HR-MAS NMR Metabolomics Profile of Vero Cells under the Influence of Virus Infection and nsP2 Inhibitor: A Chikungunya Case Study.

The arbovirus Chikungunya (CHIKV) is transmitted by Aedes mosquitoes in urban environments, and in humans, it triggers debilitating symptoms involving long-term complications, including arthritis and Guillain-Barré syndrome. The development of antiviral therapies is relevant, as no efficacious vaccine or drug has yet been approved for clinical application. As a detailed map of molecules underlying the viral infection can be obtained from the metabolome, we validated the metabolic signatures of Vero E6 cells prior to infection (CC), following CHIKV infection (CV) and also upon the inclusion of the nsP2 protease inhibitor wedelolactone (CWV), a coumestan which inhibits viral replication processes. The metabolome groups evidenced significant changes in the levels of lactate, myo-inositol, phosphocholine, glucose, betaine and a few specific amino acids. This study forms a preliminary basis for identifying metabolites through HR-MAS NMR (High Resolution Magic Angle Spinning Nuclear Magnetic Ressonance Spectroscopy) and proposing the affected metabolic pathways of cells following viral infection and upon incorporation of putative antiviral molecules.

DNA methylation patterns suggest the involvement of DNMT3B and TET1 in osteosarcoma development.

DNA methylation may be involved in the development of osteosarcomas. Osteosarcomas commonly arise during the bone growth and remodeling in puberty, making it plausible to infer the involvement of epigenetic alterations in their development. As a highly studied epigenetic mechanism, we investigated DNA methylation and related genetic variants in 28 primary osteosarcomas aiming to identify deregulated driver alterations. Methylation and genomic data were obtained using the Illumina HM450K beadchips and the TruSight One sequencing panel, respectively. Aberrant DNA methylation was spread throughout the osteosarcomas genomes. We identified 3146 differentially methylated CpGs comparing osteosarcomas and bone tissue samples, with high methylation heterogeneity, global hypomethylation and focal hypermethylation at CpG islands. Differentially methylated regions (DMR) were detected in 585 loci (319 hypomethylated and 266 hypermethylated), mapped to the promoter regions of 350 genes. These DMR genes were enriched for biological processes related to skeletal system morphogenesis, proliferation, inflammatory response, and signal transduction. Both methylation and expression data were validated in independent groups of cases. Six tumor suppressor genes harbored deletions or promoter hypermethylation (DLEC1, GJB2, HIC1, MIR149, PAX6, and WNT5A), and four oncogenes presented gains or hypomethylation (ASPSCR1, NOTCH4, PRDM16, and RUNX3). Our analysis also revealed hypomethylation at 6p22, a region that contains several histone genes. Copy-number changes in DNMT3B (gain) and TET1 (loss), as well as overexpression of DNMT3B in osteosarcomas provide a possible explanation for the observed phenotype of CpG island hypermethylation. While the detected open-sea hypomethylation likely contributes to the well-known osteosarcoma genomic instability, enriched CpG island hypermethylation suggests an underlying mechanism possibly driven by overexpression of DNMT3B likely resulting in silencing of tumor suppressors and DNA repair genes.

First description of Candida haemulonii infecting a snake Boa constrictor: Molecular, pathological and antifungal sensitivity characteristics.

Candida haemulonii is an emergent infectious pathogen that affects human presenting comorbidities and/or immunodepression. Little is known about other possible hosts. For the first time, this fungus was found causing a cutaneous infection in a snake, Boa constrictor, characterized by scale opacity and several ulcerative lesions. This C. haemulonii was isolated, identified using molecular techniques and a phylogenetic study, and had its growth totally inhibited by all the drugs tested; however, no fungicide effect was seen for fluconazole and itraconazole. The B. constrictor clinical signals subsided after a treatment using a biogenic silver nanoparticle-based ointment. These findings, along with the B. constrictor presence near human habitats, warn for the necessity of wildlife health monitoring for emergent and opportunistic diseases in peri-urban environments.

A serum NMR metabolomic analysis of the Corynebacterium pseudotuberculosis infection in goats.

