Detection of buffalo milk adulteration with cow milk by capillary electrophoresis analysis.

The addition of cow milk during the production of buffalo mozzarella is a common fraud in dairy industries because of the lower price and greater availability of cow milk throughout the year. The aim of this study was to develop a new, rapid, and robust capillary electrophoresis method for detecting and quantifying cow milk in buffalo milk by exploiting cow α-lactalbumin as a marker of adulteration. In particular, a linear calibration curve was generated, using a training set of calibrators consisting of 7 series of 17 buffalo/bovine whey mixtures, obtained after casein precipitation, with increasing percentages of cow whey. The capillary electrophoresis method showed high linearity (R2 = 0.968), repeatability [relative standard deviation (RSD) = 2.11, 3.02, 4.38, and 1.18%, respectively for 5, 10, 20, and 50% of buffalo/bovine whey mixtures], and intermediate precision (RSD = 2.18, 2.49, 5.09, and 3.19%, respectively, for 5, 10, 20, and 50% buffalo/bovine whey mixtures). Moreover, the minimum amount of detectable fraudulent cow milk was 1%, and the limit of quantification was 3.1%.

Protective effects of some statins on epileptogenesis and depressive-like behavior in WAG/Rij rats, a genetic animal model of absence epilepsy.

OBJECTIVE: Statins (3-hydroxy-3-methylglutaryl-coenzyme A [HMG-CoA] reductase inhibitors) exert cholesterol-independent pleiotropic effects that include antithrombotic, antiinflammatory, and antioxidative properties. Moreover, both in vitro and in vivo studies have shown neuroprotective, antiseizure, and antiexcitotoxic effects of statins, suggesting their potential role in the treatment of neurologic diseases. Only a few studies have investigated whether statins modulate absence seizure activity and epileptogenesis. METHODS: We investigated the effects of atorvastatin (5 and 10 mg/kg/day), simvastatin (10 mg/kg/day), and pravastatin (10 and 30 mg/kg/day), given orally for 17 consecutive weeks (starting at 45 days of age), on the development of absence seizures (electroencephalography [EEG] recordings), depressive-like behavior (forced swimming test [FST]), and anxiety levels (open field test [OF]) in Wistar Albino Glaxo/Rijswijk rats (WAG/Rij) rats at the age of 6 months (1 month after suspension). WAG/Rij rats are a genetic animal model of absence epilepsy, epileptogenesis, and mild-depression comorbidity. The effects of statins were also studied after acute intraperitoneal injection for 4 h after administration of various doses in 6-months-old rats. Plasma cholesterol levels were measured throughout drug treatment. RESULTS: We found that early long-term statin treatment possesses antiepileptogenic properties, reduced immobility time in the FST, and reduced anxiety in the OF, whereas they were not effective against established absence seizures when acutely administered. The observed effects were not related to changes in plasma cholesterol levels, which remained unchanged during drug treatment. SIGNIFICANCE: Our results suggest that statins administration might be a possible intervention and promising strategy for preventing the epileptogenesis and/or behavioral comorbidity.

Human immunodeficiency virus-1 Tat activates NF-κB via physical interaction with IκB-α and p65.

Nuclear factor (NF)-κB is a master regulator of pro-inflammatory genes and is upregulated in human immunodeficiency virus 1 (HIV-1) infection. Mechanisms underlying the NF-κB deregulation by HIV-1 are relevant for immune dysfunction in AIDS. We report that in single round HIV-1 infection, or single-pulse PMA stimulation, the HIV-1 Tat transactivator activated NF-κB by hijacking the inhibitor IκB-α and by preventing the repressor binding to the NF-κB complex. Moreover, Tat associated with the p65 subunit of NF-κB and increased the p65 DNA-binding affinity and transcriptional activity. The arginine- and cysteine-rich domains of Tat were required for IκB-α and p65 association, respectively, and for sustaining the NF-κB activity. Among an array of NF-κB-responsive genes, Tat mostly activated the MIP-1α expression in a p65-dependent manner, and bound to the MIP-1α NF-κB enhancer thus promoting the recruitment of p65 with displacement of IκB-α; similar findings were obtained for the NF-κB-responsive genes CSF3, LTA, NFKBIA and TLR2. Our results support a novel mechanism of NF-κB activation via physical interaction of Tat with IκB-α and p65, and may contribute to further insights into the deregulation of the inflammatory response by HIV-1.

