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Bactericidal permeability-increasing protein (BPI) is a multifunctional cationic protein produced by neutrophils, eosinophils, fibroblasts, and macrophages with antibacterial anti-inflammatory properties. In the context of Gram-negative infection, BPI kills bacteria, neutralizes the endotoxic activity of lipopolysaccharides (LPSs), and, thus, avoids immune hyperactivation. Interestingly, BPI increases in patients with Gram-positive meningitis, interacts with lipopeptides and lipoteichoic acids of Gram-positive bacteria, and significantly enhances the immune response in peripheral blood mononuclear cells. We evaluated the antimycobacterial and immunoregulatory properties of BPI in human macrophages infected with Mycobacterium tuberculosis . Our results showed that recombinant BPI entered macrophages, significantly reduced the intracellular growth of M. tuberculosis , and inhibited the production of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-α). Furthermore, BPI decreased bacterial growth directly in vitro. These data suggest that BPI has direct and indirect bactericidal effects inhibiting bacterial growth and potentiating the immune response in human macrophages and support that this new protein's broad-spectrum antibacterial activity has the potential for fighting tuberculosis.

Mitochondria and peroxisomes are independent but functionally closely related organelles. A few proteins have been characterized as dual-organelle locating proteins with distinct or similar roles on mitochondria and peroxisomes. MARCH5 is a mitochondria-associated ubiquitin ligase best known for its regulatory role in mitochondria quality control, fission, and fusion. Here, we used a proximity tagging system, PUP-IT, and identified new interacting proteins of MARCH5. Our data uncover that MARCH5 is a dual-organelle locating protein that interacts with several peroxisomal proteins. PEX19 binds the transmembrane region on MARCH5 and targets it to peroxisomes. On peroxisomes, MARCH5 binds and mediates the ubiquitination of PMP70. Furthermore, we find PMP70 ubiquitination and pexophagy induced by mTOR inhibition are blocked in the absence of MARCH5. Our study suggests novel roles of MARCH5 on peroxisomes., (© 2021 Zheng et al.)

Seizure threshold 2 (SZT2) is a component of the KICSTOR complex which, under catabolic conditions, functions as a negative regulator in the amino acid-sensing branch of mTORC1. Mutations in this gene cause a severe neurodevelopmental and epileptic encephalopathy whose main symptoms include epilepsy, intellectual disability, and macrocephaly. As SZT2 remains one of the least characterized regulators of mTORC1, in this work we performed a systematic interactome analysis under catabolic and anabolic conditions. Besides numerous mTORC1 and AMPK signaling components, we identified clusters of proteins related to autophagy, ciliogenesis regulation, neurogenesis, and neurodegenerative processes. Moreover, analysis of SZT2 ablated cells revealed increased mTORC1 signaling activation that could be reversed by Rapamycin or Torin treatments. Strikingly, SZT2 KO cells also exhibited higher levels of autophagic components, independent of the physiological conditions tested. These results are consistent with our interactome data, in which we detected an enriched pool of selective autophagy receptors/regulators. Moreover, preliminary analyses indicated that SZT2 alters ciliogenesis. Overall, the data presented form the basis to comprehensively investigate the physiological functions of SZT2 that could explain major molecular events in the pathophysiology of developmental and epileptic encephalopathy in patients with SZT2 mutations.

Liver-expressed antimicrobial peptide 2 (LEAP-2), originally described as an antimicrobial peptide, has recently been recognized as an endogenous blocker of growth hormone secretagogue receptor 1a (GHS-R1a). GHS-R1a, also known as ghrelin receptor, is a G protein-coupled receptor (GPCR) widely distributed on the hypothalamus and pituitary gland where it exerts its major functions of regulating appetite and growth hormone (GH) secretion. The activity of GHS-R1a is controlled by two counter-regulatory endogenous ligands: Ghrelin (activation) and LEAP-2 (inhibition). Ghrelin activates GHS-R1a on the neuropeptide Y/Agouti-related protein (NPY/AgRP) neurons at the arcuate nucleus (ARC) to promote appetite, and on the pituitary somatotrophs to stimulate GH release. On the flip side, LEAP-2, acts both as an endogenous competitive antagonist of ghrelin and an inverse agonist of constitutive GHS-R1a activity. Such a biological property of LEAP-2 vigorously blocks ghrelin's effects on food intake and hormonal secretion. In circulation, LEAP-2 displays an inverse pattern as to ghrelin; it increases with food intake and obesity (positive energy balance), whereas decreases upon fasting and weight loss (negative energy balance). Thus, the LEAP-2/ghrelin molar ratio fluctuates in response to energy status and modulation of this ratio conversely influences energy intake. Inhibiting ghrelin's activity has shown beneficial effects on obesity in preclinical experiments, which sheds light on LEAP-2's anti-obesity potential. In this review, we will analyze LEAP-2's effects from a metabolic point of view with a focus on metabolic hormones (e.g., ghrelin, GH, and insulin), and discuss LEAP-2's potential as a promising therapeutic target for obesity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lu, Huang, Huang, Feng, Clark and Chen.)

We examined the effects of mTOR inhibitors on the fibrotic response induced by transforming growth factor-beta2 (TGF-β2) in cultured human trabecular meshwork (hTM) cells. TGF-β2-induced expression of fibronectin, collagen type I, alpha 1 chain (COL1A1), and alpha-smooth muscle actin (αSMA) in hTM cells was examined in the presence or absence of mTOR inhibitors using quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry. The migration rates of hTM cells were examined in the presence of TGF-β2 with or without mTOR inhibitors. An in vitro study showed that the expression of fibronectin, COL1A1, and αSMA was upregulated by TGF-β2 treatment of hTM cells; such upregulation was significantly suppressed by mTOR inhibitors. The inhibitors significantly reduced the migration rate of TGF-β2-stimulated hTM cells. mTOR inhibitors may usefully reduce the fibrotic response of hTM cells and we may have to explore if it is also effective in in vivo model.

Both weak survival ability of stem cells and hostile microenvironment are dual dilemma for cell therapy. Adropin, a bioactive substance, has been demonstrated to be cytoprotective. We therefore hypothesized that adropin may produce dual protective effects on the therapeutic potential of stem cells in myocardial infarction by employing an adropin-based dual treatment of promoting stem cell survival in vitro and modifying microenvironment in vivo. In the current study, adropin (25 ng/ml) in vitro reduced hydrogen peroxide-induced apoptosis in rat bone marrow mesenchymal stem cells (MSCs) and improved MSCs survival with increased phosphorylation of Akt and extracellular regulated protein kinases (ERK) l/2. Adropin-induced cytoprotection was blocked by the inhibitors of Akt and ERK1/2. The left main coronary artery of rats was ligated for 3 or 28 days to induce myocardial infarction. Bromodeoxyuridine (BrdU)-labeled MSCs, which were in vitro pretreated with adropin, were in vivo intramyocardially injected after ischemia, following an intravenous injection of 0.2 mg/kg adropin (dual treatment). Compared with MSCs transplantation alone, the dual treatment with adropin reported a higher level of interleukin-10, a lower level of tumor necrosis factor-α and interleukin-1β in plasma at day 3, and higher left ventricular ejection fraction and expression of paracrine factors at day 28, with less myocardial fibrosis and higher capillary density, and produced more surviving BrdU-positive cells at day 3 and 28. In conclusion, our data evidence that adropin-based dual treatment may enhance the therapeutic potential of MSCs to repair myocardium through paracrine mechanism via the pro-survival pathways.