Caseous lymphadenitis (CLA), an infectious disease caused by Corynebacterium pseudotuberculosis in small ruminants, is highly prevalent worldwide. Economic losses have already been associated with the disease, and little is known about the host-pathogen relationship associated with the disease. The present study aimed to perform a metabolomic study of the C. pseudotuberculosis infection in goats. Serum samples were collected from a herd of 173 goats. The animals were classified as controls (not infected), asymptomatic (seropositives but without detectable CLA clinical signs), and symptomatic (seropositive animals presenting CLA lesions), according to microbiological isolation and immunodiagnosis. The serum samples were analyzed using nuclear magnetic resonance (1H-NMR), nuclear Overhauser effect spectroscopy (NOESY), and Carr-Purcell-Meiboom-Gill (CPMG) sequences. The NMR data were analyzed using chemometrics, and principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) were performed to discover specific biomarkers responsible for discrimination between the groups. A high dissemination of the infection by C. pseudotuberculosis was observed, being 74.57% asymptomatic and 11.56% symptomatic. In the evaluation of 62 serum samples by NMR, the techniques were satisfactory in the discrimination of the groups, being also complementary and mutually confirming, demonstrating possible biomarkers for the infection by the bacterium. Twenty metabolites of interest were identified by NOESY and 29 by CPMG, such as tryptophan, polyunsaturated fatty acids, formic acid, NAD+, and 3-hydroxybutyrate, opening promising possibilities for the use of these results in new therapeutic, immunodiagnosis, and immunoprophylactic tools, as well as for studies of the immune response against C. pseudotuberculosis. KEY POINTS: • Sixty-two samples from healthy, CLA asymptomatic, and symptomatic goats were screened • Twenty metabolites of interest were identified by NOESY and 29 by CPMG • 1H-NMR NOESY and CPMG were complementary and mutually confirming.

NMR-Metabolomics in COVID-19 Research.

COVID-19 stands for Corona Virus Disease 2019, which starts as a viral infection that provokes illness with different symptoms and severity. The infected individuals can be asymptomatic or present with mild, moderate, severe, and critical illness with acute respiratory distress syndrome (ARDS), acute cardiac injury, and multiorgan failure. When the virus enters the cells, it replicates and provokes responses. Most diseased individuals resolve the problems in a short time but unfortunately, some may die, and almost 3 years after the first reported cases, COVID-19 still kills thousands per day worldwide. One of the problems in not curing the viral infection is that the virus passes by undetected in cells. This can be caused by the lack of pathogen-associated molecular patterns (PAMPs) that start an orchestrated immune response, such as activation of type 1 interferons (IFNs), inflammatory cytokines, chemokines, and antiviral defenses. Before all of these events can happen, the virus uses the infected cells and numerous small molecules as sources of energy and building blocks for newly synthesized viral nanoparticles that travel to and infect other host cells. Therefore, studying the cell metabolome and metabolomic changes in biofluids might give insights into the state of the viral infection, viral loads, and defense response. NMR-metabolomics can help in solving the real-time host interactions by monitoring concentration changes in metabolites. This chapter addresses the state of the art of COVIDomics by NMR analyses and presents exemplified biomolecules identified in different world regions and gravities of illness as potential biomarkers.

Untargeted Lipidomics Study of Bipolar Disorder Patients in Serbia.

The Lipidomic profiles of serum samples from patients with bipolar disorder (BD) and healthy controls (C) were explored and compared. The sample cohort included 31 BD patients and 31 control individuals. An untargeted lipidomics study applying liquid chromatography (LC) coupled with high-resolution mass spectrometry (HRMS) was conducted to achieve the lipid profiles. Multivariate statistical analyses (principal component analysis and partial least squares discriminant analysis) were performed, and fifty-six differential lipids were confirmed in BD and controls. Our results pointed to alterations in lipid metabolism, including pathways of glycerophospholipids, sphingolipids, glycerolipids, and sterol lipids, in BD patient sera. This study emphasized the role of lipid pathways in BD, and comprehensive research using the LC-HRMS platform is necessary for future application in the diagnosis and improvement of BD treatments.

Applications of Silver Nanoparticles in Dentistry: Advances and Technological Innovation.

