Quantification of whey proteins by reversed phase-HPLC and effectiveness of mid-infrared spectroscopy for their rapid prediction in sweet whey

In the dairy industry, membrane filtration, is used to reduce the amount of whey waste and, simultaneously, to recover whey proteins (WP). The composition of WP can strongly affect the filtration treatment of whey, and rapid determination of WP fractions would be of interest for dairy producers to monitor WP recovery. This study aimed to develop mid-infrared spectroscopy (MIRS) prediction models for the rapid quantification of protein in sweet whey, using a validated rapid reversed phase (RP)-HPLC as a reference method. Quantified WP included alpha-lactalbumin (alpha-LA), beta-lactoglobulin (beta-LG) A and B, bovine serum albumin, caseinomacropeptides, and proteose peptone. Validation of RP-HPLC was performed by calculating the relative standard deviation (RSD) in repeatability and reproducibility tests for WP retention time and peak areas. Samples of liquid whey (n = 187) were analyzed by RP-HPLC and scanned through MIRS to collect spectral information (900 to 4,000 cm(-1)); statistical analysis was carried out through partial least squares regression and random cross-validation procedure. Retention times in RP-HPLC method were stable (RSD between 0.03 and 0.80%), whereas the RSD of peak area (from 0.25 to 8.48%) was affected by WP relative abundance. Higher coefficients of determination in validation for MIRS model were obtained for protein fractions present in Whey in large amounts, such as beta-Lc (0.58), total identified WP (0.58), and alpha-LA (0.56). Results of this study suggest that MIRS is an easy method for rapid quantification of detail protein in sweet whey, even if better resolution was achieved with the method based on RP-HPLC. The prediction of WP in sweet whey by MIRS might be used for screening and for classifying sweet whey according to its total and individual WP contents

Senescence promotes in vivo reprogramming through p16INK4a and IL-6

Cellular senescence is a damage response aimed to orchestrate tissue repair. We have recently reported that cellular senescence, through the paracrine release of interleukin-6 (IL6) and other soluble factors, strongly favors cellular reprogramming by Oct4, Sox2, Klf4, and c-Myc (OSKM) in nonsenescent cells. Indeed, activation of OSKM in mouse tissues triggers senescence in some cells and reprogramming in other cells, both processes occurring concomitantly and in close proximity. In this system, Ink4a/Arf-null tissues cannot undergo senescence, fail to produce IL6, and cannot reprogram efficiently; whereas p53-null tissues undergo extensive damage and senescence, produce high levels of IL6, and reprogram efficiently. Here, we have further explored the genetic determinants of in vivo reprogramming. We report that Ink4a, but not Arf, is necessary for OSKM-induced senescence and, thereby, for the paracrine stimulation of reprogramming. However, in the absence of p53, IL6 production and reprogramming become independent of Ink4a, as revealed by the analysis of Ink4a/Arf/p53 deficient mice. In the case of the cell cycle inhibitor p21, its protein levels are highly elevated upon OSKM activation in a p53-independent manner, and we show that p21-null tissues present increased levels of senescence, IL6, and reprogramming. We also report that Il6-mutant tissues are impaired in undergoing reprogramming, thus reinforcing the critical role of IL6 in reprogramming. Finally, young female mice present lower efficiency of in vivo reprogramming compared to male mice, and this gender difference disappears with aging, both observations being consistent with the known anti-inflammatory effect of estrogens. The current findings regarding the interplay between senescence and reprogramming may conceivably apply to other contexts of tissue damage.L.M. was recipient of an FPU contract from the Spanish Ministry of Education (MECD). Work in the laboratory of M.S. was funded by the CNIO and by grants from the Spanish Ministry of Economy co-funded by the European Regional Development Fund (RETOS project), the European Research Council (ERC Advanced Grant), the European Union (RISK-IR project), and the Botin Foundation and Banco Santander (Santander Universities Global Division). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Effect of microparticulated whey proteins on milk coagulation properties

