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.

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.

Gastrointestinal Effects on the Antioxidant and Immunomodulatory Properties of South African Fynbos Honey.

The Fynbos biome, Western Cape Province, South Africa, produces a unique honey from Apis mellifera capensis. The bioactivity of Fynbos (FB1-FB6) honeys and Manuka, unique manuka factor 15+ (MAN UMF15+) honey subjected to simulated in vitro digestion, was compared. The effect of each phase of digestion on the antioxidant properties and nitric oxide- (NO-) associated immunomodulatory effects was determined. The total phenolic content of MAN (UMF15+) was higher than that of FB honeys, and following digestion, the percentage bioaccessibility (BA) was 68.6% and 87.1 ± 27.0%, respectively. With the Trolox equivalent antioxidant capacity assay, the activity of FB1 and FB6 was similar to MAN (UMF15+) but reduced for FB2, FB3, FB4, and FB5 with a %BA of 77.9% for MAN (UMF15+) and 78.2 ± 13.4% for FB. The oxygen radical absorbance capacity of MAN (UMF15+) and FB honeys was similar and unaltered with digestion. In a cellular environment, using colon adenocarcinoma (Caco-2) cells, both undigested and the gastric digested honey reduced 2,2'-azobis-(2-amidinopropane) dihydrochloride- (AAPH-) mediated peroxyl radical formation. In contrast, following gastroduodenal digestion, the formation of reactive oxygen species (ROS) was increased. In murine macrophage (RAW 264.7) cells, all honeys induced different levels of NO which was significantly increased with digestion for MAN (UMF15+) and FB1. In LPS/IFN-γ stimulated RAW 264.7 macrophages, only undigested MAN (UMF15+) effectively reduced NO levels, and with digestion, NO scavenging activity of MAN (UMF15+) was reduced but increased for FB5 and FB6. In a noncellular environment, MAN (UMF15+), FB1, FB2, and FB6 scavenged NO, and with digestion, this activity was maintained. This study has identified that undigested and gastric-digested FB honey has antioxidant properties with strong potential anticancer effects following gastroduodenal digestion, related to ROS formation. MAN (UMF15+) had anti-inflammatory effects which were lost postdigestion, and in contrast, FB5 and FB6 had anti-inflammatory effects postdigestion.

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.

Triterpenoids from Protorhus longifolia Exhibit Hypocholesterolemic Potential via Regulation of Cholesterol Biosynthesis and Stimulation of Low-Density Lipoprotein Uptake in HepG2 Cells.

The increasing incidence of hypercholesterolemia-related diseases even in the presence of the currently available cholesterol-lowering drugs indicates a need to discover new therapeutic drugs. This study aimed to investigate the hypocholesterolemic potential of two triterpenoids isolated from Protorhus longifolia stem bark. In silico techniques and in vitro enzyme assays were used to evaluate the potential inhibition of cholesterol esterase and HMG-CoA reductase by the triterpenoids (ARM-2 and RA-5). The toxicity, modulation of low-density lipoprotein (LDL) uptake, and associated gene expression were determined in HepG2 hepatocytes. In silico molecular docking revealed that ARM-2 compared with RA-5 has a relatively stronger binding affinity for both enzymes. Both triterpenoids further demonstrated promising in silico drug-likeness properties and favorable ADMET profiles characterized by high intestinal absorption and lack of CYP450 enzyme inhibition. The compounds further showed, to varying degrees of efficacy, inhibition of cholesterol micellization as well as both cholesterol esterase and HMG-CoA reductase activities with IC50 values ranging from 16.4 to 41.1 µM. Moreover, enhanced hepatic cellular LDL uptake and the associated upregulation of the LDL-R and SREBP-2 gene expression were observed in the triterpenoid-treated HepG2 cells. It is evident that the triterpenoids, especially ARM-2, possess hypocholesterolemic properties, and these molecules can serve as leads or structural templates for the development of new hypocholesterolemic drugs.

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.

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.

Renal Papillary Necrosis (RPN) in an African Population: Disease Patterns, Relevant Pathways, and Management.

