Evolutionary relevance of metabolite production in relation to marine sponge bacteria symbiont.

Sponges are habitats for a diverse community of microorganisms. Sponges provide shelter, whereas microbes provide a complementary defensive mechanism. Here, a symbiotic bacterium, identified as Bacillus spp., was isolated from a marine sponge following culture enrichment. Fermentation-assisted metabolomics using thin-layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS) indicated that marine simulated nutrition and temperature was the optimum in metabolite production represented by the highest number of metabolites and the diverse chemical classes when compared to other culture media. Following large-scale culture in potato dextrose broth (PDB) and dereplication, compound M1 was isolated and identified as octadecyl-1-(2',6'-di-tert-butyl-1'-hydroxyphenyl) propionate. M1, at screening concentrations up to 10 mg/ml, showed no activity against prokaryotic bacteria including Staphylococcus aureus and Escherichia coli, while 1 mg/ml of M1 was sufficient to cause a significant killing effect on eukaryotic cells including Candida albicans, Candida auris, and Rhizopus delemar fungi and different mammalian cells. M1 exhibited MIC50 0.97 ± 0.006 and 7.667 ± 0.079 mg/ml against C. albicans and C. auris, respectively. Like fatty acid esters, we hypothesize that M1 is stored in a less harmful form and upon pathogenic attack is hydrolyzed to a more active form as a defensive metabolite. Subsequently, [3-(3,5-di-tert-butyl-4-hydroxyphenyl)-propionic acid] (DTBPA), the hydrolysis product of M1, exhibited ~ 8-fold and 18-fold more antifungal activity than M1 against C. albicans and C. auris, respectively. These findings indicated the selectivity of that compound as a defensive metabolite towards the eukaryotic cells particularly the fungi, a major infectious agent to sponges. Metabolomic-assisted fermentation can provide a significant understanding of a triple marine-evolved interaction. KEY POINTS: • Bacillus species, closely related to uncultured Bacillus, is isolated from Gulf marine sponge • Metabolomic-assisted fermentations showed diverse metabolites • An ester with a killing effect against eukaryotes but not prokaryotes is isolated.

Intra-clade Heterogeneity in Candida auris: Risk of Management.

Candida auris has emerged as a significant nosocomial fungal pathogen with a high risk of pathogenicity. Since the initial detection of C. auris in 2009, it gained lots of attention with a recent alert by the Centers for Disease Control and Prevention (CDC) due to its high infectivity and drug resistance. Several studies showed the capability of C. auris to secrete lytic enzymes, germinate, and form a biofilm that eventually results in interactions with the host cells, leading to serious infections. Other studies demonstrated a decrease in susceptibility of C. auris strains to available antifungals, which may be caused by mutations within the target genes, or the drug efflux pumps. However, the contribution of C. auris heterogeneity in pathogenicity and drug resistance is not well studied. Here, we shed light on the factors contributing to the development of heterogeneity in C. auris. These include phenotypic changes, biofilm formation, mechanisms of drug resistance, host invasion, mode of transmission, and expression of virulence factors. C. auris exhibits different phenotypes, particularly aggregative, and non-aggregative forms that play an important role in fungal heterogeneity, which significantly affects drug resistance and pathogenicity. Collectively, heterogeneity in C. auris significantly contributes to ineffective treatment, which in turn affects the fungal pathogenicity and drug resistance. Therefore, understanding the underlying reasons for C. auris heterogeneity and applying effective antifungal stewardship could play a major role in controlling this pathogen.

Comparative evaluation of flavonoids reveals the superiority and promising inhibition activity of silibinin against SARS-CoV-2.

