The newly synthesized thiazole derivatives as potential antifungal compounds against Candida albicans

Recently, the occurrence of candidiasis has increased dramatically, especially in immunocompromised patients. Additionally, their treatment is often ineffective due to the resistance of yeasts to antimycotics. Therefore, there is a need to search for new antifungals. A series of nine newly synthesized thiazole derivatives containing the cyclopropane system, showing promising activity against Candida spp., has been further investigated. We decided to verify their antifungal activity towards clinical Candida albicans isolated from the oral cavity of patients with hematological malignancies and investigate the mode of action on fungal cell, the effect of combination with the selected antimycotics, toxicity to erythrocytes, and lipophilicity. These studies were performed by the broth microdilution method, test with sorbitol and ergosterol, checkerboard technique, erythrocyte lysis assay, and reversed phase thin-layer chromatography, respectively. All derivatives showed very strong activity (similar and even higher than nystatin) against all C. albicans isolates with minimal inhibitory concentration (MIC) = 0.008–7.81 µg/mL Their mechanism of action may be related to action within the fungal cell wall structure and/or within the cell membrane. The interactions between the derivatives and the selected antimycotics (nystatin, chlorhexidine, and thymol) showed additive effect only in the case of combination some of them and thymol. The erythrocyte lysis assay confirmed the low cytotoxicity of these compounds as compared to nystatin. The high lipophilicity of the derivatives was related with their high antifungal activity. The present studies confirm that the studied thiazole derivatives containing the cyclopropane system appear to be a very promising group of compounds in treatment of infections caused by C. albicans. However, this requires further studies in vivo. Key points • The newly thiazoles showed high antifungal activity and some of them — additive effect in combination with thymol. • Their mode of action may be related with the influence on the structure of the fungal cell wall and/or the cell membrane. • The low cytotoxicity against erythrocytes and high lipophilicity of these derivatives are their additional good properties. Graphical abstract

Antihaemolytic, Antihaemorrhagic and Antifibrinolytic Effects of Fractions of Buchholzia coriacea Seeds on Naja nigricollis Crude Venom

Bulchhozia coriacea (Capparaceae) seeds are used in managing snake bite in Western Nigeria were investigated against Naja nigricollis (Spitting cobra) venom-induced hemolytic, hemorrhagic and fibrinolytic effects. This study was aimed at determining the antihaemolytic, antihaemorrhagic as well as antifibrinolytic effects of B. coriacea on N. nigricollis crude venom. Microwave-assisted extraction with hexane, chloroform, ethyl acetate, and methanol was carried out. Naja nigricollis venom-induced erythrocyte lysis (100 %) was significantly reduced to 18% by the chloroform fraction at 0.625 mg/mL. At the concentration of 0.625 mg/mL, the hexane, chloroform, ethyl acetate and methanol fractions administered in combination with the venom reduced percentage hemorrhagic activity to 23%, 17%, 49%, and 87%, respectively. In conclusion, Bulchhozia coriacea seed fractions exhibited significant antihaemolytic, antihaemorrhagic and antifibrinolytic activities against N. nigricollis crude venom and may beneficial as a pre-treatment the while victim is transferred to a healthcare facility to receive the definite treatment to ensure speedy recovery. Key words: Antihaemolytic, antihaemorrhagic, fibrinolytic, venom, Bulchhozia coriacea

Revisiting the interaction of heme with hemopexin

In hemolytic disorders, erythrocyte lysis results in massive release of hemoglobin and, subsequently, toxic heme. Hemopexin is the major protective factor against heme toxicity in human blood and currently considered for therapeutic use. It has been widely accepted that hemopexin binds heme with extraordinarily high affinity of <1 pM in a 1:1 ratio. However, several lines of evidence point to a higher stoichiometry and lower affinity than determined 50 years ago. Here, we re-analyzed these data. SPR and UV/Vis spectroscopy were used to monitor the interaction of heme with the human protein. The heme-binding sites of hemopexin were characterized using hemopexin-derived peptide models and competitive displacement assays. We obtained a K D value of 0.32 ± 0.04 nM and the ratio for the interaction was determined to be 1:1 at low heme concentrations and at least 2:1 (heme:hemopexin) at high concentrations. We were able to identify two yet unknown potential heme-binding sites on hemopexin. Furthermore, molecular modelling with a newly created homology model of human hemopexin suggested a possible recruiting mechanism by which heme could consecutively bind several histidine residues on its way into the binding pocket. Our findings have direct implications for the potential administration of hemopexin in hemolytic disorders.

