Adverse neurological effects after exposure to copper, manganese, and mercury mixtures in a Spraque-Dawley rat model: an ultrastructural investigation.

Exposure to environmental metal pollutants is linked to oxidative stress and the subsequent development of neurological disease. In this study, the effects of copper, manganese, and mercury, were evaluated at X100 the World Health Organization safety limits for drinking water. Using a Sprague-Dawley rat model, following exposure for 28 days, the effects of these metals on biochemical blood parameters and tissue and cellular structure of the brain were determined. Biochemical analysis revealed no hepatocellular injury with minor changes associated with the hepatobiliary system. Minimal changes were found for renal function and the Na+/K+ ratio was reduced in the copper and manganese (Cu + Mn) and copper, manganese, and mercury (Cu, Mn + Hg) groups that could affect neurological function. Light microscopy of the brain revealed abnormal histopathology of Purkinje cells in the cerebellum and pyramidal cells in the cerebrum as well as tissue damage and fibrosis of the surface blood vessels. Transmission electron microscopy of the cerebral neurons showed microscopic signs of axonal damage, chromatin condensation, the presence of indistinct nucleoli and mitochondrial damage. Together these cellular features suggest the presence and influence of oxidative stress. Exposure to these metals at X100 the safety limits, as part of mixtures, induces changes to neurological tissue that could adversely influence neurological functioning in the central nervous system.

Antifungal activity and mode of action of synthetic peptides derived from the tick OsDef2 defensin.

Candida albicans is the principal opportunistic fungal pathogen in nosocomial settings and resistance to antifungal drugs is on the rise. Antimicrobial peptides from natural sources are promising novel therapeutics against C. albicans. OsDef2 defensin was previously found to be active against only Gram-positive bacteria, whereas derived fragments Os and its cysteine-free analogue, Os-C, are active against Gram-positive and Gram-negative bacteria at low micromolar concentrations. In this study, OsDef2-derived analogues and fragments were screened for anticandidal activity with the aim to identify peptides with antifungal activity and in so doing obtain a better understanding of the structural requirements for activity and modes of action. Os, Os-C and Os(11-22)NH2 , a Os-truncated carboxy-terminal-amidated fragment, had the most significant antifungal activities, with minimum fungicidal concentrations (MFCs) in the micromolar range (6-28 µM). C. albicans killing was rapid and occurred within 30-60 min. Further investigations showed all three peptides interacted with cell wall derived polysaccharides while both Os and Os(11-22)NH2 permeabilized fungal liposomes. Confocal laser scanning microscopy confirmed that Os-C and Os(11-22)NH2 could enter the cytosol of live cells and subsequent findings suggest that the uptake of Os and Os-C, in contrast to Os(11-22)NH2 , is energy dependent. Although Os, Os-C and Os(11-22)NH2 induced the production of reactive oxygen species (ROS), co-incubation with ascorbic acid revealed that only ROS generated by Os-C and to a lesser extent Os(11-22)NH2 resulted in cell death. Overall, Os, Os-C and Os(11-22)NH2 are promising candidacidal agents.

Stability, Morphology, and Effects of In Vitro Digestion on the Antioxidant Properties of Polyphenol Inclusion Complexes with ß-Cyclodextrin.

Polyphenols are inversely associated with the incidence of chronic diseases, but therapeutic use is limited by poor stability and bioaccessibility. Encapsulation has been shown to overcome some of these limitations. A selection of polyphenols (catechin, gallic acid, and epigallocatechin gallate) and their combinations were encapsulated in beta-cyclodextrin (ßCD). Encapsulation was characterized and the thermal and storage stability was evaluated using the 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. The samples were then subjected to in vitro digestion using a simple digestion (SD) model (gastric and duodenal phases) and a more complex digestion (CD) model (oral, gastric, and duodenal phases). Thereafter, the chemical (oxygen radical absorbance capacity assay) and cellular (dichlorofluorescein diacetate assay in Caco-2 cells) antioxidant and antiglycation (advanced glycation end-products assay) activities were determined. Inclusion complexes formed at a 1:1 molar ratio with a high encapsulation yield and efficiency. Encapsulation altered the morphology of the samples, increased the thermal stability of some and the storage stability of all samples. Encapsulation maintained the antioxidant activity of all samples and significantly improved the antiglycation and cellular antioxidant activities of some polyphenols following SD. In conclusion, the formed inclusion complexes of ßCD with polyphenols had greater storage stability, without altering the beneficial cellular effects of the polyphenols.