Renal papillary necrosis (RPN) is characterized by coagulative necrosis of the renal medullary pyramids and papillae. Multiple conditions and toxins are associated with RPN. Several RPN risk factors, or POSTCARDS, have been identified, with most patients presenting with RPN having at least two contributing risk factors. Currently, there is no specific test to diagnose and confirm RPN; however, several imaging tools can be used to diagnose the condition. RPN is currently underdiagnosed in African populations, often with fatal outcomes. In African clinical settings, there is a lack of consensus on how to define and describe RPN in terms of kidney anatomy, pathology, endourology, epidemiology, the identification of African-specific risk factors, the contribution of oxidative stress, and lastly an algorithm for managing the condition. Several risk factors are unique to African populations including population-specific genetic factors, iatrogenic factors, viral infections, antimicrobial therapy, schistosomiasis, substance abuse, and hypertension (GIVASSH). Oxidative stress is central to both GIVASSH and POSTCARDS-associated risk factors. In this review, we present information specific to African populations that can be used to establish an updated consensual definition and practical grading system for radiologists, urologists, nephrologists, nuclear physicians, and pathologists in African clinical settings.

Effect of simulated in vitro upper gut digestion of processed cowpea beans on phenolic composition, antioxidant properties and cellular protection.

The effect of simulated in vitro upper gut digestion on the phenolic composition and antioxidant properties of processed cowpea beans was studied. The samples comprised four cowpea cultivars: a cream, brownish-cream and two reddish-brown cultivars. Dry cowpea seeds were soaked in water, blended into paste and deep-fried in vegetable oil. The fried samples were taken through in vitro upper gut digestion followed by freeze-drying of the supernatant. Phenolic composition of extracts from the supernatants were determined using HPLC-MS. Radical scavenging activities were documented using the TEAC, ORAC and nitric oxide (NO) assays. In vitro digestion of the processed cowpeas resulted in phenolic-peptide complexes that were identified for the first time, and decreased extractable phenolic compounds. However, the radical scavenging activities increased. The processed cowpeas and their digests inhibited cellular NO production, and oxidative DNA and cellular damage. In conclusion, deep-fried cowpeas when consumed, could potentially help alleviate oxidative stress-related conditions.

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.

Antioxidant properties and inhibition of lipid formation in 3T3-L1 adipocytes of in vitro digested mageu, a commercial sample.

Mageu is a fermented, non-alcoholic maize-derived product unique to southern Africa. The aim of this study was to identify the health benefits of a polyphenolic extract of commercially produced mageu related to the antioxidant properties and effects on lipid accumulation in differentiated 3T3-L1 adipocytes. A pooled sample of mageu Number 1 brand (original non-flavored) was subjected to in vitro gastroduodenal digestion (GDD). Reverse phase high-performance liquid chromatography of unfractionated undigested (UD) and GDD mageu revealed that with digestion there was an increased extraction of 1.2, 1.83, 1.45, 4.86, and 3.17-fold of caffeic acid, 3,4-dihydroxybenzoic acid, p-coumaric acid, 4 hydroxybenzoic acid and ferulic acid, respectively. An associated increase in the total phenolic acid content and antioxidant activity in the <3 kDa fraction was obtained. In contrast with digestion, inhibition of advanced glycation end products formation and low-density lipoprotein oxidation was found in the <30 kDa fraction indicating the contribution of larger, possibly feruloylated polysaccharides, to activity. Cellular antioxidant activity in Caco-2 cells was >90% for all UD fractions, but with GDD was reduced. All fractions had low scavenging of nitric oxide in the lipopolysaccharide/murine cell model. Exposure of 3T3-L1 adipocytes to all the UD and GDD mageu fractions (at 1% and 10% concentrations) during differentiation resulted in at least a 35% reduction in lipid accumulation, which was not associated with a loss of cellular viability. In conclusion, mageu, UD, and subjected to GDD contains phenolic acids with beneficial bioactive properties that contribute to antioxidant activity and reduces lipid accumulation in adipocytes. PRACTICAL APPLICATIONS: Mageu is a non-alcoholic fermented maize product which when digested has increased bioactivity. Its reported health benefits are due to its caloric content therefore the practical application of this research is to validate the scientific benefits of this food and encourage increased consumption of this functional food. This is especially important in the context of the South African population where this product is widely consumed as increasing obesity is associated with an increased risk of non-communicable disease. Furthermore, as a non-alcoholic drink, consumption can be promoted for all ages' groups and religions, and a commercialized manufacture processes can be optimized to increase phenolic acid release.

