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Free-living amoebae infections are on the rise while the prognosis remains poor. Current therapies are ineffective, and there is a need for novel effective drugs which can target Naegleria, Balamuthia, and Acanthamoeba species. In this study, we determined the effects of a nano-formulation based on flavonoid patuletin-loaded gallic acid functionalized zinc oxide nanoparticles (PA-GA-ZnO) against Acanthamoeba, Balamuthia, and Naegleria trophozoites. Characterization of the nano-formulation was accomplished utilizing analytical tools, namely Fourier-transform infrared spectroscopy, drug entrapment efficiency, polydispersity index, dimensions, and surface morphologies. Anti-amoebic effects were investigated using amoebicidal assay, cytopathogenicity assay, and cytotoxicity of the nano-formulation on human cells. The findings revealed that nano-formulation (PA-GA-ZnO) displayed significant anti-amoebic properties and augmented effects of patuletin alone against all three brain-eating amoebae. When tested alone, patuletin nano-formulations showed minimal toxicity effects against human cells. In summary, the nano-formulations evaluated herein depicts efficacy versus Acanthamoeba, Balamuthia, and Naegleria. Nonetheless, future studies are needed to comprehend the molecular mechanisms of patuletin nano-formulations versus free-living amoebae pathogens, in addition to animal studies to determine their potential value for clinical applications.

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PURPOSE: The treatment of amoebic infections is often problematic, largely due to delayed diagnosis, amoebae transformation into resistant cyst form, and lack of availability of effective chemotherapeutic agents. Herein, we determined anti-Acanthamoeba castellanii properties of three metal oxide nanoparticles (TiO2, ZrO2, and Al2O3). METHODS: Amoebicidal assays were performed to determine whether metal oxide nanoparticles inhibit amoebae viability. Encystation assays were performed to test whether metal oxide nanoparticles inhibit cyst formation. By measuring lactate dehydrogenase release, cytotoxicity assays were performed to determine human cell damage. Hoechst 33342/PI staining was performed to determine programmed cell death (apoptosis) and necrosis in A. castellanii. RESULTS: TiO2-NPs significantly inhibited amoebae viability as observed through amoebicidal assays, as well as inhibited their phenotypic transformation as evident using encystation assays, and showed limited human cell damage as observed by measuring lactate dehydrogenase assays. Furthermore, TiO2-NPs altered parasite membranes and resulted in necrotic cell death as determined using double staining cell death assays with Hoechst33342/Propidium iodide (PI) observed through chromatin condensation. These findings suggest that TiO2-NPs offers a potential viable avenue in the rationale development of therapeutic interventions against Acanthamoeba infections.

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The gut microbiota and its secreted metabolites play a significant role in cardiovascular and musculoskeletal health and diseases. The dysregulation of the intestinal microbiota poses a significant threat to cardiovascular and skeletal muscle well-being. Nonetheless, the precise molecular mechanisms underlying these changes remain unclear. Furthermore, microgravity presents several challenges to cardiovascular and musculoskeletal health compromising muscle strength, endothelial dysfunction, and metabolic changes. The purpose of this review is to critically examine the role of gut microbiota metabolites on cardiovascular and skeletal muscle functions and dysfunctions. It also explores the molecular mechanisms that drive microgravity-induced deconditioning in both cardiovascular and skeletal muscle. Key findings in this review highlight that several alterations in gut microbiota and secreted metabolites in microgravity mirror characteristics seen in cardiovascular and skeletal muscle diseases. Those alterations include increased levels of Firmicutes/Bacteroidetes (F/B) ratio, elevated lipopolysaccharide levels (LPS), increased in para-cresol (p-cresol) and secondary metabolites, along with reduction in bile acids and Akkermansia muciniphila bacteria. Highlighting the potential, modulating gut microbiota in microgravity conditions could play a significant role in mitigating cardiovascular and skeletal muscle diseases not only during space flight but also in prolonged bed rest scenarios here on Earth.

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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.

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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.

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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.