In vivo targeting and growth inhibition of the A20 murine B-cell lymphoma by an idiotype-specific peptide binder.

B-cell lymphoma is a clonal expansion of neoplastic cells that may result in fatal outcomes. Here, we report the in vivo targeting and growth inhibition of aggressive A20 murine B-cell lymphoma by idiotype-specific peptide pA20-36. pA20-36 was selected from random peptide libraries and bound specifically to the B-cell receptor (BCR) of A20 cells in mice engrafted with A20 lymphoma, as shown by histology and positron emission tomographic analysis. BCR cross-linking of A20 cells with pA20-36 resulted in massive apoptosis of targeted tumor cells and in an increased survival of the diseased animals without any detectable evidence of toxicity. The pA20-36 treatment reverted the immune suppression of the tumor microenvironment as shown by reduced expression of vascular endothelial growth factor, interleukin-10, and transforming growth factor-beta cytokines together with a lower number of CD11b+Gr-1+ inhibitor myeloid-derived suppressor cells and Foxp3+CD4+ Treg cells. Furthermore, pA20-36 treatment was associated with an increased number of tumor-infiltrating, activated CD8+ T cells that exerted a tumor-specific cytolytic activity. These findings show that a short peptide that binds specifically to the complementarity-determining regions of the A20 BCR allows in vivo detection of neoplastic cells together with significant inhibition of tumor growth in vivo.

Suitability of Protein Content Measured by MilkoScan FT-Plus Milk Analyzer to Evaluate Bovine and Ovine Colostrum Quality.

The objective of this study was to evaluate MilkoScan FT-plus for the estimation of the immunoglobulin G (IgG) content in bovine and ovine colostrum. Between April and May 2016, a total of 94 colostrum samples (54 from Simmental dairy cows and 39 from Sarda ewes) were collected within 6 h (T0) and after 24 h (T24) from parturition. Colostrum samples were subjected to the radial immunodiffusion (RID) assay for the quantification of IgG and to MilkoScan FT-plus for the estimation of protein content (TP, %), which was then used as an indirect method for the evaluation of colostrum quality. To compare the two methods, correlation and regression analysis of IgG quantification by RID and protein (%) content estimation by MilkoScan FT-plus data was performed using Procedure CORR and Procedure REG of SAS, respectively (version 9.3, SAS Institute Inc., Cary, NC, USA). Thresholds for the classification of good colostrum quality (as determined by RID assay, the gold standard method) were set at 50 g of IgG/L in cows and 20 g of IgG/L in ewes. The concentration of IgG in bovine colostrum assayed by RID showed a variation ranging from 41.45 to 199.97 g/L with an average of 99.85 ± 40.84 g/L at T0, and from 2.83 to 75.93 g/L with an average of 19.76 ± 19.01 g/L at T24. Regarding ovine colostrum, the concentration of IgG assayed by RID ranged from 34.45 to 156.32 g/L with an average value of 77.82 ± 37.58 g/L at T0, and from 5.6 to 69.74 g/L with an average of 27.90 ± 19.81 g/L at T24. Colostrum TP ranged from 3.70 to 23.96% for bovine colostrum and 6.32 to 22.88% for ovine colostrum using MilkoScan FT-plus. MilkoScan FT-plus and RID data were highly and significantly correlated (r = 0.91 for bovine and r = 0.94 for ovine colostrum), and regression analysis showed a strong relationship between IgG concentration provided by RID assay and TP provided by MilkoScan FT-plus (R2 = 0.84 and 0.88 for bovine and ovine, respectively). Optimal cut-off points for the greatest accuracy of TP (%) determined by MilkoScan FT-plus were 12.8% in cows [with 88.9% sensitivity (Se) and 100% specificity (Sp)] and 9% in ewes (with 96.7% Se and 100% Sp). In conclusion, these outcomes indicate that MilkoScan FT-plus as an indirect method may be a reliable tool for the estimation of the total IgG concentration and quality in bovine and ovine colostrum. Moreover, the cut-off levels of 12.8% for bovine and 9% for ovine of TP, seem sufficient to ensure that all poor-quality colostrum can be classified as such, with only a low proportion of good-quality colostrum being misclassified as poor-colostrum, thereby increasing the probability of delivering good-quality colostrum to new-born calves and lambs.

Serum 25-hydroxyvitamin D measurement: Comparative evaluation of three automated immunoassays.