The enhancement of milk coagulation properties (MCP) and the reuse of whey produced by the dairy industry are of great interest to improve the efficiency of the cheese-making process. Native whey proteins (WP) can be aggregated and denatured to obtain colloidal microparticulated WP (MWP). The objective of this study was to assess the effect of MWP on MCP; namely, rennet coagulation time (RCT), curd-firming time, and curd firmness 30 min after rennet addition. Six concentrations of MWP (vol/vol; 1.5, 3.0, 4.5, 6.0, 7.5, and 9.0%) were added to 3 bulk milk samples (collected and analyzed during 3 d), and a sample without MWP was used as control. Within each day of analysis, 6 replicates of MCP for each treatment were obtained, changing the position of the treatment in the rack. For control samples, 2 replicates per day were performed. In addition to MCP, WP fractions were measured on each treatment during the 3 d of analysis. Milk coagulation properties were measured on 144 samples by using a Formagraph (Foss Electric, Hillerød, Denmark). Increasing the amount of MWP added to milk led to a longer RCT. In particular, significant differences were found between RCT of the control samples (13.5 min) and RCT of samples with 3.0% (14.6 min) or more MWP. A similar trend was observed for curd-firming time, which was shortest in the control samples and longest in samples with 9.0% MWP (21.4 min). No significant differences were detected for curd firmness at 30 min across concentrations of MWP. Adjustments in cheese processing should be made when recycling MWP, in particular during the coagulation process, by prolonging the time of rennet activity before cutting the curd

The lipopolysaccharide from Escherichia coli O127:B8 induces inflammation and motility disturbances in rabbit ileum

[EN] The aim of this work was to evaluate the effects of lipopolysaccharide (LPS) from Escherichia coli O127:B8 on the expression of toll-like receptor 4 (TLR4), the histology, and motor function in rabbit ileum. Rabbits were injected intravenously with saline or LPS (100 μg/kg, 2 h). The mRNA expression and localization of TLR4 were determined by reverse transcriptase-PCR and immunofluorescence, respectively. Histological damage induced by LPS was evaluated in sections of ileum stained with haematoxylin and eosin. Contractility studies of ileum were performed in an organ bath. The mRNA expression of TLR4 decreased in the muscular but not in the mucosal layer of rabbits treated with LPS. TLR4 was localised in both the mucosal and muscular layers of rabbit ileum. LPS induced intestinal inflammation and altered the spontaneous contractions and the serotonin-, acetylcholine- and KCl-induced contractions. In conclusion, LPS from E. coli O127:B8 induced a decrease in the mRNA expression of TLR4, an inflammatory response, and changes in the contractility of rabbit ileum.This work was funded by Gobierno de Aragón and European Regional Development Fund ERDF (B61/2012), Spain.Grasa, L.; Gonzalo, S.; De Martino, A.; Murillo, MD. (2017). The lipopolysaccharide from Escherichia coli O127:B8 induces inflammation and motility disturbances in rabbit ileum. World Rabbit Science. 25(2):185-191. https://doi.org/10.4995/wrs.2017.5160SWORD18519125

Involvement of fractalkine and macrophage inflammatory protein-1 alpha in moderate-severe depression.

Moderate-severe depression (MSD) is linked to overexpression of proinflammatory cytokines and chemokines. Fractalkine (FKN) and macrophage inflammatory protein-1 alpha (MIP-1alpha) are, respectively, members of CX3C and C-C chemokines, and both are involved in recruiting and activating mononuclear phagocytes in the central nervous system. We analysed the presence of FKN and MIP-1alpha in sera of untreated MSD patients and healthy donors. High FKN levels were observed in all MSD patients as compared with values only detectable in 26% of healthy donors. MIP-1alpha was measurable in 20% of patients, while no healthy donors showed detectable chemokine levels. In conclusion, we describe a previously unknown involvement of FKN in the pathogenesis of MSD, suggesting that FKN may represent a target for a specific immune therapy of this disease