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Antimicrobial and chemotherapy resistance are escalating medical problem of paramount importance. Yet, research for novel antimicrobial and anticancer agents remains lagging behind. With their reported medical applications, DNA minor groove binders (MGBs) are worthy of exploration. In this study, the approach of structure-based drug design was implemented to generate 11 MGB compounds including a novel class of bioactive alkyne-linked MGBs. The NCI screening protocol was utilized to evaluate the antitumor activity of the target MGBs. Furthermore, a variety of bactericidal, cytopathogenicity, MIC90, and cytotoxicity assays were carried out using these MGBs against 6 medically relevant bacteria: Salmonella enterica, Escherichia coli, Serratia marcescens, Bacillus cereus, Streptococcus pneumoniae and Streptococcus pyogenes. Moreover, molecular docking, molecular dynamic simulations, DNA melting, and isothermal titration calorimetry (ITC) analyses were utilized to explore the binding mode and interactions between the most potent MGBs and the DNA duplex d(CGACTAGTCG)2. NCI results showed that alkyne-linked MGBs (26 & 28) displayed the most significant growth inhibition among the NCI-60 panel. In addition, compounds MGB3, MGB4, MGB28, and MGB32 showed significant bactericidal effects, inhibited B. cereus and S. enterica-mediated cytopathogenicity, and exhibited low cytotoxicity. MGB28 and MGB32 demonstrated significant inhibition of S. pyogenes, whereas MGB28 notably inhibited S. marcescens and all four minor groove binders significantly inhibited B. cereus. The ability of these compounds to bind with DNA and distort its groove dimensions provides the molecular basis for the allosteric perturbation of proteins-DNA interactions by MGBs. This study shed light on the mechanism of action of MGBs and revealed the important structural features for their antitumor and antibacterial activities, which are important to guide future development of MGB derivatives as novel antibacterial and anticancer agents.

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Keratitis is corneal inflammatory disease which may be caused by several reason such as an injury, allergy, as well as a microbial infection. Besides these, overexposure to ultraviolet light and unhygienic practice of contact lenses are also associated with keratitis. Based on the cause of keratitis, different lines of treatments are recommended. Photodynamic therapy is a promising approach that utilizes light activated compounds to instigate either killing or healing mechanism to treat various diseases including both communicable and non-communicable diseases. This review focuses on clinically-important patent applications and the recent literature for the use of photodynamic therapy against keratitis.

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Acanthamoeba castellanii are opportunistic pathogens known to cause infection of the central nervous system termed: granulomatous amoebic encephalitis, that mostly effects immunocompromised individuals, and a sight threatening keratitis, known as Acanthamoeba keratitis, which mostly affects contact lens wearers. The current treatment available is problematic, and is toxic. Herein, an amphiphilic star polymer with AB2 miktoarms [A = hydrophobic poly(ℇ-Caprolacton) and B = hydrophilic poly (ethylene glycol)] was synthesized by ring opening polymerization and CuI catalyzed azide-alkyne cycloaddition. Characterization by 1H and 13C NMR spectroscopy, size-exclusion chromatography and fluorescence spectroscopy was accomplished. The hydrophobic drug itraconazole (ITZ) was incorporated in self-assembled micellar structure of AB2 miktoarms through co-solvent evaporation. The properties of ITZ loaded (ITZ-PCL-PEG2) and blank micelles (PCL-PEG2) were investigated through zeta sizer, scanning electron microscopy and Fourier-transform infrared spectroscopy. Itraconazole alone (ITZ), polymer (DPB-PCL), empty polymeric micelles (PCL-PEG2) alone, and itraconazole loaded in polymeric micelles (ITZ-PCL-PEG2) were tested for anti-amoebic potential against Acanthamoeba, and the cytotoxicity on human cells were determined. The polymer was able to self-assemble in aqueous conditions and exhibited low value for critical micelle concentration (CMC) 0.05-0.06 µg/mL. The maximum entrapment efficiency of ITZ was 68%. Of note, ITZ, DPB, PCL-PEG2 and ITZ-PCL-PEG2 inhibited amoebae trophozoites by 37.34%, 36.30%, 35.77%, and 68.24%, respectively, as compared to controls. Moreover, ITZ-PCL-PEG2 revealed limited cytotoxicity against human keratinocyte cells. These results are indicative that ITZ-PCL-PEG2 micelle show significantly better anti-amoebic effects as compared to ITZ alone and thus should be investigated further in vivo to determine its clinical potential.