Hydrothermal Processing and In Vitro Simulated Human Digestion Affects the Bioaccessibility and Bioactivity of Phenolic Compounds in African Pumpkin (Momordica balsamina) Leaves.

The African pumpkin (Momordica balsamina) contains bioactive phenolic compounds that may assist in reducing oxidative stress in the human body. The leaves are mainly consumed after boiling in water for a specific time; this hydrothermal process and conditions of the gastrointestinal tract may affect the presence and bioactivity of phenolics either positively or negatively. In this study, the effects of hydrothermal processing (boiling) and in vitro simulated human digestion on the phenolic composition, bioaccessibility and bioactivity in African pumpkin were investigated in comparison with those of spinach (Spinacia oleracea). A high-resolution ultra-performance liquid chromatography, coupled with diode array detection, quadrupole time-of-flight and mass spectrometer (UPLC-DAD-QTOF-MS) was used to profile phenolic metabolites. Metabolites such as 3-caffeoylquinic acid, 5-caffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid were highly concentrated in the boiled vegetable extracts compared to the raw undigested and all digested samples. The majority of African pumpkin and spinach extracts (non-digested and digested) protected Deoxyribonucleic acid (DNA), (mouse fibroblast) L929 and human epithelial colorectal adenocarcinoma (Caco-2) cells from 2,2'-Azobis(2-methylpropionamidine) dihydrochloride (AAPH)-induced oxidative damage. From these results, the consumption of boiled African pumpkin leaves, as well as spinach, could be encouraged, as bioactive metabolites present may reduce oxidative stress in the body.

Adverse cardiovascular effects of exposure to cadmium and mercury alone and in combination on the cardiac tissue and aorta of Sprague-Dawley rats.

The aim of this study was to identify cardiovascular effects of relevant concentrations of Cd and Hg alone and in combination as a mixture in water. This was achieved by administering to male Sprague-Dawley rats via gavage 0.62 mg/kg Cd or 1.23 mg/kg Hg, or a combination of 0.62 mg/kg Cd and 1.23 mg/kg Hg in the co-exposure group for 28 days. Concentrations were the rat equivalence dosages of 1,000 times the World Health Organization's limits of 0.003 mg/L and 0.006 mg/L for Cd and Hg, respectively, for water. With termination, blood levels of the metals were increased. For all metal exposed groups, histological evaluation and transmission electron microscopy of the myocardium revealed myofibrillar necrosis, increased fibrosis, vacuole formation and mitochondrial damage. Cd caused the most mitochondrial damage while Hg to a greater degree induced fibrosis. In the aorta, both Cd and Hg also increased collagen deposition adversely altering the morphology of the fenestrated elastic fibers in the tunica media. Co-exposure resulted in increased cardiotoxicity with increased mitochondrial damage, fibrosis and distortion of the aortic wall as a result of increased collagen deposition, as well as altered elastin deposition, fragmentation and interlink formation. These are typical features of oxidative damage that correlates with a phenotype of premature ageing of the CVS that potentially can lead to hypertension and premature cardiac failure.

Induction of hepatic portal fibrosis, mitochondria damage, and extracellular vesicle formation in Sprague-Dawley rats exposed to copper, manganese, and mercury, alone and in combination.

Increased anthropogenic activity and subsequent environmental exposure to heavy metals induce the production of reactive oxygen species (ROS), which increases oxidative stress and the risk of associated diseases. The aim of this study, in a subacute model of toxicity, was to investigate the effects of copper (Cu), manganese (Mn), and mercury (Hg) alone and in combination on the liver tissue of male Sprague-Dawley rats, exposed orally to 100 times the World Health Organization's acceptable water limits of each metal. General histological alterations as well as ultrastructural changes were investigated using light microscopy and transmission electron microscopy (TEM) respectively. Exposure to Cu, Mn, and Hg, alone and in combinations, caused hydropic swelling of the hepatocytes, dilation of the sinusoids, formation of binucleated hepatocytes with an increased inflammatory cell accumulation at the portal triad. Increased collagen deposition with associated fibrosis was also observed. Evaluation of hepatocyte ultrastructure revealed mitochondrial membrane damage and inner membrane swelling especially for hepatocytes exposed to Mn. Extracellular vesicle (EV) formation was observed in the liver tissue of all exposed rats. Furthermore, increased damage observed for metal combinations was possibly due to synergism. In conclusion, Cu, Mn, and Hg alone and as part of a mixture cause cellular damage, inflammation, and fibrosis increasing the risk of associated diseases.