Plk1 overexpression induces chromosomal instability and suppresses tumor development

Polo-like kinase 1 (Plk1) is overexpressed in a wide spectrum of human tumors, being frequently considered as an oncogene and an attractive cancer target. However, its contribution to tumor development is unclear. Using a new inducible knock-in mouse model we report here that Plk1 overexpression results in abnormal chromosome segregation and cytokinesis, generating polyploid cells with reduced proliferative potential. Mechanistically, these cytokinesis defects correlate with defective loading of Cep55 and ESCRT complexes to the abscission bridge, in a Plk1 kinase-dependent manner. In vivo, Plk1 overexpression prevents the development of Kras-induced and Her2-induced mammary gland tumors, in the presence of increased rates of chromosome instability. In patients, Plk1 overexpression correlates with improved survival in specific breast cancer subtypes. Therefore, despite the therapeutic benefits of inhibiting Plk1 due to its essential role in tumor cell cycles, Plk1 overexpression has tumor-suppressive properties by perturbing mitotic progression and cytokinesis.We are indebted to Stephen Taylor for the Sgo1 antibody. We thank Simone Kraut, Jessica Steiner, and the DKFZ light microscopy unit for excellent technical assistance. The results published here are in part based on data generated by TCGA pilot project (https://cancergenome.nih.gov/established by the NCI and the National Human Gen- ome Research Institute. The data were retrieved through dbGaP authorization (accession no. phs000178.v9.p8). S.V.V. was supported by the Marie Curie Network Ploidynet, funded by the European Union Seventh Framework Programme (FP7/2007–2013) under Grant Agreement #316964. L.S. is supported by a postdoctoral fellowship from Funda- cion Ramon Areces. Work in the R.S. laboratory was supported by an ERC starting grant (#281614), Marie Curie PCIG09-GA-2011 –293745 and the Howard Hughes Medical Institute. G.d.C. is funded by AECC Scientific Foundation (LABAE16017DECA). Work in the M.M. laboratory was supported by grants from the MINECO (SAF2015 –69920-R cofunded by ERDF-EU), Worldwide Cancer Research (WCR no. 150278), and Comunidad de Madrid (iLUNG-CM; B2017/BMD3884). The CNIO is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0510).S

Self-organized arrays of dislocations in thin smectic liquid crystal films

International audienceCombining optical microscopy, synchrotron X-ray diffraction and ellipsometry, we studied the internal structure of linear defect domains (oily streaks) in films of smectic liquid crystal 8CB with thickness 100-300 nm confined between air and a rubbed PVA polymer substrate which impose hybrid anchoring conditions (normal and unidirectional planar, respectively). We show how the presence or absence of dislocations control the structure of highly deformed thin smectic films. Each domain contains smectic layers curved in the shape of flattened hemicylinders to satisfy both anchoring conditions, together with grain boundaries whose size and shape are controlled by the presence of dislocation lines. A flat grain boundary normal to the interface connects neighboring hemicylinders, while a rotating grain boundary (RGB) is located near the axis of curvature of the cylinders. The RGB shape appears such that dislocation lines are concentrated at its summit close to the air interface. The smectic layers reach the polymer substrate via a transition region where the smectic layer orientation satisfies the planar anchoring condition over the entire polymer substrate and whose thickness does not depend on the one of the film. The strength of the planar anchoring appears to be high, larger than 10 −2 J/m 2 , compensating for the high energy cost of creating an additional 2D defect between an horizontal smectic layer and perpendicular ones. This 2D defect may be melted, in order to avoid the creation of a transition region structure composed of a large number of dislocations. As a result, linear defect domains can be considered as arrays of oriented defects, straight dislocations of various Burger vectors, whose location is now known and 2D nematic defects. The possibility of easy variation between the present structure with a moderate amount of dislocations and a structure with a large number of dislocations is also demonstrated

