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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.)

Cyclotides are plant-derived peptides with complex structures shaped by their head-to-tail cyclic backbone and cystine knot core. These structural features underpin the native bioactivities of cyclotides, as well as their beneficial properties as pharmaceutical leads, including high proteolytic stability and cell permeability. However, their inherent structural complexity presents a challenge for cyclotide engineering, particularly for accessing libraries of sufficient chemical diversity to design potent and selective cyclotide variants. Here, we report a strategy using mRNA display enabling us to select potent cyclotide-based FXIIa inhibitors from a library comprising more than 10 12 members based on the cyclotide scaffold of Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II). The most potent and selective inhibitor, cMCoFx1, has a pM inhibitory constant toward FXIIa with greater than three orders of magnitude selectivity over related serine proteases, realizing specific inhibition of the intrinsic coagulation pathway. The cocrystal structure of cMCoFx1 and FXIIa revealed interactions at several positions across the contact interface that conveyed high affinity binding, highlighting that such cyclotides are attractive cystine knot scaffolds for therapeutic development.

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.

Can a minor difference in the nonmetal binding sequence of antimicrobial clavanins explain the drastic change in the coordination environment and antimicrobial efficiency? This study answers the question with a definite "yes", showing the details of the bioinorganic chemistry of Zn(II) and Cu(II) complexes with clavanins, histidine-rich, antimicrobial peptides from hemocytes of the tunicate Styela clava .

Bivalve mollusks are continuously exposed to potentially pathogenic microorganisms living in the marine environment. Not surprisingly, these filter-feeders developed a robust innate immunity to protect themselves, which includes a broad panel of antimicrobial peptides. Among these, myticalins represent a recently discovered family of linear cationic peptides expressed in the gills of Mytilus galloprovincialis. Even though myticalins and insect and mammalian proline-rich antimicrobial peptides (PrAMPs) share a similar amino acid composition, we here show that none of the tested mussel peptides use a non-lytic mode of action relying on the bacterial transporter SbmA. On the other hand, all the tested myticalins perturbed and permeabilized the membranes of E. coli BW25113, as shown by flow-cytometry and atomic force microscopy. Circular dichroism spectra revealed that most myticalins did not adopt recognizable secondary structures in the presence of amphipathic environments, such as biological membranes. To explore possible uses of myticalins for biotech, we assessed their biocompatibility with a human cell line. Non-negligible cytotoxic effects displayed by myticalins indicate that their optimization would be required before their further use as lead compounds in the development of new antibiotics., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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.)

In biological fluids, micro- or nano-size particles are prone to adsorb proteins and form a layer. The ambient air fine particulate matter (PM 2.5 ) is inhaled via the lung, penetrates biological barriers and eventually reaches systemic blood circulation. However, there are very few data available regarding the adsorption of proteins on PM 2.5 . Here, we compared protein corona formed in plasma after bronchoalveolar lavage fluid (BALF) exposure with those formed in plasma alone. Using purified coronal proteins, we explored their adsorption behaviors on PM 2.5 and their influence on biological reactivity of PM 2.5. Liquid-chromatography tandem mass-spectrometry (LC-MS/MS) analysis revealed that exposure to BALF significantly changed the blood protein profile on PM 2.5 . Regardless of the presence of BALF, the protein corona on PM 2.5 contained an abundance of serum albumin, hemoglobin (Hb) and fibrinogen (Fg) proteins. Using Fg as a corona surrogate, we found that van der Waals interactions, hydrophobic interactions, π-π stacking and electrostatic attractions contributed to the Fg adsorption and led to the conformational changes of Fg. In addition, Fg decoration decreased cellular internalization of PM 2.5 and corresponding subsequent oxidative stress responses in a murine RAW264.7 macrophage. These results support the view that the formation of PM 2.5 corona should be considered for toxicity assessment of PM 2.5., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

