Millet-based supplement restored gut microbial diversity of acute malnourished pigs.

The tight association between malnutrition and gut microbiota (GM) dysbiosis enables microbiota-targeting intervention to be a promising strategy. Thus, we used a malnourished pig model to investigate the host response and GM alterations under different diet supplementation strategies. Pigs at age of 4 weeks were fed with pure maize diet to induce malnutrition symptoms, and followed by continuous feeding with maize (Maize, n = 8) or re-feeding using either corn-soy-blend (CSB+, n = 10) or millet-soy-blend based (MSB+, n = 10) supplementary food for 3 weeks. Meanwhile, 8 pigs were fed on a standard formulated ration as control (Ref). The effect of nutritional supplementation was assessed by the growth status, blood chemistry, gastrointestinal pathology, mucosal microbiota composition and colon production of short-chain fatty acids. Compared with purely maize-fed pigs, both CSB+ and MSB+ elevated the concentrations of total protein and globulin in blood. These pigs still showed most malnutrition symptoms after the food intervention period. MSB+ had superior influence on the GM development, exhibiting better performance in both structural and functional aspects. MSB+ pigs were colonized by less Proteobacteria but more Bacteroidetes, Firmicutes and Lachnospira spp. Pearson's correlation analysis indicated a strong correlation between the abundance of mucosal e.g., Faecalibacterium and Lachnospira spp. and body weight, crown-rump length and total serum protein. In conclusion, the malnutrition symptoms were accompanied by an aberrant GM, and millet-based nutritional supplementation showed promising potentials to restore the reduced GM diversity implicated in pig malnutrition.

Ghrelin and LEAP-2: Rivals in Energy Metabolism.

Liver-expressed antimicrobial peptide 2 (LEAP-2), the endogenous noncompetitive allosteric antagonist of the growth hormone secretagogue receptor 1a (GHSR1a), was recently identified as a key endocrine factor regulating systemic energy metabolism. This antagonist impairs the ability of ghrelin to activate GHSR1a and diminishes ghrelin-induced Ca2+ release in vitro. The physiological relevance of the molecular LEAP-2-GHSR1a interaction was subsequently demonstrated in vivo. LEAP-2 is therefore a promising therapeutic target in the treatment of obesity and other metabolic diseases. Here, we discuss not only the current understanding of LEAP-2 in metabolic regulation, but also the potential of this peptide in the treatment of obesity and other diseases that involve dysregulation of the ghrelin system.

Lipid oxidation inactivates the anticoagulant function of protein Z-dependent protease inhibitor (ZPI).

Lipid oxidation due to oxidative stress plays an important role in the pathogenesis of inflammatory and thrombotic cardiovascular diseases. Several findings suggest that lipid peroxidation can alter the function of coagulation proteins and contribute to a hypercoagulable state, but the molecular mechanisms are unclear. Here, we report that oxidized phospholipids suppress the anticoagulant function of the serpin, protein Z-dependent protease inhibitor (ZPI), a specific inhibitor of membrane-associated factor Xa (FXa) that requires protein Z (PZ), phospholipid, and calcium as cofactors. We found that this suppression arises from a diminished ability of the oxidized membrane to function as a cofactor to promote ZPI inhibition of membrane-bound FXa, due fully or in part to the susceptibility of the bound ZPI-PZ complex to oxidative inactivation. Surprisingly, free ZPI was also susceptible to inactivation by oxidized membrane vesicles in the absence of calcium. Oxidized vesicles containing both phosphatidylserine and polyunsaturated fatty acids were required to promote inactivation of the ZPI-PZ complex or free ZPI, indicating that binding of the PZ-complexed or free ZPI to peroxide-modified phospholipid vesicles mediates the inactivation. Heparin protected the ZPI-PZ complex and free ZPI from inactivation, suggesting that blocking the heparin-binding site on ZPI interferes with ZPI binding to lipid or to PZ. This was confirmed by direct lipid-binding experiments. Native PAGE indicated that oxidization induced dissociation of the ZPI-PZ complex and increased the negative charge of ZPI. We conclude that compromised ZPI anticoagulant function could contribute to thrombus initiation and growth in oxidative stress-induced cardiovascular diseases.

Suppressive effect of CORM-2 on LPS-induced platelet activation by glycoprotein mediated HS1 phosphorylation interference.