The dual functionality of antimicrobial peptides Os and Os-C in human leukocytes.

Antimicrobial peptides (AMPs), Os and Os-C, have been identified as multifunctional peptides with antibacterial, antiendotoxin, and anti-inflammatory properties. For further development of Os and Os-C as therapeutic peptides, it is essential to evaluate these effects in human mononuclear (MN) and polymorphonuclear (PMN) leukocytes. The cytotoxicity and the effects of both peptides on MN and PMN morphology were determined with the Alamar-Blue assay and scanning electron microscopy, respectively. The ability of Os and Os-C to induce reactive oxygen species (ROS) and to protect against 2,2'-azobis(2-amidinopropane) dihydrochloride-induced oxidative damage in both cell populations was evaluated using 2',7'-dichlorofluorescin diacetate (DCFH-DA). Using fluorescently labeled peptides, the ability of the peptides to cross the cell membranes of MN and PMN was also evaluated. At the minimum bactericidal concentrations of Os and Os-C, neither peptide was cytotoxic. Os caused morphological features of toxicity at 100 µM, entered MN cells, and also protected these cells against oxidative damage. Os-C caused MN and PMN leukocyte activation associated with ROS formation and was unable to penetrate cell membranes, indicating extracellular membrane interactions. This study confirms that both Os and Os-C at less than 100 µM are not cytotoxic. The MN-specific uptake of Os identifies it as a cell-specific cargo-carrier peptide, with additional anti-inflammatory properties. In contrast, the ability of Os-C to activate MN and PMN cells implies that this peptide should be further evaluated as an AMP, which, in addition to its ability to eradicate infection, can further enhance host immunity. These novel characteristics of Os and Os-C indicate that these AMPs as peptides can be further developed for specific applications.

Antimicrobial function of short amidated peptide fragments from the tick-derived OsDef2 defensin.

Previously Os, a 22 amino acid sequence of a defensin from the soft tick Ornithodoros savignyi, was found to kill Gram-positive and Gram-negative bacteria at low micromolar concentrations. In this study, we evaluated synthetic peptide analogues of Os for antibacterial activity with an aim to identify minimalized active peptide sequences and in so doing obtain a better understanding of the structural requirements for activity. Out of eight partially overlapping sequences of 10 to 12 residues, only Os(3-12) and Os(11-22) exhibit activity when screened against Gram-positive and Gram-negative bacteria. Carboxyamidation of both peptides increased membrane-mediated activity, although carboxyamidation of Os(11-22) negatively impacted on activity against Staphylococcus aureus. The amidated peptides, Os(3-12)NH2 and Os(11-22)NH2 , have minimum bactericidal concentrations of 3.3 µM against Escherichia coli. Killing was reached within 10 minutes for Os(3-12)NH2 and only during the second hour for Os(11-22)NH2 . In an E. coli membrane liposome system, both Os and Os(3-12)NH2 were identified as membrane disrupting while Os(11-22)NH2 was less active, indicating that in addition to membrane permeabilization, other targets may be involved in bacterial killing. In contrast to Os, the membrane disruptive effect of Os(3-12)NH2 did not diminish in the presence of salt. Neither Os nor its amidated derivatives caused human erythrocyte haemolysis. The contrasting killing kinetics and effects of amidation together with structural and liposome leakage data suggest that the 3-12 fragment relies on a membrane disruptive mechanism while the 11-22 fragment involves additional target mechanisms. The salt-resistant potency of Os(3-12)NH2 identifies it as a promising candidate for further development.

Ultrastructural, Confocal and Viscoelastic Characteristics of Whole Blood and Plasma After Exposure to Cadmium and Chromium Alone and in Combination: An Ex Vivo Study.

