Influence of rumen protein degradability on productive and reproductive performance in buffalo cows

The present study aimed to ascertain the influence of crude protein (CP) digestibility in the rumen on the quantity and quality of milk production and reproductive performance, blood (BU) and milk (MU) urea, haematological profile and vaginal mucus urea, ammonia and potassium of buffalo cows. Lactating buffaloes (n = 84), 60 days in milk, were randomly subdivided into Group C (control, n = 42) and Group T (fed a diet supplemented with Aspergillus oryzae, n = 42). In three fistulated buffalo, the diet supplemented with Aspergillus oryzae showed a decrease (P < 0.01) in protein digestibility in the rumen (79.3 vs. 45.9%). No differences were registered in productive performance. Nine buffaloes not in oestrus during the dietary treatment (Groups T1 and C1), 30 days in milk, were used to study the haematological profile and to determine milk urea and ammonia in the vaginal mucus. The animals in Group T1 had higher ammonia values in the blood (P < 0.01) but not in the vaginal mucus than Group C1. A relationship was found between MU and BU. MU was influenced by CP intake and dry matter intake. No differences between the treatments were observed in reproductive performance and the conception rate and calving interval were 37.9% and 41.4% (90 trial-day) and 449 and 419 days respectively in Groups T and C. Reproductive performance was not influenced by high levels of BU nor by blood ammonia levels, although the latter were higher in the group fed the diet supplemented with Aspergillus oryzae.     

Quantitative determination of anti-fungal and insecticide amides in adult plants, plantlets and callus from Piper tuberculatum by reverse-phase high-performance liquid chromatography

A rapid, sensitive and reliable reverse-phase HPLC method was used for the quantitative determination of the anti-fungal and insecticide amides, dihydropiplartine (1), piplartine (2), deltaalpha,beta-dihydropiperine (3) and pellitorine (4) in plants in natura, in plantlets in vitro and ex vitro, and in callus of Piper tuberculatum. Well-resolved peaks were obtained with good detection response and linearity in the range of 15.0-3000 microg/mL. The plants in natura contained compounds 1-4, the plantlets ex vitro and in vitro accumulated compounds 1-2 and 1-4, respectively, while only amide 4 was found in callus.     

Involvement of ezrin/moesin in de novo actin assembly on phagosomal membranes

The current study focuses on the molecular mechanisms responsible for actin assembly on a defined membrane surface: the phagosome. Mature phagosomes were surrounded by filamentous actin in vivo in two different cell types. Fluorescence microscopy was used to study in vitro actin nucleation/polymerization (assembly) on the surface of phagosomes isolated from J774 mouse macrophages. In order to prevent non-specific actin polymerization during the assay, fluorescent G-actin was mixed with thymosin beta4. The cytoplasmic side of phagosomes induced de novo assembly and barbed end growth of actin filaments. This activity varied cyclically with the maturation state of phagosomes, both in vivo and in vitro. Peripheral membrane proteins are crucial components of this actin assembly machinery, and we demonstrate a role for ezrin and/or moesin in this process. We propose that this actin assembly process facilitates phagosome/endosome aggregation prior to membrane fusion.     

(T2AG3)n Telomeric Sequence Hybridization Suggestive of Centric Fusion in Karyotype Marsupials Evolution

It has been suggested that the karyotype of the marsupials derived from a low diploid number (2n = 14) which originated, through fissions of biarmed chromosomes, the karyotypes with a higher 2n. The telomeric sequence (T₂AG₃)_nwas in situhybridized to the chromosomes of Gracilinanus microtarsusand G. emiliae, Micoureus demeraraeand Marmosa murina, species with 2n = 14, in Monodelphissp., M. domestica, M. kunsiand M. brevicaudatawith 2n = 18, and in Lutreolina crassicaudata, Didelphis albiventris, Chironectes minimus, Philander opossumand P. frenata, all of them with 2n = 22. The probe hybridization occurred in the telomeric regions of both arms, short and long, of all chromosomes of the complement of all individuals of all species analysed. However, in some pairs of the karyotypes of Gracilinanus microtarsusand Micoureus demerarae(with 2n = 14), and in Monodelphissp., M. domestica, M. kunsiand M. brevicaudata(2n = 18) ectopic signs of hybridization were detected proximal to the centromeres, suggesting the retention of this telomeric sequence in the centromeric regions of some chromosomes of these species. Based on these results, it is proposed that the karyotype of marsupials evolved from a 2n = 22 to a 2n = 14, by means of chromosomal fusions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Palmitoylethanolamide Treatment Reduces Blood Pressure in Spontaneously Hypertensive Rats: Involvement of Cytochrome P450-Derived Eicosanoids and Renin Angiotensin System