Gram-negative bacteria are some of the biggest threats to public health due to a large prevalence of antibiotic resistance. The difficulty in treating bacterial infections, stemming from their double membrane structure combined with efflux pumps in the outer membrane, has resulted in a much greater need for antimicrobials with activity against these pathogens. Tunicate host defense peptide (HDP), Clavanin A, is capable of not only inhibiting Gram-negative growth but also potentiating activity in the presence of Zn(II). Here, we provide evidence that the improvements of Clavanin A activity in the presence of Zn(II) are due to its novel mechanism of action. We employed E. coli TD172 (Δ recA::kan ) and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to show in cellulae that DNA damage occurs upon treatment with Clavanin A. In vitro assays demonstrated that Zn(II) ions are required for the nuclease activity of the peptide. The quantum mechanics/molecular mechanics (QM/MM) calculations were used to investigate the mechanism of DNA damage. In the rate-determining step of the proposed mechanism, due to its Lewis acidity, the Zn(II) ion activates the scissile P-O bond of DNA and creates a hydroxyl nucleophile from a water molecule. A subsequent attack by this group to the electrophilic phosphorus cleaves the scissile phosphoester bond. Additionally, we utilized bacterial cytological profiling (BCP), circular dichroism (CD) spectroscopy in the presence of lipid vesicles, and surface plasmon resonance combined with electrical impedance spectroscopy in order to address the apparent discrepancies between our results and the previous studies regarding the mechanism of action of Clavanin A. Finally, our approach may lead to the identification of additional Clavanin A like HDPs and promote the development of antimicrobial peptide based therapeutics.

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.

Background: Thrombosis represents as the prime contributor to the burden of diseases, worldwide. Conventional anticoagulants for thrombosis therapy have a common bleeding side effect. Bioactive peptides are studied to be an effective alternative for currently available therapeutic drugs., Objective: In this study, VITPOR AI peptide, a previously reported coagulation FXIIa inhibitor from Nori (Porphyra yezoensis), was assessed for its inhibitory activity against FXIIa and its in vivo mode of action., Methods: In vivo efficacy as well as the antithrombotic property of the peptide was evaluated in mice model by ex vivo activated Partial Thromboplastin Time assay, tail transection model and whole blood clotting time. The enzyme kinetics was studied using chromogenic substrate assay., Results: The kinetic behaviour of VITPOR AI showed that the peptide is a competitive inhibitor of FXIIa. Peptide showed significant inhibition of platelet adhesion and aggregation. VITPOR AI exhibited significant antithrombotic activity. Furthermore, ex vivo activated Partial Thromboplastin Time assay revealed that VITPOR AI exhibited potent anticoagulant activity in vivo. Tail bleeding assay revealed that the peptide did not prolong bleeding time in mice even at a higher dose of 5 mg/kg. Cytotoxicity studies of the peptide against human blood leukocytes indicated the safety of the peptide., Conclusion: VITPOR AI could be prospected as a potent anticoagulant with Factor XIIa inhibition, antiplatelet aggregation and antithrombotic activity. It was also studied to have no bleeding side effect., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Tachylectin-5, a 41-kDa protein with a common fold of the C-terminal globular domain of the γ-chain of fibrinogen, is purified from horseshoe crab hemolymph plasma by affinity column chromatography, using acetyl-group-immobilized resin. Two types of isolectins, tachylectin-5A and tachylectin-5B, are obtained by stepwise elution with GlcNAc at 25 and 250 mM, respectively. Tachylectins-5A and -5B exhibit extraordinarily strong hemagglutinating activity against all types of human erythrocytes (the minimum agglutinating concentration of 0.004-0.008 μg/mL for tachylectin-5A and 0.077-0.27 μg/mL for tachylectin-5B). Their hemagglutinating activities are inhibited by acetyl group-containing sugars and noncarbohydrates such as sodium acetate, acetylcholine, and acetyl CoA (the minimum inhibitory concentrations of 1.3-1.6 mM), indicating that the acetyl group is required and sufficient for recognition by tachylectins-5A and -5B. EDTA inhibits their hemagglutinating activity, whereas the inhibition is overcome by adding an excess amount of Ca 2+ . Tachylectins-5A and -5B also exhibit bacterial agglutinating activity against both Gram-negative bacteria (the minimum agglutinating concentrations of 0.04-0.08 μg/mL for tachylectin-5A and 0.05-0.11 μg/mL for tachylectin-5B) and Gram-positive bacteria (the minimum agglutinating concentrations of 0.3-2.4 μg/mL for tachylectin-5A and 15.1-26.8 μg/mL for tachylectin-5B). Interestingly, tachylectins-5A and -5B enhance the antimicrobial activity of a hemocyte-derived peptide, big defensin.