Chemotherapy-induced peripheral neuropathy (CIPN) is a severe dose-limiting side effect of taxanes such as paclitaxel and docetaxel. Despite the high medical needs, insufficient understanding of the complex mechanism underlying CIPN pathogenesis precludes any endorsed causal therapy to prevent or relieve CIPN. In this study, we report that elevation of plasma galectin-3 level is a pathologic change common to both patients with taxane-treated breast cancer with CIPN and a mouse model of taxane-related CIPN. Following multiple intraperitoneal injections of paclitaxel in mice, galectin-3 levels were elevated in Schwann cells within the sciatic nerve but not in other peripheral organs or cells expressing galectin-3. Consistent with this, paclitaxel treatment of primary cultures of rat Schwann cells induced upregulation and secretion of galectin-3. In vitro migration assays revealed that recombinant galectin-3 induced a chemotactic response of the murine macrophage cell line RAW 264.7. In addition, perineural administration of galectin-3 to the sciatic nerve of naive mice mimicked paclitaxel-induced macrophage infiltration and mechanical hypersensitivity. By contrast, chemical depletion of macrophages by clodronate liposomes suppressed paclitaxel-induced mechanical hypersensitivity despite the higher level of plasma galectin-3. Deficiency ( Galectin-3 -/- mice) or pharmacologic inhibition of galectin-3 inhibited paclitaxel-induced macrophage infiltration and mechanical hypersensitivity. In conclusion, we propose that Schwann cell-derived galectin-3 plays a pronociceptive role via macrophage infiltration in the pathogenesis of taxane-induced peripheral neuropathy. Therapies targeting this phenomenon, which is common to patients with CIPN and mouse models, represent a novel approach to suppress taxane-related CIPN. SIGNIFICANCE: These findings demonstrate that the elevation of plasma galectin-3 is a CIPN-related pathologic change common to humans and mice, and that targeting galectin-3 is a therapeutic option to delay CIPN progression., (©2021 American Association for Cancer Research.)

Diabetes mellitus (DM) is a hyperglycemia-related multifactorial condition with an elevated risk of microvascular and microvascular complications associated with this disease. The current experimental study was to examine the antidiabetic activity of streptozotocin (STZ)-induced adropin against diabetic rats by altering the PI3K/Akt and insulin signaling pathways. STZ (60 mg/kg) was used for the induction of DM and rats were divided into different groups and received the adropin (20, 40 and 80 mg/kg) and glibenclamide (10 mg/kg) till 28 days. Body weight, plasma insulin, blood glucose and food intake were estimated, respectively. Biochemical enzymes, carbohydrate enzymes, lipid parameters, AMPK and insulin signalling pathway parameters were estimated. GLUT4 and PPARγ expression were also estimated. Oral administration of adropin significantly (p < 0.001) increased the glycogen, glucose-6-phosphatase dehydrogenase, insulin, hexokinase and belittled the blood glucose level, fructose 1-6-biphosphatase, glucose-6-phosphatase at dose dependent manner. Adropin significantly (p < 0.001) reduced the level of triglyceride, cholesterol, low density lipoprotein, very low density lipoprotein and increased the level of high density lipoprotein at dose dependent manner. Adropin significantly (p < 0.001) activated the Akt, IRS-2, IRS-1, IR, p-AKT and PI3k, which are the key modulator molecules of PI3K/Akt, AMPK and insulin signalling pathway in DM rats. The current experimental study confirms the anti-diabetic effect of adropin on DM rats induced by AMPK and insulin signalling pathway against STZ.

Chicken blood has limited utilization despite its high protein content. Production of a blood hydrolysate exhibiting angiotensin I-converting enzyme (ACE)-inhibitory activity would be means of valorizing chicken blood. The optimized conditions used to produce chicken blood corpuscle hydrolysate (BCH) by Alcalase were 51.1°C, 4% enzyme, and pH 9.6 for 6 h, resulting in a 35.8% degree of hydrolysis and 37.7% ACE inhibition at a peptide concentration of 0.2 mg/mL. The permeate of a 1-kDa membrane, BCH-III, showed a 2.5-fold increase in ACE inhibition compared with that of BCH. BCH-III was resistant to in vitro gastrointestinal digestion, whereas the BCH digesta exhibited an increased ACE-inhibitory activity after digestion. Both BCH and BCH-III were rich in hydrophobic amino acids. A single administration of BCH and BCH-III to spontaneously hypertensive rats at concentrations of 600 and 100 mg/kg, respectively, lowered the systolic blood pressure by -57.7 and -70.9 mmHg, respectively, 6 h after oral administration compared with the control group. The blood pressure-lowering effect of the 600 mg/kg BCH dose was comparable with that of the 100 mg/kg BCH-III dose after 4 wk of oral administration. Both BCH and BCH-III could be developed for use as nutraceutical products with antihypertensive effects., (Copyright © 2020. Published by Elsevier Inc.)

Dietary supplementation with spray-dried porcine plasma (SDP) can modulate the immune response of gut-associated lymphoid tissue. SDP supplementation reduces acute mucosal inflammation, as well as chronic inflammation associated with aging. The aim of this study was to analyze if SDP supplementation could ameliorate colitis in a genetic mouse model of inflammatory bowel disease (IBD). Wild-type mice and Mdr1a knockout (KO) mice were administered a control diet or an SDP-supplemented diet from day 21 (weaning) until day 56. The histopathological index, epithelial barrier, and intestinal immune system were analyzed in the colonic mucosa. KO mice had higher epithelial permeability, increased Muc1 and Muc4 expression, and lower abundance of E-cadherin and Muc2 (all p < 0.001). SDP prevented these effects (all p < 0.05) and decreased the colonic inflammation observed in KO mice, reducing neutrophil and monocyte infiltration and activation and the percentage of activated T helper lymphocytes in the colonic mucosa (all p < 0.05). SDP also diminished proinflammatory cytokine expression and increased the anti-inflammatory IL-10 concentration in the colonic mucosa (all p < 0.05). In conclusion, dietary supplementation with SDP enhances colon barrier function and reduces mucosal inflammation in a mouse model of IBD.

Response regulators are a critical part of the two-component system of gene expression regulation in bacteria, transferring a signal from a sensor kinase into DNA binding activity resulting in alteration of gene expression. In this study, we investigated a previously uncharacterized response regulator in Francisella novicida , FTN&#95;1452 that we have named BfpR (Biofilm-regulating Francisella protein Regulator, FTN&#95;1452 ). In contrast to another Francisella response regulator, QseB/PmrA, BfpR appears to be a negative regulator of biofilm production, and also a positive regulator of antimicrobial peptide resistance in this bacterium. The protein was crystallized and X-ray crystallography studies produced a 1.8 Å structure of the BfpR N-terminal receiver domain revealing interesting insight into its potential interaction with the sensor kinase. Structural analysis of BfpR places it in the OmpR/PhoP family of bacterial response regulators along with WalR and ResD. Proteomic and transcriptomic analyses suggest that BfpR overexpression affects expression of the critical Francisella virulence factor iglC, as well as other proteins in the bacterium. We demonstrate that mutation of bfpR is associated with an antimicrobial peptide resistance phenotype, a phenotype also associated with other response regulators, for the human cathelicidin peptide LL-37 and a sheep antimicrobial peptide SMAP-29. F. novicida with mutated bfpR replicated better than WT in intracellular infection assays in human-derived macrophages suggesting that the down-regulation of iglC expression in bfpR mutant may enable this intracellular replication to occur. Response regulators have been shown to play important roles in the regulation of bacterial biofilm production. We demonstrate that F. novicida biofilm formation was highly increased in the bfpR mutant, corresponding to altered glycogen synthesis. Waxworm infection experiments suggest a role of BfpR as a negative modulator of iglC expression with de-repression by Mg 2+ . In this study, we find that the response regulator BfpR may be a negative regulator of biofilm formation, and a positive regulator of antimicrobial peptide resistance in F. novicida ., (Copyright © 2020 Dean, Milton, Cavanagh and van Hoek.)

Adropin is a peptide hormone which is produced in brain and peripheral tissues such as liver. It was found that adropin modulates lipid and glucose homeostasis by interacting with hepatocytes and myocytes. Adropin enhances insulin sensitivity and alleviates hyperinsulinemia in animal models with high-fat diet-induced insulin resistance. However, it is unknown whether adropin regulates insulin secretion and proliferation of beta cells. Therefore, we studied the effects of adropin on insulin secretion in INS-1E cells as well as isolated pancreatic islets. Furthermore, we assessed the influence of adropin on insulin mRNA expression, cell viability and proliferation in INS-1E cells. Pancreatic islets were isolated from male Wistar rats. mRNA expression was evaluated using real-time PCR and cell viability by MTT assay. Cell replication was measured by BrdU incorporation and insulin secretion by RIA. We found that adropin suppresses insulin mRNA expression in INS-1E cells. Moreover, adropin attenuates glucose-induced insulin secretion in INS-1E cells as well as in isolated pancreatic islets. In addition, using INS-1E cells we found that adropin suppresses glucose-induced cAMP production. However, adropin fails to modulate INS-1E cell viability and proliferation. In summary, we found adropin suppresses insulin mRNA expression and secretion, without affecting beta cell viability or proliferation.