Palmitoylethanolamide (PEA), a peroxisome proliferator-activated receptor-α agonist, has been demonstrated to reduce blood pressure and kidney damage secondary to hypertension in spontaneously hypertensive rat (SHR). Currently, no information is available concerning the putative effect of PEA on modulating vascular tone. Here, we investigate the mechanisms underpinning PEA blood pressure lowering effect, exploring the contribution of epoxyeicosatrienoic acids, CYP-dependent arachidonic acid metabolites, as endothelium-derived hyperpolarizing factors (EDHF), and renin angiotensin system (RAS) modulation. To achieve this aim SHR and Wistar-Kyoto rats were treated with PEA (30 mg/kg/day) for five weeks. Functional evaluations on mesenteric bed were performed to analyze EDHF-mediated vasodilation. Moreover, mesenteric bed and carotid were harvested to measure CYP2C23 and CYP2J2, the key isoenzymes in the formation of epoxyeicosatrienoic acids, and the soluble epoxide hydrolase, which is responsible for their degradation in the corresponding diols. Effect of PEA on RAS modulation was investigated by analyzing angiotensin converting enzyme and angiotensin receptor 1 expression. Here, we showed that EDHF-mediated dilation in response to acetylcholine was increased in mesenteric beds of PEA-treated SHR. Western blot analysis revealed that the increase in CYP2C23 and CYP2J2 observed in SHR was significantly attenuated in mesenteric beds of PEA-treated SHR, but unchanged in the carotids. Interestingly, in both vascular tissues, PEA significantly decreased the soluble epoxide hydrolase protein level, accompanied by a reduced serum concentration of its metabolite 14-15 dihydroxyeicosatrienoic acid, implying a reduction in epoxyeicosatrienoic acid hydrolisis. Moreover, PEA treatment down-regulated angiotensin receptor 1 and angiotensin converting enzyme expression, indicating a reduction in angiotensin II-mediated effects. Consistently, a damping of the activation of angiotensin receptor 1 underlying pathways in mesenteric beds was shown in basal conditions in PEA-treated SHR. In conclusion, our data demonstrate the involvement of epoxyeicosatrienoic acids and renin angiotensin system in the blood pressure lowering effect of PEA.     

Development and Validation of a High-Performance Liquid Chromatography–Tandem Mass Spectrometry Method for the Simultaneous Determination of Irinotecan and Its Main Metabolites in Human Plasma and Its Application in a Clinical Pharmacokinetic Study

Irinotecan is currently used in several cancer regimens mainly in colorectal cancer (CRC). This drug has a narrow therapeutic range and treatment can lead to side effects, mainly neutropenia and diarrhea, frequently requiring discontinuing or lowering the drug dose. A wide inter-individual variability in irinotecan pharmacokinetic parameters and pharmacodynamics has been reported and associated to patients’ genetic background. In particular, a polymorphism in the UGT1A1 gene (UGT1A1*28) has been linked to an impaired detoxification of SN-38 (irinotecan active metabolite) to SN-38 glucuronide (SN-38G) leading to increased toxicities. Therefore, therapeutic drug monitoring of irinotecan, SN-38 and SN-38G is recommended to personalize therapy. In order to quantify simultaneously irinotecan and its main metabolites in patients’ plasma, we developed and validated a new, sensitive and specific HPLC–MS/MS method applicable to all irinotecan dosages used in clinic. This method required a small plasma volume, addition of camptothecin as internal standard and simple protein precipitation. Chromatographic separation was done on a Gemini C18 column (3 μM, 100 mm x 2.0 mm) using 0.1% acetic acid/bidistilled water and 0.1% acetic acid/acetonitrile as mobile phases. The mass spectrometer worked with electrospray ionization in positive ion mode and selected reaction monitoring. The standard curves were linear (R² ≥0.9962) over the concentration ranges (10–10000 ng/mL for irinotecan, 1–500 ng/mL for SN-38 and SN-38G and 1–5000 ng/mL for APC) and had good back-calculated accuracy and precision. The intra- and inter-day precision and accuracy, determined on three quality control levels for all the analytes, were always <12.3% and between 89.4% and 113.0%, respectively. Moreover, we evaluated this bioanalytical method by re-analysis of incurred samples as an additional measure of assay reproducibility. This method was successfully applied to a pharmacokinetic study in metastatic CRC patients enrolled in a genotype-guided phase Ib study of irinotecan administered in combination with 5-fluorouracil/leucovorin and bevacizumab.     