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.)

An antimicrobial peptide tachycitin (73 amino acids) is purified by steps of chromatography, including Sephadex G-50 and S Sepharose FF, from the acid extract of hemocyte debris of horseshoe crabs. Tachycitin is present in monomer form in solution, revealed by ultracentrifugation analysis. Tachycitin exhibits bacterial agglutination activity and inhibits the growth of both Gram-negative bacteria, Gram-positive bacteria, and fungus Candida albicans. Interestingly, tachycitin shows synergistic antimicrobial activity in corporation with another antimicrobial peptide, big defensin. Tachycitin shows a specific binding activity to chitin but not to cellulose, mannan, xylan, and laminarin. Tachycitin is composed of the N-terminal three-stranded β-sheet and the C-terminal two-stranded β-sheet following a short helical turn, and the C-terminal structural motif shares a significant structural similarity with the chitin-binding domain derived from a plant chitin-binding protein, hevein.

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.)

Introduction: Clinical pharmacology studies are one of the major types of regulatory data submitted for review of therapeutic proteins regulated by the Center for Biologics Evaluation and Research (CBER)., Aim: The primary objective of the current study is to provide an overview of the role of clinical pharmacology including pharmacokinetics (PK), pharmacodynamics (PD) and exposure-response analysis at CBER. Furthermore, we aim to provide a baseline estimate for the use of quantitative clinical pharmacology studies prior to implementation of FDA's model-informed drug development (MIDD) pilot programme., Methods: We survey original Biologics License Applications (BLAs) for plasma-derived and related recombinant therapeutic protein products approved by CBER/FDA (2008-2017)., Results: There were 37 original BLAs that met our inclusion criteria, and 34 of these products (92%) contained human PK data as part of the biological licensing. The products were broadly classified as coagulation factors (54%), IgG and related proteins (24%), and other therapeutic proteins (22%). Coagulation factor VIII and IX products constitute 32% of the BLAs and indicated for treatment of haemophilia A and B, respectively. Twelve products (35%) used model-based approaches (population PK/PD and exposure-response). Over the past 5 years (2013 to 2017), there is a trend for increased application of MIDD approaches as compared to the previous cohort years (2008 to 2012)., Conclusion: In conclusion, clinical pharmacology has played a major role in regulatory review of plasma-derived products, and we expect that the application of quantitative methods will further evolve for these products under the FDA MIDD programme., (Published 2019. This article is a U.S. Government work and is in the public domain in the USA.)

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.)

Background: Leptospirosis, caused by spirochetes of the genus Leptospira, is one of the most widespread zoonoses worldwide. Efficient diagnostic methods for early diagnosis of leptospirosis are still lacking, and acute disease presents with nonspecific symptomatology and is often misdiagnosed. The leptospires pathogenic processes and virulence mechanisms remain virtually unknown. In severe infections, hemostatic impairment is frequently observed, and pathophysiological complications often develop when the host response is modulated by the pathogen. The neutrophil heparin-binding protein (HBP) is an inflammatory mediator and potent inducer of vascular leakage., Results: In this study, we found that leptospires and their secreted products induce the release of HBP from stimulated neutrophils through a controlled degranulation mechanism. We acknowledged 2 leptospiral proteins as able to induce HBP degranulation. These findings have clinical implications, as high levels of HBP were detected in serum from patients with leptospirosis, especially at the early phase of the disease., Conclusion: In conclusion, we describe a new mechanism by which the leptospirosis pathophysiological complications may arise, such as vascular leakage and edema formation. We also propose HBP as a new early screening biomarker for human leptospirosis., (© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Reactive oxygen species (ROS) play a critical role in total body irradiation (TBI)-induced hematopoietic system injury. However, the mechanisms involved in ROS production in hematopoietic stem cells (HSCs) post TBI need to be further explored. In this study, we demonstrated that hematopoietic system injury in mice radiated with TBI was effectively alleviated when the blood circulation environment was changed via the injection of serum from non-radiated mice. Serum injection increased the survival of radiated mice and ameliorated TBI-induced hematopoietic system injury through attenuating myeloid skew, increasing HSC frequency, and promoting the reconstitution of radiated HSCs. Serum injection also decreased ROS levels in HSCs and regulated oxidative stress-related proteins. A serum proteome sequence array showed that proteins related to tissue injury and oxidative stress were regulated, and a serum-derived exosome microRNA sequence assay showed that the PI3K-Akt and Hippo signaling pathways were affected in radiated mice injected with serum from non-radiated mice. Furthermore, a significant increase in cell viability and a decrease in ROS were observed in radiated lineage - c-kit + cells treated with serum-derived exosomes. Similarly, an improvement in the impaired differentiation of HSCs was observed in radiated mice injected with serum-derived exosomes. Taken together, our observations suggest that serum from non-radiated mice alleviates HSC injury in radiated mice by improving the systemic environment after radiation, and exosomes contribute to this radioprotective effect as important serum active component., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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.