Flavonoids are phenolic compounds naturally found in plants and commonly consumed in diets. Herein, flavonoids were sequentially evaluated by a comparative in silico study associated with systematic literature search. This was followed by an in vitro study and enzyme inhibition assays against vital SARS-CoV-2 proteins including spike (S) protein, main protease (Mpro ), RNA-dependent RNA-polymerase (RdRp), and human transmembrane serine protease (TMPRSS2). The results obtained revealed 10 flavonoids with potential antiviral activity. Out of them, silibinin showed promising selectivity index against SARS-CoV-2 in vitro. Screening against S protein discloses the highest inhibition activity of silibinin. Mapping the activity of silibinin indicated its excellent binding inhibition activity against SARS-CoV-2 S protein, Mpro and RdRP at IC50 0.029, 0.021, and 0.042 µM, respectively, while it showed no inhibition activity against TMPRSS2 at its IC50(SARS-CoV-2) . Silibinin was tested safe on human mammalian cells at >7-fold its IC50(SARS-CoV-2) . Additionally, silibinin exhibited >90% virucidal activity at 0.031 µM. Comparative molecular docking (MD) showed that silibinin possesses the highest binding affinity to S protein and RdRP at -7.78 and -7.15 kcal/mol, respectively. MDs showed that silibinin exhibited stable interaction with key amino acids of SARS-CoV-2 targets. Collectively, silibinin, an FDA-approved drug, can significantly interfere with SARS-CoV-2 entry and replication through multi-targeting activity.

Caspofungin-resistance in Candida auris is cell wall-dependent phenotype and potential prevention by zinc oxide nanoparticles.

Candida auris is an emergent nosocomial multi-drug-resistant yeast that represents a global health threat. Recently, C. auris clinical isolates with caspofungin resistance were identified. Mutation in FKS1 gene was determined as a mechanism of resistance. However, the ability of C. auris to develop acquired and cross-resistance has never been investigated. Herein, this resistance ability due to caspofungin and associate mechanisms were investigated. C. auris clinical isolate was successively cultured for ten generations in the presence of caspofungin compared to fluconazole-treatment and untreated controls. This was followed by the analysis of target gene expression and phenotypic changes. The obtained results showed that caspofungin-treated C. auris exhibited elevated MIC50(caspofungin), slower growth, elevated chitin content, overexpression of caspofungin target genes, and cross-resistance to fluconazole. Interestingly, caspofungin exposure induced cell-cell adhesion and biofilm formation. C. auris gradually lost caspofungin resistance after removal of antifungal pressure, while keeping the overexpression of fungal cell wall-related genes including ALS5. We propose that C. auris ageing in the presence of caspofungin caused the development of persistent phenotypic changes in the fungal cell wall, leading to acquired and physical cross-resistance mechanisms. Surprisingly, formulation of caspofungin in zinc oxide nanoparticles prevented the aforementioned behavioral changes regardless of the pathogen generations. LAY SUMMARY: Candida auris developed resistance against caspofungin. Our data indicated that this resistance mechanism is unique because of changes in the genes related to cell wall adhesions. Formulation of caspofungin in ZnO nanoparticles was able to overcome these phenotypic changes.

Promising anti-SARS-CoV-2 drugs by effective dual targeting against the viral and host proteases.

SARS-CoV-2 caused dramatic health, social and economic threats to the globe. With this threat, the expectation of future outbreak, and the shortage of anti-viral drugs, scientists were challenged to develop novel antivirals. The objective of this study is to develop novel anti-SARS-CoV-2 compounds with dual activity by targeting valuable less-mutated enzymes. Here, we have mapped the binding affinity of >500,000 compounds for potential activity against SARS-CoV-2 main protease (Mpro), papain protease (PLpro) and human furin protease. The enzyme inhibition activity of most promising hits was screened and tested in vitro on SARS-CoV-2 clinical isolate incubated with Vero cells. Computational modelling and toxicity of the compounds were validated. The results revealed that 16 compounds showed potential binding activity against Mpro, two of them showed binding affinity against PLpro and furin protease. Respectively, compounds 7 and 13 showed inhibition activity against Mpro at IC50 0.45 and 0.11 µM, against PLpro at IC50 0.085 and 0.063 µM, and against furin protease at IC50 0.29 µM. Computational modelling validated the binding affinity against all proteases. Compounds 7 and 13 showed significant inhibition activity against the virus at IC50 0.77 and 0.11 µM, respectively. Both compounds showed no toxicity on mammalian cells. The data obtained indicated that compounds 7 and 13 exhibited potent dual inhibition activity against SARS-CoV-2. The dual activity of both compounds can be of great promise not only during the current pandemic but also for future outbreaks since the compounds' targets are of limited mutation and critical importance to the viral infection.

