses were subjected to two simulated races. Blood samples were collected by jugular venipuncture before and after the first simulated race (T0PRE and T0POST), every 7 days at rest condition for a month (T1R-T2R-T3R), and before and after the second simulated race (T4PRE and T4POST) in order to assess platelet aggregation, albumin and nonesterified fatty acids (NEFAs) levels. One-way analysis of variance showed a significant effect of exercise (P<0.01) on platelet aggregation, albumin and NEFAs values. A negative correlation between platelet aggregation and albumin or NEFAs values, and a positive correlation between albumin and NEFAs values, were found both at T0POST and T4POST (P<0.05). These findings are likely related to dynamic physiological adaptations to exercise that allow re-establishment of the homeostatic equilibrium of the organism.

Epigenetic regulation of the secreted frizzled-related protein family in human glioblastoma multiforme.

Glioblastoma multiforme (GBM) are intracranial tumors of the central nervous system and the most lethal among solid tumors. Current therapy is palliative and is limited to surgical resection followed by radiation therapy and temozolomide treatment. Aberrant WNT pathway activation mediates not only cancer cell proliferation but also promotes radiation and chemotherapeutic resistance. WNT antagonists such as the secreted frizzled-related protein (sFRP) family have an ability to sensitize glioma cells to chemotherapeutics, decrease proliferation rate and induce apoptosis. During tumor development, sFRP genes (1-5) are frequently hypermethylated, causing transcriptional silencing. We investigated a possible involvement of methylation-mediated silencing of the sFRP gene family in human GBM using four human glioblastoma cell lines (U87, U138, A172 and LN18). To induce demethylation of the DNA, we inhibited DNA methyltransferases through treatment with 5-azacytidine. Genomic DNA, RNA and total protein were isolated from GBM cells before and after treatment. We utilized bisulfite modification of genomic DNA to examine the methylation status of the respective sFRP promoter regions. Pharmacological demethylation of the GBM cell lines demonstrated a loss of methylation in sFRP promoter regions, as well as an increase in sFRP gene-specific mRNA abundance. Western blot analysis demonstrated an increased protein expression of sFRP-4 and increased levels of phosphorylated-ß-catenin. These data indicate an important role of methylation-induced gene silencing of the sFRP gene family in human GBM.

Cancer stem-like cells from head and neck cancers are chemosensitized by the Wnt antagonist, sFRP4, by inducing apoptosis, decreasing stemness, drug resistance and epithelial to mesenchymal transition.

Cancer stem cells (CSCs) of head and neck squamous cell carcinoma (HNSCC) are defined by high self-renewal and drug refractory potential. Involvement of Wnt/ß-catenin signaling has been implicated in rapidly cycling cells such as CSCs, and inhibition of the Wnt/ß-catenin pathway is a novel approach to target CSCs from HNSCC. In this study, we found that an antagonist of FrzB/Wnt, the secreted frizzled-related protein 4 (sFRP4), inhibited the growth of CSCs from two HNSCC cell lines, Hep2 and KB. We enriched the CD44(+) CSC population, and grew them in spheroid cultures. sFRP4 decreased the proliferation and increased the sensitivity of spheroids to a commonly used drug in HNSCC, namely cisplatin. Self-renewal in sphere formation assays decreased upon sFRP4 treatment, and the effect was reverted by the addition of Wnt3a. sFRP4 treatment of spheroids also decreased ß-catenin, confirming its action through the Wnt/ß-catenin signaling pathway. Quantitative PCR demonstrated a clear decrease of the stemness markers CD44 and ALDH, and an increase in CD24 and drug-resistance markers ABCG2 and ABCC4. Furthermore, we found that after sFRP4 treatment, there was a reversal in the expression of epithelial to mesenchymal (EMT) markers with the restoration of the epithelial marker E-cadherin, and depletion of EMT-specific markers twist, snail and N-cadherin. This is the first report demonstrating that the naturally occurring Wnt inhibitor, sFRP4, can be a potential drug to destroy CSC-enriched spheroids from HNSCCs. The repression of EMT and the decrease in stemness profile further strengthen the use of sFRP4 as a potent therapeutic against CSCs.

Apoptosis does not affect the vasculature of the corpus luteum of pregnancy in the rat.

There has been much research into the mechanics of angiogenesis and many studies have demonstrated that newly formed vessels regress during angiogenesis. This vascular involution has been shown to involve basement membrane dissolution and endothelial cell apoptosis. The corpus luteum provides an ideal in vivo model to study physiologic angiogenesis and studies have shown that involution of newly formed vessels occurs during corpus luteum regression. However, few studies to date have investigated the role of apoptosis on the vasculature which develops during pregnancy. By the use of the TUNEL technique to detect apoptotic cells and immunohistochemistry to distinguish between endothelial cells and pericytes, this present study demonstrated that the vasculature of the corpus luteum of pregnancy in the rat does not undergo apoptosis.