Inflammation is characterized by early influx of polymorphonuclear neutrophils (PMNs), followed by a second wave of monocyte recruitment. PMNs mediate monocyte recruitment via their release of heparin binding protein (HBP), which activates CCR2 (CC-chemokine receptor 2) on monocytes. However, the pathways for such signal transmission remain unknown. Accumulating evidences have highlighted the importance of leukocyte-endothelial cell interactions in the initiation of inflammation. In this study, an interesting finding is that HBP enhances the secretion of monocyte chemotactic protein 1(MCP-1), ligand of CCR2, from a third party, the endothelial cells (ECs). HBP-induced increase in MCP-1 production was demonstrated at the protein, mRNA and secretion levels. Exposure of ECs to HBP elicited rapid phosphorylation of FAK/PI3K/AKT and p38 MAPK/NF-κB signaling. MCP-1 levels were attenuated during the response to HBP stimulation by pretreatment with a FAK inhibitor (or siRNA), a PI3K inhibitor, an AKT inhibitor, a p38 inhibitor (or siRNA) and two NF-κB inhibitors. Additionally, pretreatment with inhibitors to FAK, PI3K and AKT led to a decrease in HBP-induced phosphorylation of p38/NF-κB axis. These results showed that HBP induced MCP-1 expression via a sequential activation of the FAK/PI3K/AKT pathway and p38 MAPK/NF-κB axis. Interestingly, the patterns of HBP regulation of the expression of the adhesion molecular VCAM-1 were similar to those seen in MCP-1 after pretreatment with inhibitors (or not). These findings may help to determine key pharmacological points of intervention, thus slowing the progress of inflammatory-mediated responses in certain diseases where inflammation is detrimental to the host., (Copyright © 2017. Published by Elsevier Inc.)

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.

Platelet-rich plasma (PRP), an autologous blood derived product is a concentrated mix of multiple growth factors and cytokines. Direct injections of PRP are clinically used for treatment of various musculoskeletal disorders and in wound healing. However, PRP therapy has met with limited clinical success possibly due to unpredictable and premature bolus delivery of PRP growth factors. The objective of this study was to predictably control the bioavailability of PRP growth factors using a hydrolytically degradable polyethylene glycol (PEG) hydrogel. We used a step-growth polymerization based on a Michael-type addition reaction between a 6-arm PEG-acrylate and a dithiol crosslinker, which led to the formation of a homogenous hydrogel network under mild, physiologically relevant conditions. Specifically, to model the release of multicomponent PRP through PEG hydrogels, we examined bulk diffusion of PRP as well as model proteins in a size range corresponding to that of growth factors found in PRP. Our results indicated that protein size and hydrogel degradation controlled diffusion of all proteins and that secondary structure of proteins encapsulated during gelation remained unaffected post-release. Analysis of specific PRP proteins released from the hydrogel showed sustained release until complete hydrogel degradation. PRP released from hydrogels promoted proliferation of human dermal fibroblast, indicating retained bioactivity upon encapsulation and release. The versatile hydrogel system holds clinical potential as a therapeutic drug delivery depot of multicomponent mixtures like PRP. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3304-3314, 2017., (© 2017 Wiley Periodicals, Inc.)

