Dilution Assay Research Articles

Cannabidiol (CBD) Acts as an Antioxidant on Gardnerella vaginalis, Resulting in Reduced Metabolic Activity, Loss of Survivability, and Elimination of Biofilms

Background: Gardnerella vaginalis is a natural inhabitant of the vagina, but when an imbalance occurs in the vaginal microbiota, this bacterium can cause vaginosis, a condition that must be treated when symptomatic and prior to a gynecological intervention. Cannabidiol (CBD) is an anti-inflammatory compound that also has antibacterial activities against several Gram-positive and certain Gram-negative bacteria. Objectives: Since G. vaginalis is an opportunistic pathogenic Gram-variable bacterium, we investigated its response to CBD. Methods: The antibacterial activity of CBD was studied by broth dilution assay, changes in intracellular ATP levels, and the ability of bacteria to recover on chocolate agar plates. The antibiofilm activity was investigated by MTT metabolic assay, crystal violet staining, and HR-SEM. Flow cytometric analyses were performed to measure changes in membrane potential, membrane perforation, and metabolic activity. Reactive oxygen species (ROS) production was analyzed using the nitro blue tetrazolium (NBT) reagent. Gene expression was determined by semi-quantitative real-time PCR, while protein composition was determined by LC-MS/MS analysis. Results: We observed that G. vaginalis clinical isolates exhibited high susceptibility to CBD with a minimum inhibitory concentration (MIC) of 2.5 µg/mL CBD. CBD induced rapid membrane hyperpolarization and caused cytoplasmic leakage of ATP without increasing propidium iodide uptake. This was accompanied by reduced metabolic activity and loss of survivability. Proteomic analysis revealed decreased expression of some ribosomal-associated proteins. CBD exhibited antioxidant activity by reducing intracellular ROS levels in a dose-dependent manner. The antibacterial effect was neutralized by the free radical scavenger α-tocopherol, suggesting the involvement of radicals in executing the antibacterial effect. Importantly, CBD not only prevented the biofilm formation of G. vaginalis but also reduced the metabolic activity and biofilm biomass of preformed, mature biofilms. Real-time PCR analysis of G. vaginalis treated with CBD for 6 h showed an increase in the expression of biofilm-associated genes, suggesting that the antibiofilm activity of CBD is mainly due to its antibacterial effect. CBD did not alter the ability of G. vaginalis to adhere to HeLa cervical carcinoma cells and CBD-treated bacteria were still phagocytosed by RAW264.7 macrophages. Conclusions: Our study shows that CBD exhibits antibacterial and antibiofilm activities against G. vaginalis clinical isolates and is thus a potential drug for the treatment of vaginosis caused by this bacterium.

Citrus maxima extract-coated versatile gold nanoparticles display ROS-mediated inhibition of MDR-Pseudomonas aeruginosa and cancer cells.

The expanding prevalence of microbial resistance to conventional treatments has triggered a race to develop alternative/improved strategies to combat drug-resistant microorganisms in an efficient manner. Here, the lethal impact of the biosynthesized gold nanoparticles (AuNPs) against multi-drug resistant (MDR) bacteria has been elucidated. AuNPs, synthesized from the extracts of the fruit, leaf and peel of the Citrus maxima plant, were physicochemically characterized by UV-Vis spectrophotometry, Dynamic Light Scattering (DLS), electron microscopy and spectroscopic techniques not only confirmed the production of AuNPs of size below 100nm but also identified the phytochemicals adsorbed onto the surface of NPs. AuFeNP not only showed excellent antioxidant activity (∼95% at 1mg/mL) but also exhibited a commendable antimicrobial activity against MDR-Pseudomonas aeruginosa as assessed by the zone of inhibition (13.5mm) and microwell broth dilution assays (9.5μg/mL, MIC). Transmission electron microscopy (TEM) displayed bacterial cell membrane destruction post-AuNPs exposure. The killing mechanism of AuNPs elucidated the permeabilization of the cell membrane and generation of reactive oxygen species (ROS), ∼10-fold high depletion of GSH, and eventually leaching protein out of the cell. DNA damage, as a marker of apoptosis, was also noticed, which could be an implication of ROS accumulation in MDR-PA. AuNPs displayed significant toxicity at∼10μg/mL on various cancer cells (HT-1080, MRC-5, MDA-MB-231 and B16-F10) and relatively low toxicity on normal cells (MRC-5 and HaCaT). Scratch assay to identify the migration capability of breast cancer cells on treatment with AuNPs deciphered hampering of migration potential of breast cancer cells. Apoptotic topographies in B16-F10 cells were confirmed using AO/EtBr dual dye staining, DNA fragmentation, Caspase-3 assay and cell cycle analysis using flow cytometry. Hemolysis revealed minimal toxicity of AuNPs on human red blood cells. Nominal toxicity (∼70% survival at 500μg/mL of AuNPs) on mammalian cells was evaluated using Cell Titer-Glo cell viability assay. Overall results advocate the promising potential of biosynthetic AuFeNP against multi-drug-resistant pathogens and for further formulation into anticancer agents.