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.

Inhibition of α-glucosidase and α-amylase by herbal compounds for the treatment of type 2 diabetes: A validation of in silico reverse docking with in vitro enzyme assays.

BACKGROUND: α-Amylase and α-glucosidase are important therapeutic targets for the management of type 2 diabetes mellitus. The inhibition of these enzymes decreases postprandial hyperglycemia. In the present study, compounds found in commercially available herbs and spices were tested for their ability to inhibit α-amylase and α-glucosidase. These compounds were acetyleugenol, apigenin, cinnamic acid, eriodictyol, myrcene, piperine, and rosmarinic acid. METHODS: The enzyme inhibitory nature of the compounds was evaluated using in silico docking analysis with Maestro software and was further confirmed by in vitro α-amylase and α-glucosidase biochemical assays. RESULTS: The relationships between the in silico and in vitro results were well correlated; a more negative docking score was associated with a higher in vitro inhibitory activity. There was no significant (P > .05) difference between the inhibition constant (Ki ) value of acarbose, a widely prescribed α-glucosidase and α-amylase inhibitor, and those of apigenin, eriodictyol, and piperine. For α-amylase, there was no significant (P > .05) difference between the Ki value of acarbose and those of apigenin, cinnamic acid, and rosmarinic acid. The effect of the herbal compounds on cell viability was assessed with the sulforhodamine B (SRB) assay in C2C12 and HepG2 cells. Acetyleugenol, cinnamic acid, myrcene, piperine, and rosmarinic acid had similar (P > .05) IC50 values to acarbose. CONCLUSIONS: Several of the herbal compounds studied could regulate postprandial hyperglycemia. Using herbal plants has several advantages including low cost, natural origin, and easy cultivation. These compounds can easily be consumed as teas or as herbs and spices to flavor food.

The dipeptidyl peptidase IV inhibitory activity and multifunctional antidiabetic properties of SQSPA: Structure - Activity relationship evaluated with alanine scanning.

Type 2 diabetes is a multifactorial disease and drugs with multifunctional properties are required. The peptide, SQSPA, was reported to be a potent and gastrointestinally stable α-glucosidase inhibitory peptide. In this study, the structure-activity relationship of this peptide was studied using alanine scanning. Four analogs; AQSPA, SASPA, SQAPA and SQSAA were designed and investigated for multifunctional antidiabetic effects. Molecular docking studies on human dipeptidyl peptidase-IV (DPP-IV) suggested that the binding affinities were in the order; AQSPA>SASPA>SQSPA>SQSAA>SQAPA while for in vitro DPP-IV inhibitory activity, it was SQSPA>SQSAA>AQSPA>SASPA>SQAPA. Enzyme kinetic studies revealed that the peptides are uncompetitive inhibitors with the exception of SQSAA and SQSPA. In 3T3-L1 differentiated adipocytes, SASPA was the only analog that significantly (p < 0.05) reduced and prevented lipid accumulation and did not induce cytotoxicity to differentiated 3T3-L1 cells. All peptides, especially SASPA scavenged methylglyoxal and peroxyl radicals thereby preventing advanced glycosylated end products formation and oxidative stress. The nitric oxide scavenging activity of all peptides was comparable to IPI and glutathione. Findings indicate that the amide side chain of Q2 is probably the most critical functional group for modulating the multifunctional antidiabetic effects of SQSPA while SASPA has been identified, as a novel peptide with enhanced multifunctional antidiabetic activity.

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.

Ultrastructural alterations of whole blood by copper, manganese and mercury metal mixtures using a chronic in vivo model of coagulation.