BACKGROUND/AIMS: Heavy metal pollution is increasing in the environment, contaminating water, food and air supplies. This can be linked to many anthropogenic activities. Heavy metals are absorbed through the skin, inhalation and/or orally. Irrespective of the manner of heavy metal entry in the body, the blood circulatory system is potentially the first to be affected following exposure and adverse effects on blood coagulation can lead to associated thrombotic disease. Although the plasma levels and the effects of cadmium (Cd) and chromium (Cr) on erythrocytes and lymphocytes have been described, the environmental exposure to heavy metals are not limited to a single metal and often involves metal mixtures, with each metal having different rates of absorption, different cellular, tissue, and organ targets. Therefore the aim of this study is to investigate the effects of the heavy metals Cd and Cr alone and whether Cr synergistically increases the effect of Cd on physiological important processes such as blood coagulation. METHODS: Human blood was exposed to the heavy metals ex vivo, and thereafter morphological analysis was performed with scanning electron- and confocal laser scanning microscopy (CLSM) in conjunction with thromboelastography®. RESULTS: The erythrocytes, platelets and fibrin networks presented with ultrastructural changes, including varied erythrocytes morphologies, activated platelets and significantly thicker fibrin fibres in the metal-exposed groups. CLSM analysis revealed the presence of phosphatidylserine on the outer surface of the membranes of the spherocytic erythrocytes exposed to Cd and Cr alone and in combination. The viscoelastic analysis revealed only a trend that indicates that clots that will form after heavy metal exposure, will likely be fragile and unstable especially for Cd and Cr in combination. CONCLUSION: This study identified the blood as an important target system of Cd and Cr toxicity.

Effects of chronic exposure to mercury and cadmium alone and in combination on the coagulation system of Sprague-Dawley rats.

Water contamination with heavy metals may adversely affect our health. High metal levels lead to changes in blood coagulation processes, increasing the risk for cardiovascular disease. Exposure is not limited to a single metal but usually involves a mixture of metals. In this study 24 male Sprague-Dawley rats were exposed to cadmium (Cd) and mercury (Hg), alone and in combination, for 28 days at dosages equivalent to 1000 times the World Health Organization water limits. Scanning electron microscopy analysis revealed that both metals caused platelet activation. Cd significantly increased fibrin fibers thickness and caused aggregation and formation of dense matted deposits (DMDs). Hg reduced fibrin network formation. In the combination group, Hg appeared to augment the effect of Cd, and the presence of extensive DMDs or aggregates between the fibers, with no changes to the actual fibrin thickness, was observed.

Anti-inflammatory and anti-endotoxin properties of peptides derived from the carboxy-terminal region of a defensin from the tick Ornithodoros savignyi.

Antimicrobial peptides are small cationic peptides that possess a large spectrum of bioactivities, including antimicrobial, anti-inflammatory and antioxidant activities. Several antimicrobial peptides are known to inhibit lipopolysaccharide (LPS)-induced inflammation in vitro and to protect animals from sepsis. In this study, the cellular anti-inflammatory and anti-endotoxin activities of Os and Os-C, peptides derived from the carboxy-terminal of a tick defensin, were investigated. Both Os and Os-C were found to bind LPS in vitro, albeit to a lesser extent than polymyxin B and melittin, known endotoxin-binding peptides. Binding to LPS was found to reduce the bactericidal activity of Os and Os-C against Escherichia coli confirming the affinity of both peptides for LPS. At a concentration of 25 µM, the nitric oxide (NO) scavenging activity of Os was higher than glutathione, a known NO scavenger. In contrast, Os-C showed no scavenging activity. Os and Os-C inhibited LPS/IFN-γ induced NO and TNF-α production in RAW 264.7 cells in a concentration-dependent manner, with no cellular toxicity even at a concentration of 100 µM. Although inhibition of NO and TNF-α secretion was more pronounced for melittin and polymyxin B, significant cytotoxicity was observed at concentrations of 1.56 µM and 25 µM for melittin and polymyxin B, respectively. In addition, Os, Os-C and glutathione protected RAW 264.7 cells from oxidative damage at concentrations as low as 25 µM. This study identified that besides previously reported antibacterial activity of Os and Os-C, both peptides have in addition anti-inflammatory and anti-endotoxin properties.

How methylglyoxal kills bacteria: An ultrastructural study.