Silver nanoparticles (AgNPs) have been successfully applied in several areas due to their significant antimicrobial activity against several microorganisms. In dentistry, AgNP can be applied in disinfection, prophylaxis, and prevention of infections in the oral cavity. In this work, the use of silver nanoparticles in dentistry and associated technological innovations was analyzed. The scientific literature was searched using PubMed and Scopus databases with descriptors related to the use of silver nanoparticles in dentistry, resulting in 90 open-access articles. The search for patents was restricted to the A61K code (International Patent Classification), using the same descriptors, resulting in 206 patents. The results found were ordered by dental specialties and demonstrated the incorporation of AgNPs in different areas of dentistry. In this context, the search for patents reaffirmed the growth of this technology and the dominance of the USA pharmaceutical industry over AgNPs product development. It could be concluded that nanotechnology is a promising area in dentistry with several applications.

Multi-target drug with potential applications: violacein in the spotlight.

The aim of the current review is to address updated research on a natural pigment called violacein, with emphasis on its production, biological activity and applications. New information about violacein's action mechanisms as antitumor agent and about its synergistic action in drug delivery systems has brought new alternatives for anticancer therapy. Thus, violacein is introduced as reliable drug capable of overcoming at least three cancer hallmarks, namely: proliferative signaling, cell death resistance and metastasis. In addition, antimicrobial effects on several microorganisms affecting humans and other animals turn violacein into an attractive drug to combat resistant pathogens. Emphasis is given to effects of violacein combined with different agents, such as antibiotics, anticancer agents and nanoparticles. Although violacein is well-known for many decades, it remains an attractive compound. Thus, research groups have been making continuous effort to help improving its production in recent years, which can surely enable its pharmaceutical and chemical application as multi-task compound, even in the cosmetics and food industries.

Hepatoblastomas exhibit marked NNMT downregulation driven by promoter DNA hypermethylation.

Hepatoblastomas exhibit the lowest mutational burden among pediatric tumors. We previously showed that epigenetic disruption is crucial for hepatoblastoma carcinogenesis. Our data revealed hypermethylation of nicotinamide N-methyltransferase, a highly expressed gene in adipocytes and hepatocytes. The expression pattern and the role of nicotinamide N-methyltransferase in pediatric liver tumors have not yet been explored, and this study aimed to evaluate the effect of nicotinamide N-methyltransferase hypermethylation in hepatoblastomas. We evaluated 45 hepatoblastomas and 26 non-tumoral liver samples. We examined in hepatoblastomas if the observed nicotinamide N-methyltransferase promoter hypermethylation could lead to dysregulation of expression by measuring mRNA and protein levels by real-time quantitative polymerase chain reaction, immunohistochemistry, and Western blot assays. The potential impact of nicotinamide N-methyltransferase changes was evaluated on the metabolic profile by high-resolution magic angle spinning nuclear magnetic resonance spectroscopy. Significant nicotinamide N-methyltransferase downregulation was revealed in hepatoblastomas, with two orders of magnitude lower nicotinamide N-methyltransferase expression in tumor samples and hepatoblastoma cell lines than in hepatocellular carcinoma cell lines. A specific TSS1500 CpG site (cg02094283) of nicotinamide N-methyltransferase was hypermethylated in tumors, with an inverse correlation between its methylation level and nicotinamide N-methyltransferase expression. A marked global reduction of the nicotinamide N-methyltransferase protein was validated in tumors, with strong correlation between gene and protein expression. Of note, higher nicotinamide N-methyltransferase expression was statistically associated with late hepatoblastoma diagnosis, a known clinical variable of worse prognosis. In addition, untargeted metabolomics analysis detected aberrant lipid metabolism in hepatoblastomas. Data presented here showed the first evidence that nicotinamide N-methyltransferase reduction occurs in hepatoblastomas, providing further support that the nicotinamide N-methyltransferase downregulation is a wide phenomenon in liver cancer. Furthermore, this study unraveled the role of DNA methylation in the regulation of nicotinamide N-methyltransferase expression in hepatoblastomas, in addition to evaluate the potential effect of nicotinamide N-methyltransferase reduction in the metabolism of these tumors. These preliminary findings also suggested that nicotinamide N-methyltransferase level may be a potential prognostic biomarker for hepatoblastoma.

Insights into Interactions of Flavanones with Target Human Respiratory Syncytial Virus M2-1 Protein from STD-NMR, Fluorescence Spectroscopy, and Computational Simulations.

