Assay Microscopy Research Articles

High-Efficiency CRISPR-Cas9 Genome Editing Unveils Biofilm Insights and Enhances Antimicrobial Activity in Bacillus velezensis FZB42.

Bacillus velezensis FZB42 is a prominent plant growth-promoting rhizobacterium and biocontrol agent known for producing a wide array of antimicrobial compounds. The capability to genetically manipulate this strain would facilitate understanding its metabolism and enhancing its sustainable agriculture applications. In this study, we report the first successful implementation of high-efficiency CRISPR-Cas9 genome editing in B. velezensis FZB42, enabling targeted genetic modifications to gain insights into its plant growth-promotion and biocontrol properties. Deletion of the slrR gene, a key regulator of biofilm formation, resulted in significant alterations in biofilm structure and development, as demonstrated by scanning electron microscopy and quantitative biofilm assays. These findings provide valuable insights into the mechanisms of biofilm formation, which are critical for root colonization and plant growth promotion. Additionally, we overexpressed the bac gene cluster responsible for bacilysin biosynthesis by replacing its native promoter with the strong constitutive promoter P43 and integrating an additional copy of the bacG gene. This genetic manipulation led to a 2.7-fold increase in bacB gene expression and significantly enhanced antibacterial activity against Escherichia coli and Lactobacillus diolivorans. The successful implementation of the CRISPR-Cas9 system for genome editing in FZB42 provides a valuable tool for genetic engineering, with the potential to improve its biocontrol efficacy and broaden its applications in agriculture and other biotechnology areas. Our principles and procedures are broadly applicable to other agriculturally significant microorganisms.

Comprehensive safety and toxicity analysis of 2,2’-Bipyridine derivatives in combating MRSA biofilm formation and persistence

IntroductionMethicillin-resistant Staphylococcus aureus (MRSA) infections have become arduous to treat due to their capacity to form biofilms, develop persistence, and exhibit significant antimicrobial resistance. These factors contribute to the complexity of managing MRSA infections and highlight the urgent need for innovative treatment strategies.ObjectivesThis endeavor aims to evaluate the safety of 2,2’-Bipyridine (2,2’-Bipy) derivatives and their antimicrobial, anti-biofilm, and anti-persister activities in treating MRSA Infections.MethodsSix derivatives were screened for their ADMET properties and tested for minimum inhibitory concentrations against various bacterial strains using agar well diffusion and broth dilution. Safety studies were conducted through hemolysis tests, cell viability assays, and in vivo acute oral toxicity examinations. Bactericidal mechanisms and biofilm disruption effects were analyzed using crystal violet staining and confocal microscopy assays. The murine thigh infection model was also used to investigate the in vivo efficacy.ResultsAll derivatives exhibited favorable physicochemical profiles and ADMET properties and are predicted to be safe based on their drug-like properties. in vitro studies demonstrated that derivatives are non-toxic to 3T3 L1, and in vivo studies confirmed their safety in mice at a dose of 300 mg/kg and their non-hemolytic nature against rabbit red blood cells. All compounds showed potent antibacterial activity against the tested bacteria, including the resistant MRSA strain 831. They inhibited biofilm formation and eradicated biofilms in a dose-dependent manner against MTCC 737 and MRSA 831, and they effectively eliminated MRSA persister cells, outperforming the reference antibiotic vancomycin. These derivatives were found to depolarize the mitochondrial membrane and accumulate intracellular reactive oxygen species. These derivatives significantly reduced the bacterial load in the murine thigh infection model.ConclusionThe study concluded that 2,2’-Bipy derivatives possess significant antimicrobial activity, are non-toxic, and are effective in inhibiting biofilm formation and killing persister cells.

Macranthoside B Suppresses the Growth of Adenocarcinoma of Esophagogastric Junction by Regulating Iron Homeostasis and Ferroptosis through NRF2 Inhibition.