OBJECTIVES: the different analytical methods for measurement of serum 25-hydroxyvitamin D (25(OH)D) are not yet fully harmonized and no consensus exists on a threshold of 25(OH)D defining a deficiency status. In this study, we compared the results from the assays of serum 25(OH)D performed with three different methods to evaluate the presence of potential biases and how much these biases can influence the assignment of patients to specific 25(OH)D deficiency/sufficiency categories. DESIGN AND METHODS: Liaison 25(OH) Vitamin D Total (DiaSorin Liaison XL), Elecsys Vitamin D Total II (Roche Elecsys) and Lumipulse G25(OH) Vitamin D (Fujirebio Lumipulse G1200) were used. Methods comparability was established performing Passing-Bablok regression and Bland-Altman analysis to prove whether the differences found were lower than the preliminarily pre-established maximum acceptable bias. RESULTS: all Passing-Bablok regressions exhibited the presence of a proportional and constant systematic error. Bland-Altman analysis revealed biases well above the maximum acceptable bias, so the 25(OH)D concentrations measured were not comparable. To evaluate whether the three methods had the same ability to classify patients into different categories of vitamin D levels, we categorized results obtained by each method in reference classes. Lumipulse categorized most patients into the class with the lowest 25(OH)D concentrations (<20 ng/mL) whereas Elecsys ranked the lowest number. CONCLUSIONS: Liaison XL and Elecsys have shown good accuracy compared to Lumipulse in measuring 25(OH)D levels. Nevertheless, the assays were not interchangeable due to the lack of comparability of results as well as to the disagreement in classification of hormone deficiency or sufficiency.

Outcomes from Experimental Testing of Nonsteroidal Anti-Inflammatory Drug (NSAID) Administration during the Transition Period of Dairy Cows.

During the transition period, dairy cows experience great physiological stress caused by changes in metabolism and in the immune and endocrine systems. A pro-inflammatory state is another difficulty faced by even apparently healthy animals. The most significant negative consequences of inflammation in dairy cows are substantial impairment of milk production and deleterious effects on cows' health in extreme cases. Nonetheless, a certain degree of inflammation is necessary to sustain physiological adaptations. In recent years, many studies have attempted to determine whether the use of nonsteroidal anti-inflammatory drugs (NSAID) in the transition period of dairy cows could positively affect milk production and cows' health by controlling the inflammation status. This literature indicates that NSAIDs that act as preferential inhibitors of cyclooxygenase-1 (COX-1) activity show important side effects (e.g., increased risk of retained placenta, culling, or metritis) even if milk production is, on average, ameliorated. In contrast, preferential inhibitors of cyclooxygenase-2 (COX-2) activity have overall positive effects on cows' health, with potential beneficial effects on milk production. Furthermore, it is important to note that with certain NSAID treatments, milk discarding is mandatory to prevent contamination with drug residues, but increased milk production can compensate for the loss of milk revenue during the withdrawal period.

Reference Ranges for Hematological and Biochemical Profile of Martina Franca Donkeys.

The Martina Franca donkey (MFd) is one of the largest Italian donkey breeds, considered as endangered breed. To support the conservation strategies, knowledge about the physiologic hematological parameters of MFds is needed. The aims of the study were to determine reference value for hematological and major serum parameters in a population of healthy MFds and to estimate the influence of age on these parameters. Eighty-one clinically healthy MFds (17 males and 64 females) in different ages were enrolled: group A (foals, n° 16, animals < 1 year old) group B (young animals, n° 36, from 1 to 3 years old), and group C (adult animals, n° 29, over 3 years old). Red blood cell count (RBC); hematocrit value (HCT); hemoglobin concentration (HGB); mean corpuscular volume (MCV); mean corpuscular hemoglobin (MCH); hemoglobin concentration distribution width (HDW); RBC distribution width (RDW); total white blood cell (WBC); WBC differential count for neutrophils, lymphocytes, monocytes, eosinophils and basophils, and platelets (PLT); mean platelet volume (MPV); platelet volume distribution width; and plateletcrit (PCT) were analyzed. The biochemistry panel included aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), total serum protein (TP), albumin (ALB), cholesterol (CHOL), triglyceride (TGL), blood urea nitrogen (BUN), creatinine (CREA), glucose (GLU), Ca2+, phosphorus, Na+, Cl-, K+, and Mg2+. The effect of age on hematological parameters was investigated using one-way ANOVA test. Age of donkeys does not influence total WBC, HGB, HCT, platelet count and MPV, and PCT (P > 0.05). Some leukocyte populations such as eosinophils, monocytes, and basophils showed age-linked variations (P < 0.05). RBC count, RDW, and HDW decrease with age whereas MCV and MCH increase. Na+, K+, Cl-, Ca2+, phosphorus, ALP, GGT, CREA, GLUC, and CHOL decrease with age (P < 0.05), while AST and TP showed an increase with aging (P < 0.05). ALB reaches the lowest values in young donkeys and returns to values of foals in older animals (P < 0.05). Finally, a difference among groups for BUN and TGL was not found (P < 0.05). The results suggest how even for the MFd breed, age is a variable that affects different hematological and biochemical parameters. Compared to other donkey and horses, the MFd breed showed some differences that clinicians involved during conservation strategies need to be consider.