CD271high cancer stem cells regulate macrophage polarization in head and neck squamous cell carcinoma.

Cancer stem cells (CSCs) are considered key drivers of progression in head and neck squamous cell carcinoma (HNSCC). Our single-cell RNA sequencing (scRNA-seq) analysis revealed predominant expression of CD271 in CSCs, however, its role as a CSC marker in HNSCC requires further elucidation. We investigated the stemness characteristics of CD271high HNSCC cells and their interactions with the tumor immune microenvironment. scRNA-seq data from hypopharyngeal squamous cell carcinoma (HPSCC) tissues were analyzed to identify expression profile of CSCs. Overall survival was compared between CD271high and CD271low patients based on immunostaining of HPSCC samples. The stemness of CD271high HNSCC cells was evaluated via an in vivo limiting dilution assay. In a C57BL/6 mice model, the percentage of immune cells and macrophage subtypes were analyzed by flow cytometry. The role of CD271 in macrophage polarization was further examined by in vitro coculture of CD271high cells with CD14+ monocytes. Gene expressions were analyzed by qPCR. CD271 is predominantly expressed in CSCs identified by scRNA-seq analysis. CD271 enhances HNSCC cell proliferation and is negatively correlated with patient prognosis in HPSCC. CD271 knockdown suppressed HNSCC tumor growth and regulated macrophage polarization within the TME. CD271high cells exhibited stemness features and enhanced tumor growth in vivo. CD271high HNSCC cells exhibit CSC characteristics and regulate macrophage polarization. Targeting CD271 may improve the immunosuppressive TME to inhibit tumor growth. Combining CD271-targeting agents with other therapies presents a promising strategy that may enhance therapeutic efficacy and prognosis in HNSCC.

Quantitative proteomic analysis unveils a critical role of VARS1 in hepatocellular carcinoma aggressiveness through the modulation of MAGI1 expression

BackgroundHepatocellular carcinoma (HCC) genetic/transcriptomic signatures have been widely described. However, its proteomic characterization is incomplete. We performed non-targeted quantitative proteomics of HCC samples and explored its clinical, functional, and molecular consequences.MethodsNon-targeted quantitative proteomics were performed on cytosolic and nuclear fractions of liver samples [HCC vs. non-tumour adjacent tissue (NTAT), n = 42 patients]. Changes were confirmed in 7 in silico HCC cohorts. Functional and molecular implications were evaluated on HCC-derived cell lines after silencing/overexpressing VARS1 and/or MAGI1. VARS1-overexpressing Hep3B cells were used for in vivo studies [Extreme Limiting Dilution Assay (ELDA) and orthotopic tumour formation]. Quantitative proteomics were performed on VARS1-overexpressing HCC cell lines.ResultsQuantitative proteomics revealed the dysregulation of the cytosolic and nuclear proteomes in HCC, and defined two proteomic HCC subgroups, the most aggressive associated to the dysregulation of the aminoacyl-tRNA synthetases (ARSs). ARSs dysregulation was corroborated in in silico HCC cohorts and associated to poor prognosis. Patients with ARSs upregulation had genomic/transcriptomic characteristics of the proliferative HCC. Valine tRNA-aminoacyl synthetase (VARS1) was the ARSs most consistently overexpressed and associated to aggressiveness. VARS1 modulation (silencing/overexpression) altered tumour establishment-associated parameters in vitro and/or in vivo. Quantitative proteomics on cells overexpressing VARS1 and rescue experiments identified the downregulation of MAGI1, a tumour suppressor in HCC, as a mediator of VARS1 function.ConclusionsQuantitative proteomics defines two prognosis-related proteomic HCC subgroups. ARSs machinery is dysregulated in the aggressive subgroup, bearing potential as prognostic biomarkers. VARS1 promotes aggressiveness through the modulation of MAGI1, representing a novel targetable vulnerability in HCC.

