Next generation imidazothiazole and imidazooxazole derivatives as potential drugs against brain-eating amoebae.

Managing primary amoebic meningoencephalitis, induced by Naegleria fowleri poses a complex medical challenge. There is currently no specific anti-amoebic drug that has proven effectiveness against N. fowleri infection. Ongoing research endeavours are dedicated to uncovering innovative treatment strategies, including the utilization of drugs and immune modulators targeting Naegleria infection. In this study, we explored the potential of imidazo[2,1-b]thiazole and imidazooxazole derivatives that incorporate sulfonate and sulfamate groups as agents with anti-amoebic properties against N. fowleri. We assessed several synthesized compounds (1f, 1m, 1q, 1s, and 1t) for their efficacy in eliminating amoebae, their impact on cytotoxicity, and their influence on the damage caused to human cerebral microvascular endothelial (HBEC-5i) cells when exposed to the N. fowleri (ATCC 30174) strain. The outcomes revealed that, among the five compounds under examination, 1m, 1q, and 1t demonstrated notable anti-parasitic effects against N. fowleri (P ≤ 0.05). Compound 1t exhibited the highest anti-parasitic activity, reducing N. fowleri population by 80%. Additionally, three compounds, 1m, 1q, and 1t, significantly mitigated the damage inflicted on host cells by N. fowleri. However, the results of cytotoxicity analysis indicated that while 1m and 1q had minimal cytotoxic effects on endothelial cells, compound 1t caused moderate cytotoxicity (34%). Consequently, we conclude that imidazo[2,1-b]thiazole and imidazooxazole derivatives containing sulfonate and sulfamate groups exhibit a marked capacity to eliminate amoebae viability while causing limited toxicity to human cells. In aggregate, these findings hold promise that could potentially evolve into novel therapeutic options for treating N. fowleri infection.

Phosphonium chloride-based deep eutectic solvents inhibit pathogenic Acanthamoeba castellanii belonging to the T4 genotype.

Herein, we investigated the anti-amoebic activity of phosphonium-chloride-based deep eutectic solvents against pathogenic Acanthamoeba castellanii of the T4 genotype. Deep eutectic solvents are ionic fluids composed of two or three substances, capable of self-association to form a eutectic mixture with a melting point lower than each substance. In this study, three distinct hydrophobic deep eutectic solvents were formulated, employing trihexyltetradecylphosphonium chloride as the hydrogen bond acceptor and aspirin, dodecanoic acid, and 4-tert-butylbenzoic acid as the hydrogen bond donors. Subsequently, all three deep eutectic solvents, denoted as DES1, DES2, DES3 formulations, underwent investigations comprising amoebicidal, adhesion, excystation, cytotoxicity, and cytopathogenicity assays. The findings revealed that DES2 was the most potent anti-amoebic agent, with a 94% elimination rate against the amoebae within 24 h at 30 °C. Adhesion assays revealed that deep eutectic solvents hindered amoebae adhesion to human brain endothelial cells, with DES2 exhibiting 88% reduction of adhesion. Notably, DES3 exhibited remarkable anti-excystation properties, preventing 94% of cysts from reverting to trophozoites. In cytopathogenicity experiments, deep eutectic solvent formulations and dodecanoic acid alone reduced amoebae-induced human brain endothelial cell death, with DES2 showing the highest effects. Lactate dehydrogenase assays revealed the minimal cytotoxicity of the tested deep eutectic solvents, with the exception of trihexyltetradecylphosphonium chloride, which exhibited 35% endothelial cell damage. These findings underscore the potential of specific deep eutectic solvents in combating pathogenic Acanthamoeba, presenting promising avenues for further research and development against free-living amoebae.

Azole-based compounds as potential anti-Acanthamoeba agents.