Revisiting the interaction of heme with hemopexin: Recommendations for the responsible use of an emerging drug

In hemolytic disorders, erythrocyte lysis results in massive release of hemoglobin and, subsequently, toxic heme. Hemopexin is the major protective factor against heme toxicity in human blood and currently considered for therapeutic use. It has been widely accepted that hemopexin binds heme with extraordinarily high affinity in a 1:1 ratio. Here we show that hemopexin binds heme with lower affinity than previously assumed and that the interaction ratio tends to 2:1 (heme:hemopexin) or above. The heme-binding sites of hemopexin were characterized using hemopexin-derived peptide models and competitive displacement assays. In addition, in silico molecular modelling with a newly created homology model of human hemopexin allowed us to propose a recruiting mechanism by which heme consecutively binds to several histidine residues and is finally funnelled into the high-affinity binding pocket. Our findings have direct implications for the biomedical application of hemopexin and its potential administration in hemolytic disorders.

Toxicity Characterisation of Gambierdiscus Species from the Canary Islands

In the last decade, several outbreaks of ciguatera fish poisoning (CFP) have been reported in the Canary Islands (central northeast Atlantic Ocean), confirming ciguatera as an emerging alimentary risk in this region. Five Gambierdiscus species, G. australes, G. excentricus, G. silvae, G. carolinianus and G. caribaeus, have been detected in macrophytes from this area and are known to produce the ciguatoxins (CTXs) that cause CFP. A characterization of the toxicity of these species is the first step in identifying locations in the Canary Islands at risk of CFP. Therefore, in this study the toxicity of 63 strains of these five Gambierdiscus species were analysed using the erythrocyte lysis assay to evaluate their maitotoxin (MTX) content. In addition, 20 of the strains were also analysed in a neuroblastoma Neuro-2a (N2a) cytotoxicity assay to determine their CTX-like toxicity. The results allowed the different species to be grouped according to their ratios of CTX-like and MTX-like toxicity. MTX-like toxicity was especially high in G. excentricus and G. australes but much lower in the other species and lowest in G. silvae. CTX-like toxicity was highest in G. excentricus, which produced the toxin in amounts ranging between 128.2 ± 25.68 and 510.6 ± 134.2 fg CTX1B equivalents (eq) cell−1 (mean ± SD). In the other species, CTX concentrations were as follows: G. carolinianus (100.84 ± 18.05 fg CTX1B eq cell−1), G. australes (31.1 ± 0.56 to 107.16 ± 21.88 fg CTX1B eq cell−1), G. silvae (12.19 ± 0.62 to 76.79 ± 4.97 fg CTX1B eq cell−1) and G. caribaeus (<LOD to 90.37 ± 15.89 fg CTX1B eq cell−1). Unlike the similar CTX-like toxicity of G. australes and G. silvae strains from different locations, G. excentricus and G. caribaeus differed considerably according to the origin of the strain. These differences emphasise the importance of species identification to assess the regional risk of CFP.

Potential of Cocoa Extract (Theobroma cacao) in Inhibiting Erythrocyte Damage Induced by Physalia utriculus Venom