Macranthoside B (MB) is a saponin compound extracted from hon-eysuckle that has been reported to exhibit significant medicinal values, particularly anti-tumor activities. This study aimed to evaluate the anticancer efficacy of MB in treating adenocarci-noma of the esophagogastric junction (AEG) and elucidate its underlying mechanisms. Three AEG cell lines and normal gastric epithelial cells were used to assess the an-ticancer activity of MB in vitro. A series of experiments, including RNA sequencing (RNA-seq) analysis, transmission electron microscopy (TEM), immunofluorescence, and western blot assay, were conducted to validate the molecular mechanisms by which MB may mediate these physiological changes. Finally, we used shRNA assays to silence the key gene driving these changes and examined the expression of molecules involved in the affected pathways. MB exhibited significant anti-AEG cell activity with IC50 values ranging from 9.5 to 12.7 μM. RNA-seq results indicated that MB treatment in AEG cells significantly altered mRNA levels of autophagy- and ferroptosis-related genes. Further experiments revealed that MB treatment led to the up-regulation of lipid reactive oxygen species (Lip-ROS), oxidative stress-related pathway genes, and LC3B-labeled autophagic vesicles in AEG cells. Moreover, MB mediated NCOA4-dependent ferritinophagy, disrupting iron homeostasis and causing subsequent ferroptosis. We further confirmed that the intrinsic connection between autophagy and ferroptosis was due to the inhibition of NRF2 by MB. The inhibition of NRF2 by MB triggered transcriptional repression of its downstream effector molecules HERC2 and VAMP8, thus stabilizing NCOA4. This study demonstrated MB to inhibit AEG cell growth by regulating iron ho-meostasis and inducing ferroptosis through the inhibition of NRF2, providing a basis for the development of novel drugs for AEG treatment.

Extracellular vesicle-mediated VEGF-A mRNA delivery rescues ischaemic injury with low immunogenicity.

Lackluster results from recently completed gene therapy clinical trials of VEGF-A delivered by viral vectors have heightened the need to develop alternative delivery strategies. This study aims to demonstrate the pre-clinical efficacy and safety of extracellular vesicles (EVs) loaded with VEGF-A mRNA for the treatment of ischaemic vascular disease. After encapsulation of full-length VEGF-A mRNA into fibroblast-derived EVs via cellular nanoporation (CNP), collected VEGF-A EVs were delivered into mouse models of ischaemic injury. Target tissue delivery was verified by in situ analysis of protein and gene expression. Functional rescue was confirmed by in vivo imaging and histology. The safety of single and serial delivery was demonstrated using immune-based assays. VEGF-A EVs were generated with high mRNA content using a CNP methodology. VEGF-A EV administration demonstrated expression of exogenous VEGF-A mRNA by in situ RNA hybridization and elevated protein expression by western blot, microscopy, and enzyme-linked immunosorbent assay. Mice treated with human VEGF-A EVs after femoral or coronary artery ligation exhibited heightened neovascularization in ischaemic tissues with increased arterial perfusion and improvement in left ventricular function, respectively. Serial delivery of VEGF-EVs in injured skin showed improved wound healing with repeat administration. Importantly, as compared with adeno-associated viral and lipid nanoparticle VEGF-A gene therapy modalities, murine VEGF-A EV delivery did not trigger innate or adaptive immune responses at the injection site or systemically. This study demonstrated that VEGF-A EV therapy offers efficient, dose-dependent VEGF-A protein formation with low immunogenicity, resulting in new vessel formation in murine models of ischaemic vascular disease.

Bacterial Nanovesicles as Interkingdom Signaling Moieties Mediating Pain Hypersensitivity.

Gut dysbiosis contributes to multiple pathologies, yet the mechanisms of the gut microbiota-mediated influence on systemic and distant responses remain largely elusive. This study aimed to identify the role of nanosized bacterial extracellular vesicles (bEVs) in mediating allodynia, i.e., pain hypersensitivity, in a diet-induced obesity (DIO) gut dysbiosis model. bEVs were enriched from the feces of lean (bEVLean) and DIO (bEVDIO) mice by an approach combining ultracentrifugation and immunoprecipitation and then extensively analyzed for purity and bacterial characteristics. Next, bEVs were injected, either intraplantarly or intravenously, in mice to assess pain sensitivity. Fluorescence-labeled bEVs were injected in mice by enema to assess biodistribution. The effect of bEV on immune cells and inflammation was analyzed by array, immunophenotyping, microscopy, NF-κB activation, and cellular uptake assays. Results showed that bEVDIO administration in wild-type mice replicated the allodynia phenotype observed in DIO mice for both mechanical and thermal stimuli. Importantly, this effect was compromised in TRPA1/TRPV1 double-knockout mice. Biodistribution analyses showed bEV entry into systemic circulation with subsequent localization at distant sites. Multiple analyses revealed that bEVDIO exposure incited systemic inflammation, primarily through modulating the innate immune system. This inflammatory mechanism involved LPS on the bEV surface, activating TLR2- and TLR4-related pathways, as confirmed using TLR2 and TLR4 inhibitors and shaving bEV surface proteins. Interestingly, the enhanced cellular uptake of bEVDIO was contingent on interactions involving LPS and proteins on bEVs and TLR2/TLR4 on monocytes. These findings illuminate the hitherto unexplored role of bEV as pivotal mediators of allodynia and inflammation linked to gut dysbiosis.