In recent years, it has been discovered that septic patients display coagulation abnormalities. Platelets play a major role in the coagulation system. Studies have confirmed that carbon monoxide (CO) has important cytoprotective and anti-inflammatory function. However, whether CO could alter abnormal activation of platelets and coagulation and thereby reduce the incidence of mortality during sepsis has not been defined. In this report, we have used CO-releasing molecules (CORM-2) to determine whether CO inhibits LPS-induced abnormal activation of platelets and have explored the potential mechanisms. LPS was used to induce activation of platelets in vitro, which were purified from the peripheral venous blood of healthy adult donors. CORM-2 was applied as a potential therapeutic agent. CORM-2 preconditioning and delayed treatment were also studied. We found that in the LPS groups, the function of platelets such as spreading, aggregation, and release were enhanced abnormally. By contrast, the platelets in the CORM-2 group were gently activated. Further studies showed that the expression of platelet membrane glycoproteins increased in the LPS group. Coincidently, both hematopoietic lineage cell-specific protein 1 and its phosphorylated form also increased dramatically. These phenomena were less dramatically seen in the CORM-2 groups. Taken together, we conclude that during LPS stimulation, platelets were abnormally activated, and this functional state may be associated with the signal that is transmitted between membrane glycoproteins and HS1. CORM-released CO suppresses the abnormal activation of platelets by interfering with glycoprotein-mediated HS1 phosphorylation.

Secretion of heparin-binding protein from human neutrophils is determined by its localization in azurophilic granules and secretory vesicles.

Human neutrophils have an important role in host defense against microbial infection. At different stages of an infectious process, neutrophils progressively up-regulate receptors and release various effector molecules. These are stored in several distinct types of granules with varying propensity to be secreted. Heparin-binding protein (HBP), also known as CAP37 or azurocidin, is a multifunctional, inactive serine-protease homologue. The present work shows that HBP is released from neutrophils on stimulation with secretagogues that do not trigger the secretion of azurophilic granule content. Therefore, the subcellular localization of HBP was investigated in more detail. Immunofluorescence microscopy revealed that HBP was localized close to the plasma membrane. Further analysis by fractionation of postnuclear supernatants from cavitated neutrophils showed that HBP is stored in azurophilic granules and secretory vesicles but that it is also detected to a minor extent in the plasma membrane. These findings were confirmed by immunoelectron microscopy showing that HBP colocalized with marker proteins of azurophilic granules and secretory vesicles. The presence of HBP in secretory vesicles possibly depends on the stage of cell differentiation, since the promyelocytic cell line HL-60 contains less HBP than mature neutrophils, stored exclusively in the less easily mobilized azurophilic granules. Our findings suggest that HBP can be synthesized or targeted to easily mobilized compartments at a late stage of neutrophil maturation. The ability of neutrophils to secrete HBP from secretory vesicles may be important for proinflammatory functions of this protein, such as the alteration of vascular permeability.

Eosinophils maintain their capacity to signal and release eosinophil cationic protein upon repetitive stimulation with the same agonist.

Eosinophils contain in their granules eosinophil cationic protein (ECP) and other basic proteins that have been implicated in immunity to parasites and pathophysiology of chronic allergic responses. In a model of eosinophil degranulation, we show that eosinophils release ECP upon short-term GM-CSF priming and stimulation with either platelet-activating factor (PAF) or the anaphylatoxin C5a, but not eotaxin. Restimulation with the same agonist (PAF or C5a) was unsuccessful as assessed by monitoring intracellular calcium concentration and ECP release. In contrast, upon an intermediate washing step, eosinophils rapidly transduced PAF and C5a signals followed by significant ECP releases. Ligand-binding studies demonstrated that only a proportion of PAF receptors is internalized upon cell stimulation and that washing of the cells removes the agonist from the cell surface. Upon repetitive stimulation, eosinophils with less than 50% of the original ECP content were obtained. Such eosinophils did not increase cellular ECP levels even in the presence of the eosinophil survival factor GM-CSF in overnight cultures. In vivo studies revealed that eosinophils always express detectable amounts of ECP under chronic inflammatory conditions. In conclusion, we have shown that eosinophils maintain their capacity to degranulate upon repetitive stimulation with the same agonist as long as the receptor is not occupied from a previous stimulation. The cellular content of ECP appears to be a no limiting factor in the case of repetitive stimulation, implying that mature eosinophils may not require a significant ECP resynthesis.