Acanthamoeba castellanii is an opportunistic pathogen with public health implications, largely due to its invasive nature and non-specific symptoms. Our study focuses on the potential of azole compounds, particularly those with triazole scaffolds, as anti-amoebic agents. Out of 10 compounds, compounds T1 and T8 exhibited effective anti-Acanthamoeba activity with MIC50 values of 125.37 and 143.92 µg mL-1, respectively. Interestingly, compounds T1, T4, T5 and T8 revealed profound anti-excystation activity with MIC50 at 32.01, 85.53, 19.54 and 80.57 µg mL-1, respectively, alongside limited cytotoxicity to human cells. The study underscores the potential of T1, T4, T5, and T8, thiazole-based compounds, as anti-Acanthamoeba agents by both eliminating amoeba viability and preventing excystation, via preserving the amoeba in its latent cyst form, exposing them to elimination by the immune system. Notably, compounds T1, T4, T5, and T8 showed optimal molecular properties, moderate oral bioavailability, and stable complex formation with Acanthamoeba CYP51. They also display superior binding interactions. Further research is needed to understand their mechanisms and optimize their efficacy against Acanthamoeba infections.

One pot synthesis of 5-hydroxyalkylated thiadiazine thiones: Implication in pain management and bactericidal properties.

The synthesis of a new series of thiadiazine thiones including 5-(2-hydroxyethyl)-3-alkyl/aryl-1, 3, 5-thiadiazine-2-thiones (1-5), 5-(2-hydroxypropyl)-3-alkyl/aryl-1, 3, 5-thiadiazine-2-thiones (6-8), 3,5-dipropyl-1, 3, 5-thiadiazine-2-thione (9) and (2-(5-alkyl/aryl-6-thioxo-1, 3, 5-thiadiazine-3-yl) alkyl acetate/benzoate) (10-17) was accomplished via one pot reaction. The structures of the synthesized compounds were characterized through NMR and Mass spectrometry. The anti-nociceptive activity of compounds was performed on BALB/C mice by hot plate method, where compounds 3, 5 (50 µg/kg), and 8 (50, 100 µg/kg) exhibited significant effect (P < 0.01, P < 0.05) in latency time of 15, 30, and 60 min, while compounds 6 and 16 (100 µg/kg) exhibited significant effect (P < 0.01, P < 0.05) in latency time interval of 15 and 30 min. Compounds 1, 12-13, and 15 showed moderate activity. Among the tested hits, compounds 5 (17.3 ± 2.2), 11 (16.2 ± 2.1), and 8 (16.1 ± 2.1) showed significant anti-nociceptive potential. Molecular docking studies on the most active anti-nociceptive hits indicated that the activity might be attributed to the ability of the compounds to target µ-opioid receptor (µOR) effectively. Furthermore, compounds 14 and 11 showed anti-bacterial activity against Pseudomonas aeruginosa and MSRA with MIC of 40.97 and 54.77 µg/mL, respectively. In addition, the predicted ADMET profile of 5, 9, and 11 indicates that these molecules follow the drug-likeness criteria, and their activity can be enhanced through structural optimization.

Nanomedicine: Patuletin-conjugated with zinc oxide exhibit potent effects against Gram-negative and Gram-positive bacterial pathogens.

With the emergence of drug-resistance, there is a need for novel anti-bacterials or to enhance the efficacy of existing drugs. In this study, Patuletin (PA), a flavanoid was loaded onto Gallic acid modified Zinc oxide nanoparticles (PA-GA-ZnO), and evaluated for antibacterial properties against Gram-positive (Bacillus cereus and Streptococcus pneumoniae) and Gram-negative (Samonella enterica and Escherichia coli) bacteria. Characterization of PA, GA-ZnO and PA-GA-ZnO' nanoparticles was accomplished utilizing fourier-transform infrared spectroscopy, efficiency of drug entrapment, polydispersity index, zeta potential, size, and surface morphology analysis through atomic force microscopy. Using bactericidal assays, the results revealed that ZnO conjugation displayed remarkable effects and enhanced Patuletin's effects against both Gram-positive and Gram-negative bacteria, with the minimum inhibitory concentration observed at micromolar concentrations. Cytopathogenicity assays exhibited that the drug-nanoconjugates reduced bacterial-mediated human cell death with minimal side effects to human cells. When tested alone, drug-nanoconjugates tested in this study showed limited toxic effects against human cells in vitro. These are promising findings, but future work is needed to understand the molecular mechanisms of effects of drug-nanoconjugates against bacterial pathogens, in addition to in vivo testing to determine their translational value. This study suggests that Patuletin-loaded nano-formulation (PA-GA-ZnO) may be implicated in a multi-target mechanism that affects both Gram-positive and Gram-negative pathogen cell structures, however this needs to be ascertained in future work.