Pharmacokinetic analysis using single dilution assays: enhancing precision, reducing errors and increasing throughput.

Technologies such as ELISA, MSD, and Gyrolab have been employed for quantifying protein therapeutics in clinical trials. However, these technologies have limitations with dynamic range often requiring multiple dilution steps, introducing potential errors and variability. A pharmacokinetics assay was successfully developed on the NUcleic acid Linked Immuno-Sandwich Assay (NULISA) platform with a concentration dynamic range exceeding 6 logs. This enabled assessment of all clinical samples across different concentrations with a single dilution, yielding results with good correlation to ELISA and Gyrolab. NULISA technology offers high sensitivity, full automation, and a wide dynamic range, streamlining assay development and optimization, simplifying sample analysis, minimizing errors, and increasing throughput.

Anti-Bacterial Activity of an Ancient Remedy Against Pathogenic Skin Infections

Background/Objectives Skin and soft-tissue infections are among the most common infections and can develop severe local and systemic complications. Bacterial species: Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa and Escherichia coli usually cause skin infections. Studies of plants and natural materials used in ancient medicine to treat infections may assist us in further antibiotic discoveries. In this study, we tested the bactericidal activity of a 1000-years-old Arabic Al-Razi remedy against common skin infections Gram-positive and Gram-negative bacterial strains. Methods The antimicrobial activity was tested by agar diffusion method, after adding specific test sample, zone of inhibition was measured. Minimum inhibitory concentrations (MIC) of Al-Razi full remedy, dropout batches and single ingredients were performed by broth micro dilution assay. Results The mixture of all ingredients of this remedy (vinegar, gum arabic, boswellia, vitriol and aloe) has shown highest antibacterial activity against S. aureus, S. pyogenes, multi drug resistance (MDR) P. aeruginosa and clinical strain of P. aeruginosa. However, the presence of a single ingredient in water has shown no activity against tested bacterial strains except vitriol. Conclusion The AR remedy comprising several ingredients (vinegar, gum arabic, boswellia, vitriol and aloe) had shown great potential to inhibit skin infection bacterial strains ( S. aureus, S. pyogenes, MDR P. aeruginosa and the clinical strain of P. aeruginosa). In contrast, single ingredients (except vitriol) had shown no activity against all tested bacterial strains. These results suggest that combination of natural products has more potential against bacterial infections.

Indole phytochemical camalexin as a promising scaffold for AcrB efflux pump inhibitors against Escherichia coli.

Escherichia coli is amongst the most frequent causative agent of nosocomial infections and the overexpression of the efflux pump gene acrB plays a major role in its resistance to various antibiotics. In this study, we evaluated two indole phytochemicals, camalexin and brassinin, as potential AcrB efflux pump inhibitors. Among these two phytochemicals, camalexin increased the accumulation of ethidium in acrB proficient E.coli with no membrane permeabilization effect observed, indicating a direct interaction of camalexin with the pump. Camalexin also showed up to 64-fold MIC reduction for drugs in the acrB proficient strain. Brassinin was less effective, showing up to 4-fold MIC reduction for the same drugs. Camalexin did not potentiate drugs in the AcrB inactive strain D407N. Plate dilution assays in E. coli acrB variants further corroborated the effect of camalexin in diminishing pump activity. Blind docking results suggested that camalexin and brassinin may enter mainly via CH3, one of the channels present in AcrB, and camalexin showed a more stable binding mode than brassinin in the distal binding pocket of AcrB. Camalexin, therefore, holds potential as a scaffold for further development as a potent AcrB inhibitor to tackle antimicrobial resistance in the gram-negative bacterium E. coli.

Quantification of mulberrin and morusin in mulberry and other food plants via stable isotope dilution analysis using LC-MS/MS.

Mulberry (Morus) is an important food and medicinal plant primarily used in sericulture. It is rich in prenylated flavonoids like morusin and mulberrin, which have shown promising bioactivities in vitro and in vivo but so far rely on HPLC methods for their quantification in plant material. Hence, a more sensitive LC-MS/MS stable isotope dilution assay for morusin and mulberrin was developed and validated. The analytes were quantified in 17 plant samples, with up to three plant compartments investigated (branches, leaves, fruits). The highest amounts were found in branches of a German mulberry tree (43μg/g morusin, 910μg/g mulberrin). Limits of detection reached 0.02μg/g for morusin and 0.06μg/g for mulberrin, which is significantly lower than existing methods, allowing for the detection and quantification of the analytes in leaves and fruits of mulberry, as well as common snowball (Viburnum opulus), a common food and medicine plant.