Myticin C is the most studied antimicrobial peptide in the marine mussel Mytilus galloprovincialis . Although it is constitutively expressed in mussel hemocytes and displays antibacterial, antiviral, and chemotactic functions, recent work has suggested that this molecule is mainly activated after tissue injury. Therefore, the main objective of this work was to characterize the hemocytes' transcriptomic response after a myticin C treatment, in order to understand the molecular changes induced by this cytokine-like molecule. The transcriptome analysis revealed the modulation of genes related to cellular movement, such as myosin, transgelin, and calponin-like proteins, in agreement with results of functional assays, where an implication of myticin C in the in vitro activation of hemocytes and migration was evidenced. This was also observed in vivo after a tissue injury, when hemocytes, with high concentrations of myticin C, migrated to the damaged area to heal the wound. All these properties allowed us to think about the biotechnological application of these molecules as wound healers. Human keratinocytes and larvae zebrafish models were used to confirm this hypothesis. Accelerated regeneration after a wound or tail fin amputation was observed after treatment with the myticin C peptide, supporting the chemotactic and healing activity of myticin C., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Serious juvenile oyster disease induced by pathogenic Vibrio splendidus has resulted in tremendous economic loss, but the molecular mechanisms underlying this killing mechanism remain unclear. The resistance of adult oyster to V. splendidus or its virulence factors might provide a possible access to cognize the interaction between pathogen and host. In the present study, the extracellular products (ECP) from less virulent V. splendidus JZ6 were injected into adult Pacific oyster Crassostrea gigas, and the cellular and humoral immune response induced by ECP were investigated. The phagocytosis rate of hemocytes was significantly up-regulated (30.57%) at 6 h after ECP injection compared with that (21%) of control groups. And significantly high level of ROS production was also observed from 3 h to 12 h in ECP-injected oysters, concomitant with increased apoptosis rate of hemocytes (16.4% in ECP-injected group, p < 0.01) compared with control group (6.7%). By RT-PCR analysis, the expression level of antioxidant CgSOD in hemocytes significantly increased to 6.41-fold of that in control groups (p < 0.01) at 12 h post ECP injection. The expression levels of anti-toxic metalloprotease inhibitors CgTIMP629 and CgTIMP628 were also significantly up-regulated at the early (3-6 h) and late (6-24 h) stage of immune response, respectively. Moreover, after the ECP were incubated with serum proteins isolated from the ECP-injected oysters in vitro, the metalloprotease activity of ECP significantly declined by 21.39%, and less degraded serum proteins were detected by SDS-PAGE. When the primarily cultured hemocytes were stimulated with heat-inactivated ECP or fragments derived from ECP-degraded serum proteins, the expressions of CgTIMP629 (13.64 and 7.03-fold of that in saline group, respectively, p < 0.01) and CgTIMP628 (5.07 and 6.08-fold of that in saline group, respectively, p < 0.01) in hemocytes were all significantly induced. All the results indicated that the adult oysters could launch phagocytosis, antioxidant and anti-toxic response to resist the virulence of ECP, possibly by sensing heterologous ECP and ECP-induced endogenous alarm signals. These results provided a possible clue for the resistance mechanism of adult oysters towards the ECP of less virulent V. splendidus, which might be valuable for exploring strategies for the control of oyster disease., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Medicinal plants of Pakistan are known for their curative properties against snake bite as rural people have been using natural herbs for such injuries for hundreds to thousands of years. People of rural areas of Pakistan are prone to snakebite, and on the whole death due to snakebite has been increasing worldwide. The objective of this study was to test the neutralizing potential of 17 Pakistani medicinal plant extracts against phospholipase A2 activity in Echis carinatus venom. Plant material was extracted by simple maceration and fractionation of active plant extracts. Venom was collected by manual massage of the venom glands. The PLA2 enzymatic assay was performed to map out the venomous activity of Echis carinatus envenomation. Snake venom released fatty acids at different concentrations (0.1-5 mg/ml) of venom in a dose-dependent manner. Reduction of pH by 01 correlated with 133 μmol of fatty acids released at 5mg/ml of venom. All plants extract inhibited PLA2 activity, however, Curcuma longa, Citrullus colocynthis and Rubia cordifolia inhibited maximum of PLA2 activity (⁓78%) comparable to the standard antidote (p>0.5). Medicinal plants possess secondary metabolites and many active compounds that may have neutralizing or inhibiting properties against the PLA2 activity of Echis venom. Further studies such as compound analysis could provide an alternative against snakebites injuries resulting from Echis carinatus venom.

Hypochlorous acid (HOCl), a powerful antimicrobial oxidant, is produced by neutrophils to fight infections. Here, we show that N -chlorination, induced by HOCl concentrations encountered at sites of inflammation, converts blood plasma proteins into chaperone-like holdases that protect other proteins from aggregation. This chaperone-like conversion was reversible by antioxidants and was abrogated by prior methylation of basic amino acids. Furthermore, reversible N -chlorination of basic amino acid side chains is the major factor that converts plasma proteins into efficient activators of immune cells. Finally, HOCl-modified serum albumin was found to act as a pro-survival molecule that protects neutrophils from cell death induced by highly immunogenic foreign antigens. We propose that activation and enhanced persistence of neutrophils mediated by HOCl-modified plasma proteins, resulting in the increased and prolonged generation of ROS, including HOCl, constitutes a potentially detrimental positive feedback loop that can only be attenuated through the reversible nature of the modification involved., Competing Interests: AU, AS, AM, NL, LL No competing interests declared, (© 2019, Ulfig et al.)

In the study, the protective effect of plasma protein from Tachypleus tridentatus (PPTT) on acute kidney injury (AKI) and the related molecular mechanisms were first investigated by Western blotting analyses, TdT-mediated dUTP Nick-End Labeling (TUNEL) assay, and immunohistochemistry. It was found that PPTT had an obviously inhibitory effect on Reactive oxygen species (ROS) in cyclophosphamide (CTX)-exposed mice. Furthermore, results demonstrated that the renal cell death mode is due to inducing apoptosis and autophagy inhibited by dose-dependent PPTT in mice treated with CTX by decreasing the protein expression of bax, beclin-1, and LC3 and increasing the expression of bcl-2. Moreover, the p38 MAPK and PI3K/Akt signaling pathways were observed to take part in the PPTT-induced renal cell growth effect by enhancing the upregulation of the expression of Akt and p-Akt as well as the downregulation of the expression of p38 and p-p38, which indicated a PPTT ameliorating effect on AKI CTX-induced in mice through p38 MAPK and PI3K/Akt signaling pathways. Briefly, this article preliminarily studies the mechanism of the PPTT ameliorating effect on AKI CTX-induced in mice, which helps to provide a reference for PPTT clinical application in AKI therapy.

Bleeding complications secondary to surgery, trauma, or coagulation disorders are important causes of morbidity and mortality. Although fibrin sealants are considered to minimize blood loss, this is not widely adopted because of its high cost and/or risk for infection. We present a novel methodology employing nonantibody fibrinogen-binding proteins, termed Affimers, to stabilize fibrin networks with the potential to control excessive bleeding. Two fibrinogen-specific Affimer proteins, F5 and G2, were identified and characterized for their effects on clot structure/fibrinolysis, using turbidimetric and permeation analyses and confocal and electron microscopy. Binding studies and molecular modeling identified interaction sites, whereas plasmin generation assays determined effects on plasminogen activation. In human plasma, F5 and G2 prolonged clot lysis time from 9.8 ± 1.1 minutes in the absence of Affimers to 172.6 ± 7.4 and more than 180 minutes ( P < .0001), respectively, and from 7.6 ± 0.2 to 28.7 ± 5.8 ( P < .05) and 149.3 ± 9.7 ( P < .0001) minutes in clots made from purified fibrinogen. Prolongation in fibrinolysis was consistent across plasma samples from healthy control patients and individuals at high bleeding risk. F5 and G2 had a differential effect on clot structure and G2 profoundly altered fibrin fiber arrangement, whereas F5 maintained physiological clot structure. Affimer F5 reduced fibrin-dependent plasmin generation and was predicted to bind fibrinogen D fragment close to tissue plasminogen activator (tPA; residues γ312-324) and plasminogen (α148-160) binding sites, thus interfering with tPA-plasminogen interaction and representing 1 potential mechanism for modulation of fibrinolysis. Our Affimer proteins provide a novel methodology for stabilizing fibrin networks with potential future clinical implications to reduce bleeding risk., (© 2019 by The American Society of Hematology.)