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Pathogenic Naegleria fowleri (N. fowleri) are opportunistic free-living amoebae and are the causative agents of a very rare but severe brain infection called primary amoebic meningoencephalitis (PAM). The fatality rate of PAM in reported cases is more than 95%. Most of the drugs used againstN. fowleri infections are repurposed drugs. Therefore, a large number of compounds have been tested againstN. fowleri in vitro, but most of the tested compounds showed high toxicity and an inability to cross the blood-brain barrier. Andrographolide, forskolin, and borneol are important natural compounds that have shown various valuable biological properties. In the present study, the nanoconjugates (AND-AgNPs, BOR-AgNPs, and FOR-AgNPs) of these compounds were synthesized and assessed against both stages (trophozoite and cyst) ofN. fowleri for their antiamoebic and cysticidal potential in vitro. In addition, cytotoxicity and host cell pathogenicity were also evaluated in vitro. FOR-AgNPs were the most potent nanoconjugate and showed potent antiamoebic activity againstN. fowleriwith an IC50 of 26.35 µM. Nanoconjugates FOR-AgNPs, BOR-AgNPs, and AND-AgNPs also significantly inhibit the viability of N. fowleri cysts. Cytotoxicity assessment showed that these nanoconjugates caused minimum damage to human keratinocyte cells (HaCaT cells) at 100 µg/mL, while also effectively reducing the cytopathogenicity of N. fowleri trophozoites to the HaCaT cells. The outcomes of our experiments have unveiled substantial potential for AND-AgNPs, BOR-AgNPs, and FOR-AgNPs in the realm of developing innovative alternative therapeutic agents to combat infections caused by N. fowleri. This study represents a significant step forward in the pursuit of advanced strategies for managing such amoebic infections, laying the foundation for the development of novel and more effective therapeutic modalities in the fight against free-living amoebae.

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A rare but lethal central nervous system disease known as granulomatous amoebic encephalitis (GAE) and potentially blinding Acanthamoeba keratitis are diseases caused by free-living Acanthamoeba. Currently, no therapeutic agent can completely eradicate or prevent GAE. Synthetic compounds are a likely source of bioactive compounds for developing new drugs. This study synthesized seventeen 1,4-benzothiazine derivatives (I -XVII) by a base-catalyzed one-pot reaction of 2-amino thiophenol with substituted bromo acetophenones. Different spectroscopic techniques, such as EI-MS, 1H-, and 13C NMR (only for the new compounds), were used for the structural characterization and conformation of compounds. These compounds were assessed for the first time against Acanthamoeba castellanii. All compounds showed anti-amoebic potential in vitro against A. castellanii, reducing its ability to encyst and excyst at 100 µM. Compounds IX, X, and XVI showed the most potent activities among all derivatives and significantly reduced the viability to 5.3 × 104 (p < 0.0003), 2 × 105 (p < 0.006), and 2.4 × 105 (p < 0.002) cells/mL, respectively. The cytotoxicity profile revealed that these molecules showed lower to moderate cytotoxicity, i.e., 36 %, 2 %, and 21 %, respectively, against human keratinocytes in vitro. These results indicate that 1,4-benzothiazines showed potent in vitro activity against trophozoites and cysts of A. castellanii. Hence, these 1,4-benzothiazine derivatives should be considered to develop new potential therapeutic agents against Acanthamoeba infections.

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During space travel, the gut microbiota is changed which can lead to health-related issues. Previously, we utilized the hind-limb unloaded (HU) mouse, which is an established ground-based in-vivo model of microgravity and observed altered gut microbiota. In this study, we evaluated the beneficial effects of novel bacterial conditioned media in HU mice to understand if they can offset the effects of unloading in the HU mouse model. We aimed to explore the influence of bacterial conditioned media on diversity and quantity of intestinal microbes in HU mice, and investigated the microarchitecture of mice retinas and kidneys to evaluate the potential systemic effects of bacterial conditioned media in HU mice. Four-month-old, male C57/Bl6 mice were separated into groups: including the ground-based control group, the HU group mice fed with vehicle as placebo (HU-placebo mice), and the HU group fed with bacterial conditioned media (HU-CP mice) and kept under controlled environmental conditions for three weeks. Next, mice were sacrificed; gut dissections were conducted, and metagenomic analysis of bacterial species was performed via DNA extraction and 16S rRNA analysis. The results revealed an HU-induced reduction in intestinal microbial diversity, and an increase in pathogenic bacteria dominated by Firmicutes (45%). In contrast, supplementation with bacterial conditioned media for three weeks led to a significant increase in gut microbial diversity with noticeable changes in the OTUs abundance in the HU mice. Additionally, HU-induced muscle weakness and structural abnormalities in the retina and kidney were partially prevented with bacterial conditioned media. Moreover, a greater diversity of several bacteria in the HU-CP was observed including, Bacteriodota, Firmicutes, Proteobacteria, Actionobacteriota, Verrucomicorbiota, Cyanobacteria, Gemmatimonadota, Acidobacteriota, Chloroflexi, Myxococcota, and others. Prospective research involving molecular mechanistic studies are needed to comprehend the systemic effects of bacterial metabolites conditioned media on experimental animal models under chronic stress.

