Beta vulgaris Assisted Fabrication of Novel Ag-Cu Bimetallic Nanoparticles for Growth Inhibition and Virulence in Candida albicans.

Beta vulgaris extract contains water-soluble red pigment betanin and is used as a food colorant. In this study, the biogenic Ag-Cu bimetallic nanoparticles were synthesized and characterized by different spectroscopic and microscopic techniques, including UV-Visible, FTIR, TEM. SEM-EDX, XRD, and TGA. Further, Ag-Cu bimetallic nanoparticles capped with Beta vulgaris biomolecules were evaluated for their antifungal activity against Candida albicans via targeting its major virulence factors, including adherence, yeast to hyphae transition, extracellular enzyme secretion, biofilm formation, and the expression of genes related to these pathogenic traits by using standard methods. C. albicans is an opportunistic human fungal pathogen that causes significant morbidity and mortality, mainly in immunocompromised patients. The current antifungal therapy is limited with various shortcomings such as host toxicity and developing multidrug resistance. Therefore, the development of novel antifungal agents is urgently required. Furthermore, NPs were screened for cell viability and cytotoxicity effect. Antifungal susceptibility testing showed potent antifungal activity of the Ag-Cu bimetallic NPs with a significant inhibitory effect on adherence, yeast to hyphae transition, extracellular enzymes secretion, and formation of biofilms in C. albicans at sub-inhibitory and inhibitory concentrations. The RT-qPCR results at an MIC value of the NPs exhibited a varying degree of downregulation in expression levels of virulence genes. Results also revealed the dose-dependent effect of NPs on cellular viability (up to 100%) using MUSE cell analyzer. Moreover, the low cytotoxicity effect of bimetallic NPs has been observed using haemolytic assay. The overall results indicated that the newly synthesized Ag-Cu bimetallic NPs capped with Beta vulgaris are proven to possess a potent anticandidal activity, by affecting the vital pathogenic factors of C. albicans.

Cholesterol-loaded cyclodextrin attenuates dilution effect and improves quality of bovine low sperm insemination doses during cryopreservation.

In the present study, the effect of cholesterol-loaded cyclodextrin (CLC) on the quality of low sperm doses at post-thaw was evaluated. Twenty four ejaculates (6 from each bull) were collected and split into eight aliquots. First four aliquots were diluted up to 20-, 15-, 10- and 5-million sperm/0.25 ml, and remaining four were treated with CLC at the rate of 1 mg/120 million spermatozoa, followed by dilution up to 20-, 15-, 10- and 5-million sperm/0.25 ml. The diluted semen was equilibrated, cryopreserved and evaluated post-thaw. The averages of total motility, progressive motility, average path velocity, straight linear velocity, membrane intact spermatozoa and noncapacitated spermatozoa were higher (p < .05) in CLC-treated sperm doses compared to control ones. However, the moribund spermatozoa, capacitated spermatozoa and acrosome-reacted spermatozoa were reduced (p < .05) in CLC-treated spermatozoa compared to control. The curvilinear velocity and linearity did not differ (p > .05) between control and CLC-treated sperm doses. In conclusion, treatment of spermatozoa with CLC at the rate of 1 mg/120 million spermatozoon attenuates the dilution effect and improves the quality of bovine low sperm insemination doses during cryopreservation; hence it could be a favourable cryoprotectant for preserving bovine semen at higher dilutions.

Citral and its derivatives inhibit quorum sensing and biofilm formation in Chromobacterium violaceum.

