Search Results (177)

Salinity serves as a critical environmental factor influencing the physiological and morphological characteristics of Spirulina, a filamentous cyanobacterium used for food production and commercial purposes. This study examined a Spirulina strain’s responses to different salinity levels (10–45 ppt) through three independent laboratory experiments that determined growth, productivity, and size structure. Growth across salinity treatments was assessed by monitoring optical density in 24-well microplates over 20 days and estimating specific growth rates using a logistic growth model. Primary productivity under different salinity and light conditions was measured using light and dark bottle experiments to calculate gross primary productivity (GPP) and to estimate photosynthetic efficiency through linear regression of GPP against light intensity. The size structure was assessed through tube-based experiments and image analysis, with organism sizes categorized and analyzed to identify salinity-induced patterns in filament structure. The study demonstrated that the Spirulina strain achieved its greatest growth at 10 ppt yet produced the highest photosynthetic efficiency between 27 and 45 ppt because it reallocated energy during salinity stress. The morphological analysis revealed that the Spirulina strain produced medium-sized filaments between 400 and 799 µm at elevated salinity levels, and our analysis confirmed substantial variations in size structure. The Spirulina strain demonstrates both physiological and morphological plasticity when exposed to salinity changes. The cultivation of the Spirulina strain at 27 ppt provides conditions that support moderate growth, enhanced productivity, and manageable morphological shifts while using its natural salinity tolerance to improve the efficiency and scalability of production for diverse biotechnological applications. Full article

Overexploitation has led to a rise in pathogenic bacteria within aquaculture, increasing reliance on antibiotics, and developing microorganism resistance. This situation underscores the need to explore alternatives with a reduced ecological impact. Metabolites derived from essential oils have demonstrated antimicrobial properties that can inhibit or diminish the activity of various microorganisms. In this study, the antimicrobial efficacy of cinnamon (Cinnamomum zeylanicum), tea tree (Melaleuca alternifolia), and thyme (Thymus vulgaris) essential oils against pathogenic bacteria (Aeromonas, Pseudomonas, Shewanella, Comamonas, Vibrio, Acinetobacter, and Empedobacter) isolated from tilapia (Oreochromis spp.) brooded in Hidalgo State, Mexico, were investigated. Diffusion tests were conducted using discs infused with 12 different antibiotics and discs infused with essential oils at concentrations of 15, 10, and 5 μL each. Minimal inhibitory concentration tests were performed using a 96-well microplate format. All bacterial strains exhibited multi-resistance to various antibiotics; however, thyme and cinnamon effectively inhibited the tested bacteria at the lowest concentrations, while tea tree oil was the least effective. The findings suggest the potential incorporation of thyme and cinnamon as an alternative prevention to decrease the use of antibiotic treatment. Full article

Improved in vitro models are needed to reduce costs and delays in central nervous system (CNS) drug discovery. The FDA Modernization Acts 2.0 and 3.0 require human-centered alternative testing methods to mitigate animal-based experiments and discovery delays, and to ensure human safety. Developing cost-efficient, flexible microfluidic chips is essential to advance organ-on-chip (OoC) technology for drug discovery and disease modeling. While CNC micromilling shows promise for fabricating microfluidic devices, it remains underutilized due to limited accessibility. We present a simple CNC-milled flexible microfluidic chip fabricated from thermoplastic poly (methyl methacrylate) (PMMA). The structure of the microplate included drilled openings for connecting the wells. The chip’s biocompatibility was evaluated with isolated primary neuronal cultures from postnatal Wistar rat pups (p1). Primary cells cultured in the device showed high viability, differentiation, and 3D neurosphere formation, similar to conventional well-plate cultures. Neuronal cultures showed neurite growth and functional markers. Although cleanroom-based methods provide higher accuracy, the chip effectively promotes cell viability, differentiation, and alignment, offering an ideal platform for tissue modeling and OoC applications. It allows cell biologists to quickly create prototypes at lower cost and in less time than required for soft lithography and is a viable alternative to the current manufacturing methods. Full article