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In view of the critical role the gut microbiome plays in human health, it has become clear that astronauts' gut microbiota composition changes after spending time in space. Astronauts are exposed to several risks in space, including a protracted period of microgravity, radiation, and mechanical unloading of the body. Several deleterious effects of such an environment are reported, including orthostatic intolerance, cardiovascular endothelial dysfunction, cellular and molecular changes, and changes in the composition of the gut microbiome. Herein, the correlation between the gut microbiome and cardiovascular disease in a microgravity environment is evaluated. Additionally, the relationship between orthostatic hypotension, cardiac shrinkage and arrhythmias during spaceflight, and cellular alterations during spaceflight is reviewed. Given its impact on human health in general, modifying the gut microbiota may significantly promote astronaut health and performance. This is merited, given the prospect of augmented human activities in future space missions.

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Microgravity, in space travel and prolonged bed rest conditions, induces cardiovascular deconditioning along with skeletal muscle mass loss and weakness. The findings of microgravity research may also aid in the understanding and treatment of human health conditions on Earth such as muscle atrophy, and cardiovascular diseases. Due to the paucity of biomarkers and the unknown underlying mechanisms of cardiovascular and skeletal muscle deconditioning in these environments, there are insufficient diagnostic and preventative measures. In this study, we employed hindlimb unloading (HU) mouse model, which mimics astronauts in space and bedridden patients, to first evaluate cardiovascular and skeletal muscle function, followed by proteomics and metabolomics LC-MS/MS-based analysis using serum samples. Three weeks of unloading caused changes in the function of the cardiovascular system in c57/Bl6 mice, as seen by a decrease in mean arterial pressure and heart weight. Unloading for three weeks also changed skeletal muscle function, causing a loss in grip strength in HU mice and atrophy of skeletal muscle indicated by a reduction in muscle mass. These modifications were partially reversed by a two-week recovery period of reloading condition, emphasizing the significance of the recovery process. Proteomics analysis revealed 12 dysregulated proteins among the groups, such as phospholipid transfer protein, Carbonic anhydrase 3, Parvalbumin alpha, Major urinary protein 20 (Mup20), Thrombospondin-1, and Apolipoprotein C-IV. On the other hand, metabolomics analysis showed altered metabolites among the groups such as inosine, hypoxanthine, xanthosine, sphinganine, l-valine, 3,4-Dihydroxyphenylglycol, and l-Glutamic acid. The joint data analysis revealed that HU conditions mainly impacted pathways such as ABC transporters, complement and coagulation cascades, nitrogen metabolism, and purine metabolism. Overall, our results indicate that microgravity environment induces significant alterations in the function, proteins, and metabolites of these mice. These observations suggest the potential utilization of these proteins and metabolites as novel biomarkers for assessing and mitigating cardiovascular and skeletal muscle deconditioning associated with such conditions.

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The free living Acanthamoeba spp. are ubiquitous amoebae associated with potentially blinding disease known as Acanthamoeba keratitis (AK) and a fatal central nervous system infection granulomatous amoebic encephalitis (GAE). With the inherent ability of cellular differentiation, it can phenotypically transform to a dormant cyst form from an active trophozoite form. Acanthamoeba cysts are highly resistant to therapeutic agents as well as contact lens cleaning solutions. One way to tackle drug resistance against Acanthamoeba is by inhibiting the formation of cysts from trophozoites. The biochemical analysis showed that the major component of Acanthamoeba cyst wall is composed of carbohydrate moieties such as galactose and glucose. The disaccharide of galactose and glucose is lactose. In this study, we analyzed the potential of lactase enzyme to target carbohydrate moieties of cyst walls. Amoebicidal assessment showed that lactase was ineffective against trophozoite of A. castellanii but enhanced amoebicidal effects of chlorhexidine. The lactase enzyme did not show any toxicity against normal human keratinocyte cells (HaCaT) at the tested range. Hence, lactase can be used for further assessment for development of potential therapeutic agents in the management of Acanthamoeba infection as well as formulation of effective contact lens disinfectants.