Adropin is expressed in the CNS and plays a crucial role in the development of stroke. However, little is currently known about the effects of adropin on the blood-brain barrier (BBB) function after intracerebral hemorrhage (ICH). In this study, the role of adropin in collagenase-induced ICH was investigated in mice. At 1-h post-ICH, mice were administered with recombinant human adropin by intranasal. Brain water +content, BBB permeability, and neurological function were measured at different time intervals. Proteins were quantified using western blot analysis, and the localizations of adropin and Notch1 were visualized via immunofluorescence staining. It is shown that adropin reduced brain water content and improved neurological functions. Adropin preserved the functionality of BBB by increasing N-cadherin expression and reducing extravasation of albumin. Moreover, in vivo knockdown of Notch1 and Hes1 both abolished the protective effects of adropin. Taken together, our data demonstrate that adropin constitutes a potential treatment value for ICH by preserving BBB and improving functional outcomes through the Notch1 signaling pathway., (© 2017 International Society for Neurochemistry.)

There currently is renewed interest in blood clotting Factor XII as a potential target for thrombosis inhibition. Historically untargeted, there is little drug information with which to start drug candidate searches. Typical high-throughput screening can identify potential drug candidates, but is inefficient. Virtual high-throughput screening can be used to raise efficiency by focusing experimental efforts on compounds predicted to be active and is applied here to identify new Factor XIIa inhibitors. We combine principal component analysis, genetic algorithm and support vector machine to create the models used in the virtual high-throughput screening. In this work, experimental data from a PubChem Bioassay was used to train predictive models of Factor XIIa inhibition activity. The models created were then used to virtually screen the entire 72 million PubChem Compound database. Experimental validation of select candidates identified by this process resulted in a 42.9% hit-rate in the first-pass and 100% hit-rate in the second-pass, suggesting the effectiveness of the approach., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Amniotic membrane (AM) is frequently used in ophthalmologic surgery for rapid ocular surface reconstruction. Sometimes it may create a major problem with associated infections after biofilm formation over the membrane. To overcome this problem, AM was coated with the antimicrobial peptide clavanin A. The antifungal activity of clavanin A in the native and self-assembled form was determined against the common ocular surface pathogens Candida albicans, Aspergillus fumigatus, Alternaria sp. and Fusarium sp. Biofilm formation over the coated surface was significantly reduced in comparison with the uncoated membrane. The coated membrane revealed effectiveness in terms of biocompatibility, cell attachment colonization when tested in non-cancerous 3T3 and human embryonic kidney (HEK)-293 cell lines. Clavanin A-coated AM also exhibited excellent physical, morphological and antifungal characteristics, indicating potential applicability for ocular surface infection control.

In previous clinical trials, we showed that remote ischemic preconditioning (rIPC) reduced myocardial damage in children undergoing treatment for congenital heart defects and postoperative renal failure in patients undergoing abdominal aortic aneurysm surgery. In rabbit experiments, pre-treatment with plasma and plasma dialysate (obtained using 15-kDa cut-off dialysis membrane) from donor rabbits subjected to rIPC similarly protected against cardiac infarction. However, the protective substances containing in rIPC plasma have been unknown. In the present study, we showed that rIPC plasma exerted anti-apoptotic and anti-oxidative effects on human neural stem cells under oxygen glucose deprivation (OGD) that mimics brain ischemia. Additionally, we applied the sample to the liquid chromatography integrated with mass spectrometry to identify candidate key molecules in the rIPC plasma and determine its role in protecting neural stem cells from OGD-induced cell death. Thioredoxin increased significantly after rIPC compared to pre-IPC. Pretreatment with thioredoxin, the antioxidant protein, markedly protected human neural stem cells from OGD-induced cell death. The effect of thioredoxin on brain ischemia in animals should be further evaluated. However, the present study first evaluated the effect of rIPC in the ischemic cellular model.