Microbial Analysis of Vended Fruit Juices and Sauces Consumed in Restaurants in Narsingdi City, Bangladesh

Fresh fruit juices from cafes and restaurants are a popular beverage in Narsingdi city of Bangladesh during summer season. Food-borne outbreaks may arise from them since harmful germs from the unsanitary surroundings of the food preparation area easily infect them. The purpose of the current study was to identify the microbiological characteristics of various fruit juices and sauces consumed in different cafes and restaurants in Narsingdi City, Bangladesh. The detection of total viable bacterial load, total fungi count, coliforms, and some other pathogenic bacteria was checked in a total of thirty-nine juice and sauce samples, specifically of six categories of juices (orange, lemon, pineapple, sugarcane, papaya, and apple) and three categories of sauces (tomato sauce, mustard sauce, and tamarind sauce). Total viable bacteria in these samples ranged from 104 to 108 CFU/mL. Sixty percent of the samples had total coliforms (including Escherichia coli and Klebsiella spp.), all of which alarmingly exceeded the conventional bacteriological limits (1.0×104 CFU/mL) advised for fruit juices. Vibrio spp., Salmonella spp., and Staphylococcus spp. were identified as harmful microorganisms. Standard biochemical, microscopic, and cultural assays were used to identify each of these bacterial isolates. Sulfomethoxazole-trimethoprim, Ciprofloxacin, Tetracycline, Nalidixic Acid, Gentamicin, Ceftriazone, Ampicillin, and Netilmicin were among the drugs against which the isolates showed variable levels of drug resistance. The microbiological evaluation of these well-known ready-to-drink goods is therefore constantly required; otherwise, they could pose health risks.

Ultrasonic-Assisted Synthesis and Cytocompatibility Assessment of TiO2/SiO2 Nanoparticles-Impregnated Gum Arabic Nanocomposite: Edible Coating of Dates for Shelf-Life Extension.

The post-harvest management of fruit is crucial to preventing its decay and loss. Generally, edible coatings are applied to fruit to avoid decay and microbial contamination. We have used ultrasonication to synthesize TiO2 and Pennisetum glaucum residue-derived biosilica embedded in gum arabic nanocomposite. The SiO2/TiO2/gum arabic nanocomposite morphological and crystalline features were investigated using a scanning electron microscope and X-ray diffractometer, respectively. The SiO2/TiO2/gum arabic cytocompatibility was assessed using cell viability and microscopic assay. The SEM images revealed that 70-90 nm biosilica and 70-100 nm TiO2 nanostructures were present on the gum arabic. According to MTT assay and microscopic examination results, SiO2/TiO2/gum arabic do not inhibit cell viability and modulate cellular structural features; it inferred that SiO2/TiO2/gum arabic possess good cytocompatibility on human mesenchymal stem cells even up to 400 µg/mL. The date fruits were immersed in SiO2/TiO2/gum arabic-based coating mixtures and stored at 6 °C for 4 weeks. When date fruits were examined during storage, it was found that the applied coatings contributed to maintaining physicochemical features (e.g., color and texture). These findings suggest that the SiO2/TiO2/gum arabic-based coating can be applied to extend the shelf life of dates.

Mechanism of female CHH caused by compound heterozygous mutations in the LHB gene.