Epidemiology and phylogeny of Haemonchus contortus through internal transcribed spacer 2 gene in small ruminants.

Introduction: Haemonchus contortus (H. contortus) is a blood-feeding nematode causing infectious disease haemonchosis in small ruminants of tropical and subtropical regions around the world. This study aimed to explore the prevalence and phylogeny of H. contortus in small ruminants using the internal transcribed spacer-2 (ITS-2) gene. In addition, a comprehensive review of the available literature on the status of H. contortus in Pakistan was conducted. Methods: Fecal samples were collected from sheep and goats (n = 180). Microscopically positive samples were subjected to DNA extraction followed by PCR using species-specific primers. Results: The overall prevalence of H. contortus was 25.55% in small ruminants. The prevalence of H. contortus was significantly associated with months and area. The highest occurrence of haemonchosis was documented in July (38.70%), whereas the lowest occurred in December (11.11%), with significant difference. The prevalence was highest in the Ghamkol camp (29.4%) and lowest in the arid zone of the Small Ruminant Research Institute (17.5%) (p = 0.01). The results of the systematic review revealed the highest prevalence of haemonchosis (34.4%) in Khyber Pakhtunkhwa (p = 0.001). Discussion: Phylogenetic analysis revealed a close relationship between H. contortus and isolates from Asia (China, India, Iran, Bangladesh, Malaysia, and Mongolia) and European countries (Italy and the United Kingdom). It has been concluded that H. contortus is prevalent in small ruminants of Kohat district and all over Pakistan, which could be a potential threat to food-producing animals, farmers, dairy, and the meat industry. Phylogenetic analysis indicates that H. contortus isolates share close phylogenetic relationships with species from Asia and Europe.

Consanguineous marriages increase the incidence of recurrent tuberculosis: Evidence from whole exome sequencing.

BACKGROUND: In this study, we have identified multiple mutations in the IL-12R1 gene among Pakistani patients who have inherited them through consanguineous marriages. These patients have experienced severe Bacille-Calmette-Guérin (BCG) infection as well as recurrent tuberculosis. We will demonstrate the pivotal role of interleukin (IL)-12/interferon (IFN)-γ axis in the regulation of mycobacterial diseases. METHODOLOGY: First, we checked the patients' medical records, and then afterward, we assessed interferon-gamma (IFN-γ) production through ELISA. Following that, DNA was extracted to investigate IL-12/IFN- abnormalities. Whole exome sequencing was conducted through Sanger sequencing. Secretory cytokine levels were compared from healthy control of the same age groups and they were found to be considerably less in the disease cohort. To evaluate the probable functional impact of these alterations, an in silico study was performed. RESULTS: The study found that the patients' PBMCs produced considerably less IFN-γ than expected. Analysis using flow cytometry showed that activated T cells lacked surface expression of IL-12Rß1. Exon 7 of the IL-12Rß1 gene, which encodes a portion of the cytokine binding region (CBR), and exon 10, which encodes the fibronectin-type III (FNIII) domain, were found to have the mutations c.641 A > G; p.Q214R and c.1094 T > C; p.M365T, respectively. In silico analysis showed that these mutations likely to have a deleterious effect on protein function. CONCLUSION: Our findings indicate the significant contribution of the IL-12/IFN-γ is in combating infections due to mycobacterium. Among Pakistani patients born to consanguineous marriages, the identified mutations in the IL-12Rß-1 gene provide insights into the genetic basis of severe BCG infections and recurrent tuberculosis. The study highlights the potential utility of newborn screening in regions with mandatory BCG vaccination, enabling early detection and intervention for primary immunodeficiencies associated with mycobacterial infections. Moreover, the study suggests at the potential role of other related genes such as IL-23Rß1, TYK2, or JAK2 in IFN-γ production, warranting further investigation.