Antibacterial activity of honey is due to the presence of methylglyoxal (MGO), H2O2, bee defensin as well as polyphenols. High MGO levels in manuka honey are the main source of antibacterial activity. Manuka honey has been reported to reduce the swarming and swimming motility of Pseudomonas aeruginosa due to de-flagellation. Due to the complexity of honey it is unknown if this effect is directly due to MGO. In this ultrastructural investigation the effects of MGO on the morphology of bacteria and specifically the structure of fimbriae and flagella were investigated. MGO effectively inhibited Gram positive (Bacillus subtilis; MIC 0.8 mM and Staphylococcus aureus; MIC 1.2 mM) and Gram negative (P. aeruginosa; MIC 1.0 mM and Escherichia coli; MIC 1.2 mM) bacteria growth. The ultrastructural effects of 0.5, 1.0 and 2 mM MGO on B. substilis and E. coli morphology was then evaluated. At 0.5 mM MGO, bacteria structure was unaltered. For both bacteria at 1 mM MGO fewer fimbriae were present and the flagella were less or absent. Identified structures appeared stunted and fragile. At 2 mM MGO fimbriae and flagella were absent while the bacteria were rounded with shrinkage and loss of membrane integrity. Antibacterial MGO causes alterations in the structure of bacterial fimbriae and flagella which would limit bacteria adherence and motility.

Anti-proliferative properties of commercial Pelargonium sidoides tincture, with cell-cycle G0/G1 arrest and apoptosis in Jurkat leukaemia cells.

Context Pelargonium sidoides DC (Geraniaceae) is an important medicinal plant indigenous to South Africa and Lesotho. Previous studies have shown that root extracts are rich in polyphenolic compounds with antibacterial, antiviral and immunomodulatory activities. Little is known regarding the anticancer properties of Pelargonium sidoides extracts. Objective This study evaluates the anti-proliferative effects of a Pelargonium sidoides radix mother tincture (PST). Materials and methods The PST was characterized by LC-MS/MS. Anti-proliferative activity was evaluated in the pre-screen panel of the National Cancer Institute (NCI-H460, MCF-7 and SF-268) and the Jurkat leukaemia cell line at concentrations of 0-150 µg/mL. The effect on cell growth was determined with sulphorhodamine B and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays after 72 h. The effect on cell cycle and apoptosis induction in Jurkat cells was determined by flow cytometry with propidium iodide and Annexin V: fluorescein isothiocyanate staining. Results Dihydroxycoumarin sulphates, gallic acid as well as gallocatechin dimers and trimers were characterized in PST by mass spectrometry. Moderate anti-proliferative effects with GI50 values between 40 and 80 µg/mL were observed in the NCI-pre-screen panel. Strong activity observed with Jurkat cells with a GI50 value of 6.2 µg/mL, significantly better than positive control 5-fluorouracil (GI50 value of 9.7 µg/mL). The PST arrested Jurkat cells at the G0/G1 phase of the cell cycle and increased the apoptotic cells from 9% to 21%, while the dead cells increased from 4% to 17%. Conclusion We present evidence that P. sidoides has cancer cell type-specific anti-proliferative effects and may be a source of novel anticancer molecules.

Premature collagen fibril formation, fibroblast-mast cell interactions and mast cell-mediated phagocytosis of collagen in keloids.

Keloids are benign hyper-proliferative growths of fibrous tissue where increased fibroblast activity results in abnormal collagen deposition. Excessive inflammation is a characteristic feature of keloids, but little is known about the underlying ultrastructural features of keloids related to collagen processing, fibril and fiber formation, the interaction between fibroblasts and associated collagen fibers and mast cells. In this study, the ultrastructure of the dermis of keloid patients was evaluated using light and transmission electron microscopy techniques. Abnormal intracellular premature collagen fibril formation was observed. Phagocytosis of collagen fibrils by mast cells was a common ultrastructural feature of keloid tissue as was a close or direct association between fibroblasts and mast cells. Based on these findings and recent advances in knowledge related to collagen synthesis, fibril formation and processing, we hypothesize that keloid formation is primarily due to abnormal collagen synthesis where the consequent accumulation of collagen fibers causes increased mast cell recruitment and collagen phagocytosis. Subsequent release of mast cell-derived mediators then promotes further collagen synthesis. The observation of early formation in keloid tissue of premature insoluble collagen fibrils supports previous studies that enzymes such as procollagen C-proteinase are important early therapeutic targets.

An in ovo investigation into the hepatotoxicity of cadmium and chromium evaluated with light- and transmission electron microscopy and electron energy-loss spectroscopy.