The use of central venous catheters (CVC) is highly associated with nosocomial blood infections and its use largely requires a systematic assessment of benefits and risks. Bacterial contamination of these tubes is frequent and may result in development of microbial consortia also known as biofilm. The woven nature of biofilm provides a practical defense against antimicrobial agents, facilitating bacterial dissemination through the patient's body and development of antimicrobial resistance. In this work, the authors describe the modification of CVC tubing by immobilizing Fe 3 O 4 -aminosilane core-shell nanoparticles functionalized with antimicrobial peptide clavanin A (clavA) as an antimicrobial prophylactic towards Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. Its anti-biofilm-attachment characteristic relies in clavA natural activity to disrupt the bacterial lipidic membrane. The aminosilane shell prevents iron leaching, which is an important nutrient for bacterial growth. Fe 3 O 4 -clavA-modified CVCs showed to decrease Gram-negative bacteria attachment up to 90% when compared to control clean CVC. Additionally, when hyperthermal treatment is triggered for 5 min at 80 °C in a tubing that already presents bacterial biofilm (CVC-BF), the viability of attached bacteria reduces up to 88%, providing an efficient solution to avoid changing catheter., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Spray-dried plasma protein (SDP) compared with blood meal (BM) may contain various functional and active components that may benefit animal health. The objective of this experiment was to investigate the effects of feeding SDP or BM on production and blood profile in dairy cows during the transition and early-lactation periods. Seventy-two Holstein cows at 14 d before calving were used in a randomized block design. During the prepartum period, cows were fed a typical late-gestation diet containing BM (100 g/cow per day; 100BM, n = 24) or SDP (100 g/cow per day; 100SDP; n = 48). After calving, cows that were fed BM prepartum were fed a typical lactation diet formulated to provide 100 g/d of BM (100BM). Half the cows that were fed 100SDP prepartum were fed a lactation diet formulated to provide 100 g/d of SDP (100SDP; n = 24), and half were fed a diet formulated to provide 400 g/d of SDP (400SDP; n = 24) on a dry matter basis where SDP replaced BM (100SDP) or BM and soybean products (400SDP). All diets were balanced for crude protein concentration and metabolizable protein supply assuming BM and SDP were equal in rumen-degradable protein and rumen-undegradable protein. All data were analyzed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC) as a randomized block design where contrasts were made for 100BM versus 100SDP for prepartum variables and 100BM versus 100SDP and 100SDP versus 400SDP for postpartum variables. Prepartum supplementation of SDP had no effect on plasma fatty acids and β-hydroxybutyrate (2 d before calving). Plasma fatty acids (255 ± 29 µEq/mL) and β-hydroxybutyrate (675 ± 70 µmol/L) at 8 and 14 d of lactation were not affected by SDP in the diet. Feeding SDP at 100 g/d compared with 100BM increased or tended to increase milk fat, protein, and lactose contents for 16 wk after parturition. Providing SDP at 400 g/d in the diet increased milk yield (42 vs. 39 kg/d), energy-corrected milk (44 vs. 41 kg/d), energy-corrected milk per kilogram of dry matter intake, and yields of milk fat (1.60 vs. 1.48 kg/d), protein (1.21 vs. 1.16 kg/d), and lactose compared with 100SDP. Body weight losses tended to be lower for 100SDP compared with 100BM without a difference between 100SDP and 400SDP. Plasma histidine concentration (d 14 of lactation) was lower for SDP compared with 100BM. In addition, plasma 1-methyl-l-histidine tended to be lower as inclusion rate of SDP increased. In conclusion, SDP at 400 g/d increased milk and milk component yields without an increase in feed intake. Studies evaluating effects of functional and active compounds in SDP on gut microbiome, gut health, and immune functions may be needed to determine mode of action., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

This study was aimed to analyze the effects of spray-dried porcine plasma (SDPP) on the health of post weaning piglets challenged with diets contaminated with aflatoxins and fumonisins. Fifty-six male piglets (7.15 ± 0.61 kg) were allocated in four groups: CTL group received a regular diet; SDPP group received a regular diet and 6% SDPP; MYC group received a diet containing 300 µg/kg aflatoxins and 8,000 µg/kg fumonisins; group MYC+SDPP received 300 µg/kg aflatoxins, 8,000 µg/kg fumonisins and 6% SDPP. The animals that received the experimental diet containing mycotoxins (MYC group) had lower weight gain at the end of the experiment compared to the other treatments. Animals receiving SDPP showed decreased urea levels throughout the experiment (P<0.05). Animals from MYC group presented an increased on reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) levels and decreased catalase activity (P<0.05). In contrast, SDPP prevented the increase of ROS and TBARS and stimulated superoxide dismutase activity (P<0.05). In conclusion, diet contaminated with mycotoxins (group MYC) caused subclinical intoxication in the piglets, as observed by the increase on free radical's production and lipid peroxidation. Conversely, SDPP presented a protective effect, minimizing the effects of oxidative stress caused by aflatoxins and fumonisins ingestion.

The elder ( Sambucus spp.) tree has a number of uses in traditional medicine. Previous studies have demonstrated the antimicrobial properties of elderberry liquid extract against human pathogenic bacteria and also influenza viruses. These properties have been mainly attributed to phenolic compounds. However, other plant defense molecules, such as antimicrobial peptides (AMPs), may be present. Here, we studied peptide extracts from flowers of Sambucus nigra L. The mass spectrometry analyses determined peptides of 3 to 3.6 kDa, among them, cysteine-rich peptides were identified with antimicrobial activity against various Gram-negative bacteria, including recurrent pathogens of Chilean aquaculture. In addition, membrane blebbing on the bacterial surface after exposure to the cyclotide was visualized by SEM microscopy and SYTOX Green permeabilization assay showed the ability to disrupt the bacterial membrane. We postulate that these peptides exert their action by destroying the bacterial membrane.

Increased life expectancy has promoted research on healthy aging. Aging is accompanied by increased non-specific immune activation (inflammaging) which favors the appearance of several disorders. Here, we study whether dietary supplementation with spray-dried animal plasma (SDP), which has been shown to reduce the activation of gut-associated lymphoid tissue (GALT) in rodents challenged by S. aureus enterotoxin B (SEB), and can also prevent the effects of aging on immune system homeostasis. We first characterized GALT in a mouse model of accelerated senescence (SAMP8) at different ages (compared to mice resistant to accelerated senescence; SAMR1). Second, we analyzed the SDP effects on GALT response to an SEB challenge in SAMP8 mice. In GALT characterization, aging increased the cell number and the percentage of activated Th lymphocytes in mesenteric lymph nodes and Peyer's patches (all, p < 0.05), as well as the expression of IL-6 and TNF-α in intestinal mucosa (both, p < 0.05). With respect to GALT response to the SEB challenge, young mice showed increased expression of intestinal IL-6 and TNF-α, as well as lymphocyte recruitment and activation (all, p < 0.05). However, the immune response of senescent mice to the SEB challenge was weak, since SEB did not change cell recruitment or the percentage of activated Th lymphocytes. Mice supplemented with SDP showed improved capacity to respond to the SEB challenge, similar to the response of the young mice. These results indicate that senescent mice have an impaired mucosal immune response characterized by unspecific GALT activation and a weak specific immune response. SDP supplementation reduces non-specific basal immune activation, allowing for the generation of specific responses.