Physalia utriculus is one of the invertebrate marine biota that is often found in Indonesia. Some symptoms of venoming due to jellyfish stings cause pain, itching, and hemolysis. In Indonesia, 13 cases of jellyfish stings were reported in 2005-2009 with three people dying in Java, Bali, and Bangka. Cocoa beans (Theobroma cacao L.) contain fat, carbohydrates, proteins, and polyphenol compounds that are useful as antioxidants. Polyphenols in the form of epicathechins, catechins, and procyanidins serve to provide protection to hemolysis. The purpose of this study was to determine the potential of ethanol extract of cacao (Theobroma cacao L.) in inhibiting the damage of erythrocyte induced by Physalia utriculus in vitro. This study used 28 samples of erythrocytes divided into seven groups, namely the normal control group, negative controls, and treatment with cocoa ethanol extract 0.2%, 0.1%, 0.04%, and 0.02%. Each subsequent group induced venom Physalia utriculus. The results showed that the average speed of erythrocyte lysis in the treatment group by giving cocoa ethanol extract 0.2%, 0.1%, 0.04%, and 0.02% respectively (seconds ± standard deviation) was 858,25 ± 94,44; 1.000,5 ± 159,93; 678,5 ± 19,71; and 1.006 ± 159,50. The mean speed of erythrocyte lysis in the negative control group was 1,025 ± 164.63 and the positive control group with the administration of N-Acetylcystein can last up to one hour after administration of venoms. Test for normality and homogeneity shows that data is normally distributed and homogeneous. One Way Annova analysis shows the significance value of p <0.05, then a post hoc analysis test was performed with the Bonferoni method to find out the differences in significance in each group. In this study it can be concluded that the administration of cocoa ethanol extract has no potential to inhibit erythrocyte damage that has been venomed by Physalia utriculus in vitro. Keywords: Physalia utriculus, cacao, erythrocyte damage

The potential of neurofilaments analysis using dry-blood and plasma spots

The lack of biomarkers for an early diagnosis of neurodegenerative disorders (NDs) has hampered the development of therapeutics whose effect would be enhanced by a timely intervention. Neurofilaments light chain (Nf-L), an integral part of the axonal structure, has emerged as a robust fluid biomarker for fatal neurodegenerative disorders like amyotrophic lateral sclerosis (ALS). To facilitate large-scale studies into early-stage neurodegeneration, reduce costs of samples collection/processing and cold-chain storage, we describe the measurement of Nf-L in blood fractions obtained from dry blood spots (DBS) and dry plasma spots (DPS), two filter paper-based remote blood collection tools. To test the feasibility of using this approach, Nf-L analysis in DBS/DPS is compared to that in plasma obtained from the same blood sample, looking at Nf-L discriminatory power in the clinical stratification of ALS compared to healthy controls. With the best pre-analytical treatment for total protein recovery and using highly sensitive immunoassays, we report the detection of different Nf-L levels in DBS elute compared to reference plasma and DPS from the same blood samples. However, Nf-L measurement in DBS elutes provides a very good discrimination of ALS from healthy controls which is comparable to that obtained using plasma Nf-L assays. With the available immunodetection methods, we show that Nf-L measurement based on DPS microsampling is similar to that in plasma. The filter-paper biophysical characteristics and the interference of high haemoglobin concentration released by erythrocyte lysis is likely to perturb Nf-L detection in DBS elute. Further studies into DBS-based Nf-L detection and its analytical optimization are needed to make this method suitable for routine Nf-L blood analyses in neurodegeneration.

Machine learning algorithms for the detection of spurious white blood cell differentials due to erythrocyte lysis resistance

AimsRed blood cell (RBC) lysis resistance interferes with white blood cell (WBC) count and differential; still, its detection relies on the identification of an abnormal scattergram, and this is not clearly adverted by specific flags in the Beckman-Coulter DXH-800. The aims were to analyse precisely the effect of RBC lysis resistance interference in WBC counts, differentials and cell population data (CPD) and then to design, develop and implement a novel diagnostic machine learning (ML) model to optimise the detection of samples presenting this phenomenon.MethodsWBC counts, differentials and CPD from 232 patients (anaemia or liver disease) were compared with 100 healthy controls (HC) using analysis of variance. The data were analysed after a corrective action, and the analyser differentials were also compared with the digital leucocyte differentials. The ML support vector machine (SVM) algorithm was trained with 70% of the samples (n=233) and the 30% remaining (n=99) were employed exclusively during the validation phase.ResultsWe identified that impedance WBC was not affected by the RBC lysis resistance interference while the DXH-800 differentials overestimated lymphoid subpopulations (17.6%), sometimes even yielding spurious lymphocytosis, and the latter were corrected when sample dilution was performed. The ML-SVM algorithm allowed the classification of the pathological groups when compared with HC with validation accuracies corresponding to 97.98%, 100% and 88.78% for the global, anaemia and liver disease groups, respectively.ConclusionsThe proposed algorithm has an impressive discriminatory potential and its application would be a valuable support system to detect spurious results due to RBC lysis resistance.