Potential targeting of Hep3B liver cancer cells by lupeol isolated from Avicennia marina.

Medicinal plants are valuable sources of different active constituents that are known to have important pharmacological activities including anticancer effects. Lupeol, a pentacyclic triterpenoid, present in many medicinal plants, has a wide range of biological activities. Although the anticancer activity of lupeol was reported, the published data are inconsistent and the clear mechanism of action has never been assigned. The current study aims at investigating the anticancer specificity and mechanism of lupeol isolated from Avicennia marina, which grows in the desert of the United Arab Emirates. The compound was purified by chromatography and identified by spectroscopy. Compared with a negative control, lupeol caused significant (p < .001) growth inhibitory activity on MCF-7 and Hep3B parental and resistant cells by 45%, 46%, 72%, and 35%, respectively. The mechanism of action of lupeol was further explored by measuring its effect on key players in cancer development and progression, BCL-2 anti-apoptotic and BAX pro-apoptotic proteins. Lupeol significantly (p < .01) downregulated BCL-2 gene expression in parental and resistant Hep3B cells by 33 and 3.5 times, respectively, contributing to the induction of apoptosis in Hep3B cells, whereas it caused no effect on BAX. Furthermore, the immunoblotting analysis revealed that lupeol cleaved the executioner caspase-3 into its active form. Interestingly, lupeol showed no significant effect on the proliferation of monocytes, whereas it caused an increase in the sub-G1 population and a reduction in the apoptosis rates of monocytes at 48 and 72 h, indicative of no immuno-inflammatory responses. Collectively, lupeol can be considered as promising effective and safe anticancer agent, particularly against Hep3B cancer cells.

Iterated Virtual Screening-Assisted Antiviral and Enzyme Inhibition Assays Reveal the Discovery of Novel Promising Anti-SARS-CoV-2 with Dual Activity.

Unfortunately, COVID-19 is still a threat to humankind and has a dramatic impact on human health, social life, the world economy, and food security. With the limited number of suggested therapies under clinical trials, the discovery of novel therapeutic agents is essential. Here, a previously identified anti-SARS-CoV-2 compound named Compound 13 (1,2,5-Oxadiazole-3-carboximidic acid, 4,4'-(methylenediimino) bis,bis[[(2-hydroxyphenyl)methylene]hydrazide) was subjected to an iterated virtual screening against SARS-CoV-2 Mpro using a combination of Ligand Designer and PathFinder. PathFinder, a computational reaction enumeration tool, was used for the rapid generation of enumerated structures via default reaction library. Ligand designer was employed for the computerized lead optimization and selection of the best structural modification that resulted in a favorable ligand-protein complex. The obtained compounds that showed the best binding to Mpro were re-screened against TMPRSS2, leading to the identification of 20 shared compounds. The compounds were further visually inspected, which resulted in the identification of five shared compounds M1-5 with dual binding affinity. In vitro evaluation and enzyme inhibition assay indicated that M3, an analogue of Compound 13 afforded by replacing the phenolic moiety with pyridinyl, possesses an improved antiviral activity and safety. M3 displayed in vitro antiviral activity with IC50 0.016 µM and Mpro inhibition activity with IC50 0.013 µM, 7-fold more potent than the parent Compound 13 and potent than the antivirals drugs that are currently under clinical trials. Moreover, M3 showed potent activity against human TMPRSS2 and furin enzymes with IC50 0.05, and 0.08 µM, respectively. Molecular docking, WaterMap analysis, molecular dynamics simulation, and R-group analysis confirmed the superiority of the binding fit to M3 with the target enzymes. WaterMap analysis calculated the thermodynamic properties of the hydration site in the binding pocket that significantly affects the biological activity. Loading M3 on zinc oxide nanoparticles (ZnO NPs) increased the antiviral activity of the compound 1.5-fold, while maintaining a higher safety profile. In conclusion, lead optimized discovery following an iterated virtual screening in association with molecular docking and biological evaluation revealed a novel compound named M3 with promising dual activity against SARS-CoV-2. The compound deserves further investigation for potential clinical-based studies.