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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.

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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.

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Several antimicrobial agents are commonly included in contact lens disinfectant solutions including chlorhexidine diacetate (CHX), polyhexamethylene biguanide (PHMB) or myristamidopropyl dimethylamine (MAPD); however, their mode of action, i.e. necrosis versus apoptosis is incompletely understood. Here, we determined whether a mechanism of cell death resembling that of apoptosis was present in Acanthamoeba castellanii of the T4 genotype (NEFF) following exposure to the aforementioned antimicrobials using the anticoagulant annexin V that undergoes rapid high affinity binding to phosphatidylserine in the presence of calcium, making it a sensitive probe for phosphatidylserine exposure. The results revealed that under the conditions employed in this study, an apoptotic pathway of cell death in this organism at the tested conditions does not occur. Our findings suggest that necrosis is the likely mode of action; however, future mechanistic studies should be accomplished in additional experimental conditions to further comprehend the molecular mechanisms of cell death in Acanthamoeba.

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Naegleria fowleri is one of the free-living amoebae and is a causative agent of a lethal and rare central nervous system infection called primary amoebic meningoencephalitis. Despite the advancement in antimicrobial chemotherapy, the fatality rate in the reported cases is more than 95%. Most of the treatment drugs used against N. fowleri infection are repurposed drugs. Therefore, a large number of compounds have been tested against N. fowleri in vitro, but most of the compounds showed high toxicity. To overcome this, we evaluated the effectiveness of naturally occurring terpene compounds against N. fowleri. In this study, we evaluated the antiamoebic potential of natural compounds including Thymol, Borneol, Andrographolide, and Forskolin againstN. fowleri. Thymol showed the highest amoebicidal activity with IC50/24 h at 153.601 ± 19.6 µM. Two combinations of compounds Forskolin + Thymol and Forskolin + Borneol showed a higher effect on the viability of trophozoites as compared to compounds alone and hence showed a synergistic effect. The IC50 reported for Forskolin + Thymol was 81.30 ± 6.86 µM. Borneol showed maximum cysticidal activity with IC50/24 h at 192.605 ± 3.01 µM. Importantly, lactate dehydrogenase release testing revealed that all compounds displayed minimal cytotoxicity to human HaCaT, HeLa, and SH-SY5Y cell lines. The cytopathogenicity assay showed that Thymol and Borneol also significantly reduced the host cell cytotoxicity of pretreated amoeba toward the human HaCaT cell line. So, these terpene compounds hold potential as therapeutic agents against infections caused by N. fowleri and are potentially a step forward in drug development against this deadly pathogen as these compounds have also been reported to cross the blood-brain barrier. Therefore, an in vivo study using animal models is necessary to assess the efficacy of these compounds and the need for further research into the intranasal route of delivery for the treatment of these life-threatening infections.

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Cardiovascular diseases (CVDs) are highly associated with both vitamin D deficiency and obesity, two prevalent health conditions worldwide. Arterial stiffness, an independent predictor of CVDs, is particularly elevated in both conditions, yet the molecular mechanisms underlying this phenomenon remain elusive, hindering effective management of CVDs in this population. We recruited 20 middle-aged Emiratis, including 9 individuals with vitamin D deficiency (Vit D level ≤20 ng) and obesity (BMI ≥30) and 11 individuals as control with Vit D level >20 ng and BMI <30. We measured arterial stiffness using pulse wave velocity (PWV) and performed whole transcriptome sequencing to identify differentially expressed genes (DEGs) and enriched pathways. We validated these findings using qRT-PCR, Western blot, and multiplex analysis. PWV was significantly higher in the vitamin D deficient and obese group relative to controls (p ≤ 0.05). The DEG analysis revealed that pathways related to interleukin 1 (IL-1), nitrogen metabolism, HIF-1 signaling, and MAPK signaling were over-activated in the vitamin D deficient and obese group. We found that HIF-1alpha, NOX-I, NOX-II, IL-1b, IL-8, IL-10, and VEGF were significantly upregulated in the vitamin D deficient and obese group (p < 0.05). Our study provides new insights into the molecular mechanisms of arterial stiffness in vitamin D deficiency and obesity, demonstrating the role of oxidative stress and inflammation in this process. Our findings suggest that these biomarkers may serve as potential therapeutic targets for early prevention of CVDs. Further studies are needed to investigate these pathways and biomarkers with larger cohort.