Collective cell migration is crucial in various biological processes, including tumor progression and metastasis. The widely used scratch assay (wound healing assay) has limitations in throughput, reproducibility, and data analysis. To overcome these challenges, we previously developed the Transient Agarose Spot (TAS) assay, which enhanced assay precision and reproducibility. In this study, we present an improved microplate-based TAS assay. By using a microplate reader, we automated data acquisition, enabling the detection of cell migration in a 96-well plate format with greater throughput and accuracy. The new method applies Hoechst staining to label viable cells, providing a stable signal for kinetic analysis without compromising cell viability. We validated this approach with fluorophore-expressing cancer cells and demonstrated its ability to monitor dose-dependent effects of fetal bovine serum on cell migration. Additionally, we applied the microplate-based TAS assay to assess the anti-migratory effects of kinase inhibitors and mesenchymal stem cell-derived extracellular vesicles (EVs) on lung cancer cells. The assay accurately quantified migration inhibition and revealed the concentration-dependent effects of EVs, highlighting their potential as therapeutic agents. This microplate-based TAS assay provides a scalable, efficient, and cost-effective platform for high-throughput screening of cell migration and drug discovery, offering a robust alternative to traditional microscopy-based methods. Full article

Background/Objectives: A preventative vaccine against human cytomegalovirus (HCMV) infection and disease remains an unmet medical need. Several attenuated virus and antigen-based HCMV vaccine candidates have been proposed; however, development challenges have limited their progression through the clinical pipeline. Method: A high-throughput and robust relative potency assay, Imaging of Relative Viral Expression (IRVE), was developed and applied to measure the infection of a live-attenuated HCMV vaccine candidate in ARPE-19 epithelial cells. The IRVE assay measures HCMV infection by immunostaining Immediate Early 1 (IE1) protein and enumeration of IE1-positive, infected cells against total cells. Increased throughput was accomplished using 384-well plate automation on a custom-designed integrated robotic system. Results: The IRVE assay effectively measures relative potency changes in an HCMV vaccine candidate under different upstream processes, downstream processes, and formulation conditions. Key assay parameters including microplate format, cell density, serum concentration, infection time and influence of cell age were evaluated and optimized. The IRVE assay was correlated to historical, lower throughput HCMV potency assays, including plaque and Infectivity of Early Gene Expression (IEE), validating its application as a potency screening tool. Conclusions: The IRVE assay has been successfully implemented to support HCMV vaccine development over several years of clinical development. Full article

This study on Foeniculum vulgare Mill., derived from seeds collected in Meknes (Morocco), evaluates in vitro and in silico the therapeutic potential of its extracts and essential oil through a comprehensive analysis of its phytochemical composition, as well as its antioxidant and antimicrobial activities. Aqueous extracts (E0), hydroethanolic extract (E1) obtained via Soxhlet, decoction (E2), and essential oil (EO) obtained through hydrodistillation were analyzed using HPLC/UV-ESI-MS and GC-MS, revealing a richness in phenolic and terpenic compounds. The quantification of total polyphenols, flavonoids, and tannins in aqueous and organic extracts was performed using spectrophotometric methods. Antioxidant activity was assessed through three methods: DPPH, FRAP, and Total Antioxidant Capacity (TAC). The antimicrobial activity of the essential oil and decoction was evaluated by microdilution in microplate assays. The aqueux extract was dominated by butyl ferulate (14.33%), while hydroethanolic extract contained chlorogenic acid (14.79%) and quercetin-3-glucuronide (13%). The extract (E₂) was characterized by dihydrocaffeic acid (11.25%) and 3-O-caffeoylshikimic acid (11.08%), whereas the EO was primarily composed of fenchone (24.72%), trans-anethole (22.22%), and limonene (20.48%). Antioxidant assays (DPPH/FRAP/TAC) demonstrated decreasing efficacy as follows: EO exhibited the highest efficiency (IC₅₀ = 51.45 μg/mL), followed by E₁ (93.71 μg/mL), E₀ (212.86 μg/mL), and E₂ (397.41 μg/mL), confirming a correlation between phenolic composition and antioxidant activity. Furthermore, antimicrobial tests highlighted a pronounced fungicidal effect against Candida albicans (MIC = 3.13 mg/mL) and Aspergillus niger (6.25 mg/mL), contrasting with a more moderate inhibition of Escherichia coli and Staphylococcus aureus. Molecular docking simulations identified stable interactions between chlorogenic acid, quercetin-3-glucuronide, and microbial proteases, suggesting a synergistic inhibitory mechanism. This research validates the potential of F. vulgare as a source of bioactive molecules with promising applications in phytotherapy for managing oxidative stress and fungal infections, while emphasizing the need for clinical studies to confirm these effects in vivo. Full article