Excessive agriculture, transport and mining often lead to the contamination of valuable water resources. Communities using this water for drinking, washing, bathing and the irrigation of crops are continuously being exposed to these heavy metals. The most vulnerable is the developing fetus. Cadmium (Cd) and chrome (Cr) were identified as two of the most prevalent heavy metal water contaminants in South Africa. In this study, chicken embryos at the stage of early organogenesis were exposed to a single dosage of 0.430 µM physiological dosage (PD) and 430 µM (×1000 PD) CdCl2, as well as 0.476 µM (PD) and 746 µM (×1000 PD) K2Cr2O7. At day 14, when all organ systems were completely developed, the embryos were terminated and the effect of these metals on liver tissue and cellular morphology was determined with light- and transmission electron microscopy (TEM). The intracellular localization of these metals was determined using electron energy-loss spectroscopy (EELS). With light microscopy, the PD of both Cd and Cr had no effect on liver tissue or cellular morphology. At ×1000 PD both Cd and Cr caused sinusoid dilation and tissue necrosis. With TEM analysis, Cd exposed hepatocytes presented with irregular chromatin condensation, ruptured cellular membranes and damaged or absent organelles. In contrast Cr caused only slight mitochondrial damage. EELS revealed the bio-accumulation of Cd and Cr along the cristae of the mitochondria and chromatin of the nuclei.

The effect of sibutramine, a serotonin-norepinephrine reuptake inhibitor, on platelets and fibrin networks of male Sprague-Dawley rats: a descriptive study.

Sibutramine is used in the treatment of obesity due to its ability to influence feelings of hunger and satiety by inhibiting the re-uptake of serotonin and noradrenalin in the central nervous system (CNS). Sibutramine use has been associated with numerous adverse events in particular cardiovascular complications possibly due to the formation of thrombi. This ultrastructural descriptive study investigated the effect of sibutramine on blood coagulation, specifically the effect on morphology of platelets and fibrin networks using scanning electron microscopy. Male Sprague-Dawley rats treated with either a recommended therapeutic dose [low dosage 1.32 mg/kg] or a toxicological higher dose [high dosage 13.2 mg/kg] of sibutramine for 28 days were used and compared to control animals. Blood samples were collected and plasma smears were prepared for platelet evaluation. Following the addition of thrombin to the plasma samples, the morphology of the fibrin clots was evaluated. Platelet evaluation by scanning electron microscopy revealed morphology typical of a prothrombotic state with a characteristic excessive platelet activation in both low-dose (LD) and high-dose (HD) rats. The fibrin clots of sibutramine-treated rats, LD and HD revealed fused thick fibers with thin fibers forming a net-like structure over the thick fibers which differ considerably from the organized structure of the control animals. It can be concluded that sibutramine alters the ultrastructure of platelets and fibrin networks creating a prothrombotic state.

Tryptophan End-Tagging Confers Antifungal Activity on a Tick-Derived Peptide by Triggering Reactive Oxygen Species Production.

WHO has identified several Candida species including Candida albicans as critical priority fungal pathogens due to greater infection prevalence and formation of recalcitrant biofilms. Novel antifungal agents are urgently needed, and antimicrobial peptides (AMPs) are being considered as potential alternatives, but inactivity in physiological salt environments, serum, and plasma often limits further therapeutic development. Tryptophan end-tagging is a strategy to overcome these limitations and is thought to selectively enhance membrane permeabilization in both fungal and bacterial plasma membranes. Here, we show that C-terminal tryptophan end-tagging of the tick-derived peptide Os-C transforms an inactive peptide into Os-C(W5), an antifungal peptide capable of preventing the formation of C. albicans biofilms. Mechanistic insight is provided by circular dichroism spectroscopy and molecular dynamics simulations, which demonstrate that tryptophan end-tagging alters the secondary structure of Os-C, while the latter reveals that end-tagging reduces interactions with, and insertion into, a model C. albicans membrane but promotes peptide aggregation on its surface. Interestingly, this leads to the induction of reactive oxygen species production rather than membrane permeabilization, and consequently, oxidative stress leads to cell wall damage. Os-C(W5) does not induce the hemolysis of human erythrocytes. Reduced cell adhesion and viability contribute to decreased biofilm extracellular matrix formation which, although reduced, is retained in the serum-containing medium. In this study, tryptophan end-tagging was identified as a promising strategy for enhancing the antifungal activity, including the biofilm inhibitory activity of Os-C against C. albicans in physiological salt environments.

QSAR Reveals Decreased Lipophilicity of Polar Residues Determines the Selectivity of Antimicrobial Peptide Activity.