Essential Oil-Based Design and Development of Novel Anti-Candida Azoles Formulation.

Candida is the most common fungal class, causing both superficial and invasive diseases in humans. Although Candida albicans is the most common cause of fungal infections in humans, C. auris is a new emergent serious pathogen causing complications similar to those of C. albicans. Both C. albicans and C. auris are associated with high mortality rates, mainly because of their multidrug-resistance patterns against most available antifungal drugs. Although several compounds were designed against C. albicans, very few or none were tested on C. auris. Therefore, it is urgent to develop novel effective antifungal drugs that can accommodate not only C. albicans, but also other Candida spp., particularly newly emergent one, including C. auris. Inspired by the significant broad-spectrum antifungal activities of the essential oil cuminaldehyde and the reported wide incorporation of azoles in the antifungal drugs, a series of compounds (UoST1-11) was designed and developed. The new compounds were designed to overcome the toxicity of the aldehyde group of cuminaldehyde and benefit from the antifungal selectivity of azoles. The new developed UoST compounds showed significant anti-Candida activities against both Candida species. The best candidate compound, UoST5, was further formulated into polymeric nanoparticles (NPs). The new formula, UoST5-NPs, showed similar activities to the nanoparticles-free drug, while providing only 25% release after 24 h, maintainng prolonged activity up to 48 h and affording no toxicity. In conclusion, new azole formulations with significantly enhanced activities against C. albicans and C. auris, while maintaining prolonged action and no toxicities at lower concentrations, were developed.

Multiplexed integrating plasmids for engineering of the erythromycin gene cluster for expression in Streptomyces spp. and combinatorial biosynthesis.

Bacteria in the genus Streptomyces and its close relatives are prolific producers of secondary metabolites with antibiotic activity. Genome sequencing of these bacteria has revealed a rich source of potentially new antibiotic pathways, whose products have never been observed. Moreover, these new pathways can provide novel genes that could be used in combinatorial biosynthesis approaches to generate unnatural analogues of existing antibiotics. We explore here the use of multiple orthologous integrating plasmid systems, based on the int/attP loci from phages TG1, SV1, and ϕBT1, to express the polyketide synthase (PKS) for erythromycin in a heterologous Streptomyces host. Streptomyces strains containing the three polyketide synthase genes eryAI, eryAII, and eryAIII expressed from three different integrated plasmids produced the aglycone intermediate, 6-deoxyerythronolide B (6-dEB). A further pair of integrating plasmids, both derived from the ϕC31 int/attP locus, were constructed carrying a gene cassette for glycosylation of the aglycone intermediates, with or without the tailoring gene, eryF, required for the synthesis of erythronolide B (EB). Liquid chromatography-mass spectrometry of the metabolites indicated the production of angolosaminyl-6-dEB and angolosaminyl-EB. The advantages of using multiplexed integrating plasmids for engineering expression and for combinatorial biosynthesis were demonstrated.

A novel Streptomyces spp. integration vector derived from the S. venezuelae phage, SV1.

BACKGROUND: Integrating vectors based on the int/attP loci of temperate phages are convenient and used widely, particularly for cloning genes in Streptomyces spp. RESULTS: We have constructed and tested a novel integrating vector based on g27, encoding integrase, and attP site from the phage, SV1. This plasmid, pBF3 integrates efficiently in S. coelicolor and S. lividans but surprisingly fails to generate stable integrants in S. venezuelae, the natural host for phage SV1. CONCLUSION: pBF3 promises to be a useful addition to the range of integrating vectors currently available for Streptomyces molecular genetics.