Adropin is a peptide hormone that has been implicated in insulin resistance and as a potential regulator of growth. The aim of this study is to determine the effect of calorie restriction on circulating levels of adropin in the MMTV-TGFα breast cancer mouse model and investigate the effects of adropin peptide on the viability of MCF-7 and MDA-231 breast cancer cells in culture. Ten-week-old mice were assigned to either ad libitum-fed (AL), chronic calorie-restricted, or intermittent calorie-restricted groups. Concentrations of serum adropin were measured using an enzyme-linked immunosorbent assay. Results showed an inverse correlation between serum adropin levels and mouse age that was attenuated by calorie restriction. In the AL group the level of adropin was significantly lower at week 50 compared to levels at week 10. However, among the calorie-restricted groups, serum levels of adropin remained high at week 50. The cell-line-specific effects were observed after treatment of cancer cell lines with a series of adropin concentrations (5, 10, 25, 50 ng/mL). Flow cytometry analysis showed that MCF-7 cells entered the early phase of apoptosis after treatment with 50 ng/mL for 24 h. Adropin may be involved in the protective effects that calorie restriction has on breast cancer risk.

Currently, echinocandins are first-line drugs for treatment of invasive candidiasis. However, data on how serum influences killing activity of echinocandins against uncommon Candida species are limited. Therefore, the killing activity of micafungin in RPMI-1640 and in 50% serum was compared against Candida guilliermondii, Candida lusitaniae, and Candida kefyr. Minimum inhibitory concentration (MIC) ranges in RPMI-1640 were 0.5-1, 0.12-0.25, and 0.06-0.12 mg/L, respectively. In 50% serum, MICs increased 32- to 256-fold. In RPMI-1640 ≥ 0.25, ≥4, and 32 mg/L micafungin was fungicidal against all four C. kefyr (≤4.04 hours), two of three C. lusitaniae (≤16.10 hours), and two of three C. guilliermondii (≤12.30 hours), respectively. In 50% serum, all three species grew at ≤4 mg/L. Micafungin at 16-32 mg/L was fungicidal against all C. kefyr isolates (≤3.03 hours) and at 32 mg/L was fungistatic against one of three C. lusitaniae isolates. Two C. lusitaniae isolates and all three C. guilliermondii grew at all tested concentrations. Adding human serum to susceptibility test media drew attention to loss of fungicidal or fungistatic activity of micafungin in the presence of serum proteins, which is not predicted by MICs in case of C. kefyr and C. lusitaniae in RPMI-1640. Our results strongly suggest that micafungin and probably other echinocandins should be used with caution against rare Candida species.

With the development of a wide range of new biomaterials for the sensing of different cell behaviour, it is important to consider whether the cells tested in vitro are in direct contact with the material or whether cell-biomaterial contact is mediated by an interfacial layer of proteins originating from the culture medium or from the cells themselves. Thus, this study describes the differences between the cell adhesion mediated by proteins originating from foetal bovine serum and without the presence of such proteins 2 h following cell seeding exemplarily with different cell types (an osteoblastic cell line, primary fibroblasts, and mesenchymal stem cells). Three of the examined cell types were found to react differently to differing conditions in terms of cell shape, area, and number. Nevertheless, the expression and localization of the various proteins involved in cell adhesion and signalling (CD44, vinculin, talin, actin, focal adhesion kinase, Rho-GTPases and extracellular signal-regulated kinases 1 and 2) were, in general, similar with respect to all the cell types tested, albeit varying according to the presence or absence of serum. Moreover, no classical focal adhesions were formed during cell adhesion without serum proteins, while different signalling pathways were involved in this process. The study systematically describes and discusses the cell adhesion of three different human cell types to a well-known substrate without the presence of external proteins and it is hoped that this knowledge will be subsequently applied in biomaterial applications in which the presence of external proteins is undesirable (e.g. for biosensing purposes).