The human Respiratory Syncytial Virus (hRSV) is the most frequent agent of respiratory infections in infants and children with no currently approved vaccine. The M2-1 protein is an important transcriptional antitermination factor and a potential target for viral replication inhibitor development. Hesperetin (HST) and hesperidin (HSD) are flavonoids from the flavanone group, naturally found in citrus and have, as one of their properties, antiviral activity. The present study reports on the interactions between hRSV M2-1 and these flavanones using experimental techniques in association with computational tools. STD-NMR results showed that HST and HSD bind to M2-1 by positioning their aromatic rings into the target protein binding site. Fluorescence quenching measurements revealed that HST had an interaction affinity greater than HSD towards M2-1. The thermodynamic analysis suggested that hydrogen bonds and van der Waals interactions are important for the molecular stabilization of the complexes. Computational simulations corroborated with the experimental results and indicated that the possible interaction region for the flavonoids is the AMP-binding site in M2-1. Therefore, these results point that HST and HSD bind stably to a critical region in M2-1, which is vital for its biological function, and thus might play a possible role antiviral against hRSV.

New Sustainable Process for Hesperidin Isolation and Anti-Ageing Effects of Hesperidin Nanocrystals.

Hesperidin, a secondary orange (Citrus sinensis) metabolite, was extracted from orange bagasse. No organic solvents or additional energy consumption were used in the clean and sustainable process. Hesperidin purity was approximately 98% and had a yield of 1%. Hesperidin is a known supplement due to antioxidant, chelating, and anti-ageing properties. Herein, hesperidin application to eliminate dark eye circles, which are sensitive and thin skin regions, was studied. In addition, the proposed method for its aqueous extraction was especially important for human consumption. Further, the most effective methods for hesperidin nanonization were explored, after which the nanoemulsions were incorporated into a cream formulation that was formulated for a tropical climate. Silky cream formulations (oil in water) were tested in vitro on artificial 3D skin from cultured cells extracted from skin residues after plastic surgery. The proposed in vitro assay avoided tests of the different formulations in human volunteers and animals. It was shown that one of the nanonized hesperidin formulations was the most skin-friendly and might be used in cosmetics.

Insights into the Effects of Crack Abuse on the Human Metabolome Using a NMR Approach.

Approximately 255 million people consume illicit drugs every year, among which 18 million use cocaine. A portion of this drug is represented by crack, but it is difficult to estimate the number of users since most are marginalized. However, there are no recognized efficacious pharmacotherapies for crack-cocaine dependence. Inflammation and infection in cocaine users may be due to behavior adopted in conjunction with drug-related changes in the brain. To understand the metabolic changes associated with the drug abuse disorder and identify biomarkers, we performed a 1H NMR-based metabonomic analysis of 44 crack users' and 44 healthy volunteers' blood serum. The LDA model achieved 98% of accuracy. From the water suppressed 1H NMR spectra analyses, it was observed that the relative concentration of lactate was higher in the crack group, while long chain fatty acid acylated carnitines were decreased, which was associated with their nutritional behavior. Analyses of the aromatic region of CPMG 1H NMR spectra demonstrated histidine and tyrosine levels increased in the blood serum of crack users. The reduction of carnitine and acylcarnitines and the accumulation of histidine in the serum of the crack users suggest that histamine biosynthesis is compromised. The tyrosine level points to altered dopamine concentration.

Metabolomic Biomarkers in Mental Disorders: Bipolar Disorder and Schizophrenia.

Psychiatric disorders are some of the most impairing human diseases. Among them, bipolar disorder and schizophrenia are the most common. Both have complicated diagnostics due to their phenotypic, biological, and genetic heterogeneity, unknown etiology, and the underlying biological pathways, and molecular mechanisms are still not completely understood. Given the multifactorial complexity of these disorders, identification and implementation of metabolic biomarkers would assist in their early detection and diagnosis and facilitate disease monitoring and treatment responses. To date, numerous studies have utilized metabolomics to better understand psychiatric disorders, and findings from these studies have begun to converge. In this chapter, we briefly describe some of the metabolomic biomarkers found in these two disorders.

Development and characterisation of polymeric microparticle of poly(d,l-lactic acid) loaded with holmium acetylacetonate.