1H-NMR metabolomics analysis identifies hypoxanthine as a novel metastasis-associated metabolite in breast cancer.

Breast cancer is one of the leading causes of death in females, mainly because of metastasis. Oncometabolites, produced via metabolic reprogramming, can influence metastatic signaling cascades. Accordingly, and based on our previous results, we propose that metabolites from highly metastatic breast cancer cells behave differently from less-metastatic cells and may play a significant role in metastasis. For instance, we aim to identify these metabolites and their role in breast cancer metastasis. Less metastatic cells (MCF-7) were treated with metabolites secreted from highly metastatic cells (MDA-MB-231) and the gene expression of three epithelial-to-mesenchymal transition (EMT) markers including E-cadherin, N-cadherin and vimentin were examined. Some metabolites secreted from MDA-MB-231 cells significantly induced EMT activity. Specifically, hypoxanthine demonstrated a significant EMT effect and increased the migration and invasion effects of MCF-7 cells through a hypoxia-associated mechanism. Hypoxanthine exhibited pro-angiogenic effects via increasing the VEGF and PDGF gene expression and affected lipid metabolism by increasing the gene expression of PCSK-9. Notably, knockdown of purine nucleoside phosphorylase, a gene encoding for an important enzyme in the biosynthesis of hypoxanthine, and inhibition of hypoxanthine uptake caused a significant decrease in hypoxanthine-associated EMT effects. Collectively for the first time, hypoxanthine was identified as a novel metastasis-associated metabolite in breast cancer cells and represents a promising target for diagnosis and therapy.

Nano-combination for Reviving the Activity of Fluconazole against Rhizopus delemar.

BACKGROUND: Rhizopus delemar, the main causative pathogen for the lethal mucormycosis and a severe threat during the COVID-19 pandemic, is resistant to most antifungals, including fluconazole, a known selective antifungal drug. On the other hand, antifungals are known to enhance fungal melanin synthesis. Rhizopus melanin plays an important role in fungal pathogenesis and in escaping the human defense mechanism, thus complicating the use of current antifungal drugs and fungal eradication. Because of drug resistance and the slow discovery of effective antifungals, sensitizing the activity of older ones seems a more promising strategy. METHODS: In this study, a strategy was employed to revive the use and enhance the effectiveness of fluconazole against R. delemar. UOSC-13, a compound synthesized in-house to target the Rhizopus melanin, was combined with fluconazole either as is or after encapsulation in poly (lactic-coglycolic acid) nanoparticles (PLG-NPs). Both combinations were tested for the growth of R. delemar, and the MIC50 values were calculated and compared. RESULTS: The activity of fluconazole was found to be enhanced several folds following the use of both combined treatment and nanoencapsulation. The combination of fluconazole with UOSC-13 caused a 5-fold reduction in the MIC50 value of fluconazole. Furthermore, encapsulating UOSC-13 in PLG-NPs enhanced the activity of fluconazole by an additional 10 folds while providing a wide safety profile. CONCLUSION: Consistent with previous reports, the encapsulation of fluconazole without sensitization showed no significant difference in activity. Collectively, sensitization of fluconazole represents a promising strategy to revive the use of outdated antifungal drugs back in the market.

Influence of Fabrication Technique on Adhesion and Biofilm Formation of Candida albicans to Conventional, Milled, and 3D-Printed Denture Base Resin Materials: A Comparative In Vitro Study.