Human rhinoviruses (HRVs) are the most common cause of viral respiratory tract infections, and are associated with significant morbidity and mortality in immunocompromised individuals and patients with pre-existing pulmonary conditions. The therapeutic options available are extremely limited and therefore novel therapeutics for HRV infections are of significant interest. Cathelicidins have been shown to have potent antiviral activity against a range of pathogens and are known to be key immunomodulatory mediators during infection. We therefore assessed the antiviral potential of cathelicidins from humans and other mammalian species against HRV, together with the potential for the human cathelicidin to modulate apoptotic pathways and alter cell viability during HRV infection. We demonstrate that LL-37, the porcine cathelicidin Protegrin-1, and the ovine cathelicidin SMAP-29 display potent antiviral activity towards HRV and that this activity is visible when either the virus is exposed to the peptides prior to cell infection or after cells have been infected. We further demonstrate that, in contrast to established findings with bacterial infection models, LL-37 does not induce apoptosis or necrosis in HRV-infected lung epithelial cells at physiological or superphysiological concentrations, but does reduce the metabolic activity of infected cells compared to uninfected cells treated with similar peptide concentrations. Collectively, the findings from this study demonstrate that the mechanism of action of cathelicidins against rhinovirus is by directly affecting the virus and we propose that the delivery of exogenous cathelicidins, or novel synthetic analogues, represent an exciting and novel therapeutic strategy for rhinovirus infection., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Water molecules play an essential role for mediating interactions between ligands and protein binding sites. Displacement of specific water molecules can favorably modulate the free energy of binding of protein-ligand complexes. Here, the nature of water interactions in protein binding sites is investigated by 3D RISM (three-dimensional reference interaction site model) integral equation theory to understand and exploit local thermodynamic features of water molecules by ranking their possible displacement in structure-based design. Unlike molecular dynamics-based approaches, 3D RISM theory allows for fast and noise-free calculations using the same detailed level of solute-solvent interaction description. Here we correlate molecular water entities instead of mere site density maxima with local contributions to the solvation free energy using novel algorithms. Distinct water molecules and hydration sites are investigated in multiple protein-ligand X-ray structures, namely streptavidin, factor Xa, and factor VIIa, based on 3D RISM-derived free energy density fields. Our approach allows the semiquantitative assessment of whether a given structural water molecule can potentially be targeted for replacement in structure-based design. Finally, PLS-based regression models from free energy density fields used within a 3D-QSAR approach (CARMa - comparative analysis of 3D RISM Maps) are shown to be able to extract relevant information for the interpretation of structure-activity relationship (SAR) trends, as demonstrated for a series of serine protease inhibitors.

Cancer cells exhibit specific physiological differences compared to normal cells. Most surface membranes of cancer cells are characterized by high expression of given protein receptors, such as albumin, transferrin, and growth factors that are also present in the plasma of patients themselves, but are lacking on the surface of normal cells. These distinct features between cancer and normal cells can serve as a niche for developing specific treatment strategies. Near-infrared (NIR)-light-triggered therapy platforms are an interesting novel avenue for use in clinical nanomedicine. As a photothermal agent, conducting polymer nanoparticles, such as polypyrrole (PPy), of great NIR light photothermal effects and good biocompatibility, show promising applications in cancer treatments through the hyperthermia mechanism. Autologous plasma proteins coated PPy nanoparticles for hyperthermia therapy as a novel core technology platform to treat cancers through secreted protein acid and rich in cysteine targeting are developed here. This approach can provide unique features of specific targeting toward cancer cell surface markers and immune transparency to avoid recognition and attack by defense cells and achieve prolonged circulation half-life. This technology platform unveils new clinical options for treatment of cancer patients, supporting the emergence of innovative clinical products., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ethyl pyruvate (EP) is a stable lipophilic pyruvate derivative. Studies demonstrated that EP shows potent anti-oxidation, anti-inflammatory and anti-coagulant effects. Inflammation and coagulation are closely interacted with platelet activation. However, it is unclear whether EP has anti-platelet effects. Therefore, we investigated the anti-platelet effect of EP in this study in vitro. We found that EP inhibited agonists induced platelets aggregation, ATP release and adhesion to collagen. Flow cytometric analysis revealed that EP inhibited agonist induced platelets PAC-1 binding, as well as P-selectin and CD40L expression. The underlying mechanism of action may involve the inhibition of platelet PI3K/Akt and Protein Kinase C (PKC) signaling pathways. Additionally, EP dose dependently inhibited platelet PS exposure induced by high concentration thrombin. Lactate dehydrogenase (LDH) activity assay and mice platelet count implied that EP may have no toxic effect on platelets. Therefore, we are the first to report that EP has potent anti-platelet activity and attenuates platelet PS exposure in vitro, suggesting that the inhibitory effects of EP on platelets may also play important roles in improvement of inflammation and coagulation disorder in related animal models., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The objectives of this study is to evaluate the efficacy of protein-free media in the preparation, holding and crypreservation of spermatazoa for use in ART. Normozoospermic semen samples (N=71) were used to compare the effects of media on the survival and quality of spermatozoa when washed and cultured with different media with and without added proteins at 4°C, 15°C, 22°C and 37°C for 0, 4-7 and 24h. Survival and quality of spermatozoa were assessed after freeze-thaw with synthetic cryoprotectant with and without proteins. Ethics/IRB approval was obtained (Ref. 1073.52). Spermatozoa parameters were similar in all media after washing and culture for 24h. Post-thaw survival and quality of spermatozoa was not significantly different 24h after thawing of samples frozen in all cryoprotectant medium. In conclusion synthetic protein-free culture and cryoprotectant media are equal in efficacy to protein-containing media in culture and cryopreservation of spermatozoa . Use of these synthetic media are anticipated to significantly reduce the risk, potentially associated with conventional protein-containing media, of transmission of disease and possibly harmful undeclared proteins to the patient, baby and the healtcare worker. Synthetic media also ensure consistency of quality between batches of media., (Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