With an upsurge in multidrug resistant bacteria backed by biofilm defence armours, there is a desperate need of new antibiotics with a non-traditional mechanism of action. Targeting bacteria by misguiding them or halting their communication is a new approach that could offer a new way to combat the multidrug resistance problem. Quorum sensing is considered to be the achilles heel of bacteria that has a lot to offer. Since, both quorum sensing and biofilm formation have been related to drug resistance and pathogenicity, in this study we synthesised new derivatives of citral with antiquorum sensing and biofilm disrupting properties. We previously reported antimicrobial and antiquorum sensing activity of citral and herein we report the synthesis and evaluation of citral and its derivatives (CD1-CD3) for antibacterial, antibiofilm and antiquorum sensing potential against Chromobacterium violaceum using standard methods. Preliminary results revealed that CD1 is the most active of all the derivatives. Qualitative and quantitative evaluation of antiquorum sensing activity at sub-inhibitory concentrations of these compounds also revealed high activity for CD1 followed by CD2, CD3 and citral. These compounds also inhibit biofilm formation at subinhibitory concentrations without causing any bacterial growth inhibition. These results were replicated by RT-qPCR with down regulation of the quorum sensing genes when C. violaceum was treated with these test compounds. Overall, the results are quite encouraging, revealing that biofilm and quorum sensing are interrelated processes and also indicating the potential of these derivatives to impede bacterial communication and biofilm formation.

Modification of French mini-straw plug position for cryopreservation of small doses of bull sperm.

Greater than optimal diluting of semen for producing a large number of doses containing relatively small numbers of sperm can lead to compromised quality of sperm, post-thawing. In the present study the French mini-straw plug position was modified and the effect of re-positioning was evaluated on the quality of sperm after thawing subsequent to cryopreservation of small doses of sperm. Four types of mini-straws were used based on the position of cotton plug including no plug displacement (Type 1; Manufacturers location for plug-placement in 0.25 mL French mini-straws), and Type II, III, and IV with re-positioning the cotton plug being 2.5, 5, and 7.5 cm, respectively, further from the manufacturer's placement location. Each ejaculate was proportioned into four Aliquots (I, II, III, and IV) and diluted to 80, 60, 40, and 20, million sperm/mL, respectively. Aliquot I was placed in all types of straws, while Aliquots II, III, IV were placed only in Type I straws. Semen straws were equilibrated, cryopreserved and sperm kinetic and functional variables were evaluated post-thawing. The results indicate that in Aliquots III and IV there were lesser (P < 0.05) values for sperm kinetic and function variables compared with sperm from Aliquot I (i.e., unmodified mini-straw). In conclusion, cryopreservation of small doses of sperm in modified French mini-straws resulted in acceptable values for kinetic and function variables, post-thawing.

Inhibitory effect of novel Eugenol Tosylate Congeners on pathogenicity of Candida albicans.

BACKGROUND: The global prevalence of fungal diseases is increasing rapidly, which affects more than a billion people every year with significant mortality rate. On the other hand, the development of new drugs to treat these fungal infections is slow, while the current antifungal therapy is insufficient and associated with adverse side effects and emerging multidrug resistance. Therefore, development of novel antifungal drugs with least or no toxicity and multi-target mechanisms of action is an immediate priority. Natural products have long been known to possess antimicrobial activities and are source of new drugs. Currently, modifying natural products to synthesize derivatives/analogues are of great scientific focus for discovering novel drugs with improved potency and safety. Modifications in eugenol to synthesize eugenol derivatives with enhanced antifungal activity have already been reported. METHODS: In this study, three most active novel eugenol tosylate congeners (ETC-5, ETC-6 and ETC-7) were selected from our previous study to investigate their effect on major virulence factors of Candida albicans which include adherence, morphogenesis, hydrolytic enzymes secretion, biofilm formation and on expression of genes related to these virulence factors. Adherence and biofilm formation were studied by alamarBlue dye and XTT reduction assays respectively, hydrolytic enzyme secretion was evaluated by plate assays. Further, morphological transition was monitored microscopically and RT-qPCR was used to assess the gene expression levels. RESULTS: ETCs significantly inhibited adherence in C. albicans with an inhibition range of 16-66%, and completely inhibited the morphogenesis at MIC values. Inhibition of proteinase and phospholipase activity was in the range of 2-48% and 8-34% respectively. Test compounds also significantly inhibit biofilm formation in C. albicans in the range of 7-77%. Furthermore, RT-qPCR results indicated a significant down regulation in expression levels of genes (ALS1, ALS2, ALS3, ALS9, CPH1, HWP1, SAP1, SAP2, SAP3 and PLB1) in C. albicans cells after treated with ETCs. CONCLUSION: The results indicated that these novel ETCs target major virulence factors of C. albicans and avert this commensal microbe to turn into pathogenic. However, further in-depth studies may facilitate the mechanisms involved by ETCs in targeting these virulence factors.