The aim of this study was to evaluate the adhesion and biofilm formation of Candida albicans (C. albicans) on conventionally fabricated, milled, and 3D-printed denture base resin materials in order to determine the susceptibility of denture contamination during clinical use. Specimens were incubated with C. albicans (ATCC 10231) for 1 and 24 h. Adhesion and biofilm formation of C. albicans were assessed using the field emission scanning electron microscopy (FESEM). The XTT (2,3-(2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) assay was used for the quantification of fungal adhesion and biofilm formation. The data were analyzed using GraphPad Prism 8.02 for windows. One-way ANOVA with Tukey's post hoc testing were performed with a statistical significance level set at α = 0.05. The quantitative XTT biofilm assay revealed significant differences in the biofilm formation of C. albicans between the three groups in the 24 h incubation period. The highest proportion of biofilm formation was observed in the 3D-printed group, followed by the conventional group, while the lowest candida biofilm formation was observed in the milled group. The difference in biofilm formation among the three tested dentures was statistically significant (p < 0.001). The manufacturing technique has an influence on the surface topography and microbiological properties of the fabricated denture base resin material. Additive 3D-printing technology results in increased candida adhesion and the roughest surface topography of maxillary resin denture base as compared to conventional flask compression and CAD/CAM milling techniques. In a clinical setting, patients wearing additively manufactured maxillary complete dentures are thus more susceptible to the development of candida-associated denture stomatitis and accordingly, strict oral hygiene measures and maintenance programs should be emphasized to patients.

Peptide-functionalized zinc oxide nanoparticles for the selective targeting of breast cancer expressing placenta-specific protein 1.

Functionalized metal oxide nanoparticles (NPs) have demonstrated specific binding affinity to antigens or receptors presented on the cancer cell surface, favouring selective targeting and minimizing side effects during the chemotherapy. Placenta-specific protein 1 (PLAC-1) is a small cell surface protein overexpressed in certain types of breast cancer (BC); therefore, it can be used as a therapeutic target. The objective of this study is to develop NPs that can bind PLAC-1 and hence can inhibit the progression and metastatic potential of BC cells. Zinc oxide (ZnO) NPs were coated with a peptide (GILGFVFTL), which possesses a strong binding ability to PLAC-1. The physical attachment of the peptide to ZnO NPs was verified through various physicochemical and morphological characterization techniques. The selective cytotoxicity of the designed NPs was investigated using PLAC-1-bearing MDA-MB 231 human BC cell line and compared to LS-180 cells that do not express PLAC-1. The anti-metastatic and pro-apoptotic effects of the functionalized NPs on MDA-MB 231 cells were examined. Confocal microscopy was used to investigate the mechanism of NPs uptake by MDA-MB 231 cells. Compared to non-functionalized NPs, peptide functionalization significantly improved the targeting and uptake of the designed NPs by PLAC-1-expressing cancer cells with significant pro-apoptotic and anti-metastatic effects. The uptake of peptide functionalized ZnO NPs (ZnO-P NPs) occurred via peptide-PLAC1 interaction-assisted clathrin-mediated endocytosis. These findings highlight the potential targeted therapy of ZnO-P NPs against PLAC-1-expressing breast cancer cells.

Co-administration of amoxicillin-loaded chitosan nanoparticles and inulin: A novel strategy for mitigating antibiotic resistance and preserving microbiota balance in Helicobacter pylori treatment.

Helicobacter pylori (H. pylori) is a causative agent of various gastrointestinal diseases and eradication mainly relies on antibiotic treatment, with (AMX) being a key component. However, rising antibiotic resistance in H. pylori necessitates the use of antibiotics combination therapy, often disrupting gut microbiota equilibrium leading to further health complications. This study investigates a novel strategy utilizing AMX-loaded chitosan nanoparticles (AMX-CS NPs), co-administered with prebiotic inulin to counteract H. pylori infection while preserving microbiota health. Following microbroth dilution method, AMX displayed efficacy against H. pylori, with a MIC50 of 48.34 ± 3.3 ng/mL, albeit with a detrimental impact on Lactobacillus casei (L. casei). The co-administration of inulin (500 µg/mL) with AMX restored L. casei viability while retaining the lethal effect on H. pylori. Encapsulation of AMX in CS-NPs via ionic gelation method, resulted in particles of 157.8 ± 3.85 nm in size and an entrapment efficiency (EE) of 86.44 ± 2.19 %. Moreover, AMX-CS NPs showed a sustained drug release pattern over 72 h with no detectable toxicity on human dermal fibroblasts cell lines. Encapsulation of AMX into CS NPs also reduced its MIC50 against H. pylori, while its co-administration with inulin maintained L. casei viability. Interestingly, treatment with AMX-CS NPs also reduced the expression of the efflux pump gene hefA in H. pylori. This dual treatment strategy offers a promising approach for more selective antimicrobial treatment, minimizing disruption to healthy microbial communities while effectively addressing pathogenic threats.