Pegbovigrastim Treatment around Parturition Enhances Postpartum Immune Response Gene Network Expression of whole Blood Leukocytes in Holstein and Simmental Cows.

Pegbovigrastim is a commercial long-acting analog of bovine granulocyte colony-stimulating factor (rbG-CSF) that promotes the increased count and functionality of polymorphonuclear cells in dairy cows around the time of parturition. We hypothesized that pegbovigrastim administered to periparturient cows at approximately seven days before parturition and within 24 hours after calving could affect the profiles of gene networks involved in leukocyte function. Blood was collected on Day 3 after calving from treated groups (pegbovigrastim (PEG); 13 Simmental (seven multiparous and six primiparous) and 13 Holstein (seven multiparous and six primiparous) cows) that received pegbovigrastim (Imrestor; Elanco Animal Health) and controls (CTR; 13 Simmental (seven multiparous and six primiparous) and 13 Holstein (six multiparous and seven primiparous) cows) that received saline solution. Blood from all cows was sampled from the jugular vein in a PAXgene Blood RNA System tube (Preanalytix, Hombrechtikon, Switzerland) for RNA extraction. The RT-qPCR analysis was performed to investigate a panel of 34 genes of interest, representing recognition, immune mediation, migration, cell adhesion, antimicrobial strategies, inflammatory cascade, oxidative pattern, and leukotrienes in whole blood leukocytes. Normalized data were subjected to the MIXED model of SAS (ver. 9.4) with treatment, breed, parity, and their interaction as fixed effects. Compared with CTR, whole blood leukocytes of PEG cows had higher expression of genes involved in recognition and immune modulation (CD14, CD16, MYD88, TLR2, and TLR4), cell adhesion (ITGB2, ITGAL, TLN1, SELL, SELPLG, and CD44), antimicrobial activity (MMP9, LTF, and LCN2), and inflammatory cascade (CASP1, TNFRSF1A, IL1B, IL1R, IL18, IRAK1, NLRP3, and S100A8). This suggested an improvement of migration, adhesion, and antimicrobial ability and an enhanced inflammatory response, which in turn could trigger immune cell activation and enhance function. Expression of SOD2 and ALOX5 was also greater in the PEG group. In contrast, compared with CTR cows, PEG led to lower expression of RPL13A, ALOX15, IL8, and TNF. Overall, leukocytes from Simmental compared with Holstein cows had greater expression of IDO1, RPL13A, ALOX5, CD44, CX3CR1, ITGB2, and TNFA, whereas expression of CD16 and TLR2 was lower. Overall, compared with multiparous cows, primiparous cows had higher expression of IL1B, IL18, MYD88, SELL, and TLR2 and lower expression of MMP9. Simmental cows seemed more sensitive to induction of the immune system after calving, as revealed by the greater abundance of genes involved in immune system adaptation, regardless of pegbovigrastim treatment. Primiparous cows undergoing a new stress condition with respect to older cows were characterized by leukocytes with a higher inflammatory response. In conclusion, pegbovigrastim led to higher expression levels of most genes involved in the processes investigated, suggesting a thorough activation of the immune machinery during the critical post-partum period.

Evaluation of freezing point in milk from buffalos reared in Calabria, Italy.