COVID-19 pandemic - an African perspective.

The recently emerged novel coronavirus, "severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)", caused a highly contagious disease called coronavirus disease 2019 (COVID-19). The virus was first reported from Wuhan city in China in December, 2019, which in less than three months spread throughout the globe and was declared a global pandemic by the World Health Organization (WHO) on 11th of March, 2020. So far, the ongoing pandemic severely damaged the world's most developed countries and is becoming a major threat for low- and middle-income countries. The poorest continent, Africa with the most vulnerable populations to infectious diseases, is predicted to be significantly affected by the ongoing COVID-19 outbreak. Therefore, in this review we collected and summarized the currently available literature on the epidemiology, etiology, vulnerability, preparedness and economic impact of COVID-19 in Africa, which could be useful and provide necessary information on ongoing COVID-19 pandemics in the continent. We also briefly summarized the concomitance of the COVID-19 pandemic and global warming.

S-benzyldithiocarbazate imine coordinated metal complexes kill Candida albicans by causing cellular apoptosis and necrosis.

Development of new chemotherapeutic agents and strategies are urgently needed to curb and halt the growing menace caused by hard-to-treat microbes. Coordination of metals to bioactive organic ligands is now considered to be an efficient strategy for delivering bioactive compounds inside the microbial cell membranes. Metal complexes have been effectively used to treat many dreadful diseases were other treatment modalities had failed. Use of metal complexes to treat microbial infections is now conceived to be an alternative and efficient strategy. Towards this, some new homoleptic transition metal complexes, obtained by coordination of metal ions to bioactive S-benzyldithiocarbazate Schiff-base ligands were evaluated for their anti-Candida activity and their potential to disrupt the membrane architecture. The complexes displayed remarkable antifungal activity against a wide spectrum of fluconazole susceptible and resistant Candida albicans isolates, with Ni complex (dtc3) being highly active with minimum inhibitory concentration (MIC) values ranging from 1 to 32 µg/mL. Cell viability assay confirmed the fungicidal activity of these metal complexes, especially the complex dtc3. These metal complexes kill Candida albicans by inducing cellular apoptosis and necrosis thereby causing phosphatidylserine externalization as revealed by Annexin V-FITC and propidium iodide staining assays.

Cellular apoptosis and necrosis as therapeutic targets for novel Eugenol Tosylate Congeners against Candida albicans.

Despite the rise of new Candida species, Candida albicans tops the list with high morbidity and mortality rates. To tackle this problem there is a need to explore new antifungals that could replace or augment the current treatment options. We previously reported that tosylation of eugenol on hydroxyl group resulted in molecules with enhanced antifungal potency. In line with that work, we synthesized new eugenol tosylate congeners (ETC-1-ETC-7) with different substituents on pendent sulfonyl group and tested their susceptibility against different fluconazole susceptible and resistant C. albicans strains. We evaluated physiology and mode of cell death in response to the most active derivatives by analyzing major apoptotic markers in yeast such as phosphatidylserine externalization, DNA fragmentation, mitochondrial depolarization and decrease in cytochrome c oxidase activity. The results demonstrated that all C. albicans strains were variably susceptible to the test compounds with MIC ranging from 0.125-512 µg/ml, and the most active compounds (ETC-5, ETC-6 and ETC-7) actuate apoptosis and necrosis in Candida cells in a dose-dependent manner via metacaspase-dependent pathway. Furthermore haemolytic assay showed low cytotoxicity effect of these ETCs. Overall the results indicated that ETCs exhibit potential antifungal activity against C. albicans by activating apoptotic and necrotic pathways.