Biodegradable polymers containing radioactive isotopes such as Holmium 166 (166Ho) have potential applications as beta particle emitters in tumour tissues. It is also a gamma ray emitter, allowing nuclear imaging of any tissue to be acquired. It is frequently used in the form of complexes such as holmium acetylacetonate (HoAcAc), which may cause damages in tissues next to the targets cancer cells, as it is difficult to control its linkage or healthy tissues radiotherapy effects. Poly(d,l-lactic acid), PDLLA, was used to encapsulate holmium acetylacetonate (HoAcAc) using an emulsion solvent extraction/evaporation technique. Microspheres with sizes between 20-53 µm were extensively characterised. HoAcAc release from the microspheres was assessed through studies using Inductively Coupled Plasma - Optical Emission Spectroscopy, and the microspheres showed no holmium leakage after a period of 10 half-lives and following gamma irradiation. Thus, HoAcAc loaded microspheres are here presented as a potential system for brachytherapy and imaging purposes.

Discovery of small molecule inhibitors for the snake venom metalloprotease BaP1 using in silico and in vitro tests.

Snakebites represent an important public health problem, with a great number of victims with permanent sequelae or fatal outcomes, particularly in rural, agriculturally active areas. The snake venom metalloproteases (SVMPs) are the principal proteins responsible for some clinically-relevant effects, such as local and systemic hemorrhage, dermonecrosis, and myonecrosis. Because of the difficulties in neutralizing them rapidly and locally by antivenoms, the search and design of small molecules as inhibitors of SVMPs are proposed. The Bothrops asper metalloprotease P1 (BaP1) is hereby used as a target protein and by High Throughput Virtual Screening (HTVS) approach, the free access virtual libraries: ZINC, PubChem and ChEMBL, were searched for potent small molecule inhibitors. Results from the aforementioned approaches provided strong evidences on the structural requirements for the efficient BaP1 inhibition such as the presence of the pyrimidine-2,4,6-trione moiety. The two proposed compounds have also shown excellent results in performed in vitro interaction studies against BaP1.

Lipidomics, Biomarkers, and Schizophrenia: A Current Perspective.

Lipidomics is a lipid-targeted metabolomics approach aiming at comprehensive analysis of lipids in biological systems. Recent technological progresses in mass spectrometry, nuclear magnetic resonance spectroscopy, and chromatography have significantly enhanced the developments and applications of metabolic profiling of lipids in more complex biological samples. As many diseases reveal a notable change in lipid profiles compared with that of healthy people, lipidomics have also been broadly introduced to scientific research on diseases. Exploration of lipid biochemistry by lipidomics approach will not only provide insights into specific roles of lipid molecular species in health and disease, but it will also support the identification of potential biomarkers for establishing preventive or therapeutic approaches for human health. This chapter aims to illustrate how lipidomics can contribute for understanding the biological mechanisms inherent to schizophrenia and why lipids are relevant biomarkers of schizophrenia. The application of lipidomics in clinical studies has the potential to provide new insights into lipid profiling and pathophysiological mechanisms underlying schizophrenia. The future perspectives of lipidomics in mental disorders are also discussed herein.

Enhanced materials from nature: nanocellulose from citrus waste.

Nanocellulose is a relatively inexpensive, highly versatile bio-based renewable material with advantageous properties, including biodegradability and nontoxicity. Numerous potential applications of nanocellulose, such as its use for the preparation of high-performance composites, have attracted much attention from industry. Owing to the low energy consumption and the addition of significant value, nanocellulose extraction from agricultural waste is one of the best alternatives for waste treatment. Different techniques for the isolation and purification of nanocellulose have been reported, and combining these techniques influences the morphology of the resultant fibers. Herein, some of the extraction routes for obtaining nanocellulose from citrus waste are addressed. The morphology of nanocellulose was determined by Scanning Electron Microscopy (SEM) and Field Emission Scanning Electron Microscopy (FESEM), while cellulose crystallinity indexes (CI) from lyophilized samples were determined using solid-state Nuclear Magnetic Resonance (NMR) and X-Ray Diffraction (XRD) measurements. The resultant nanofibers had 55% crystallinity, an average diameter of 10 nm and a length of 458 nm.

Investigation of pectin deficiency in modulating the bioflavonoid profile of orange processing waste: A sustainable valorization of industrial waste.