Peptide-functionalized graphene oxide quantum dots as colorectal cancer theranostics.

Colorectal cancer (CRC) accounts for approximately 10% of all new cancer cases worldwide with significant morbidity and mortality. The current imaging techniques are lacking diagnostic precision while traditional chemotherapeutic strategies are limited by their adverse side effects and poor response in advanced stages. Targeted nanoparticles (NPs) can specifically bind to surface antigens on cancer cells and provide effective delivery of diagnostic and chemotherapeutic agent. Placenta-specific protein 1 (PLAC-1) is overexpressed in CRC and can be used as a target for detection and treatment of the disease. The aim of this work was to develop a targeted nanotheranostic agent for early diagnosis and inhibition of the malignant progression and metastasis of CRC. Graphene oxide quantum dots (QD) were covalently labeled with a peptide (GILGFVFTL) having high affinity to PLAC-1. The covalent coupling between the QD and the peptide was confirmed using a series of physicochemical and morphological characterization techniques. Confocal microscopy was used to evaluate the uptake of QD and QD-P in HCT-29, HT-116 and LS-180 CRC cell lines. Selective targeting of antigen PLAC-1 overexpressed on HT-29 and HCT-116 cells was measured by immunofluorescence. Cell proliferation, cell invasion and extent of PLAC-1 expression in CRC cells after treatment with QD and QD-P were determined. The prepared QD-P showed a significant increase in targeting and specific uptake in cells expressing the antigen PLAC-1 compared to non-functionalized QD. Treatment with QD-P also increased the cell cytotoxicity, reduced the invasiveness of HT-29 and HCT-116 cells by 38% and 62%, respectively, and downregulated the expression of PLAC-1 by 53% and 33%, respectively. These results highlight the potential use of QD-P as a theranostic agent for the detection and treatment of CRC cells expressing the antigen PLAC-1.

Novel erythropoietin-loaded nanoparticles with prolonged in vivo response.

The aim of this study was to incorporate human recombinant erythropoietin (EPO) in biodegradable polymeric nanoparticles targeting a prolonged-release effect. EPO-loaded poly(DL-lactide-co-glycolide) nanoparticles were prepared using double emulsion method (w/o/w) with least process-related stress on the encapsulated drug. The nanoparticles have been fully characterized including in vitro release profile. The biological activity was assessed in vivo using BALB-c mice. The produced particles appeared spherical in shape with smooth regular surfaces and had an average particle size of 225.9 ± 3.8 nm. The entrapment efficiency was 33.3%. The in vitro release profile exhibited a biphasic mode with a burst of 50% cumulative drug release, followed by a slow rate of release over 24 h, reaching a maximum of 82%. The bioassay results showed that EPO-loaded nanoparticles were able to maintain the physiological activity of EPO for 14 days after single subcutaneous injection compared with pure and marketed EPO formulae (EPREX®).

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of Candida auris Metabolites.

Unique metabolic features allow fungi to colonize and persist within the human host. Investigations of unique metabolic fingerprints of a pathogenic fungus can provide a more complete understanding of the infection process and an interpretation of associations between genotype and phenotype. Gas chromatography-mass spectrometry (GC-MS) has proved to be one of the most powerful analytical techniques used for qualitative and quantitative detection of cellular metabolites. This technique has been used for comparative metabolomic analyses of both intracellular and secreted metabolites under variable conditions. This book chapter describes the use of GC-MS in the detection of both intracellular and secreted metabolites from Candida auris, a newly emerging fungal pathogen representing a serious global health threat due to its multidrug resistance profile. The identified fungal metabolites are compared using available software in order to assign a correlation between the pattern of accumulation of metabolites and behavior of the organism.