Evaluation of freezing point is one of the most common technique used to detect milk adulteration such as addition of external water to increase volume. The aim of this study was to evaluate the freezing point of buffalo milk using infrared spectroscopy and to assess how it is influenced by other milk components. A total of 361 individual buffalo milk samples were collected monthly from March to August of 2017 in a dairy farm in Catanzaro district, Italy. Samples were tested for freezing point, urea, acetone and beta-hydroxybutyrate, percent of fat, protein, lactose, casein, by Fourier Transformed Spectroscopy. The pH and daily milk production were also recorded. Freezing point ranged from -0.574°C to - 0.512°C and the mean values was -0.545°C ±0.010. According to lactation stage, freezing point decreased until 210 days post-partum reaching the minimum value of -0.550°C, then it slightly increased during lactation; according to sampling month the highest and lowest values were recorded in August and June, respectively. A positive correlation between freezing point and lactose content were evidenced (r=0.1806, P<0.05). Moreover, a faintly positive correlation was also found between freezing point and beta-idroxibutirrate (r=0.0869, P<0.05) and acetone (r=0.0096, P<0.05), whereas a negative correlation with fat (r=-0.2356, P<0.05), protein (r=-0.1855, P<0.05), casein (r=-0.2127, P<0.05) and urea (r=-0.1229, P<0.05) was evidenced.

Effect of Pegbovigrastim on Hematological Profile of Simmental Dairy Cows during the Transition Period.

Pegbovigrastim is a long-acting analog of recombinant bovine granulocyte colony-stimulating factor, that promotes and increases the count and functionality of polymorphonuclear cells in dairy cows. The present study aimed to explore, for the first time in Simmental cows, the clinical and hematological effect of pegbovigrastim during the transition period (TP). Cows were randomly assigned into two groups: treated group (PEG; n = 16) received pegbovigrastim at approximately 7 days before expected parturition and within 6 h after calving, and control group (CTR; n = 16) received saline solution. Blood samples were obtained at -7, 0, 1, 3, 7, 14, 21, and 30 days relative to calving. PEG group showed white blood cells (WBC) count consistently higher compared with CTR group (p < 0.001) until to 3 weeks after calving. Neutrophils remained higher in PEG group (p < 0.001) up to three weeks after calving, compared with CTR group, with slight increment of band cells. Moreover, PEG group displayed a lower index of myeloperoxidase at 1, 3, and 7 days after calving (p < 0.01) compared with CTR. Basophils and lymphocytes showed a similar trend to those observed for neutrophils at 1 day after calving in PEG group. Finally, monocytes remained markedly elevated until 3 days after calving in PEG compared to CTR group (p < 0.001), whereas in PEG group, eosinophils population showed lower percentage values at 1 and 3 days after calving but higher values at 30 days compared with CTR group. PEG group was characterized by lower red blood cells (RBCs) count compared with CTR group (p < 0.05) and higher % of red cell volume distribution width (RDW) from week 2 and mean corpuscular volume (MCV) at 30 days after calving. In addition, the mean platelet volume (MPV) was significantly higher in PEG group at calving, 1, 3, and 7 days after calving compared with CTR group (p < 0.05). For the first time, we described the effect of pegbovigrastim in a breed not specialized exclusively in milk production as Holstein, but with dual purpose (meat and milk), evaluating the complete hematological profile in cows during the transition period. These results provide evidence on the proliferative effect of pegbovigrastim on WBC in Simmental breed highlighting its possible side effect on RBCs.

Bergamot Polyphenols Boost Therapeutic Effects of the Diet on Non-Alcoholic Steatohepatitis (NASH) Induced by "Junk Food": Evidence for Anti-Inflammatory Activity.

Wrong alimentary behaviors and so-called "junk food" are a driving force for the rising incidence of non-alcoholic fatty liver disease (NAFLD) among children and adults. The "junk food" toxicity can be studied in "cafeteria" (CAF) diet animal model. Young rats exposed to CAF diet become obese and rapidly develop NAFLD. We have previously showed that bergamot (Citrus bergamia Risso et Poiteau) flavonoids, in the form of bergamot polyphenol fraction (BPF), effectively prevent CAF diet-induced NAFLD in rats. Here, we addressed if BPF can accelerate therapeutic effects of weight loss induced by a normocaloric standard chow (SC) diet. 21 rats fed with CAF diet for 16 weeks to induce NAFLD with inflammatory features (NASH) were divided into three groups. Two groups were switched to SC diet supplemented or not with BPF (CAF/SC±BPF), while one group continued with CAF diet (CAF/CAF) for 10 weeks. BPF had no effect on SC diet-induced weight loss, but it accelerated hepatic lipid droplets clearance and reduced blood triglycerides. Accordingly, BPF improved insulin sensitivity, but had little effect on leptin levels. Interestingly, the inflammatory parameters were still elevated in CAF/SC livers compared to CAF/CAF group after 10 weeks of dietary intervention, despite over 90% hepatic fat reduction. In contrast, BPF supplementation decreased hepatic inflammation by reducing interleukin 6 (Il6) mRNA expression and increasing anti-inflammatory Il10, which correlated with fewer Kupffer cells and lower inflammatory foci score in CAF/SC+BPF livers compared to CAF/SC group. These data indicate that BPF mediates a specific anti-inflammatory activity in livers recovering from NASH, while it boosts lipid-lowering and anti-diabetic effects of the dietary intervention.