Antimicrobial resistance has increased rapidly, causing daunting morbidity and mortality rates worldwide. Antimicrobial peptides (AMPs) have emerged as promising alternatives to traditional antibiotics due to their broad range of targets and low tendency to elicit resistance. However, potent antimicrobial activity is often accompanied by excessive cytotoxicity toward host cells, leading to a halt in AMP therapeutic development. Here, we present multivariate analyses that correlate 28 peptide properties to the activity and toxicity of 46 diverse African-derived AMPs and identify the negative lipophilicity of polar residues as an essential physiochemical property for selective antimicrobial activity. Twenty-seven active AMPs are identified, of which the majority are of scorpion or frog origin. Of these, thirteen are novel with no previously reported activities. Principal component analysis and quantitative structure-activity relationships (QSAR) reveal that overall hydrophobicity, lipophilicity, and residue side chain surface area affect the antimicrobial and cytotoxic activity of an AMP. This has been well documented previously, but the present QSAR analysis additionally reveals that a decrease in the lipophilicity, contributed by those amino acids classified as polar, confers selectivity for a peptide to pathogen over mammalian cells. Furthermore, an increase in overall peptide charge aids selectivity toward Gram-negative bacteria and fungi, while selectivity toward Gram-positive bacteria is obtained through an increased number of small lipophilic residues. Finally, a conservative increase in peptide size in terms of sequence length and molecular weight also contributes to improved activity without affecting toxicity. Our findings suggest a novel approach for the rational design or modification of existing AMPs to increase pathogen selectivity and enhance therapeutic potential.

Structural and functional characterization of peptides derived from the carboxy-terminal region of a defensin from the tick Ornithodoros savignyi.

Tick defensins may serve as templates for the development of multifunctional peptides. The purpose of this study was to evaluate shorter peptides derived from tick defensin isoform 2 (OsDef2) in terms of their antibacterial, antioxidant, and cytotoxic activities. We compared the structural and functional properties of a synthetic peptide derived from the carboxy-terminal of the parent peptide (Os) to that of an analogue in which the three cysteine residues were omitted (Os-C). Here, we report that both peptides were bactericidal (MBC values ranging from 0.94-15 µg/ml) to both Gram-positive and Gram-negative bacteria, whereas the parent peptide only exhibited Gram-positive antibacterial activity. The Os peptide was found to be two-fold more active than Os-C against three of the four tested bacteria but equally active against Staphylococcus aureus. Os showed rapid killing kinetics against both Escherichia coli and Bacillus subtilis, whereas Os-C took longer, suggesting different modes of action. Scanning electron microscopy showed that in contrast to melittin for which blebbing of bacterial surfaces was observed, cells exposed to either peptide appeared flattened and empty. Circular dichroism data indicated that in a membrane-mimicking environment, the cysteine-containing peptide has a higher α-helical content. Both peptides were found to be non-toxic to mammalian cells. Moreover, the peptides displayed potent antioxidant activity and were 12 times more active than melittin. Multifunctional peptides hold potential for a wide range of clinical applications and further investigation into their mode of antibacterial and antioxidant properties is therefore warranted.

Rats on a high-energy diet showing no weight gain present with ultrastructural changes associated with liver fibrosis.

Sibutramine is widely used as a weight-loss substance in the treatment of obesity and is a selective inhibitor of the neuronal reuptake of serotonin and noradrenaline. Although banned, it is often a hidden ingredient in herbal and dietary supplements that are widely used by the general public. Various weight loss products, including sibutramine, have successfully been tested in animal models of diet-induced obesity. In the female Sprague-Dawley rat model, fed a high-energy diet that did not produce a significant increase in BMI, the cellular structure of the liver was evaluated using transmission electron microscopy. Compared to controls showing no damage, the livers of rats fed a high-energy diet were found to have increased fibrosis without steatosis, while for rats fed high-energy diet with sibutramine, fibrosis was increased and steatosis had developed. In conclusion, in female rats fed a high-energy diet that does not result in weight gain hepatic fibrosis occurs without steatosis. In these rats the co-administration of sibutramine increases the degree of fibrosis and steatosis develops. Although it has been widely believed that sibutramine is not hepatotoxic, this study clearly shows that at an ultrastructural level, rats fed a high-energy diet treated with sibutramine show signs of hepatotoxicity.