Ageing drives changes in neuronal and cognitive function, the decline of which is a major feature of many neurological disorders. The hippocampus, a brain region subserving roles of spatial and episodic memory and learning, is sensitive to the detrimental effects of ageing at morphological and molecular levels. With advancing age, synapses in various hippocampal subfields exhibit impaired long-term potentiation, an electrophysiological correlate of learning and memory. At the molecular level, immediate early genes are among the synaptic plasticity genes that are both induced by long-term potentiation and downregulated in the aged brain. In addition to revitalizing other aged tissues, exposure to factors in young blood counteracts age-related changes in these central nervous system parameters, although the identities of specific cognition-promoting factors or whether such activity exists in human plasma remains unknown. We hypothesized that plasma of an early developmental stage, namely umbilical cord plasma, provides a reservoir of such plasticity-promoting proteins. Here we show that human cord plasma treatment revitalizes the hippocampus and improves cognitive function in aged mice. Tissue inhibitor of metalloproteinases 2 (TIMP2), a blood-borne factor enriched in human cord plasma, young mouse plasma, and young mouse hippocampi, appears in the brain after systemic administration and increases synaptic plasticity and hippocampal-dependent cognition in aged mice. Depletion experiments in aged mice revealed TIMP2 to be necessary for the cognitive benefits conferred by cord plasma. We find that systemic pools of TIMP2 are necessary for spatial memory in young mice, while treatment of brain slices with TIMP2 antibody prevents long-term potentiation, arguing for previously unknown roles for TIMP2 in normal hippocampal function. Our findings reveal that human cord plasma contains plasticity-enhancing proteins of high translational value for targeting ageing- or disease-associated hippocampal dysfunction.

Hematopoietic stem cells (HSCs) are considered one of the most promising therapeutic targets for the treatment of various blood disorders. However, due to difficulties in establishing stable maintenance and expansion of HSCs in vitro, their insufficient supply is a major constraint to transplantation studies. To solve these problems we have developed a fully defined, all-recombinant protein-based culture system. Through this system, we have identified hemopexin (HPX) and interleukin-1α as responsible for HSC maintenance in vitro. Subsequent molecular analysis revealed that HPX reduces intracellular reactive oxygen species levels within cultured HSCs. Furthermore, bone marrow immunostaining and 3D immunohistochemistry revealed that HPX is expressed in non-myelinating Schwann cells, known HSC niche constituents. These results highlight the utility of this fully defined all-recombinant protein-based culture system for reproducible in vitro HSC culture and its potential to contribute to the identification of factors responsible for in vitro maintenance, expansion, and differentiation of stem cell populations., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Mitochondria form close physical associations with the endoplasmic reticulum (ER) that regulate a number of physiological functions. One mechanism by which regions of ER are recruited to mitochondria involves binding of the ER protein VAPB to the mitochondrial protein PTPIP51, which act as scaffolds to tether the two organelles. Here, we show that the VAPB-PTPIP51 tethers regulate autophagy. We demonstrate that overexpression of VAPB or PTPIP51 to tighten ER-mitochondria contacts impairs, whereas small interfering RNA (siRNA)-mediated loss of VAPB or PTPIP51 to loosen contacts stimulates, autophagosome formation. Moreover, we show that expression of a synthetic linker protein that artificially tethers ER and mitochondria also reduces autophagosome formation, and that this artificial tether rescues the effects of siRNA loss of VAPB or PTPIP51 on autophagy. Thus, these effects of VAPB and PTPIP51 manipulation on autophagy are a consequence of their ER-mitochondria tethering function. Interestingly, we discovered that tightening of ER-mitochondria contacts by overexpression of VAPB or PTPIP51 impairs rapamycin- and torin 1-induced, but not starvation-induced, autophagy. This suggests that the regulation of autophagy by ER-mitochondria signaling is at least partly dependent upon the nature of the autophagic stimulus. Finally, we demonstrate that the mechanism by which the VAPB-PTPIP51 tethers regulate autophagy involves their role in mediating delivery of Ca 2+ to mitochondria from ER stores. Thus, our findings reveal a new molecular mechanism for regulating autophagy., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The mechanistic target of Rapamycin (MTOR) protein is a crucial signaling regulator in mammalian cells that is extensively involved in cellular biology. The function of MTOR signaling in keratinocytes remains unclear. In this study, we detected the MTOR signaling and autophagy response in the human keratinocyte cell line HaCaT and human epidermal keratinocytes treated with MTOR inhibitors. Moreover, we detected the impact of MTOR inhibitors on keratinocytes exposed to the common carcinogenic stressors ultraviolet B (UVB) and UVA radiation. As a result, keratinocytes were sensitive to the MTOR inhibitors Rapamycin, everolimus, Torin 1, and pp242, but the regulation of MTOR downstream signaling was distinct. Next, autophagy induction only was observed in HaCaT cells treated with Rapamycin. Furthermore, we found that MTOR signaling was insensitive to UVB but sensitive to UVA radiation. UVB treatment also had no impact on the inhibition of MTOR signaling by MTOR inhibitors. Finally, MTOR inhibition by Rapamycin, everolimus, or pp242 did not affect the series of biological events in keratinocytes exposed to UVB, including the downregulation of BiP and PERK, activation of Histone H2A and JNK, and cleavage of caspase-3 and PARP. Our study demonstrated that MTOR inhibition in keratinocytes cannot always induce autophagy, and the MTOR pathway does not play a central role in the UVB triggered cellular response.

Antibiotic-resistant infections are predicted to kill 10 million people per year by 2050, costing the global economy $100 trillion. Therefore, there is an urgent need to develop alternative technologies. We have engineered a synthetic peptide called clavanin-MO, derived from a marine tunicate antimicrobial peptide, which exhibits potent antimicrobial and immunomodulatory properties both in vitro and in vivo. The peptide effectively killed a panel of representative bacterial strains, including multidrug-resistant hospital isolates. Antimicrobial activity of the peptide was demonstrated in animal models, reducing bacterial counts by six orders of magnitude, and contributing to infection clearance. In addition, clavanin-MO was capable of modulating innate immunity by stimulating leukocyte recruitment to the site of infection, and production of immune mediators GM-CSF, IFN-γ and MCP-1, while suppressing an excessive and potentially harmful inflammatory response by increasing synthesis of anti-inflammatory cytokines such as IL-10 and repressing the levels of pro-inflammatory cytokines IL-12 and TNF-α. Finally, treatment with the peptide protected mice against otherwise lethal infections caused by both Gram-negative and -positive drug-resistant strains. The peptide presented here directly kills bacteria and further helps resolve infections through its immune modulatory properties. Peptide anti-infective therapeutics with combined antimicrobial and immunomodulatory properties represent a new approach to treat antibiotic-resistant infections.

Emerging evidence is implicating abnormal activation of the mechanistic target of rapamycin (mTOR) pathway in several monogenetic neuropsychiatric disorders, including Angelman syndrome (AS), which is caused by deficiency in maternally inherited UBE3A. Using an AS mouse model, we show that semi-chronic rapamycin treatment improves long-term potentiation (LTP) and actin polymerization in hippocampal slices, spine morphology, and fear-conditioning learning. Activity of mTORC1 and of its downstream substrate, S6K1, was increased in hippocampus of AS mice. However, mTORC2 activity, as reflected by PKCα levels, was decreased. Both increased mTORC1 and decreased mTORC2 activities were reversed by semi-chronic rapamycin treatment. Acute treatment of hippocampal slices from AS mice with rapamycin or an S6K1 inhibitor, PF4708671, improved LTP, restored actin polymerization, and normalized mTORC1 and mTORC2 activity. These treatments also reduced Arc levels in AS mice. Treatment with Torin 1, an inhibitor of both mTORC1 and mTORC2, partially rescued LTP and actin polymerization in hippocampal slices from AS mice, while partially impairing them in wild-type (WT) mice. Torin 1 decreased mTORC1 and increased mTORC2 activity in slices from AS mice but inhibited both mTORC1 and mTORC2 in WT mice. Finally, an mTORC2 activator, A-443654, increased hippocampal LTP in AS mice and actin polymerization in both WT and AS mice. Collectively, these results indicate that events set in motion by increased mTORC1 and decreased mTORC2 activities, including increased Arc translation and impaired actin remodeling, are crucial in AS pathogenesis. Therefore, selectively targeting these two master kinase complexes may provide new therapeutic approaches for AS treatment., Competing Interests: The authors declare no conflict of interest.