This work develops a unified size-dependent shear deformation theory (SDSDT) to analyze the free vibration behavior of a functionally graded (FG) magneto-electro-elastic (MEE) microplate under fully simply supported conditions, open- or closed-circuit surface conditions, biaxial compression, magnetic and electric potentials, and uniform temperature changes based on consistent couple stress theory (CCST). The FG-MEE microplate is composed of BaTiO₃ (a piezoelectric material) and CoFe₂O₄ (a magnetostrictive material). Various CCST-based SDSDTs, considering couple stress and thickness stretching effects, can be reproduced by employing a generalized shape function that characterizes shear deformation distributions along the thickness direction within the unified SDSDT. These CCST-based SDSDTs encompass the size-dependent classical plate theory (CPT), first-order shear deformation theory (SDT), Reddy’s refined SDT, exponential SDT, sinusoidal SDT, and hyperbolic SDT. The unified SDSDT is validated by comparing its solutions with relevant three-dimensional solutions available in the literature. After validation and comparison studies, we conduct a parametric study, whose results indicate that the effects of thickness stretching, material length-scale parameter, inhomogeneity index, and length-to-thickness ratio, as well as the magnitude of biaxial compressive forces, electric potential, magnetic potential, and uniform temperature changes significantly impact the microplate’s natural frequency. Full article

Neutrophil extracellular traps (NETs) are not only promising biomarkers of disease, but also potential therapeutic targets. Overproduction or the improper clearance of NETs has been linked to disease severity. In vitro NET degradation assays can reveal mechanisms and degradation efficiency differences in diseased serum samples. There is a need for more convenient assays to increase the speed of NET degradation studies. This paper describes a simplified, lower variability mimetic assay with DNA–histone structures, referred to as surface webs, that performs functionally similarly to traditional NET degradation assays with increased scalability, ease of use, shorter preparation time, and lowered costs. The surface webs are created and dehydrated in a 96-well microplate that is shelf-stable, transportable, and viable for 30 days of storage at room temperature. The surface webs, compared to NETs, have similar shapes and distribution but lower intraplate variability while degrading with healthy serum and DNase I within the same timeframe. The assay can identify patient serum with reduced degradation capabilities. This assay opens new opportunities for NET-targeted drug discovery and studies on the role of NETs as modulators of disease. Full article

Objective: Fungal corneal infections are challenging to treat due to delayed diagnostic procedures, bacterial co-infections, and limited antifungal efficacy. This study investigates the therapeutic potential of ultraviolet C (UVC) light alone and combined with antifungal drugs. Methods: A subsurface infection model was developed in semi-solid agar droplets, with Candida albicans cells or Aspergillus brasiliensis spores inoculated into 0.75% w/v yeast peptone dextrose (YPD) agar in a 96-well microplate (5 µL per well). Two treatment groups were tested: (1) UVC exposure (265 nm, 1.93 mW/cm²) for durations of 0 s, 5 s, 10 s, 15 s, 30 s, 60 s, or 120 s, and (2) UVC combined with antifungal drugs (Amphotericin B and Natamycin) at their minimum inhibitory concentrations (MICs), determined in YPD broth. After treatment, agar droplets were homogenized, diluted, and plated for microbial enumeration. The most effective UVC doses were further tested in an ex vivo C. albicans porcine keratitis model, where the corneal epithelium was debrided, infected with C. albicans, and exposed to UVC. Corneas were then homogenized and plated to evaluate treatment efficacy. Results: UVC exposure of ≥15 s inhibited C. albicans, and ≥10 s inhibited A. brasiliensis (all p < 0.05). The broth MICs were 0.1875 µg/mL for Amphotericin B against C. albicans, 6.25 µg/mL against A. brasiliensis, and 0.78125 µg/mL for Natamycin against C. albicans, 7.8125 µg/mL against A. brasiliensis. The broth MIC did not eradicate fungi in the subsurface model. Combined treatments enhanced inhibition (all p < 0.05), with 30 s UVC + amphotericin B for C. albicans (p = 0.0218) and 30 s UVC + natamycin for A. brasiliensis (p = 0.0017). Ex vivo, 15 s and 30 s UVC inhibited growth (p = 0.0476), but no differences were seen between groups (all p > 0.05). Conclusion: UVC demonstrated strong antifungal efficacy, with supplementary benefits from combining UVC with low doses of antifungal drugs. Full article