Antibacterial effects of quercetagetin are significantly enhanced upon conjugation with chitosan engineered copper oxide nanoparticles.

The development of antibiotic alternatives that entail distinctive chemistry and modes of action is necessary due to the threat posed by drug resistance. Nanotechnology has gained increasing attention in recent years, as a vehicle to enhance the efficacy of existing antimicrobials. In this study, Chitosan copper oxide nanoparticles (CHI-CuO) were synthesized and were further loaded with Quercetagetin (QTG) to achieve the desired (CHI-CuO-QTG). Size distribution, zeta potential and morphological analysis were accomplished. Next, the developed CHI-CuO-QTG was assessed for synergistic antibacterial properties, as well as cytotoxic attributes. Bactericidal assays revealed that CHI-CuO conjugation showed remarkable effects and enhanced QTG effects against a range of Gram + ve and Gram - ve bacteria. The MIC50 of QTG against S. pyogenes was 107 µg/mL while CHI-CuO-QTG reduced it to 9 µg/mL. Similar results were observed when tested against S. pneumoniae. Likewise, the MIC50 of QTG against S. enterica was 38 µg/mL while CHI-CuO-QTG reduced it to 7 µg/mL. For E. coli K1, the MIC50 of QTG was 42 µg/mL while with CHI-CuO-QTG it was 23 µg/mL. Finally, the MIC50 of QTG against S. marcescens was 98 µg/mL while CHI-CuO-QTG reduced it to 10 µg/mL. Notably, the CHI-CuO-QTG nano-formulation showed limited damage when tested against human cells using lactate dehydrogenase release assays. Importantly, bacterial-mediated human cell damage was reduced by prior treatment of bacteria using drug nano-formulations. These findings are remarkable and clearly demonstrate that drug-nanoparticle formulations using nanotechnology is an important avenue in developing potential therapeutic interventions against microbial infections.

Potential anti-amoebic activity of sulfonate- and sulfamate-containing carboxamide derivatives against pathogenic Acanthamoeba castellanii belonging to the genotype T4.

Acanthamoeba are ubiquitously distributed in the environment and can cause infection of the central nervous system as well a sight-threatening eye infection. Herein, the potential anti-amoebic activity of a series of sulfonate/sulfamate derivatives against pathogenic A. castellanii was evaluated. These compounds were tested using several assays namely amoebicidal, adhesion, excystation, cytotoxic, and cytopathogenicity. Amoebicidal assays revealed that the selected compounds reduced amoebae viability significantly (P < 0.05), and exhibited IC50 values at two-digit micromolar concentrations. Sulfamate derivatives 1j & 1k inhibited 50% of amoebae at 30.65 µM and 27.21 µM, respectively. The tested compounds blocked amoebae binding to host cells as well as inhibited amoebae excystation. Notably, the selected derivatives exhibited minimal human cell cytotoxicity but reduced parasite-mediated host cell damage. Overall, our study showed that sulfamate derivatives 1j & 1k have anti-amoebic potential and offer a promising avenue in the development of potential anti-amoebic drug candidates.

The anti-amoebic potential of carboxamide derivatives containing sulfonyl or sulfamoyl moieties against brain-eating Naegleria fowleri.

Naegleria fowleri is a free-living thermophilic flagellate amoeba that causes a rare but life-threatening infection called primary amoebic meningoencephalitis (PAM), with a very high fatality rate. Herein, the anti-amoebic potential of carboxamide derivatives possessing sulfonyl or sulfamoyl moiety was assessed against pathogenic N. fowleri using amoebicidal, cytotoxicity and cytopathogenicity assays. The results from amoebicidal experiments showed that derivatives dramatically reduced N. fowleri viability. Selected derivatives demonstrated IC50 values at lower concentrations; 1j showed IC50 at 24.65 µM, while 1k inhibited 50% amoebae growth at 23.31 µM. Compounds with significant amoebicidal effects demonstrated limited cytotoxicity against human cerebral microvascular endothelial cells. Finally, some derivatives mitigated N. fowleri-instigated host cell death. Ultimately, this study demonstrated that 1j and 1k exhibited potent anti-amoebic activity and ought to be looked at in future studies for the development of therapeutic anti-amoebic pharmaceuticals. Further investigation is required to determine the clinical relevance of our findings.