Optimum inhibition of MCF-7 breast cancer cells by efficient targeting of the macropinocytosis using optimized paclitaxel-loaded nanoparticles.

AIMS: Breast cancer (BC) is the third leading cause of death among other cancer types. Worldwide, it is the most common harmful disease in women, representing 1/4 of all cancers. Treatment of BC remains an ongoing challenge to most researchers. Understanding how cancer cells differ from normal cells can enhance drug targeting and overall disease progression. Endocytosis is a major physiological process modified in cancer cells and affects the cellular uptake of chemotherapeutic agents. MCF-7 breast cancer cells exhibit constitutive macropinocytic activity in comparison to normal non-macropinocytic MCF-10A breast cells. Therefore, we hypothesized that blocking the macropinocytosis mechanism in MCF-7 cells may inhibit the cancer progression while maintaining the safety of normal cells. MAIN METHODS: Using nano-precipitation technique, paclitaxel-PLGA-NPs were successfully prepared in the size range and charge required to opt for macropinocytosis in MCF-7 cells. KEY FINDINGS: Uptake and endocytosis inhibitor assays indicated that the developed NPs acquired size and surface charges that efficiently target macropinocytosis of MCF-7 cells. Paclitaxel-loaded PLGA-NPs showed higher efficacy against MCF-7 cells, while providing no toxicity on normal MCF-10A cells. Metabolomics analysis indicated the nutrients deprivation because of occupying the macropinocytosis. However, treatment of fresh MCF-7 cancer cells by metabolites secreted from PLGA-NPs-treated MCF-7 cells showed a potential metastatic activity. Thus, co- administration with an anti-metastatic drug is advised. SIGNIFICANCE: Collectively, adjusting the size and surface characteristics of a drug can critically control its cellular uptake, affecting the efficacy of drugs and the microenvironment of cancer cells.

Novel Secreted Peptides From Rhizopus arrhizus var. delemar With Immunomodulatory Effects That Enhance Fungal Pathogenesis.

Secreted fungal peptides are known to influence the interactions between the pathogen and host innate immunity. The aim of this study is to screen and evaluate secreted peptides from the fungus Rhizopus arrhizus var. delemar for their immunomodulatory activity. By using mass spectrometry and immuno-informatics analysis, we identified three secreted peptides CesT (S16), Colicin (S17), and Ca2+/calmodulin-dependent protein kinase/ligand (CAMK/CAMKL; S27). Culturing peripheral blood-derived monocytic macrophages (PBMMs) in the presence of S16 or S17 caused cell clumping, while culturing them with S27 resulted in the formation of spindle-shaped cells. S27-treated PBMMs showed cell cycle arrest at G0 phase and exhibited alternatively activated macrophage phenotype with pronounced reduction in scavenger receptors CD163 and CD206. Homology prediction indicated that IL-4/IL-13 is the immunomodulatory target of S27. Confirming this prediction, S27 initiated macrophage activation through phosphorylation of STAT-6; STAT-6 inhibition reversed the activity of S27 and reduced the formation of spindle-shaped PBMMs. Lastly, S27 treatment of PBMMs was associated with altered expression of key iron regulatory genes including hepcidin, ferroportin, transferrin receptor 1, and ferritin in a pattern consistent with increased cellular iron release; a condition known to enhance Rhizopus infection. Collectively, R. arrhizus var. delemar secretes peptides with immunomodulatory activities that support fungal pathogenesis. Targeting the IL-4/IL-13R/STAT-6 axis is a potential therapeutic approach to enhance the PBMM-mediated fungal phagocytosis. This represents a potential new approach to overcome lethal mucormycosis.