Tetramethylpyrazine attenuates the cancer stem cell like-properties and doxorubicin resistance by targeting HMGCR in breast cancer.

Tetramethylpyrazine (TMP), a key bioactive constituent derived from Ligusticum wallichii Franchat, has demonstrated efficacy in mitigating multidrug resistance (MDR) in human breast cancer (BC) cells. However, the precise mechanisms underlying its action remain poorly understood. Cancer stem cells (CSCs) are widely recognized as the primary contributors to MDR. This investigation seeks to elucidate the role and mechanisms through which TMP counteracts MDR by attenuating CSC-like characteristics. Various assays, including flow cytometry, sphere formation, and Western blotting, were employed to evaluate TMP's effects on breast cancer stem cell (BCSC)-like phenotypes in vitro. In vivo, extreme limiting dilution assays and immunohistochemistry (IHC) were executed to assess the impacts of TMP on BCSC frequency and the levels of stemness markers. Mechanistically, RNA sequencing was performed to uncover the key biological processes involved in TMP's effects on BCSCs. Further experiments, encompassing micro scale thermophoresis (MST), drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA) and amino acid mutation analyses, were utilized to identify the essential targets and corresponding binding sites of TMP. Finally, the effects of TMP on BCSC-like phenotypes were confirmed using cells with mutated amino acid residues, which allowed us to investigate the specificity of TMP's binding sites. To further evaluate the impact of TMP on drug resistance, doxorubicin-resistant MCF7 (MCF-7ADR) cells, along with corresponding cell lines harboring mutated amino acid residues, were employed. TMP was found to inhibit BCSC-like properties both in vitro and in vivo, evidenced by a reduction in the CD44+/CD24- population, sphere formation capability, and expression of stemness markers. Mechanistic studies revealed that TMP targets 3‑hydroxy-3-methylglutaryl-CoA reductase (HMGCR), a rate-limiting enzyme in cholesterol biosynthesis. TMP binds to Asp-767 of HMGCR, thereby inhibiting its activity and reducing cholesterol synthesis. The influence of TMP on BCSC-like phenotypes was nullified by overexpression of wild-type HMGCR, while mutations in the binding site of HMGCR had no effect on TMP's inhibition of BCSC-like properties. Additionally, TMP mitigated MDR by targeting HMGCR. These findings suggest that TMP alleviates MDR by reducing BCSC-like traits through targeting HMGCR and disruption of cholesterol biosynthesis in BC. This provides new insights into the mechanisms through which TMP alleviates MDR and offers new lead compound for exploring HMCGR antagonists.

Monitoring Fusarium toxins from barley to malt: Targeted inoculation with Fusarium culmorum

Molds of the genus Fusarium infect nearly all types of grain, causing significant yield and quality losses. Many species of this genus produce mycotoxins, which pose significant risks to human and animal health. In beer production, the complex interaction between primary fungal metabolites and secondarily modified mycotoxins in barley, malt, and beer complicates the situation, highlighting the need for effective analytical methods to quickly and accurately monitor these toxins. We developed and validated a liquid chromatography-tandem mass spectrometry (LC–MS/MS) method to simultaneously analyze 14 Fusarium toxins, including modified forms (deoxynivalenol (DON), DON-3-glucoside, 3-acetyl-DON, 15-acetyl-DON, nivalenol, fusarenone X, HT-2 toxin, T-2 toxin, the enniatins A, A1, B, B1, beauvericin, and zearalenone) in barley and throughout the malting process. Stable isotope dilution assays (SIDAs) and matrix-matched calibration were used for quantification. A micro-malting setup was established to produce Fusarium-contaminated barley malt under reproducible conditions using targeted inoculation with F. culmorum. Mycotoxins were quantified throughout the malting process and compared to the content of fungal DNA. Further, the impact of various malting parameters was investigated, thus revealing that different malting scenarios exhibited different toxin enrichment patterns. We demonstrated that mycotoxin concentration and the ratio of DON to DON-3-glucoside changed throughout the malting processes, depending on fungal spore concentrations, germination temperature, and malting temperature. The study highlights the complexity of mycotoxin dynamics in malt production and the importance of optimized processing conditions to minimize toxin levels in final malt products.

Investigation on L-rhamnose metabolism of Loigolactobacillus coryniformis subsp. coryniformis DSM 20001 and its propionate-containing fermentates.