Macrophage dysfunction in obesity and diabetes is associated with persistent inflammation and poor wound healing responses. Relevant to these phenotypes, we have previously shown that macrophage activation in a high-fat environment results in cell death via a mechanism that involves lysosome damage. While searching for signaling pathways that were required for this response, we discovered that mTOR inhibitors, torin and rapamycin, were protective against lipotoxic cell death in primary peritoneal macrophages. The protective effect of mTOR inhibition was also confirmed by using genetic loss-of-function approaches. Given the importance of mTOR in regulation of autophagy we hypothesized that this pathway would be important in protection from cell death. We first demonstrated that autophagy was disrupted in response to palmitate and LPS as a consequence of impaired lysosome function. Conversely, the mTOR inhibitor, torin, increased macrophage autophagy and protected against lysosome damage; however, the beneficial effects of torin persisted in autophagy-deficient cells. Inhibition of mTOR also triggered nuclear localization of TFEB, a transcription factor that regulates lysosome biogenesis and function, but the rescue phenotype did not require the presence of TFEB. Instead, we demonstrated that mTOR inhibition reduces mitochondrial oxidative metabolism and attenuates the negative effects of palmitate on LPS-induced mitochondrial respiration. These results suggest that inhibition of mTOR is protective against lipotoxicity via an autophagy-independent mechanism that involves relieving mitochondrial substrate overload. On the basis of these findings, we suggest that therapies to reduce macrophage mTOR activation may protect against dysfunctional inflammation in states of overnutrition, such as diabetes., (© Society for Leukocyte Biology.)

Background: Extracellular hemoglobin and cell-free heme are toxic breakdown products of hemolyzed erythrocytes. Mammals synthesize the scavenger proteins haptoglobin and hemopexin, which bind extracellular hemoglobin and heme, respectively. Transfusion of packed red blood cells is a lifesaving therapy for patients with hemorrhagic shock. Because erythrocytes undergo progressive deleterious morphological and biochemical changes during storage, transfusion of packed red blood cells that have been stored for prolonged intervals (SRBCs; stored for 35-40 days in humans or 14 days in mice) increases plasma levels of cell-free hemoglobin and heme. Therefore, in patients with hemorrhagic shock, perfusion-sensitive organs such as the kidneys are challenged not only by hypoperfusion but also by the high concentrations of plasma hemoglobin and heme that are associated with the transfusion of SRBCs., Methods: To test whether treatment with exogenous human haptoglobin or hemopexin can ameliorate adverse effects of resuscitation with SRBCs after 2 hours of hemorrhagic shock, mice that received SRBCs were given a coinfusion of haptoglobin, hemopexin, or albumin., Results: Treatment with haptoglobin or hemopexin but not albumin improved the survival rate and attenuated SRBC-induced inflammation. Treatment with haptoglobin retained free hemoglobin in the plasma and prevented SRBC-induced hemoglobinuria and kidney injury. In mice resuscitated with fresh packed red blood cells, treatment with haptoglobin, hemopexin, or albumin did not cause harmful effects., Conclusions: In mice, the adverse effects of transfusion with SRBCs after hemorrhagic shock are ameliorated by treatment with either haptoglobin or hemopexin. Haptoglobin infusion prevents kidney injury associated with high plasma hemoglobin concentrations after resuscitation with SRBCs. Treatment with the naturally occurring human plasma proteins haptoglobin or hemopexin may have beneficial effects in conditions of severe hemolysis after prolonged hypotension., Competing Interests: Disclosures: In accordance with institutional policy, HSW has declared hemopexin to MGH as a potential candidate molecule to help decrease inflammation, and the institution has filed for patent protection., (© 2016 American Heart Association, Inc.)

The bactericidal/permeability-increasing protein (BPI) with bactericidal and endotoxin-neutralizing activity is of considerable interest in clinical applications. However, the crucial residues responsible for the bactericidal activity of BPI remain elusive. In previous study, we identified the mutation of mBPI5 associated with the male infertility of mice. Here, the effects of Asp190Ala mutation on the antibacterial activity of mBPI5 have been determined. Substitution of Asp190 by alanine caused significant improvement in cytotoxic effect toward both E.coli J5 and P.aeruginosa. Liposome co-sedimentation assay showed that the ratio of Asp190Ala mutant binding to lipids increased by 8 folds. These results were well consistent with known fact that antibacterial activity of BPI is attributed to its high affinity for lipid moiety of lipopolysaccharides (LPS). The constructed structure of mBPI5 revealed that Asp190 was located close to 6 positively charged residues on the surface of N-terminal domain. When replacing Asp190 with alanine, salt linkages with Arg188 were broken, making the side chain of Arg188 be free to move and form tighter contacts with negatively charged LPS. These findings suggest that residue 190 combined with surrounding positively charged residues largely contribute to bactericidal and endotoxin-neutralizing activities of mBPI5., Competing Interests: There are no conflicts of interest.

The chicken egg resists most environmental microbes suggesting that it potentially contains efficient antimicrobial molecules. Considering that some heparin-binding proteins in mammals are antibacterial, we investigated the presence and the antimicrobial activity of heparin-binding proteins from chicken egg white. Mass spectrometry analysis of the proteins recovered after heparin-affinity chromatography, revealed 20 proteins, including known antimicrobial proteins (avidin, lysozyme, TENP, ovalbumin-related protein X and avian bêta-defensin 11). The antibacterial activity of three new egg candidates (vitelline membrane outer layer protein 1, beta-microseminoprotein-like (LOC101750704) and pleiotrophin) was demonstrated against Listeria monocytogenes and/or Salmonella enterica Enteritidis. We showed that all these molecules share the property to inhibit bacterial growth through their heparin-binding domains. However, vitelline membrane outer layer 1 has additional specific structural features that can contribute to its antimicrobial potential. Moreover, we identified potential supplementary effectors of innate immunity including mucin 5B, E-selectin ligand 1, whey acidic protein 3, peptidyl prolyl isomerase B and retinoic acid receptor responder protein 2. These data support the concept of using heparin affinity combined to mass spectrometry to obtain an overview of the various effectors of innate immunity composing biological milieus, and to identify novel antimicrobial candidates of interest in the race for alternatives to antibiotics.

Clavanins is a class of peptides (23aa) histidine-rich, free of post-translational modifications. Clavanins have been studied largely for their ability to disrupt bacterial membranes. In the present study, the interaction of clavanin A with membranes was assessed by dynamic light scattering, zeta potential and permeabilization assays. We observed through those assays that clavanin A lysis bacterial cells at concentrations corresponding to its MIC. Further, the structure and function of clavanin A was investigated. To better understand how clavanin interacted with bacteria, its NMR structure was elucidated. The solution state NMR structure of clavanin A in the presence of TFE-d3 indicated an α-helical conformation. Secondary structures, based on circular dichroism measurements in anionic sodium dodecyl sulfate (SDS) and TFE (2,2,2-trifluorethanol), in silico lipid-peptide docking and molecular simulations with lipids DPPC and DOPC revealed that clavanin A can adopt a variety of folds, possibly influencing its different functions. Microcalorimetry assays revealed that clavanin A was capable of discriminating between different lipids. Finally, clavanin A was found to eradicate bacterial biofilms representing a previously unrecognized function.

In addition to their well-known antibacterial activity some antimicrobial peptides and proteins (AMPs) display also antiviral effects. A 27 aa peptide from the N-terminal part of human bactericidal/permeability-increasing protein (BPI) previously shown to harbour antibacterial activity inhibits the infectivity of multiple Influenza A virus strains (H1N1, H3N2 and H5N1) the causing agent of the Influenza pneumonia. In contrast, the homologous murine BPI-peptide did not show activity against Influenza A virus. In addition human BPI-peptide inhibits the activation of immune cells mediated by Influenza A virus. By changing the human BPI-peptide to the sequence of the mouse homologous peptide the antiviral activity was completely abolished. Furthermore, the human BPI-peptide also inhibited the pathogenicity of the Vesicular Stomatitis Virus but failed to interfere with HIV and measles virus. Electron microscopy indicate that the human BPI-peptide interferes with the virus envelope and at high concentrations was able to destroy the particles completely.

Bactericidal/permeability-increasing protein (BPI) is an important factor of innate immunity that in mammals is known to take part in the clearance of invading Gram-negative bacteria. In teleost, the function of BPI is unknown. In the present work, we studied the function of tongue sole (Cynoglossus semilaevis) BPI, CsBPI. We found that CsBPI was produced extracellularly by peripheral blood leukocytes (PBL). Recombinant CsBPI (rCsBPI) was able to bind to a number of Gram-negative bacteria but not Gram-positive bacteria. Binding to bacteria led to bacterial death through membrane permeabilization and structural destruction, and the bound bacteria were more readily taken up by PBL. In vivo, rCsBPI augmented the expression of a wide arrange of genes involved in antibacterial and antiviral immunity. Furthermore, rCsBPI enhanced the resistance of tongue sole against bacterial as well as viral infection. These results indicate for the first time that a teleost BPI possesses immunoregulatory effect and plays a significant role in antibacterial and antiviral defense.