Bacteria are the primary culprits of global foodborne diseases, making bacterial detection one of the most critical aspects of food safety. The quantification of viable and dead bacteria is typically achieved through distinct methodologies, such as culture-based methods and molecular biological techniques. These approaches often have non-overlapping requirements in terms of sample pre-treatment and detection equipment. However, in this presented work, bacterial extracellular nucleases and DNase I were utilized to achieve the simultaneous quantification of both live and dead bacteria in a single well of a microplate. The detection limits of the method for live and dead bacteria are estimated to be 7.13 × 10⁵ CFU/mL and 3.54 × 10⁵ CFU/mL, respectively. In the application of detecting bacteria in pickled pork stewed bamboo shoot soup, the detection limit for live bacteria can be reduced to as low as 10² CFU/mL within 24 h after enrichment cultivation. Full article

Enterococcus faecalis (E. faecalis) is a ubiquitous microbe occurring in the environment and in the intestinal tract of poultry. E. faecalis has been identified in cases of egg infertility and/or decreased hatchability and can cause amyloid arthropathy in older laying chickens. E. faecalis produces cytolysin, a bacterial exotoxin that can cause lysis of erythrocytes. It has been difficult to demonstrate this virulence trait using conventional culture methods with sheep blood agar. A 96-well microplate hemolysis assay, along with a culture method incorporating glucose and L-arginine into the culture media, is described that demonstrates the production of cytolysin in E. faecalis isolates of avian origin. Additionally, the results show that horse and sheep erythrocytes were susceptible to lysis by the E. faecalis cytolysin, but cow and chicken erythrocytes were less susceptible. Full article

Methicillin-resistant Staphylococcus aureus (MRSA) continues to represent a significant clinical challenge, characterized by consistently elevated rates of morbidity and mortality. Care regimen success is still difficult and necessitates assessing new antibiotics as well as supplemental services, including source control and searching for alternative approaches to combating it. Hence, we propose to synthesize silver nanoparticles (Ag-NPs) by employing a cell-free filter (CFF) of Streptomyces sp. to augment antibiotic activity and combat biofilm-forming MRSA. Seven bacterial isolates from clinical samples were identified, antibiotics were profiled with Vitek-2, and the phenotypic detecting of biofilm with Congo red medium and microplate assay was carried out. The PCR technique was used for detecting genes (icaA and icaD) coded in biofilm forming. The characterization of Ag-NPs was performed using several analytical methods, such as UV spectroscopy, dynamic light scattering (DLS), zeta potential measurement, transmission electron microscopy (TEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). The antibacterial properties of Ag-NPs and oxacillin–Ag-NPs were assessed against standard strains and clinical isolates by employing the agar well diffusion technique and the microdilution assay. The biogenic synthesis Ag-NPs resulted in uniformly spherical particles, with an average size of 20 nm. These Ag-NPs demonstrated significant activity against biofilm-forming MRSA, with minimum inhibitory concentrations (MICs) ranging from 12 to 15 μg/mL. Additionally, Ag-NPs completely impede biofilm formation by MRSA at sublethal doses of 0.75 MICs. The expression levels of the icaA and icaD genes were reduced by 1.9- to 2.2- and 2.4- to 2.8-fold, respectively. A significant synergistic effect was noted when Ag-NPs were used in combination with oxacillin, leading to reduced MICs of 1.87 μg/mL for oxacillin and 4.0 μg/mL for Ag-NPs against MRSA. The FICi of 0.375 further validated the synergistic relationship between oxacillin and Ag-NPs at the concentrations of 1.87 and 4 μg/mL. Findings from the time-kill test demonstrated the highest reduction in log₁₀ (CFU)/mL of the initial MRSA inoculum after 12-hour exposure. The cytotoxicity analysis of Ag-NPs revealed no significant cytotoxic effects on the human skin cell line HFB-4 at low concentrations, with IC₅₀ values of 61.40 µg/mL for HFB-4 and 34.2 µg/mL for HepG-2. Comparable with oxacillin–Ag-NPs, Ag-NPs showed no cytotoxic effects on HFB-4 at different concentrations and exhibited an IC₅₀ value of 31.2 against HepG-2-cells. In conclusion, the biosynthesis of Ag-NPs has demonstrated effective antibacterial activity against MRSA and has completely hindered biofilm formation, suggesting a valuable alternative for clinical applications. Full article