Propionate is an important short-chain carboxylic acid (SCCA) that serves as an effective antimicrobial agent for food preservation. Previous research has highlighted that few Lactobacillaceae can synthesize propionate by metabolizing deoxyhexoses via the fermentation intermediate 1,2-propanediol (1,2-PD). In this study, we investigated propionate production by Loigolactobacillus coryniformis subsp. coryniformis DSM 20001 while utilizing L-rhamnose as the primary carbon source. We cultivated L. coryniformis in small-scale anaerobic bioreactors at 30°C and pH 6.5 for 72 h and monitored the expression of key genes associated with deoxyhexose metabolism using quantitative PCR. In addition, we assessed the contribution of individual SCCA to the antimicrobial activity of the fermentate against common foodborne pathogens, including Escherichia coli, Salmonella enterica, Klebsiella oxytoca, Staphylococcus aureus, Candida albicans, Aspergillus niger, Penicillium roqueforti, and Penicillium purpurogenum using broth dilution assays. During cultivation, we observed the production of up to 16 mM propionate, alongside other metabolites such as lactate (26 mM), formate (2 mM), and acetate (4 mM), derived from 32 mM L-rhamnose. Genes related to L-rhamnose utilization were upregulated within the initial 48 h, while genes involved in 1,2-PD utilization remained highly transcribed throughout fermentation. Comparing the antimicrobial efficacy of the fermentates to synthetic SCCA mixtures, bacterial indicator strains were more sensitive than molds and yeast. Propionate was the primary SCCA responsible for inhibitory activity; inhibition was reduced if indicator strains were able to use lactate.IMPORTANCEWorldwide, approximately 30% of food produced is lost. Despite the application of complementary treatment methods, microbial food spoilage can occur along the entire value chain. The rising concern about food waste has led to increasing interest in natural preservation approaches. Lactobacillaceae fermentative systems produce a variety of short-chain carboxylic acid (SCCA) with antimicrobial potential, and we present here fundamental insight into the only recently discovered deoxyhexose metabolism of Loigolactobacillus coryniformis producing the antimicrobial SCCA propionate. We developed a bioprocess to produce propionate from L-rhamnose under controlled conditions as a first step toward the exploitation of L-rhamnose metabolism in the production of antimicrobial fermentates for use in the food industry, potentially replacing chemical alternatives. Our investigations highlight the major contribution of propionate in antimicrobial activity but also indicate the issue of co-occurring fermentable metabolites, which can affect the efficiency of fermentates.

Plant Species with Antimicrobial Effects and the Importance of a Standardization of Protocols for Antimicrobial Agents

In modern times, microbial resistance is a global threat to health and development. The misuse and inappropriate use of antimicrobials is the main cause of developing drugresistant pathogens. It requires multiple areas in direction to attain sustainable advance goals. As a result of microbial resistance, the necessity for costly medications and expenses are obstructed worldwide. Due to rising attention in the research of new antimicrobial medicaments from a variety of natural or synthetic sources to fight microbial resistance. Thus, natural antimicrobial agents have been used to a great extent nowadays because plant-derived antimicrobial agents are considered to be safer alternatives for health as compared to those synthetic antimicrobial agents. Overall, the active ingredients, water, essential oils, and ethanolic extracts from selected plants and the mixture of a variety of these natural extracts have been used for centuries, because they possess antimicrobial activity which inhibits the growth of microbes. Natural plants as an antimicrobial agent, like extracts of Curcuma longa L., Piper nigrum L., Vachellia leucophloea, Eclipta prostrata, Ocimum sanctum L., Terminalia arjuna, Manihot esculenta Crantz, Lawsonia inermis L., Zingiber officinale Roscoe, Camellia sinensis (L.) Kuntze, Coriandrum sativum L., Carica papaya L., Cinnamomum tamala, and many others have been preferred and used for ages because they are easily available worldwide. They are usually of low cost and have little or no side effects. Several antimicrobial screening approaches like the disk-diffusion method, well diffusion method, micro broth dilution assay, sterile disk method, and agar diffusion method are generally cast off for measurement of reproducibility and standardization of these antimicrobial agents. This review article is a comprehensive description of natural plants like Coriandrum sativum L., Carica papaya L., Cinnamomum tamala, etc., containing those extracts used as antimicrobial agents listed, and numerous in vitro antimicrobial susceptibility testing methods are reported. These identified plant species and antimicrobial screening techniques hold the potential for formulating these plants into antimicrobial drugs, warranting further study and exploration in the field of medicine.