Rapid capillary electrophoresis approach for the quantification of ewe milk adulteration with cow milk.

The substitution of ewe milk with more economic cow milk is a common fraud. Here we present a capillary electrophoresis method for the quantification of ewe milk in ovine/bovine milk mixtures, which allows for the rapid and inexpensive recognition of ewe milk adulteration with cow milk. We utilized a routine CE method for human blood and urine proteins analysis, which fulfilled the separation of skimmed milk proteins in alkaline buffer. Under this condition, ovine and bovine milk exhibited a recognizable and distinct CE protein profiles, with a specific ewe peak showing a reproducible migration zone in ovine/bovine mixtures. Based on ewe specific CE peak, we developed a method for ewe milk quantification in ovine/bovine skimmed milk mixtures, which showed good linearity, precision and accuracy, and a minimum amount of detectable fraudulent cow milk equal to 5%.

Oral Administration of Oleuropein and Its Semisynthetic Peracetylated Derivative Prevents Hepatic Steatosis, Hyperinsulinemia, and Weight Gain in Mice Fed with High Fat Cafeteria Diet.

The high consumption of olive tree products in the Mediterranean diet has been associated with a lower incidence of metabolic disorders and cardiovascular diseases. In particular, the protective effects of olive oil have been attributed to the presence of polyphenols such as oleuropein (Ole) and its derivatives. We have synthesized a peracetylated derivative of Ole (Ac-Ole) which has shown in vitro antioxidant and growth-inhibitory activity higher than the natural molecule. In this study, male C57BL/6JOlaHsd mice were fed with a standard (std), cafeteria (caf) diet, and caf diet supplemented with Ole (0.037 mmol/kg/day) and Ac-Ole (0.025 mmol/kg/day) for 15 weeks. We observed a significant reduction in the caf diet-induced body weight gain and increase of abdominal adipose tissue. Also, Ole and Ac-Ole prevented the development of hepatic steatosis. Finally, Ole and Ac-Ole determined a lower increase of HDL and LDL-cholesterol levels and corrected caf diet-induced elevation of plasma glucose concentrations by improving insulin sensitivity. The observed beneficial properties of Ole and Ac-Ole make these compounds and in particular Ac-Ole promising candidates for a potential pharmaceutic use in metabolic disorders.

Proteomics-driven analysis of ovine whey colostrum.

The aim of this study was to shed light in to the complexity of the ovine colostrum proteome, with a specific focus on the low abundance proteins. The ovine colostrum is characterized by a few dominating proteins, as the immunoglobulins, but it also contains less represented protein species, equally important for the correct development of neonates. Ovine colostrum, collected immediately after lambing, was separated by 1D SDS-PAGE. Proteins bands were digested with trypsin and the resulting peptides were analyzed by LC-MS/MS. On the basis of the Swiss-Prot database, a total of 343 unique proteins were identified. To our knowledge, this study represents the most comprehensive analysis of ovine colostrum proteome.