Candida auris is a globally emerging pathogenic fungus described in Japan in 2009. This fungus has been identified mainly in nosocomial environments, associated with a high frequency of multidrug-resistant strains, and mortality rates reach 60%. C. auris is divided into 6 biogeographic clades, and there is a correlation between the clades and resistance against antifungals. In the current report, 8 strains of C. auris isolated in the Montefiore Medical Center, Bronx were analyzed to assess their clade (via ClaID) and common molecular determinants of antifungal resistance. We assessed antifungal resistance as well as the efficacy of a novel nitric oxide-donating microparticle as an alternative approach against C. auris in vitro through microplate susceptibility tests. Virulence was also determined in a Galleria mellonella model. Our results indicate that 7 out of 8 strains, belonging to clade 1, were resistant to fluconazole, while clade 2 was susceptible. Additionally, the clade 2 strain was more susceptible to treatment with the microparticle, while also being more virulent in an invertebrate model of infection. Our findings were then correlated to visualize parallels between clade identification and resistance/virulence patterns. Full article

The stem bark of black locust (Robinia pseudoacacia L.) was extracted, and nine antioxidant compounds (R1–R9) were detected by high-performance thin-layer chromatography combined with the radical scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH•) assay, multi-detection, and heated electrospray high-resolution mass spectrometry. For structure elucidation, the methanolic crude extract was fractionated by solid-phase extraction, and the compounds were isolated by reversed-phase high-performance liquid chromatography with diode array detection. The structures of isolated compounds were elucidated by nuclear magnetic resonance and attenuated total reflectance Fourier-transform infrared spectroscopy as well as gas chromatography-mass spectrometry to determine the double bond position. 3-O-Caffeoyl oleanolic acid (R1), oleyl (R2), octadecyl (R3), gadoleyl (R4), eicosanyl (R5), (Z)-9-docosenyl (R6), docosyl (R7), tetracosyl (R8), and hexacosanyl (R9) caffeates were identified. While R1 has been reported in R. pseudoacacia stem bark, the known R3, R5, R7, R8, and R9 are described for the first time in this species, and the R2, R4, and R6 are new natural compounds. All nine caffeates demonstrated antioxidant activity. The antioxidant effects of the isolated compounds R1–R8 were quantified by a microplate DPPH• assay, with values ranging from 0.29 to 1.20 mol of caffeic acid equivalents per mole of isolate. Full article

The growing antibiotic resistance of microorganisms causing postoperative infections following orthopedic surgeries underscores the urgent need for localized antiseptic and lavage delivery systems to enhance infection control. This study evaluates the in vitro effectiveness of antiseptic and lavage solutions—including polyhexanide, povidone–iodine, low-concentrated hypochlorite, Ringer’s solution, and saline—against Staphylococcus epidermidis, Staphylococcus aureus MRSA, Cutibacterium acnes, Corynebacterium amycolatum, Pseudomonas aeruginosa, and Candida albicans. Using microplate models (Minimum Inhibitory Concentration, Minimum Biofilm Eradication Concentration, and Biofilm-Oriented Antiseptic Test assays), flow-based models (Bioflux system), and surfaces relevant to orthopedic implants (e.g., stainless steel disks/screws, Co-Cr-Mo, Ti-Al-Nb orthopedic alloys, and ultra-high-molecular-weight polyethylene), as well as a bio-nano-cellulose scaffold representing tissue, we assessed the solutions’ activity. The cytotoxicity of the solutions was evaluated using osteoblast and keratinocyte cell lines, with additional in vivo insights gained through the Galleria mellonella larval model. The results show that polyhexanide-based solutions outperformed povidone–iodine in biofilm eradication in most tests applied, particularly on complex surfaces, whereas iodine demonstrated higher cytotoxicity in applied in vitro and in vivo tests. Low-concentration hypochlorite solutions exhibited minimal antibiofilm activity but also showed no cytotoxicity in cell line and G. mellonella larval models. These findings highlight the importance of careful antiseptic selection and rinsing protocols to balance infection control efficacy with tissue compatibility in orthopedic applications. Full article