Combined action of two synthetic ultrashort antimicrobial peptides exhibiting synergistic effects against clinically significant resistant bacteria.

The emergence and proliferation of multidrug-resistant bacteria pose a global health crisis. This issue arises from the overuse and misuse of antibiotics, coupled with the pharmaceutical industry's limited development of new drugs, which is constrained by financial disincentives and regulatory hurdles. This study aimed to investigate the combined antibacterial efficacy and safety profile of the combined ultrashort antimicrobial peptides (AMPs) WW-185 and WOW against antibiotic-resistant bacterial strains. The WW-185 and WOW peptides were synthesized through solid-phase methods and purified using reverse-phase high-performance liquid chromatography, and their purity was confirmed by mass spectrometry. Antibacterial activity was evaluated using broth dilution and checkerboard assays to assess both individual and combined effects of the peptides against Staphylococcus aureus (including methicillin-resistant Staphylococcus aureus [MRSA]) and Escherichia coli (including extended-spectrum beta-lactamases [ESBL]-producing strains). The synergy between the peptides was quantified using fractional inhibitory concentration indices. Hemolytic activity was also assessed to determine cytotoxicity toward red blood cells. The combination of WW-185 and WOW exerted synergistic effects against both MRSA and ESBL-producing E. coli, with reduced minimal inhibitory concentrations compared with the individual treatments. The peptides exhibited minimal hemolytic activity, indicating low toxicity. The combination of the ultrashort AMPs WW-185 and WOW shows promising synergistic antibacterial effects against resistant bacteria, with potential for further therapeutic development due to their enhanced efficacy and low toxicity.

CCN1 Promotes Mesenchymal Phenotype Transition Through Activating NF-κB Signaling Pathway Regulated byS100A8 in Glioma Stem Cells.

The presence of glioma stem cells (GSCs) and the occurrence of mesenchymal phenotype transition contribute to the miserable prognosis of glioblastoma (GBM). Cellular communication network factor 1 (CCN1) is upregulated within various malignancies and associated with cancer development and progression, while the implications of CCN1 in the phenotype transition and tumorigenicity of GSCs remain unclear. Data for bioinformatic analysis were obtained from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) databases. A range of primary GBM and GSC cell models were then used to demonstrate the regulatory role of CCN1 via the phenotype validation, tumor sphere formation assays, extreme limiting dilution assays (ELDA), and transwell assays. To screen out the downstream signaling pathway, we employed high-throughput RNA-seq. Intracranial xenograft GSC mouse models were used to investigate the role of CCN1 invivo. Among the CCN family members, CCN1 was highly expressed in MES-GBM/GSCs and was correlated with a poor prognosis. Both invitro and invivo assays indicated that knockdown of CCN1 in MES-GSCs reduced the tumor stemness, proliferation, invasion, and tumorigenicity, whereas CCN1 overexpression in PN-GSCs exhibited the opposite effects. Mechanistically, CCN1 triggered the FAK/STAT3 signaling in autocrine and paracrine manners to upregulate the expression of S100A8. Knockdown of S100A8 inactivated NF-κB/p65 pathway and significantly suppressed the tumorigenesis of MES-GSCs. Our findings reveal that CCN1 may be an important factor in the enhanced invasiveness and MES phenotype transition of GSCs and highlight the potential to target CCN1 for treating GBM.

Evaluation of Antibacterial Activity and Synergistic Interaction between Punica granatum peels and Synthetic Antibiotics on Multidrug-Resistant Bacteria

IntroductionAntibiotic resistance threatens global health, necessitating the development of new antimicrobial medications. Novel drugs can reverse drug resistance and revitalize the antibiotic arsenal. Therefore, this study aimed to evaluate the synergistic antibacterial effect of Punica granatum (P. granatum) extract and antibiotics against multidrug-resistant bacteria (MDR). MethodsPreliminary susceptibility profiling of the antibiotics, the antibacterial activity of the extract, and synergistic interactions between the extract and the three antibiotics were conducted using well diffusion and micro-broth dilution assays with resazurin dye. The inhibition zones (IZs), minimum inhibitory concentrations (MICs), and Fractional Inhibition Concentration Index (FICI) were determined. The data were analyzed using statistical software program (SPSS 26). ResultsThe bacterial isolates exhibited resistance to all antibiotics, except E. coli and P. aeruginosa were sensitive to Ciprofloxacin. P. granatum extract demonstrated an inhibitory effect on all tested bacteria. The FICI values for MDR strains ranged from (0.12 to 0.53), and the best synergistic effect was observed when Augmentin and Ciprofloxacin were combined against these strains. The FICI indices for the combination of P. granatum and Azithromycin ranged from (0.12 to 0.53). ConclusionThis study identified P. granatum extract as a potential adjunctive therapy to antibiotics for the treatment of multidrug-resistant bacteria.

