4 in 1: Antibody‐free protocol for isolating the main hepatic cells from healthy and cirrhotic single rat livers

Liver cells isolated from pre‐clinical models are essential tools for studying liver (patho)physiology, and also for screening new therapeutic options. We aimed at developing a new antibody‐free isolation method able to obtain the four main hepatic cell types (hepatocytes, liver sinusoidal endothelial cells [LSEC], hepatic macrophages [HMΦ] and hepatic stellate cells [HSC]) from a single rat liver. Control and cirrhotic (CCl₄ and TAA) rat livers (n = 6) were perfused, digested with collagenase and mechanically disaggregated obtaining a multicellular suspension. Hepatocytes were purified by low revolution centrifugations while non‐parenchymal cells were subjected to differential centrifugation. Two different fractions were obtained: HSC and mixed LSEC + HMΦ. Further LSEC and HMΦ enrichment was achieved by selective adherence time to collagen‐coated substrates. Isolated cells showed high viability (80%‐95%) and purity (>95%) and were characterized as functional: hepatocytes synthetized albumin and urea, LSEC maintained endocytic capacity and in vivo fenestrae distribution, HMΦ increased expression of inflammatory markers in response to LPS and HSC were activated upon in vitro culture. The 4 in 1 protocol allows the simultaneous isolation of highly pure and functional hepatic cell sub‐populations from control or cirrhotic single livers without antibody selection.     

A Fast and Economical Sample Preparation Protocol for Interaction Proteomics Analysis

A simple and fast immunoprecipitation (IP) protocol is designed with the sample preparation incorporated, applicable to both low and high throughput. This new protocol combines two procedures based on magnetic beads in 96‐well plate format. Protein complexes are captured by antibodies and magnetic beads conjugated with protein A. Proteins are washed and on‐bead digested by using Single‐Pot solid‐phase sample preparation (SP3). The whole IP‐SP3 approach can be completed in one day, which is considerably faster compared to the classical approach. No major quantitative differences are found between SP3 and FASP (filter‐aided sample preparation) or a longer incubation protocol. Taken together, the IP‐SP3 protocol is a fast and economical approach easily applicable for large‐scale protein interactome analysis.     

Fractionation of Heavy Metals in Mine Tailings Amended with Composted Manure

This study evaluated the effect of composted cow manure (CCM) on the chemical fractionation and retention degree of heavy metals (HMs) in mine tailings from Zimapán, México. In a greenhouse experiment, mine tailings from three deposits were incubated for 3 months; experimental units were placed in a PVC container, where increasing doses of CCM were applied. HM pseudo-total concentrations, HM extractions with ethylenediaminetetraacetic acid (EDTA, 0.05 M), and a sequential chemical extraction (SCE) were carried out. The HM concentrations were determined by atomic absorption spectrophotometry. The pseudo-total concentrations of Pb, Cu, Cd, and Ni found were up to 1506, 206, 27, and 23 mg kg^?1, respectively; extractable Pb was up to 42%; 21% for Cu; 51% for Cd; and 16% of Ni of the pseudo-total concentrations of each metal. Treatment with 12% of CCM in mine tailing decreased EDTA-extractable HM concentrations, while the SCE revealed a decrease in exchangeable fraction and an increase in the organic fraction of HM. A positive correlation between CCM application and organic fractions of HMs was found, although the highest increasements were recorded in the organic fraction.     

Assessing lignocellulosic biomass production from crop residues in the European Union: Modelling,analysis of the current scenario and drivers of interannual variability

This study assesses crop residues in the EU from major crops using empirical models to predict crop residues from yield statistics; furthermore it analyses the inter‐annual variability of those estimates over the period 1998‐2015, identifying its main drivers across Europe. The models were constructed based on an exhaustive collection of experimental data from scientific papers for the crops: wheat, barley, rye, oats, triticale, rice, maize, sorghum, rapeseed, sunflower, soybean, potato and sugarbeet. We discuss the assumptions on the relationship between yield and the harvest index, adopted by previous studies, to interpret the experimental data, quantify the uncertainties of these models, and establish the premises to implement them at regional scale –i.e., NUTS level 3– within the EU. To cope this, we created a consolidated sub‐national statistical data along with an algorithm able to aggregate (figures are provided at country level) and disaggregate (production at 25 km grid is provided assupplementary material) estimates. The total lignocellulosic biomass production in the EU28 over the review period, according to our models, is 419 Mt, from which wheat is the major contributor (155 Mt). Our results show that maize and rapeseed are the two crops with the highest residue yield, respectively 8.9 and 8.6 t ha‐1. The spatial analysis revealed that these three crops, which, according to our results, are feedstocks highly suitable a priori for second generation biofuels in the EU and are unevenly distributed across Europe. Weather fluctuation was identified as the major driver in residue production from cereals, while, in the case of starch crops and oilseeds – which are predominant in northern Europe – corresponded to the marked production trend likely influenced by the agricultural policies and agro‐management over the review period. Our results, among others, could help to understand and quantify the ecological boundaries of the bioeconomy from agriculture.     