Gut microbiome of Crocodylus porosus and cellular stress: inhibition of nitric oxide, interleukin 1-beta, tumor necrosis factor-alpha, and prostaglandin E2 in cerebrovascular endothelial cells.

Crocodiles are renowned for their resilience and capacity to withstand environmental stressors, likely influenced by their unique gut microbiome. In this study, we determined whether selected gut bacteria of Crocodylus porosus exhibit anti-inflammatory effects in response to stress, by measuring nitric oxide release, interleukin 1-beta, tumor necrosis factor-alpha, and prostaglandin E2 in cerebrovascular endothelial cells. Using the Griess assay, the findings revealed that among several C. porosus gut bacterial isolates, the conditioned media containing the metabolites of two bacterial strains (CP27 and CP36) inhibited nitric oxide production significantly, in response to the positive control, i.e., taxol-treatment. Notably, CP27 and CP36 were more potent at reducing nitric oxide production than senloytic compounds (fisetin, quercetin). Using enzyme linked immunosorbent assays, the production of pro-inflammatory cytokines (IL-1ß, TNF-α, PGE2), was markedly reduced by treatment with CP27 and CP36, in response to stress. Both CP27 and CP36 contain a plethora of metabolites to exact their effects [(3,4-dihydroxyphenylglycol, 5-methoxytryptophan, nifedipine, 4-chlorotestosterone-17-acetate, 3-phenoxypropionic acid, lactic acid, f-Honaucin A, l,l-Cyclo(leucylprolyl), 3-hydroxy-decanoic acid etc.], indicative of their potential in providing protection against cellular stress. Further high-throughput bioassay-guided testing of gut microbial metabolites from crocodiles, individually as well as in combination, together with the underlying molecular mechanisms, in vitro and in vivo will elucidate their value in the rational development of innovative therapies against cellular stress/gut dysbiosis.

Novel mutations in CYBB Gene Cause X-linked chronic Granulomatous Disease in Pakistani patients.

BACKGROUND: Chronic Granulomatous Disease (CGD) is a primary immunodeficiency that causes susceptibility to recurrent fungal and bacterial infections. The CYBB gene encodes gp91phox component of the Phagocytic Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and specifically, X-linked CGD is caused by mutations in the CYBB gene, located on the X chromosome. The aim of the study was to characterize functional and genetic mutations in X-linked CGD. METHODS: Functional analysis was conducted on the whole blood of seventeen male individuals who were suspected to have X-linked chronic granulomatous disease (CGD). Flow cytometry was employed to assess the capacity of NADPH oxidase, measuring both H2O2 production and gp91phox protein expression in neutrophils. Additionally, DNA Sanger sequencing was performed for genetic analysis. The pathogenicity of novel mutations was assessed by pathogenicity prediction tools. RESULT: Among the seventeen patients evaluated, five patients (P1, P2, P3, P4, and P5) displayed impaired H2O2 production by their neutrophils upon stimulation with Phorbol myristate acetate (PMA), accompanied by abnormal gp91phox expression. DNA sequencing of the CYBB gene identified specific mutations in each patient. In P1 and P2 (previously reported cases), a hemizygous missense mutation, c.925G > A/p.E309K was identified. In P3 and P4 (novel cases), hemizygous nonsense mutations, c.216T > A/p.C72X were found. Lastly, in P5 (also a novel case), a hemizygous missense mutation, c.732T > G/p.C244W was detected. These mutations reside in exons 9,3 and 7 of the CYBB gene, respectively. CONCLUSIONS: The current study contributes to the understanding of the clinical and genetic spectrum associated with X-linked chronic granulomatous disease (CGD). It highlights the significance of early diagnosis in CGD and emphasizes the importance of lifelong prophylaxis to prevent severe infections.

Mass spectrometric analysis of bioactive conditioned media of bacteria isolated from reptilian gut.