The inflammatory state associated with Crohn's disease (CD) and ulcerative colitis (UC) remains incompletely defined. To understand more clearly the extracellular milieu associated with inflammatory bowel disease (IBD), we employed a bioassay whereby plasma of treatment naive paediatric IBD patients (n = 22 CD, n = 15 UC) and unrelated healthy controls (uHC, n = 10) were used to induce transcriptional responses in a healthy leucocyte population. After culture, gene expression was measured comprehensively with microarrays and analysed. Relative to uHC, plasma of CD and UC patients induced distinct responses consisting, respectively, of 985 and 895 regulated transcripts [|log2 ratio| ≥ 0·5 (1·4-fold); false discovery rates (FDR) ≤ 0·01]. The CD:uHC and UC:uHC signatures shared a non-random, commonly regulated, intersection of 656 transcripts (χ(2)  = P < 0·001) and were highly correlative [Pearson's correlation coefficient = 0·96, 95% confidence interval (CI) = 0.96, 0.97]. Despite sharing common genetic susceptibility loci, the IBD signature correlated negatively with that driven by plasma of type 1 diabetes (T1D) patients (Pearson's correlation coefficient = -0·51). Ontological analyses revealed the presence of an immunoregulatory plasma milieu in IBD, as transcripts for cytokines/chemokines, receptors and signalling molecules consistent with immune activation were under-expressed relative to uHC and T1D plasma. Multiplex enzyme-linked immunosorbent assay (ELISA) and receptor blockade studies confirmed transforming growth factor (TGF)-β and interleukin (IL)-10 as contributors to the IBD signature. Analysis of CD patient signatures detected a subset of transcripts associated with responsiveness to 6-mercaptopurine treatment. Through plasma-induced signature analysis, we have defined a unique, partially TGF-β/IL-10-dependent immunoregulatory signature associated with IBD that may prove useful in predicting therapeutic responsiveness., (© 2016 British Society for Immunology.)

Adropin, a recently described peptide hormone produced in the brain and liver, has been reported to have physiologically relevant actions on glucose homeostasis and lipogenesis, and to exert significant effect on endothelial function. We describe a central nervous system action of adropin to inhibit water drinking and identify a potential adropin receptor, the orphan G protein-coupled receptor, GPR19. Reduction in GPR19 mRNA levels in medial basal hypothalamus of male rats resulted in the loss of the inhibitory effect of adropin on water deprivation-induced thirst. The identification of a novel brain action of adropin and a candidate receptor for the peptide should extend and accelerate the study of the potential therapeutic value of adropin or its mimetics for the treatment of metabolic disorders., (Copyright © 2016 the American Physiological Society.)

The serine/threonine kinase mTOR is essential for the phosphoinositide 3-kinases (PI3K) signaling pathway, and regulates the development and function of immune cells. Aberrant activation of mTOR signaling pathway is associated with many cancers including leukemia. Here, we report the contributions of mTOR signaling to growth of human leukemic cell lines and mouse T-cell acute leukemia (T-ALL) cells. Torin, an ATP-competitive mTOR inhibitor, was found to have both cytotoxic and cytostatic effects on U-937, THP-1, and RPMI-8226 cells, but not on Jurkat or K-562 cells. All cells were relatively resistant to rapamycin even with suppressed activity of mTOR complex 1. Growth of T-ALL cells induced by Notch1 was profoundly affected by torin partially due to increased expression of Bcl2l11 and Bbc3. Of note, activation of Akt or knockdown of FoxO1 mitigated the effect of mTOR inhibition on T-ALL cells. Our data provide insight on the effect of mTOR inhibitors on the survival and proliferation of leukemic cells, thus further improving our understanding on cell-context-dependent impacts of mTOR signaling.

Background: To examine the effects of adropin on glucose and lipid metabolism in a rat model of Type 2 diabetes mellitus (T2DM)., Methods: T2DM were established using high-fat diet and streptozocin (STZ; 35 mg/kg/b.w.). Seven days after STZ induction, diabetic rats were randomly treated with adropin (2.1 μg/kg/day intraperitonealy) for 10 days. The study involved the evaluation of biochemical parameters, including blood glucose, total cholesterol (TC), triglycerides (TG), low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and gamma glutamyl transferase (GGT) activities. Additionally, Tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6) and inducible nitric oxide synthase (iNOS) mRNA gene expressions in pancreas tissue were determined by reverse transcription-polymerase chain reaction., Results: The serum levels of insulin and adropin were determined by ELISA. Treatment with adropin showed a significant reduction in blood glucose levels, HbA1c (%), HOMA-IR and increase in HOMA-β, serum insulin levels. In addition, intraperitoneal adropin application can reduce serum levels of TC, TG, LDL-C, and increase level of HDL-C. Adropin also effectively ameliorated the alterations in TNF-α, IL-6 and iNOS mRNA expression., Conclusion: The present study indicates that the adropin possesses antidiabetic and antidyslipidemic effects in T2DM (Tab. 2, Fig. 3, Ref. 32).

Whereas DNA provides the information to design life and proteins provide the materials to construct it, the metabolome can be viewed as the physiology that powers it. As such, metabolomics, the field charged with the study of the dynamic small-molecule fluctuations that occur in response to changing biology, is now being used to study the basis of disease. Here, we describe a comprehensive metabolomic analysis of a systemic bacterial infection using Bacillus anthracis, the etiological agent of anthrax disease, as the model pathogen. An organ and blood analysis identified approximately 400 metabolites, including several key classes of lipids involved in inflammation, as being suppressed by B. anthracis. Metabolite changes were detected as early as 1 day postinfection, well before the onset of disease or the spread of bacteria to organs, which testifies to the sensitivity of this methodology. Functional studies using pharmacologic inhibition of host phospholipases support the idea of a role of these key enzymes and lipid mediators in host survival during anthrax disease. Finally, the results are integrated to provide a comprehensive picture of how B. anthracis alters host physiology. Collectively, the results of this study provide a blueprint for using metabolomics as a platform to identify and study novel host-pathogen interactions that shape the outcome of an infection., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The rapid increase in the incidence of multidrug-resistant infections today has led to enormous interest in antimicrobial peptides (AMPs) as suitable compounds for developing unusual antibiotics. In this study, clavanin A, an antimicrobial peptide previously isolated from the marine tunicate Styela clava, was selected as a purposeful molecule that could be used in controlling infection and further synthesized. Clavanin A was in vitro evaluated against Staphylococcus aureus and Escherichia coli as well as toward L929 mouse fibroblasts and skin primary cells (SPCs). Moreover, this peptide was challenged here in an in vivo wound and sepsis model, and the immune response was also analyzed. Despite displaying clear in vitro antimicrobial activity toward Gram-positive and -negative bacteria, clavanin A showed no cytotoxic activities against mammalian cells, and in acute toxicity tests, no adverse reaction was observed at any of the concentrations. Moreover, clavanin A significantly reduced the S. aureus CFU in an experimental wound model. This peptide also reduced the mortality of mice infected with E. coli and S. aureus by 80% compared with that of control animals (treated with phosphate-buffered saline [PBS]): these data suggest that clavanin A prevents the start of sepsis and thereby reduces mortality. These data suggest that clavanin A is an AMP that could improve the development of novel peptide-based strategies for the treatment of wound and sepsis infections., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

CAP37, a protein constitutively expressed in human neutrophils and induced in response to infection in corneal epithelial cells, plays a significant role in host defense against infection. Initially identified through its potent bactericidal activity for Gram-negative bacteria, it is now known that CAP37 regulates numerous host cell functions, including corneal epithelial cell chemotaxis. Our long-term goal is to delineate the domains of CAP37 that define these functions and synthesize bioactive peptides for therapeutic use. We report the novel finding of a multifunctional domain between aa 120 and 146. Peptide analogs 120-146 QR, 120-146 QH, 120-146 WR, and 120-146 WH were synthesized and screened for induction of corneal epithelial cell migration by use of the modified Boyden chamber assay, antibacterial activity, and LPS-binding activity. In vivo activity was demonstrated by use of mouse models of sterile and infected corneal wounds. The identity of the amino acid at position 132 (H vs. R) was important for cell migration and in vivo corneal wound healing. All analogs demonstrated antimicrobial activity. However, analogs containing a W at position 131 showed significantly greater antibacterial activity against the Gram-negative pathogen Pseudomonas aeruginosa. All analogs bound P. aeruginosa LPS. Topical administration of analog 120-146 WH, in addition to accelerating corneal wound healing, effectively cleared a corneal infection as a result of P. aeruginosa. In conclusion, we have identified a multifunctional bioactive peptide, based on CAP37, that induces cell migration, possesses antibacterial and LPS-binding activity, and is effective at healing infected and noninfected corneal wounds in vivo., (© Society for Leukocyte Biology.)