Luteinizing hormone (LH) plays a crucial role in the postnatal development and maturation of gonads. Inactivating mutations of the luteinizing hormone beta subunit (LHB)gene are extremely rare and can result in congenital hypogonadotropic hypogonadism (CHH). We conducted DNA sequencing on an 18-year-old female patient with undetectable LH and clinical symptoms of CHH. Pulsatile GnRH was administered to promote puberty development. In vitro construction of mutant genes, confocal microscopy, and protein functional assays were used to investigate the effects of genetic variants on hormone function and secretion. Experiments were conducted in HEK293T cells to examine the colocalization and dimerization of LH subunits, as well as to measure intracellular and extracellular LH concentrations. Compound heterozygous mutations of c.252C>G (p.F84L) and c.364G>A (p.G122S) were found in the patient's genome. Pulsatile GnRH therapy was effective in promoting puberty development and ovulation. The LH alpha subunit was found to co-localize with both mutant beta subunits after immunofluorescence staining, and immunoprecipitation detected the dimerization of the LH alpha subunit with both mutant beta subunits. Higher intracellular LH concentrations and lower extracellular LH concentrations compared to the wild type indicate secretion dysfunction for LH. Compound heterozygous mutations of c.252C>G (p.F84L) and c.364G>A (p.G122S) in the LHB gene may lead to CHH for female patient. These mutations do not impair the expression and dimerization of the alpha and beta subunits, but they do prevent the secretion of LH. The study expands our understanding of the clinical manifestation of LHB gene mutations in females and provides a treatment for these patients.

Collagen deposition in lung parenchyma driven by depletion of interstitial Lyve-1+ macrophages prevents cigarette smoke-induced emphysema and loss of airway function.

Collagen is essential for maintaining lung structure and function and its remodeling has been associated with respiratory diseases including chronic obstructive pulmonary disease (COPD). However, the cellular mechanisms driving collagen remodeling and the functional implications of this process in the pathophysiology of pulmonary diseases remain poorly understood. To address this question, we employed Lyve1wt/cre ; Csf1rflox/flox mice with specific depletion of Lyve-1+ macrophages and assessed the content, types and organization of collagen in lung compartments at steady state and after chronic exposure to cigarette smoke (CS). Using this mouse model, we found that the absence of this subpopulation of tissue resident macrophage led to the deposition of type I collagen fibers around the alveoli and bronchi at steady state. Further analysis by polarized light microscopy and Sircol collagen assay revealed that the collagen fibers accumulating in the lungs depleted of Lyve-1+ macrophages were thicker and crosslinked. A decrease in MMP-9 gene expression and proteolytic activity together with an increase in Col1a1, Timp-3 and Lox expression accompanied the collagen alterations. Next, we investigated the effect of the collagen remodeling on the pathophysiology of COPD and airway function in mice lacking Lyve-1+ macrophages exposed chronically to cigarette smoke (CS), a well-established animal model of COPD. We found that deposition of collagen prior CS exposure protected these mice against destruction of alveoli (emphysema), and bronchi thickening and prevented loss of airway function. Thus, we uncover that interstitial Lyve-1+ macrophages regulate the composition, amount, and architecture of collagen network in the lungs at steady state and that such collagen remodeling functionally impacts the development of COPD. This study further supports the potential of targeting collagen as promising approaches to treat respiratory diseases.

Kuiyangling Enema Alleviates Ulcerative Colitis Mice by Reducing Levels of Intestinal NETs and Promoting HuR/VDR Signaling.