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.

Objective: The aim of this study was to optimize the synthesis of the plasmin and urokinase (uPA) inhibitor benzylsulfonyl-D-Ser-homoPhe-(4-amidino-benzylamide) (BSFAB), to characterize its activity and mechanism of action and to assess its use to improve stratum corneum (SC) barrier function., Methods: Peptide coupling methods were used to synthesize BSFAB, and high-performance liquid chromatography-mass spectrometry (HPLC-MS) together with 1 H- and 13 C-nuclear magnetic resonance spectroscopy (NMR) were applied to clarify its structure and determine its purity. Its binding mode was determined by docking studies to the catalytic domains of plasmin and uPA. Inhibition constants (K i ) were determined by enzyme kinetic studies, and the effect of BSFAB on plasmin, uPA and transglutaminase 1 expression was evaluated in non-cytokine and cytokine-stimulated keratinocytes. A vehicle-controlled clinical study on SC barrier function was conducted on facial skin of subjects with self-perceived sensitive skin., Results: BSFAB was synthesized with high purity (97.3%). In silico studies indicated that the amidine moiety of BSFAB was anchored in the S1 pocket of both enzymes by binding to Asp189, Ser190 and Gly219, whereas the backbone of the D-Ser residue makes an anti-parallel β-sheet interaction with Gly216. BSFAB was shown to be an effective inhibitor of plasmin and uPA with K i values of 29 and 25 nM, respectively. BSFAB also inhibited keratinocyte-secreted protease activities in basal (plasmin inhibition 37.7%, P < 0.05 and uPA inhibition 96.6%, P < 0.01) and cytokine-induced conditions (plasmin inhibition 41.1%, P < 0.05 and uPA inhibition 97.0%, P < 0.001) and stimulated the gene expression of transglutaminase 1 in cytokine-stimulated keratinocytes (approximately 4.5 times increased expression, P < 0.01). Clinically, BSFAB was shown to improve SC barrier integrity (P < 0.02 on day 29) and subjective improvements in the perception of healthy skin (P < 0.05 on day 28)., Conclusion: BSFAB binds as a reversible competitive inhibitor to the active sites of plasmin and uPA. Additionally, BSFAB positively improved keratinocyte differentiation gene expression (transglutaminase 1). These effects were translated into improvements in SC barrier integrity clinically in subjects with dry and sensitive skin and improved their perception of having a healthy skin condition., (© 2016 Society of Cosmetic Scientists and the Société Française de Cosmétologie.)