Globally, contamination of drinking water by heavy metals is increasing and poses a potential hazard to human health. Data on heavy metal mixtures and their effects on thrombosis are limited. The objective of this study was to determine the in vivo effects that copper, manganese and mercury, alone and in mixtures, have on clotting potential. Forty-eight male Sprague-Dawley rats were divided into eight groups, dependent on the type of heavy metal/s administered. The dosages were calculated at X100 the World Health Organisation limits in drinking water and orally administered for 28 days, at the University of Pretoria in 2018. Heavy metal induced morphological alterations of erythrocytes, platelets and fibrin networks were evaluated, using scanning electron microscopy. The manganese and mercury mixture had the greatest thrombotic potential by inducing acanthocyte and echinocyte formation, generating highly activated platelets with spontaneous fibrin formation and forming a disorganised fibrin network. In conclusion, chronic or single high dosage exposure to these heavy metals can potentially induce or contribute to thrombosis.

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.

Oral exposure to cadmium and mercury alone and in combination causes damage to the lung tissue of Sprague-Dawley rats.

Environmental presence and human exposure to heavy metals in air and cigarette smoke has led to a worldwide increase in respiratory disease. The effects of oral exposure to heavy metals in liver and kidney structure and function have been widely investigated and the respiratory system as a target is often overlooked. The aim of the study was to investigate the possible structural changes in the lung tissue of Sprague-Dawley rats after oral exposure for 28 days to cadmium (Cd) and mercury (Hg), alone and in combination at 1000 times the World Health Organization's limit for each metal in drinking water. Following exposure, the general morphology of the bronchiole and lungs as well as collagen and elastin distribution was evaluated using histological techniques and transmission electron microscopy. In the lungs, structural changes to the alveoli included collapsed alveolar spaces, presence of inflammatory cells and thickening of the alveolar walls. In addition, exposure to Cd and Hg caused degeneration of the alveolar structures resulting in confluent alveoli. Changes in bronchiole morphology included an increase in smooth muscle mass with luminal epithelium degeneration, detachment and aggregation. Prominent bronchiole-associated lymphoid tissue was present in the group exposed to Cd and Hg. Ultrastructural examination confirmed the presence of fibrosis where in the Cd exposed group, collagen fibrils arrangement was dense, while in the Hg exposed group, additional prominent elastin was present. This study identified the lungs as target of heavy metals toxicity following oral exposure resulting in cellular damage, inflammation and fibrosis and increased risk of respiratory disease where Hg showed the greatest fibrotic effect, which was further, aggravated in combination with Cd.

Structure - Function Analysis of Peptide Analogs of SQSPA with Respect to α-glucosidase and α-amylase Inhibition.

BACKGROUND: Peptide-based therapeutics offer a unique avenue for the development of novel agents for the treatment of diabetes mellitus including α-glucosidase inhibitors. The peptide, SQSPA, was reported to possess to α -glucosidase inhibitory activity in addition to resistance to Gastrointestinal Tract (GIT) digestion. METHODS: In this study, the in silico and in vitro structure-activity analyses of the peptide was conducted using alanine scanning to identify key amino acid residues. RESULTS: The alanine scanning led to four analogs viz; AQSPA, SASPA, SQAPA and SQSAA which were GIT stable. Initially, the peptides were subjected to molecular docking on human α- glucosidase and α -amylase where the binding affinities to the enzymes were in the order; AQSPA>SASPA>SQSPA>SQAPA> SQSAA and AQSPA>SQSAA>SASPA>SQSPA> SQAPA, respectively. Hydrogen bond were important for the binding of all peptides but SASPA and AQSPA had the highest hydrogen bonds interactions with the α-glucosidase and α-amylase, respectively. In vitro analysis revealed that the α -glucosidase and α-amylase inhibitory activities of the peptides were in the order AQSPA>SQSPA>SQAPA>SASPA>SQSAA and AQSPA>SASPA> SQAPA>SQSPA>SQSAA, respectively. Using inhibition kinetics, SQSPA was a mixed inhibitor of α-glucosidase while AQSPA, SQAPA and SQSAA showed non-competitive inhibition. For α- amylase inhibition, SQSPA was a non-competitive inhibitor while AQSPA and SQSAA were mixed inhibitors; SASPA and SQAPA showed uncompetitive inhibition. CONCLUSION: The results indicated that P4 and Q2 are important requirements for the α-glucosidase and α-amylase inhibitory activities of the parent peptide, SQSPA. Furthermore, alanine scanning has led to the design of a novel α-glucosidase inhibitory peptide, AQSPA, with increased activities.

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.