Kuiyangling is a traditional Chinese medicine formula usedfor the treatment of ulcerative colitis, but the specific mechanism remains unclear. Imbalance in NETs regulation is one of the important factors contributing to the onset of ulcerative colitis (UC). The HuR/VDR signaling pathway plays a significant role in restoring the intestinal mucosal barrier in UC. The aim of this study is to explore the mechanism of Kuiyangling in the treatment of ulcerative colitis. A mouse model of ulcerative colitis using 3% DSS water was considered, and model, normal, Kuiyangling medium- (5 g·kg-1) and high-dose (10 g·kg-1), and mesalazine (50 mg·kg-1) groups were created. Measurements of colon length, spleen index, histopathological variances, subcellular structure observations, ROS content, and NET-related proteins (PAD4, MPO, citH3) were obtained through HE staining, electron microscopy, live imaging, and Western blotting assays. Immunohistochemistry and immunofluorescence analyses were conducted to assess the levels of HuR/VDR protein complex, ZO-1, Occludin, Claudin-7, and intestinal NETs. An ELISA kit was utilized to determine cytokine levels, LC-MS was performed to analyze the composition of Kuiyangling, and next-generation sequencing was conducted for detection of the intestinal mucosal transcriptome. Kuiyangling reduced DAI, splenic index, and ROS content; maintained mucosal structure; decreased inflammation; and increased colon length and body mass index. Western blotting indicated that Kuiyangling reduced PAD4,MPO, and citH3 levels. Kuiyangling decreased NETs and increased the expression levels of ZO-1, Occludin, and Claudin-7, as well as up-regulating HuR, VDR, and HuR/VDR proteins. Kuiyangling reduced IL-1β, IL-6, and TNF-α levels while increasing TGF-β, IL-10, and IL-37 levels. Kuiyangling reduced inflammatory response proteins and elevated the levels of anti-inflammatory and intestinal barrier proteins, possibly inhibiting the TNF and oxidative phosphorylation signaling pathways. Kuiyangling enema in treating ulcerative colitis in mice, associated with a reduction in intestinal NETs and enhancement of HuR-mediated intestinal barrier signaling pathways.

Electrospun Polycaprolactone-Curcumin Scaffolds: Optimization of fiber production for enhanced Nanotopography and improved biological cell adhesion

Curcumin, a natural polyphenolic compound with well-documented anti-inflammatory, antioxidant, and anticancer properties, has gained attention for its potential in tissue regeneration and other biomedical applications. Despite this, the integration of curcumin into polymeric scaffolds remains challenging due to its hydrophobic nature and stability concerns. This study aims to overcome these limitations by developing and characterizing curcumin-loaded polycaprolactone (PCL) scaffolds through electrospinning, a technique selected based on a comprehensive review of recent advances in the field. In this work, PCL scaffolds were fabricated with curcumin concentrations of 5, 10, and 15 mg and analyzed using advanced microscopy, spectroscopy, thermogravimetry, mechanical testing, and biological assays. Microscopy revealed that increasing curcumin concentrations improved fiber diameter uniformity and distribution. Raman spectroscopy confirmed the homogeneous incorporation of curcumin within the scaffolds, showing that up to 10 mg, the electrospinning process induces a transition of curcumin from its di-keto to its more reactive keto-enol form. This transformation facilitates hydrogen bonding with the PCL matrix, enhancing the scaffolds’ mechanical properties.In vitro cytotoxicity assays demonstrated high cell viability across all scaffolds after 24 and 72 h. Furthermore, adhesion studies with fibroblast BJ-1 cells indicated significantly improved cell adhesion on curcumin-loaded scaffolds compared to pure PCL. These findings highlight the biocompatibility and bioactivity of curcumin-loaded PCL scaffolds.This study examines the possible use of curcumin’s controlled integration into PCL scaffolds for tissue regeneration applications. The innovative approach detailed here offers a scalable and effective pathway for the development of biomaterials that combine mechanical strength, bioactivity, and biocompatibility, addressing a critical need in tissue engineering.

The Role of Polo-Like Kinase 1 (PLK1) O-GlcNAcylation in Mitosis.

Polo-like kinase 1 (PLK1) is a crucial mitotic kinase that is implicated in various aspects of cell cycle. Many post-translational modifications have been identified on PLK1 to regulate its activation, stability, and localization. PLK1 has been shown previously to colocalize with the O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT), and OGT regulates PLK1 stability. In our recent work, we show that PLK1 is O-GlcNAcylated by click chemistry. Using stepped collisional energy/higher energy collision dissociation mass spectrometry, we mapped the PLK1 O-GlcNAc site to be T291. We further utilized fluorescent activated cell sorting and time-lapse microscopy to assess the mitotic defects of PLK1 O-GlcNAc mutants. In vivo studies in mouse xenograft demonstrated that it promoted uterine cancer tumorigenesis. In this chapter, we delineate the methodologies we used in studying PLK1 O-GlcNAcylation, including click chemistry, stepped collisional energy/higher energy collision dissociation mass spectrometry, fluorescent activated cell sorting, time-lapse microscopy, and mouse xenograft assays.