Aim: To determine whether selected gut bacteria of crocodile exhibit antibacterial properties. Materials & methods: Two bacteria isolated from Crocodylus porosus gut were used, namely: Pseudomonas aeruginosa and Aeromonas dhakensis. Conditioned media were tested against pathogenic bacteria and metabolites were analyzed using liquid chromatography-mass spectrometry. Results & conclusion: Antibacterial assays revealed that conditioned media showed potent effects against pathogenic Gram-positive and Gram-negative bacteria. LC-MS revealed identity of 210 metabolites. The abundant metabolites were, N-Acetyl-L-tyrosine, Acetaminophen, Trans-Ferulic acid, N, N-Dimethylformamide, Pyrocatechol, Cyclohexanone, Diphenhydramine, Melatonin, Gamma-terpinene, Cysteamine, 3-phenoxypropionic acid, Indole-3-carbinol, Benzaldehyde, Benzocaine, 2-Aminobenzoic acid, 3-Methylindole. These findings suggest that crocodile gut bacteria are potential source of novel bioactive molecules that can be utilized as pre/post/antibiotics for the benefit of human health.

Crocodiles thrive in unsanitary conditions, feed on rotten meat, and endure conditions that are detrimental to human health. In addition to their immune system, we speculate that their microbial gut flora produce substances contributing to their "hardiness" and "longevity". Herein, we showed that selected bacteria isolated from crocodile gut produced potent antibacterial properties against multiple drug-resistant pathogenic Gram-negative and Gram-positive bacteria. LC­MS/MS revealed the identity of gut microbial metabolites. These findings suggest that analyses of crocodile gut bacteria may reveal potential drug leads that can be utilized as probiotics/pre/post/antibiotics for the benefit of human health, however intensive future research is needed to realize these expectations.

Selected Gut Bacteria from Water Monitor Lizard Exhibit Effects against Pathogenic Acanthamoeba castellanii Belonging to the T4 Genotype.

Water monitor lizards (WMLs) reside in unhygienic and challenging ecological surroundings and are routinely exposed to various pathogenic microorganisms. It is possible that their gut microbiota produces substances to counter microbial infections. Here we determine whether selected gut bacteria of water monitor lizards (WMLs) possess anti-amoebic properties using Acanthamoeba castellanii of the T4 genotype. Conditioned media (CM) were prepared from bacteria isolated from WML. The CM were tested using amoebicidal, adhesion, encystation, excystation, cell cytotoxicity and amoeba-mediated host cell cytotoxicity assays in vitro. Amoebicidal assays revealed that CM exhibited anti-amoebic effects. CM inhibited both excystation and encystation in A. castellanii. CM inhibited amoebae binding to and cytotoxicity of host cells. In contrast, CM alone showed limited toxic effects against human cells in vitro. Mass spectrometry revealed several antimicrobials, anticancer, neurotransmitters, anti-depressant and other metabolites with biological functions. Overall, these findings imply that bacteria from unusual places, such as WML gut, produce molecules with anti-acanthamoebic capabilities.

Azole and 5-nitroimidazole based nanoformulations are potential antiamoebic drug candidates against brain-eating amoebae.

AIM: Herein, the anti-parasitic activity of azoles (fluconazole and itraconazole) and 5-nitroimdazole (metronidazole) against the brain-eating amoebae: Naegleria fowleri and Balamuthia mandrillaris was elucidated. METHODS AND RESULTS: Azoles and 5-nitroimidazole based nanoformulations were synthesized and characterized using a UV-visible spectrophotometer, atomic force microscopy, and fourier transform infrared spectroscopy. H1-NMR, EI-MS, and ESI-MS were performed to determine their molecular mass and elucidate their structures. Their size, zeta potential, size distribution, and polydispersity index (PDI) were assessed. Amoebicidal assays revealed that all the drugs and their nanoformulations, (except itraconazole) presented significant anti-amoebic effects against B. mandrillaris, while all the treatments indicated notable amoebicidal properties against N. fowleri. Amoebicidal effects were radically enhanced upon conjugating the drugs with nanoparticles. The IC50 values for KM-38-AgNPs-F, KM-20-AgNPs-M, and KM-IF were 65.09, 91.27, and 72.19 µg.mL-1, respectively, against B. mandrillaris. Whereas against N. fowleri, the IC50 values were: 71.85, 73.95, and 63.01 µg.mL-1, respectively. Additionally, nanoformulations significantly reduced N. fowleri-mediated host cell death, while nanoformulations along with fluconazole and metronidazole considerably reduced Balamuthia-mediated human cell damage. Finally, all the tested drugs and their nanoformulations revealed limited cytotoxic activity against human cerebral microvascular endothelial cell (HBEC-5i) cells. CONCLUSION: These compounds should be developed into novel chemotherapeutic options for use against these distressing infections due to free-living amoebae, as currently there are no effective treatments.