Green coffee infusion: An endodontic elixir

Context: Irrigation is a critical component in accessing the labyrinth of the pulp space. Although traditional irrigants such as sodium hypochlorite (NaOCl) are widely employed, they exhibit several limitations, including cytotoxicity and complete microbial eradication. In contrast, phytochemicals derived from plants, including coffee, present promising alternatives due to their established antimicrobial properties. Aims: The aim of this study was to evaluate the efficacy of intracanal irrigants derived from locally cultivated Coffea arabica and Coffea canephora (Robusta) varieties, both recognized for their antimicrobial attributes. Subjects and Methods: A total of 70 extracted human single-rooted teeth were prepared. Coffee extracts were prepared through a decoction method, and the minimum inhibitory concentrations (MICs) of these extracts were determined using microbroth dilution assays. Microbial growth was quantitatively assessed at predetermined intervals. Results: Green Robusta extracts exhibited antimicrobial effectiveness at lower concentrations of 25% and 12.5%. All coffee extract groups demonstrated substantial reductions in microbial counts compared to the negative control, with Robusta extracts proving particularly efficacious. Notably, the Robusta green 12.5% extract exhibited sustained antimicrobial efficacy, suggesting its potential as an alternative to NaOCl. Conclusions: The findings of this study suggest a novel approach to improving endodontic therapy outcomes through the utilization of coffee extracts, potentially enhancing dental health.

Overcoming GABA-Induced Treg Suppression of Immunity by ABAT to Augment CD8+T Cell Antitumor Immune Response in Liver Cancer

Introduction: ABAT, a key enzyme in GABA catabolism, modulates antitumor immune activity across various cancers. However, the molecular mechanisms by which the ABAT/GABA axis exerts immune regulation in the liver cancer microenvironment remain unclear. Methods: ABAT expression in liver cancer tissues was scrutinized via the TCGA-LIHC database, and the 5-year survival rates of liver cancer patients were appraised through Kaplan-Meier survival analyses. The mRNA levels of ABAT in liver cancer cell lines were quantified by qRT-PCR. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), colony formation, and Transwell assays were employed to gauge the influence of ABAT overexpression on liver cancer cell growth, proliferation, migration, and invasion, respectively. Western blot evaluated epithelial-mesenchymal transition-related protein expression. ELISA quantified GABA, IL-10, TGF-β1, Granzyme B, and IFN-γ in the culture medium. Flow cytometry was used to measure the frequency of CD25+FOXP3+ cells and the expression of CD25, CD69, and PD-1 on CD8+T cells in coculture. Carboxifluorescein diacetate succinimidyl ester dilution assays were performed to assess the proliferative activity of CD8+T cells. Results: ABAT was found to be underexpressed in liver cancer tissues and cells, and such underexpression is indicative of a poorer prognosis for patients, while its overexpression was shown to curb the malignancy of liver cancer cells. Upon overexpression of ABAT, there is a decrease in GABA levels in the cell supernatant, coupled with an increase in IL-10 and TGF-β1 cell number, and an upsurge in the CD25+FOXP3+ cell ratios, all of which were restored by the addition of exogenous GABA. Furthermore, the depletion of ABAT led to a reduction in the proliferation and tumor-killing ability of CD8+T cells, effects that were reversed by the application of GABAA receptor inhibitors. Conclusion: ABAT functions to inhibit Treg differentiation in liver cancer through downregulation of GABA, thus promoting the antitumor activity of CD8+T cells.