Due to clinical efficacy and safety profile, extracorporeal photochemotherapy (ECP) is a commonly used cell treatment for patients with cutaneous T cell lymphoma (CTCL) and graft-versus-host disease (GVHD). The capacity of ECP to induce dendritic antigen-presenting cell (DC)-mediated selective immunization or immunosuppression suggests a novel mechanism involving pivotal cell signalling processes that have yet to be clearly identified as related to this procedure. In this study we employ two model systems of ECP to dissect the role of integrin signalling and adsorbed plasma proteins in monocyte-to-DC differentiation. We demonstrate that monocytes that were passed through protein-modified ECP plates adhered transiently to plasma proteins, including fibronectin, adsorbed to the plastic ECP plate and activated signalling pathways that initiate monocyte-to-DC conversion. Plasma protein adsorption facilitated 54·2 ± 4·7% differentiation, while fibronectin supported 29·8 ± 7·2% differentiation, as detected by DC phenotypic expression of membrane CD80 and CD86, as well as CD36, human leucocyte antigen D-related (HLA-DR) and cytoplasmic CD83. Further, we demonstrate the ability of fibronectin and other plasma proteins to act through cell adhesion via the ubiquitous arginine-glycine-aspartic (RGD) motif to drive monocyte-to-DC differentiation, with high-density RGD substrates supporting 54·1 ± 5·8% differentiation via αVβ3 and α5β1integrin signalling. Our results demonstrate that plasma protein binding integrins and plasma proteins operate through specific binding domains to induce monocyte-to-DC differentiation in ECP, providing a mechanism that can be harnessed to enhance ECP efficacy., (© 2013 British Society for Immunology.)

The translation of stem cell-based regenerative solutions from the laboratory to the clinic is often hindered by the culture conditions used to expand cell populations. Although fetal bovine serum (FBS) is widely used, regulatory bodies and safety concerns encourage alternative, xeno-free culturing practices. In an attempt to apply this approach to a bone-forming combination product of human periosteal progenitors (human periosteum derived cells) on a clinically used calcium phosphate carrier, FBS was substituted for human allogeneic serum (hAS) during cell expansion. It was found that cell proliferation was increased in hAS along with an apparent commitment to the osteogenic lineage, indicated by enhanced Runx2 expression, as well as alkaline phosphatase activity and matrix mineralization. Following analysis of signaling pathways, it was found that interferon-mediated signaling was downregulated, whereas JAK-STAT signaling was upregulated. STAT3 phosphorylation was enhanced in hAS-cultured human periosteum derived cells, inhibition of which ablated the proliferative effect of hAS. Furthermore, following in vivo implantation of hAS-cultured cells on NuOss scaffolds, enhanced bone formation was observed compared with FBS (71% increase, p < .001). Interestingly, the de novo-formed bone appeared to have a higher ratio of immature regions to mature regions, indicating that after 8 weeks implantation, tissue-formation processes were continuing. Integration of the implant with the environment appeared to be altered, with a decrease in calcium phosphate grain size and surface area, indicative of accelerated resorption. This study highlights the advantages of using humanized culture conditions for the expansion of human periosteal progenitors intended for bone regeneration.

Introduction: Overweight status should not be considered merely an aesthetic concern; rather, it can incur health risks since it may trigger a cascade of events that produce further fat tissue through altered levels of circulating signaling molecules., Methods: We decided to investigate the influence of overweight individuals' sera on in vitro MSC proliferation and differentiation., Results: We observed that in vitro incubation of bone marrow stromal cells with the sera of overweight individuals promotes the adipogenic differentiation of MSCs while partially impairing proper osteogenesis., Conclusions: These results, which represent a pilot study, might suggest that becoming overweight triggers further weight gains by promoting a bias in the differentiation potential of MSCs toward adipogenesis. The circulating factors involved in this phenomenon remain to be determined, since the great majority of the well known pro-inflammatory cytokines and adipocyte-secreted factors we investigated did not show relevant modifications in overweight serum samples compared with controls.

Adipocyte differentiation is a complex developmental process forming adipocytes from various precursor cells. The murine 3T3-L1 preadipocyte cell line has been most frequently used in the studies of adipocyte differentiation. Differentiation of 3T3-L1 preadipocytes includes a medium containing fetal bovine serum (FBS) with hormonal induction. In this study, we observed that differentiation medium containing adult bovine serum (ABS) instead of FBS did not support differentiation of preadipocytes. Impaired adipocyte differentiation was due to the presence of a serum protein factor in ABS that suppresses differentiation of preadipocytes. Using a proteomic analysis, alpha-2-macroglobulin and paraoxonase/arylesterase 1, which were previously shown to suppress differentiation of preadipocytes, were identified as anti-adipogenic proteins. Although their functional mechanisms have not yet been elucidated, the anti-adipogenic effects of these proteins are discussed.

Aiming for the clinical application of cartilage regeneration, the xeno-free cultivation method to obtain a scaffold-free cartilage-like disc-shaped cell sheet using mesenchymal stem cells (MSCs) derived from human bone marrow without the shrinkage of the sheet was investigated. MSCs were inoculated into Cell Culture Insert (0.3 cm(2), pore size; 0.4 μm, pore density; 1.0 × 10(8)/cm(2)) using serum-free chondrogenic differentiation medium containing TGF-β3, IGF-1 and dexamethasone or other modified media, and cultured at 37 °C in 5% CO2 for 3 weeks. Sheet thickness, cartilage specific genes expression, ECM accumulation were determined, and the sections of sheets were stained with alcian blue. A novel mixed medium consisting of a growth medium (10% FCS) with a serum-free chondrogenic differentiation medium could prevent the shrinkage of the sheet and produced a disc-shaped cell sheet. The depth of the sheet was approximately 0.7 mm and the gene expression levels were higher than those in cells in normal human cartilage. The use of human serum instead of FCS did not cause shrinkage and did not decrease the accumulation levels of sGAG and type 2 collagen in the sheet. The cultivation of MSCs grown with completely xeno-free materials using the mixed medium containing human serum in a cell culture insert showed a sheet depth of 1.0 mm and gene expression levels higher than those in normal cartilage. The scaffold-free and xeno-free cartilage-like cell sheet was successfully formed without shrinkage using human bone marrow MSCs and the chondrogenic differentiation medium containing human serum., (Copyright © 2013. Published by Elsevier B.V.)

We have measured the interaction forces between a murine melanoma cell and a poly(l-lactic acid) (PLLA) microsphere coated with/without hydroxyapatite (HAp) nanoparticles (i.e., an HAp/PLLA or a bare PLLA microsphere) in a serum-free culture medium, using atomic force microscopy (AFM) with colloid probe technique, in order to investigate how the HAp-nanoparticle coating as well as interfacial serum proteins influence the cell-microsphere adhesion. The cell adhesion force of the HAp/PLLA microspheres was 1.4-fold stronger than that of the bare PLLA microspheres. When the microspheres were pretreated with a culture medium supplemented with 10% fetal bovine serum, the cell adhesion force of the HAp/PLLA microspheres was increased by a factor of 2.1; in contrast, no change was observed in the cell adhesion force of the bare PLLA microspheres before/after the pretreatment. Indeed, the cell adhesion force of the HAp/PLLA was 2.8-fold larger than that of the bare PLLA after the pretreatment. Additionally, we have investigated the effect of interfacial serum proteins on the zeta potentials of these microspheres. On the basis of the obtained results, possible mechanism of cell adhesion to the HAp/PLLA and bare PLLA microspheres in the presence/absence of the interfacial serum proteins is discussed., (Copyright © 2013 Elsevier B.V. All rights reserved.)