Inhibition of ergosterol synthesis in Candida albicans by novel eugenol tosylate congeners targeting sterol 14α-demethylase (CYP51) enzyme.

This study is a continuation and extension of our previous study in which we synthesized seven novel eugenol tosylate congeners (ETC-1 to ETC-7) from a natural compound eugenol and checked their antifungal activity against different isolates of Candida albicans. All these ETCs showed potent antifungal activity to varying degrees. In this study, the aim is to evaluate the effect of most active compounds (ETC-5, ETC-6 and ETC-7) on ergosterol biosynthesis pathway and cellular viability in C. albicans by applying combined approach of in silico and in vitro methodologies. In silico studies were done through all atom molecular mechanics approach and free binding energy estimations, and in vitro study was done by estimating total intracellular sterol content and effect on expression of ERG11 gene. Furthermore, effect on cell viability by these compounds was also tested. Our results demonstrated that these ETCs target ergosterol biosynthesis pathway in C. albicans by inhibiting the lanosterol 14-α demethylase enzyme and also downregulates expression of its related gene ERG11. Furthermore, these ETCs exhibit potent fungicidal effect in cell viability assay, thus overall results advocating the claim that these tosylates have potential to be taken to next level of antifungal drug development.

Synthesis and synergistic studies of isatin based mixed ligand complexes as potential antifungal therapeutic agents.

Metal based drugs are important class of chemotherapeutic agents that have the potential to circumvent drug resistance. Increasing drug resistance, treatment failures and limited treatment options necessitates the development of new therapeutic drugs with different mechanisms of action. Towards this direction, we synthesized a series of isatin based mixed ligand complexes of [Cu(dbm)LClH2O] (mlc1), [Co(dbm)LCl2]‒ (mlc2) and [Ni(dbm)LClH2O] (mlc3) and evaluated their antifungal activity alone and in combination with fluconazole (FLC) against seven different Candida albicans isolates. The insight mechanism of antifungal action was revealed by studying apoptosis via terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. The study revealed that all these compounds showed antifungal activity at varying concentrations with mlc3 as the most potent compound with minimum inhibitory concentration ranging from 0.5-8 µg/mL and minimum fungicidal concentration ranging from 4-16 µg/mL. Upon combination with FLC, most of the interactions were either synergistic (54 %) or additive (32 %) with no antagonistic combination against any of the tested isolate. The study on their mechanism of action revealed that these compounds show apoptotic effect on C. albicans at sub-inhibitory concentrations, suggesting that strategies to target this process may augment the current antifungal treatment modalities.

Sperm protein carbonylation.

The cryopreservation of sperm is a well established technique that plays an essential role in dissemination of elite germplasm of livestock. Despite having numerous advantages, the cryopreservation induces certain stresses on sperm including structural and functional damages leading to impaired sperm quality and fertility, which might be associated with production of reactive oxygen species (ROS). In addition, the ROS upon reacting with sperm lipids, DNA and proteins may lead to a cascade of sperm damages. The sperm membrane contains a rich amount of polyunsaturated fatty acids, which increases their susceptibility to oxidative stress induced damages, leading to formation of secondary products. These secondary products result in oxidation of sperm proteins via carbonylation. The carbonylation could lead to disturbances in specific proteins that are involved in capacitation. The present review deals with sperm protein carbonylation.

Efficacy of Novel Schiff base Derivatives as Antifungal Compounds in Combination with Approved Drugs Against Candida Albicans.