Both human and soya bean ferritins highly improve the accumulation of bioavailable iron and contribute to extend the chronological life in budding yeast

Ferritin proteins have an enormous capacity to store iron in cells. In search for the best conditions to accumulate and store bioavailable iron, we made use of a double mutant null for the monothiol glutaredoxins GRX3 and GRX4. The strain grx3grx4 accumulates high iron concentrations in the cytoplasm, making the metal easily available for ferritin chelation. Here, we perform a comparative study between human (L and H) and soya bean ferritins (H1 and H2) function in the eukaryotic system Saccharomyces cerevisiae. We demonstrate that the four human and soya bean ferritin chains are successfully expressed in our model system. Upon coexpression of either both human or soya bean ferritin chains, respiratory conditions along with iron supplementation led us to obtain the maximum yields of iron stored in yeast described to date. Human and soya bean ferritin chains are functional and present equivalent properties as promoters of cell survival in iron overload conditions. The best system revealed that the four human and soya bean ferritins possess a novel function as anti-ageing proteins in conditions of iron excess. In this respect, both ferritin chains with oxidoreductase capacity (human-H and soya bean-H2) bear the highest capacity to extend life suggesting the possibility of an evolutionary conservation.     

The importance of polymerization and galloylation for the antiproliferative properties of procyanidin-rich natural extracts

Grape (Vitis vinifera) and pine (Pinus pinaster) bark extracts are widely used as nutritional supplements. Procyanidin-rich fractions from grape and pine bark extract showing different mean degrees of polymerization, percentage of galloylation (percentage of gallate esters) and reactive oxygen species-scavenging capacity were tested on HT29 human colon cancer cells. We observed that the most efficient fractions in inhibiting cell proliferation, arresting the cell cycle in G(2) phase and inducing apoptosis were the grape fractions with the highest percentage of galloylation and mean degree of polymerization. Additionally, the antiproliferative effects of grape fractions were consistent with their oxygen radical-scavenging capacity and their ability to trigger DNA condensation-fragmentation.     

Nasal Staphylococcus aureus and S. pseudintermedius carriage in healthy dogs and cats: a systematic review of their antibiotic resistance,virulence and genetic lineages of zoonotic relevance

The molecular ecology of Staphylococcus aureus, Staphylococcus pseudintermedius and their methicillin-resistant strains in healthy dogs and cats could serve as good models to understand the concept of bacterial zoonosis due to animal companionship. This study aims to provide insights into pooled prevalence, genetic lineages, virulence and antimicrobial resistance (AMR) among healthy dogs and cats. Original research and brief communication articles published from 2001 to 2021 that reported the nasal detection of S. aureus and S. pseudintermedius in healthy dogs and cats in the community, homes and outside veterinary clinics were examined and analysed. Forty-nine studies were eligible and included in this systematic review. The pooled prevalence of nasal carriage of S. aureus/methicillin-resistant S. aureus (MRSA) in healthy dogs and cats were 10.9% (95% CI: 10.1–11.9)/2.8% (95% CI: 2.4–3.2) and 3.2% (95% CI: 1.9–4.8)/0.5% (95% CI: 0.0–1.1), respectively. Conversely, the pooled prevalence of S. pseudintermedius/methicillin-resistant S. pseudintermedius (MRSP) in healthy dogs and cats were 18.3% (95% CI: 17.1–19.7)/3.1% (95% CI: 2.5–3.7) and 1.3% (95% CI: 0.6–2.4)/1.2% (95% CI: 0.6–2.3), respectively. Although highly diverse genetic lineages of S. aureus were detected in healthy dogs and cats, MSSA-CC1/CC5/CC22/CC45/CC121/CC398 and MRSA-CC5/CC93/CC22/CC30 were mostly reported in dogs; and MSSA-CC5/CC8/CC15/CC48 and MRSA-CC22/CC30/CC80 in cats. Of note, MSSA-CC398 isolates (spa-types t034 and t5883) were detected in dogs. Genetic lineages often associated with MSSP/MRSP were ST20/ST71, highlighting the frequent detection of the epidemic European MRSP-ST71 clone in dogs. S. aureus isolates carrying the luk-S/F-PV, tst, eta, etb and etd genes were seldomly detected in dogs, and luk-S/F-PV was the unique virulence factor reported in isolates of cats. S. pseudintermedius isolates harbouring the luk-S/F-I, seint and expA genes were frequently found, especially in dogs. High and diverse rates of AMR were noted, especially among MRSA/MRSP isolates. There is a need for additional studies on the molecular characterization of isolates from countries with under-studied nasal staphylococci isolates.     

Hormonal Profiling Reveals a Hormonal Cross-Talk During Fruit Decay in Sweet Cherries

Journal of Plant Growth Regulation - Although fruit decay in sweet cherries has been studied in some detail, information is still scarce about the possible role of phytohormones during postharvest....