Biocompatibility, Inflammatory Response, and Recannalization Characteristics of Nonradioactive Resin Microspheres: Histological Findings

Intra-arterial radiotherapy with yttrium-90 microspheres (radioembolization) is a therapeutic procedure exclusively applied to the liver that allows the direct delivery of high-dose radiation to liver tumors, by means of endovascular catheters, selectively placed within the tumor vasculature. The aim of the study was to describe the distribution of spheres within the precapillaries, inflammatory response, and recannalization characteristics after embolization with nonradioactive resin microspheres in the kidney and liver. We performed a partial embolization of the liver and kidney vessels in nine white pigs. The left renal and left hepatic arteries were catheterized and filled with nonradioactive resin microspheres. Embolization was defined as the initiation of near-stasis of blood flow, rather than total occlusion of the vessels. The hepatic circulation was not isolated so that the effects of reflux of microspheres into stomach could be observed. Animals were sacrificed at 48 h, 4 weeks, and 8 weeks, and tissue samples from the kidney, liver, lung, and stomach evaluated. Microscopic evaluation revealed clusters of 10–30 microspheres (15–30 μm in diameter) in the small vessels of the kidney (the arciform arteries, vasa recti, and glomerular afferent vessels) and liver. Aggregates were associated with focal ischemia and mild vascular wall damage. Occlusion of the small vessels was associated with a mild perivascular inflammatory reaction. After filling of the left hepatic artery with microspheres, there was some evidence of arteriovenous shunting into the lungs, and one case of cholecystitis and one case of marked gastritis and ulceration at the site of arterial occlusion due to the presence of clusters of microspheres. Beyond 48 h, microspheres were progressively integrated into the vascular wall by phagocytosis and the lumen recannalized. Eight-week evaluation found that the perivascular inflammatory reaction was mild. Liver cell damage, bile duct injury, and portal space fibrosis were not observed. In conclusion, resin microspheres (15–30 μm diameter) trigger virtually no inflammatory response in target tissues (liver and kidney). Clusters rather than individual microspheres were associated with a mild to moderate perivascular inflammatory reaction. There was no evidence of either a prolonged inflammatory reaction or fibrosis in the liver parenchyma following recannalization

Plasma extracellular vesicles reveal early molecular differences in amyloid positive patients with early-onset mild cognitive impairment

In the clinical course of Alzheimer's disease (AD) development, the dementia phase is commonly preceded by a prodromal AD phase, which is mainly characterized by reaching the highest levels of Aβ and p-tau-mediated neuronal injury and a mild cognitive impairment (MCI) clinical status. Because of that, most AD cases are diagnosed when neuronal damage is already established and irreversible. Therefore, a differential diagnosis of MCI causes in these prodromal stages is one of the greatest challenges for clinicians. Blood biomarkers are emerging as desirable tools for pre-screening purposes, but the current results are still being analyzed and much more data is needed to be implemented in clinical practice. Because of that, plasma extracellular vesicles (pEVs) are gaining popularity as a new source of biomarkers for the early stages of AD development. To identify an exosome proteomics signature linked to prodromal AD, we performed a cross-sectional study in a cohort of early-onset MCI (EOMCI) patients in which 184 biomarkers were measured in pEVs, cerebrospinal fluid (CSF), and plasma samples using multiplex PEA technology of Olink© proteomics. The obtained results showed that proteins measured in pEVs from EOMCI patients with established amyloidosis correlated with CSF p-tau181 levels, brain ventricle volume changes, brain hyperintensities, and MMSE scores. In addition, the correlations of pEVs proteins with different parameters distinguished between EOMCI Aβ( +) and Aβ(-) patients, whereas the CSF or plasma proteome did not. In conclusion, our findings suggest that pEVs may be able to provide information regarding the initial amyloidotic changes of AD. Circulating exosomes may acquire a pathological protein signature of AD before raw plasma, becoming potential biomarkers for identifying subjects at the earliest stages of AD development