Orange processing waste (OPW) generated by the processing of oranges, as well as other citrus fruits, is a major source of pectin in the market nowadays. The residues generated during the pectin extraction process may contain many phytochemicals, including flavonoids. We use state-of-the-art techniques such as liquid chromatography high-resolution mass spectrometry (LC-HRMS/MS) and feature-based molecular network (FBMN) to annotate the flavonoids in OPWs. In particular, four flavonoids, hesperidin, naringin, diosmin, and hesperetin were quantified in the samples by LC-TDQ-MS. In total, 32 flavonoids from different classes were annotated, of which 16 were polymethoxylated flavonoids, 13 were flavonoid glycosides and 3 were flavanone aglycones. The results showed that flavonoid glycosides remain in high concentrations in OPWs from pectin factories even after pectin extraction by harsh conditions. The results show an exciting opportunity to harness the untapped potential of pectin factory waste as a renewable source for the extraction of glycoside flavonoids.

Biogenic silver nanoparticles' antibacterial activity and cytotoxicity on human hepatocarcinoma cells (Huh-7).

Exploring diverse synthetic pathways for nanomaterial synthesis has emerged as a promising direction. For example, silver nanoparticles (AgNPs) are synthesized using different approaches yielding nanomaterials with distinct morphological, physical and biological properties. Hence, the present study reports the biogenic synthesis of silver nanoparticles using the aqueous secretome of the fungus Fusarium oxysporum f. sp. cubense (AgNP@Fo) and orange peel extract (AgNP@OR). The physical and morphological properties of synthesized nanoparticles were similar, with AgNP@Fo measuring 56.43 ± 19.18 nm and AgNP@OR measuring 39.97 ± 19.72 nm in size. The zeta potentials for the nanoparticles were low, -26.8 ± 7.55 and -26.2 ± 2.87 mV for AgNP@Fo and AgNP@OR, respectively, demonstrating a similar negative charge. The spherical morphologies of both nanoparticles were evidenced by Scanning Transmission Electron Microscopy (STEM) and Atomic Force Microscopy (AFM). However, despite their similar physical and morphological properties, AgNPs demonstrated different bioactivities. We evaluated and compared the antimicrobial efficacy of these nanoparticles against a range of bacteria, such as Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, and Escherichia coli. The AgNP@Fo showed Minimum Inhibitory Concentration (MIC) values ranging from 0.84 to 1.68 µg mL-1 and were around ten times more potent compared to AgNP@OR. The anticancer activities of both nanoparticles were investigated using human hepatocarcinoma cells (Huh-7), where AgNP@Fo exhibited around 20 times higher cytotoxicity than AgNP@OR with an IC50 value of 0.545 µmol L-1. Anticancer effects were demonstrated by the MTT, confirmed by the calcein-AM assay and fluorescence imaging. This study establishes solid groundwork for future exploration of molecular interactions of nanoparticles synthesized through distinct biosynthetic routes, particularly within bacterial and cancerous cell environments.

Activity of biogenic silver nanoparticles in planktonic and biofilm-associated Corynebacterium pseudotuberculosis.

Corynebacterium pseudotuberculosis is a gram-positive bacterium and is the etiologic agent of caseous lymphadenitis (CL) in small ruminants. This disease is characterized by the development of encapsulated granulomas in visceral and superficial lymph nodes, and its clinical treatment is refractory to antibiotic therapy. An important virulence factor of the Corynebacterium genus is the ability to produce biofilm; however, little is known about the characteristics of the biofilm produced by C. pseudotuberculosis and its resistance to antimicrobials. Silver nanoparticles (AgNPs) are considered as promising antimicrobial agents, and are known to have several advantages, such as a broad-spectrum activity, low resistance induction potential, and antibiofilm activity. Therefore, we evaluate herein the activity of AgNPs in C. pseudotuberculosis, through the determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), antibiofilm activity, and visualization of AgNP-treated and AgNP-untreated biofilm through scanning electron microscopy. The AgNPs were able to completely inhibit bacterial growth and inactivate C. pseudotuberculosis at concentrations ranging from 0.08 to 0.312 mg/mL. The AgNPs reduced the formation of biofilm in reference strains and clinical isolates of C. pseudotuberculosis, with interference values greater than 80% at a concentration of 4 mg/mL, controlling the change between the planktonic and biofilm-associated forms, and preventing fixation and colonization. Scanning electron microscopy images showed a significant disruptive activity of AgNP on the consolidated biofilms. The results of this study demonstrate the potential of AgNPs as an effective therapeutic agent against CL.