Bergamot polyphenol fraction prevents nonalcoholic fatty liver disease via stimulation of lipophagy in cafeteria diet-induced rat model of metabolic syndrome.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in industrialized countries. Defective autophagy of lipid droplets (LDs) in hepatocytes, also known as lipophagy, has recently been identified as a possible pathophysiological mechanism of NAFLD. Experimental and epidemiological evidence suggests that dietary polyphenols may prevent NAFLD. To address this hypothesis and analyze the underlying mechanisms, we supplemented bergamot polyphenol fraction (BPF) to cafeteria (CAF) diet-fed rats, a good model for pediatric metabolic syndrome and NAFLD. BPF treatment (50 mg/kg/day supplemented with drinking water, 3 months) potently counteracted the pathogenic increase of serum triglycerides and had moderate effects on blood glucose and obesity in this animal model. Importantly, BPF strongly reduced hepatic steatosis as documented by a significant decrease in total lipid content (-41.3% ± 12% S.E.M.), ultrasound examination and histological analysis of liver sections. The morphometric analysis of oil-red stained sections confirmed a dramatic reduction in LDs parameters such as total LD area (48.5% ± 15% S.E.M.) in hepatocytes from CAF+BPF rats. BPF-treated livers showed increased levels of LC3 and Beclin 1 and reduction of SQSTM1/p62, suggesting autophagy stimulation. Consistent with BPF stimulation of lipophagy, higher levels of LC3II were found in the LD subcellular fractions of BPF-expose livers. This study demonstrates that the liver and its lipid metabolism are the main targets of bergamot flavonoids, supporting the concept that supplementation of BPF is an effective strategy to prevent NAFLD.

Inhibition of HIV-1 replication in primary human monocytes by the IkappaB-alphaS32/36A repressor of NF-kappaB.

BACKGROUND: The identification of the molecular mechanisms of human immunodeficiency virus type 1, HIV-1, transcriptional regulation is required to develop novel inhibitors of viral replication. NF-kappaB transacting factors strongly enhance the HIV/SIV expression in both epithelial and lymphoid cells. Controversial results have been reported on the requirement of NF-kappaB factors in distinct cell reservoirs, such as CD4-positive T lymphocytes and monocytes. We have previously shown that IkappaB-alphaS32/36A, a proteolysis-resistant inhibitor of NF-kappaB, potently inhibits the growth of HIV-1 and SIVmac239 in cell cultures and in the SIV macaque model of AIDS. To further extend these observations, we have generated NL(AD8)IkappaB-alphaS32/36A, a macrophage-tropic HIV-1 recombinant strain endowed to express IkappaB-alphaS32/36A. RESULTS: In this work, we show that infection with NL(AD8)IkappaB-alphaS32/36A down-regulated the NF-kappaB DNA binding activity in cells. NL(AD8)IkappaB-alphaS32/36A was also highly attenuated for replication in cultures of human primary monocytes. CONCLUSIONS: These results point to a major requirement of NF-kappaB activation for the optimal replication of HIV-1 in monocytes and suggest that agents which interfere with NF-kappaB activity could counteract HIV-1 infection of monocytes-macrophages in vivo.

IkappaB-alpha represses the transcriptional activity of the HIV-1 Tat transactivator by promoting its nuclear export.

The long terminal repeat of human immunodeficiency virus, type 1 (HIV-1) contains an NF-kappaB enhancer and is potently inhibited by IkappaB-alphaS32/36A, a proteolysis-resistant inhibitor of NF-kappaB transacting factors. The evidence that NF-kappaB is dispensable for HIV-1 expression raises the question of whether IkappaB-alpha represses the HIV-1 transcription by mechanisms distinct from NF-kappaB inhibition. Here, we report that IkappaB-alpha negatively regulates the HIV-1 expression and replication in an NF-kappaB-independent manner by directly binding to Tat, which results in the nuclear export and cytoplasmic sequestration of the viral transactivator. The sequence of IkappaB-alpha required for Tat inhibition spans from amino acids 72 to 287 and includes the nuclear localization signal, the carboxyl-terminal nuclear export signal, and the binding site for the arginine-rich domain of Tat. This novel mechanism of cross-talk between Tat and IkappaB-alpha provides further insights into the mechanisms of HIV-1 regulation and could assist in the development of novel strategies for AIDS therapy.

Regulation of the HIV promoter/enhancer.

This unit describes adaptations of two molecular techniques that can be used to study the regulation of HIV expression. The first two protocols describe the chloramphenicol acetyltransferase (CAT) assay, in which the CAT reporter gene is attached to an HIV-1 promoter and CAT activity is measured as an indication of the promoter's activity. The basic protocol is rapid, simple, and suited to analyzing multiple samples. An alternate protocol describes an assay for CAT function that involves separating the reaction products by thin-layer chromatography (TLC). The second basic protocol describes an electrophoretic mobility shift assay for detecting proteins present in cell extracts that can bind to the HIV-1 LTR (long terminal repeat). Such studies are central to current HIV research because it is important to know what agents induce and inhibit (or "down-regulate") HIV transcription.