Antibacterial Properties of Ethacridine Lactate and Sulfmethoxazole Loaded Functionalized Graphene Oxide Nanocomposites.

The emergence of drug-resistant bacterial strains that reduce the effectiveness of antimicrobial agents has become a major ongoing health concern in recent years. It is therefore necessary to find new antibacterials with broad-spectrum activity against both Gram-positive and Gram-negative bacteria, and/or to use nanotechnology to boost the potency of already available medications. In this research, we examined the antibacterial efficacy of sulfamethoxazole and ethacridine lactate loaded two-dimensional glucosamine functionalized graphene-based nanocarriers against a range of bacterial isolates. Graphene oxide was first functionalized with glucosamine, which as a carbohydrate moiety can render hydrophilic and biocompatible characters to the GO surface, and subsequently loaded with ethacridine lactate and sulfamethoxazole. The resulting nanoformulations had distinct, controllable physiochemical properties. By analyzing the formulation using Fourier Transform Infrared Spectroscopy (FTIR), X-ray diffraction (PXRD), a thermogravimetric analysis (TGA), zetasizer, and a morphological analysis using Scanning Electron Microscopy and Atomic Force Microscopy, researchers were able to confirm the synthesis of nanocarriers. Both nanoformulations were tested against Gram-negative bacteria, including Escherichia coli K1, Serratia marcescens, Pseudomonas aeruginosa, Salmonella enterica, as well as Gram-positive bacteria, including Bacillus cereus, Streptococcus pyogenes, and Streptococcus pneumoniae. Importantly, ethacridine lactate and its nanoformulations exhibited significant antibacterial properties against all bacteria tested in this study. When tested for minimum inhibitory concentration (MIC), the results were remarkable and revealed that ethacridine lactate presented MIC90 at 9.7 µg/mL against S. enteric, and MIC90 at 6.2 µg/mL against B. cereus. Notably, ethacridine lactate and its nanoformulations showed limited toxicity effects against human cells using lactate dehydrogenase assays. Overall, the results revealed that ethacridine lactate and its nanoformulations possess antibacterial activities against various Gram-negative and Gram-positive bacteria and that nanotechnology can be employed for the targeted delivery of effective drugs without harming the host tissue.

Anti-amoebic activity of a series of benzofuran/benzothiophene derivatives against Acanthamoeba castellanii belonging to the T4 genotype.

AIMS: To determine the anti-amoebic activity of benzofuran/benzothiophene-possessing compounds against Acanthamoeba castellanii of the T4 genotype. METHOD AND RESULTS: A series of benzofuran/benzothiophene-possessing compounds were tested for their anti-amoebic activities, in particular, to block encystation and excystation processes in amoebae. Cytotoxicity of the compounds were evaluated using lactate dehydrogenase (LDH) assays. The amoebicidal assay results revealed significant anti-amoebic effects against A. castellanii. Compounds 1p and 1e showed the highest amoebicidal activity, eliminating 68% and 64% of the amoebae, respectively. These compounds remarkably repressed both the encystation and excystation processes in A. castellanii. Furthermore, the selected compounds presented minimal cytotoxic properties against human cells, as well as considerably abridged amoeba-mediated cytopathogenicity when compared to the amoebae alone. CONCLUSIONS: Our findings show that benzofuran/benzothiophene derivatives depict potent anti-amoebic activities; thus these compounds should be used as promising and novel agents in the rationale development of therapeutic strategies against Acanthamoeba infections.

Cinnamic acid and lactobionic acid based nanoformulations as a potential antiamoebic therapeutics.