In vitro Cytotoxicity of Selected Medicinal Plant Extracts used for the Management of Gastroenteritis in Northern Namibia, and their Antibacterial Activity against Multidrug-resistant Pathogens

The global challenge of antibiotic resistance among gastrointestinal pathogens is increasing daily. Gastroenteritis is a major health challenge in developing countries and is ranked 8th among the top 10 causes of death in Namibia. While medicinal plants used in Namibia could be the source of medicine for gastroenteritis, there is a gap in knowledge regarding the efficacy and safety of most medicinal plants used in Namibia. The study aimed to evaluate the antibacterial and cytotoxicity of medicinal plants used in Namibia to manage gastroenteritis. Aqueous and methanol extracts were prepared from Lantana camara, Grewia tenax, Corchorus tridens, Albizia anthelmintic, and Terminalia sericea. Agar disc diffusion and resazurin-based microtiter dilution assay were used to determine the antibacterial activity. Antibiogram assay was used to determine the susceptibility of gastrointestinal pathogens to 11 clinically used antibiotics. Normal mouse embryo fibroblast cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay were used for the cytotoxicity test. A 70% multidrug resistance to ampicillin, cephalosporin, sulphamethoxazole, erythromycin, penicillin, vancomycin, and ampicillin was observed among clinical isolates of Salmonella and Escherichia coli. Meanwhile, L. camara methanol seed extracts showed potent antibacterial activity against clinical strains of E. coli and Salmonella spp. (20.67 ± 1.53 and 21.67 ± 0.58 mm, respectively), with MICs between 31.25 and 62.5 µg/mL. The IC50 of all extracts was ≥65.85.00 ± 0.58 µg/mL. The study showed that methanol extracts of L. camara, C. tridens, and T. sericea have potent antibacterial activity against multidrug-resistant clinical Salmonella and E. coli. Moreover, methanol extracts did not show significant toxicity to NIH/3T3 cells.

Enhanced bioactivity, antimicrobial efficacy and biocompatibility of silver-doped larnite for orthopaedic applications

Bone tissue engineering is an interdisciplinary field at the forefront of regenerative medicine, aiming to develop innovative strategies for repairing and regenerating bone tissue. Biomaterials play a crucial role as it provides a supportive environment that facilitates cell attachment, proliferation, and differentiation for bone formation. The current work investigates the influence of silver doping on the physicochemical and biological properties of larnite (Ca2SiO4) for the application of bone tissue regeneration. In the current work combustion assisted sol–gel method was implemented to synthesize silver doped larnite which offers phase formation at lower temperatures. In-vitro biomineralization studies revealed that silver addition significantly improved hydroxyapatite (HAp) nucleation on the scaffold surfaces when immersed in simulated body fluid. The antibacterial studies of Ag-doped larnite powders were performed using broth dilution assay which showed bacterial inhibition up to 87 % at higher addition of silver concentrations against the clinical pathogens. The biocompatibility of the materials on human adipose-derived mesenchymal stromal cells (hAMSC’s) exhibited significant proliferation (p < 0.05) on Ca1.90Ag0.10SiO4 as compared with Ca2Ag0SiO4. The increased Ag concentration was found to have a significant influence on the antibacterial properties without affecting the biocompatibility of larnite. These findings highlight the potential of Ag-doped larnite, particularly Ca1.90Ag0.10SiO4, as a promising biomaterial for bone tissue engineering. It demonstrates excellent antibacterial efficacy while maintaining biocompatibility, addressing the critical balance between these two aspects for an optimal bone tissue regeneration.

In vitro Antiviral Activity of Eucheuma cottonii against New Castle Disease Virus

Aims: To determine the antiviral activity of Eucheuma cottonii against the New Castle disease virus. Study Design: In vitro study. Place and Duration of the Study: The research was conducted in the Pharmaceutical Chemistry, Drug Design, and Pharmacognosy department at St. John’s University of Tanzania and Virology Laboratory at Sokoine University of Agriculture between April 2022 and August 2022. Methodology: The present study used an OVO assay and hemagglutination test to investigate the In vitro antiviral activity of E. cottonii collected from Zanzibar, Tanzania, against the Newcastle disease virus (NDV). Ovo assay was assessed using 60 embryonated eggs and a hemagglutination test was performed by collecting the allantoic fluid from treated eggs and serum from hatched chicks to detect antigen titer against NDV using 96 well plate diffusion method. Results: Results of the Ovo assay showed that all the 40 embryonated eggs used as test group were alive and thus confirmed that the extract of E. cottonii (0.32 g/mL) is strong enough to destroy the virus. In which the results from the serial dilution assay (hemagglutination test) confirmed a substantial decrease in the high level of NDV as observed in the allantoic fluid of the infected embryonated eggs to range from 10^-3 to 10^-8 serial dilutions. Conclusion: The OVO assay and hemagglutination results indicate that E. cottonii possesses antiviral activity against NDV. Still further studies like isolation and characterization of bioactive compounds are needed to apply E. cottonii as an alternative source of therapy to fight against NDV.