Live Cell Analysis and Mathematical Modeling Identify Determinants of Attenuation of Dengue Virus 2’-O-Methylation Mutant

Dengue virus (DENV) is the most common mosquito-transmitted virus infecting ~390 million people worldwide. In spite of this high medical relevance, neither a vaccine nor antiviral therapy is currently available. DENV elicits a strong interferon (IFN) response in infected cells, but at the same time actively counteracts IFN production and signaling. Although the kinetics of activation of this innate antiviral defense and the timing of viral counteraction critically determine the magnitude of infection and thus disease, quantitative and kinetic analyses are lacking and it remains poorly understood how DENV spreads in IFN-competent cell systems. To dissect the dynamics of replication versus antiviral defense at the single cell level, we generated a fully viable reporter DENV and host cells with authentic reporters for IFN-stimulated antiviral genes. We find that IFN controls DENV infection in a kinetically determined manner that at the single cell level is highly heterogeneous and stochastic. Even at high-dose, IFN does not fully protect all cells in the culture and, therefore, viral spread occurs even in the face of antiviral protection of naïve cells by IFN. By contrast, a vaccine candidate DENV mutant, which lacks 2’-O-methylation of viral RNA is profoundly attenuated in IFN-competent cells. Through mathematical modeling of time-resolved data and validation experiments we show that the primary determinant for attenuation is the accelerated kinetics of IFN production. This rapid induction triggered by mutant DENV precedes establishment of IFN-resistance in infected cells, thus causing a massive reduction of virus production rate. In contrast, accelerated protection of naïve cells by paracrine IFN action has negligible impact. In conclusion, these results show that attenuation of the 2’-O-methylation DENV mutant is primarily determined by kinetics of autocrine IFN action on infected cells.     

Transection Speed and Impact on Perioperative Inflammatory Response – A Randomized Controlled Trial Comparing Stapler Hepatectomy and CUSA Resection

Background

Parenchymal transection represents a crucial step during liver surgery and many different techniques have been described so far. Stapler resection is supposed to be faster than CUSA resection. However, whether speed impacts on the inflammatory response in patients undergoing liver resection (LR) remains unclear.

Materials and Methods

This is a randomized controlled trial including 40 patients undergoing anatomical LR. Primary endpoint was transection speed (cm²/min). Secondary endpoints included the perioperative change of pro- and anti-inflammatory cytokines, overall surgery duration, length of hospital stay, morbidity and mortality.

Results

Mean transection speed was significantly higher in patients undergoing stapler hepatectomy compared to CUSA resection (CUSA: 1 (0.4) cm²/min vs. Stapler: 10.8 (6.1) cm²/min; p<0.0001). Analyzing the impact of surgery duration on inflammatory response revealed a significant correlation between IL-6 levels measured at the end of surgery and the overall length of surgery (p<0.0001, r = 0.6188). Patients undergoing CUSA LR had significantly higher increase of interleukin-6 (IL-6) after parenchymal transection compared to patients with stapler hepatectomy in the portal and hepatic veins, respectively (p = 0.028; p = 0.044). C-reactive protein levels on the first post-operative day were significantly lower in the stapler cohort (p = 0.010). There was a trend towards a reduced overall surgery time in patients with stapler LR, especially in the subgroup of patients undergoing minor hepatectomies (p = 0.020).

Conclusions

Liver resection using staplers is fast, safe and suggests a diminished inflammatory response probably due to a decreased parenchymal transection time.

Trial Registration

ClinicalTrials.gov NCT01785212     

Life-History Traits of the Model Organism Pristionchus pacificus Recorded Using the Hanging Drop Method: Comparison with Caenorhabditis elegans

The nematode Pristionchus pacificus is of growing interest as a model organism in evolutionary biology. However, despite multiple studies of its genetics, developmental cues, and ecology, the basic life-history traits (LHTs) of P. pacificus remain unknown. In this study, we used the hanging drop method to follow P. pacificus at the individual level and thereby quantify its LHTs. This approach allowed direct comparisons with the LHTs of Caenorhabditis elegans recently determined using this method. When provided with 5×10⁹ Escherichia coli cells ml^–1 at 20°C, the intrinsic rate of natural increase of P. pacificus was 1.125 (individually, per day); mean net production was 115 juveniles produced during the life-time of each individual, and each nematode laid an average of 270 eggs (both fertile and unfertile). The mean age of P. pacificus individuals at first reproduction was 65 h, and the average life span was 22 days. The life cycle of P. pacificus is therefore slightly longer than that of C. elegans, with a longer average life span and hatching time and the production of fewer progeny.