Acanthamoeba castellanii causes granulomatous amoebic encephalitis, an uncommon but severe brain infection and sight-threatening Acanthamoeba keratitis. Most of the currently used anti-amoebic treatments are not always effective, due to persistence of the cyst stage, and recurrence can occur. Here in this study we synthesize cinnamic acid and lactobionic acid-based magnetic nanoparticles (MNPs) using co-precipitation technique. These nanoformulations were characterized by Fourier transform infrared spectroscopy and Atomic form microscopy. The drugs alone (Hesperidin, Curcumin and Amphotericin B), magnetic NPs alone, and drug-loaded nano-formulations were evaluated at a concentration of 100 µg/mL for antiamoebic activity against a clinical isolate of A. castellanii. Amoebicidal assays revealed that drugs and conjugation of drugs and NPs further enhanced amoebicidal effects of drug-loaded nanoformulations. Drugs and drug-loaded nanoformulations inhibited both encystation and excystation of amoebae. In addition, drugs and drug-loaded nanoformulations inhibited parasite binding capability to the host cells. Neither drugs nor drug-loaded nanoformulations showed cytotoxic effects against host cells and considerably reduced parasite-mediated host cell death. Overall, these findings imply that conjugation of medically approved drugs with MNPs produce potent anti-Acanthamoebic effects, which could eventually lead to the development of therapeutic medications.

Antiamoebic properties of Methyltrioctylammonium chloride based deep eutectic solvents.

PURPOSE: This aim of this study was to assess anti-parasitic properties of deep eutectic solvents against eye pathogen, Acanthamoeba, often associated with the use of contact lens. METHODS: Assays were performed to investigate the effects of various Methyltrioctylammonium chloride-based deep eutectic solvents on Acanthamoeba castellanii, comprising amoebicidal assays, encystment assays, excystment assays, cytotoxicity assays by measuring lactate dehydrogenase release from human cells, and cytopathogenicity assays to determine parasite-mediated host cell death. RESULTS: In a 2 h incubation period, DES-B, DES-C, DES-D, and DES-E exhibited up to 85 % amoebicidal activity at micromolar doses, which was enhanced further following 24 h incubation. When tested in encystment assays, selected deep eutectic solvents abolished cyst formation and were able to block excystment of A. castellanii. All solvents exhibited minimal human cell cytotoxicity except DES-D. Finally, all tested deep eutectic solvents inhibited amoeba-mediated cytopathogenicity, except DES-B. CONCLUSIONS: Deep eutectic solvents show potent antiamoebic effects. These findings are promising and could lead to the development of novel contact lens disinfectants, as well as opening several avenues to explore the molecular mechanisms, various doses and incubation periods, and use of different bases against Acanthamoeba castellanii.

Antiamoebic Properties of Ceftriaxone and Zinc-Oxide-Cyclodextrin-Conjugated Ceftriaxone.

Acanthamoeba castellanii is a ubiquitous free-living amoeba capable of instigating keratitis and granulomatous amoebic encephalitis in humans. Treatment remains limited and inconsistent. Accordingly, there is a pressing need for novel compounds. Nanotechnology has been gaining attention for enhancing drug delivery and reducing toxicity. Previous work has shown that various antibiotic classes displayed antiamoebic activity. Herein, we employed two antibiotics: ampicillin and ceftriaxone, conjugated with the nanocarrier zinc oxide and ß-cyclodextrin, and tested them against A. castellanii via amoebicidal, amoebistatic, encystment, excystment, cytopathogenicity, and cytotoxicity assays at a concentration of 100 µg/mL. Notably, zinc oxide ß-cyclodextrin ceftriaxone significantly inhibited A. castellanii growth and cytopathogenicity. Additionally, both zinc oxide ß-cyclodextrin ceftriaxone and ceftriaxone markedly inhibited A. castellanii encystment. Furthermore, all the tested compounds displayed negligible cytotoxicity. However, minimal anti-excystment or amoebicidal effects were observed for the compounds. Accordingly, this novel nanoconjugation should be employed in further studies in hope of discovering novel anti-Acanthamoeba compounds.