BACKGROUND: The increasing incidence of fungal infections, especially caused by Candida albicans, and their increasing drug resistance has drastically increased in recent years. Therefore, not only new drugs but also alternative treatment strategies are promptly required. METHODS: We previously reported on the synergistic interaction of some azole and non-azole compounds with fluconazole for combination antifungal therapy. In this study, we synthesized some non-azole Schiff-base derivatives and evaluated their antifungal activity profile alone and in combination with the most commonly used antifungal drugs- fluconazole (FLC) and amphotericin B (AmB) against four drug susceptible, three FLC resistant and three AmB resistant clinically isolated Candida albicans strains. To further analyze the mechanism of antifungal action of these compounds, we quantified total sterol contents in FLC-susceptible and resistant C. albicans isolates. RESULTS: A pyrimidine ring-containing derivative SB5 showed the most potent antifungal activity against all the tested strains. After combining these compounds with FLC and AmB, 76% combinations were either synergistic or additive while as the rest of the combinations were indifferent. Interestingly, none of the combinations was antagonistic, either with FLC or AmB. Results interpreted from fractional inhibitory concentration index (FICI) and isobolograms revealed 4-10-fold reduction in MIC values for synergistic combinations. These compounds also inhibit ergosterol biosynthesis in a concentration-dependent manner, supported by the results from docking studies. CONCLUSION: The results of the studies conducted advocate the potential of these compounds as new antifungal drugs. However, further studies are required to understand the other mechanisms and in vivo efficacy and toxicity of these compounds.

New transition metal complexes with a pendent indole ring: insights into the antifungal activity and mode of action.

Development of new chemotherapeutic agents to treat multidrug-resistant fungal infections to augment the current treatment options is a must. In this direction, a series of mixed ligand complexes was synthesized from a Schiff base (L) obtained by the condensation of 2-hydroxynapthaldehyde and tryptamine, and 1,10-phenanthroline (1,10-phen) as a secondary ligand. Based on spectral characterization and physical measurements an octahedral geometry was assigned to [Co(phen)LClH2O] (C2), [Ni(phen)LClH2O](C3), and [Zn(phen)LClH2O](C4) complexes while a distorted octahedral geometry was assigned to the [Cu(phen)LClH2O](C1) complex. All the synthesized compounds were tested for antifungal activity against 11 Candida albicans isolates, including fluconazole (FLC) resistant isolates, by determining minimum inhibitory concentrations and studying growth curves. MIC results suggest that all the newly synthesized compounds have potent antifungal activity at varying levels. The rapid action of these compounds on fungal cells suggested a membrane-located target for their action.

Possible mechanisms of cholesterol-loaded cyclodextrin action on sperm during cryopreservation.

Artificial insemination (AI) with cryopreserved semen has a pivotal role in wider dissemination of germplasm of elite livestock and also for conservation of various endangered species. Cryopreservation allows storage of semen for a prolonged period of time and facilitates greater exchange of genetic material among distant populations. Cryopreservation, however, leads to certain deleterious effects on sperm including premature induction of the acrosome reaction, reduced sperm motility and viability, and impaired sperm DNA integrity and fertility. During cooling procedures, membrane phase transitions take place, which result in micro-domain formation from aggregation of lipids, leading to impaired functions of the sperm membrane, and gap formation between gel and fluid domains. Cyclodextrins are produced by enzymatic degradation of starch and possess a unique feature, that when added alone to sperm cyclodextrins facilitate the removal of cholesterol from the membrane. When preloaded with cholesterol, however, cyclodextrins stimulate the insertion of cholesterol into the sperm membrane due to presence of a hydrophobic core in addition to an outer hydrophilic face. Treating sperm of various species with cholesterol-loaded cyclodextrin improves the quality of sperm during cryopreservation. It is still not clearly known how cholesterol-loaded cyclodextrin functions at sperm cells to enhance the survival during cryopreservation. The present review, therefore, highlights possible mechanisms of cholesterol-loaded cyclodextrin action on sperm during cryopreservation.