Endothelial cell heterogeneity in arteriolar annuli: Implications in retinal blood flow regulation

Aims/Purpose: To investigate the endothelial cell heterogeneity in retinal arterioles with special focus in arteriolar annuli.Methods: We compared structural and molecular characteristics between endothelial cells located in arteriolar annuli and endothelial cells located in other regions of retinal arterioles. Immunohistochemical, histochemical, and electron transmission microscopy assays were performed.Results: Molecular analysis showed that arteriolar annuli endothelial cells showed a decreased expression of PECAM‐1 together with an increased expression of NADPH‐diaphorase in comparison with other regions of retinal arterioles. PECAM‐1 is involved cell‐cell adhesion. Therefore, this result suggests a decrease in cellular adhesion at the arteriolar annuli. NADPH diaphorase reflects nitric oxide synthase activity. Thus, its increase indicates NO synthesis suggesting localized vasodilation and decrease of platelet aggregation at arteriolar annuli. An increased expression of von Willebrand factor in concomitance with a higher presence of Weibel‐Palade bodies was also observed in endothelial cells at the arteriolar annuli, suggesting that von Willebrand factor is stored and released in a regulated manner. Furthermore, transmission electron microscopy showed the presence of two endothelial cell subtypes in arteriolar annuli: activated endothelial cells, and shear stress‐adapted endothelial cells. Activated endothelial cells exhibited enlarged nuclei with chromatin condensed in the periphery, swollen perinuclear space, and an increased number of vesicles and caveolae. By contrast, shear stress‐adapted endothelial cells were thicker than the rest of endothelial cells and showed bilobulated nuclei, a well‐developed endoplasmic reticulum, as well as large increase in the number of mitochondria.Conclusions: All our results suggest that endothelium at arteriolar annuli, a structure suggested to regulate retinal blood flow by exerting a sphincter‐like activity, is formed by a subset of endothelial cells endowed with distinctive structural and molecular features which allow them to play a key role in the maintenance of blood flow through two mechanisms: the establishment of an antithrombotic milieu, and the vasodilation of arteriolar annuli.

TEFM facilitates uterine corpus endometrial carcinoma progression by activating ROS-NFκB pathway

BackgroundMitochondrial transcription elongation factor (TEFM) is a recently discovered factor involved in mitochondrial DNA replication and transcription. Previous studies have reported that abnormal TEFM expression can disrupt the assembly of mitochondrial respiratory chain and thus mitochondrial function. However, the role of TEFM on Uterine corpus endometrial carcinoma (UCEC) progression remains unclear. The present study aims to investigate the expression of TEFM in tumor tissue of UCEC and the effect of abnormal TEFM expression on malignant phenotype of UCEC cells.MethodsThe expressions of TEFM were measured in tumor tissues and cell lines of UCEC by immunohistochemistry, Western blotting, and real-time quantitative PCR assays. Besides, the effects of TEFM knockdown or overexpression on UCEC cell growth, metastasis, apoptosis, and autophagy were also determined using EdU, colony formation, flow cytometry, TUNEL, and transmission electron microscopy assays. Xenograft model was used to confirm the role of TEFM on proliferative potential of UECE cells in vivo.ResultsOur bioinformatics analysis of CPTAC data showed that TEFM is abnormally overexpressed in UCEC and its upregulation was significantly associated with poor survival of patients with UCEC. We found that TEFM upregulation significantly promoted the growth and metastasis of UCEC cells. Mechanically, TEFM upregulation impaired the function of mitochondria, decreased their membrane potential and activated the AKT-NFκB pathway by promoting reactive oxygen species (ROS) production, leading to enhanced intracellular autophagy and thus UCEC growth and metastasis.ConclusionThis study demonstrates that TEFM positively regulates autophagy to promote the growth and metastasis of UCEC cells, which provides a potential prognostic biomarker and therapeutic target for the treatment of UCEC.Graphical

PEGylated iron oxide-gold core-shell nanoparticles for tumor-targeted delivery of Rapamycin.

Rapamycin analogs are approved by the FDA for breast and renal cancer treatment. Hence, the possibility of nanoparticle-mediated delivery ofRapamycin could be examined. In the present study, PEGylated Gold-core shell iron oxide nanoparticles were used for the targeted delivery of Rapamycin, and R-Au-IONPs were formulated. SEM, XRD, and FTIR determined the smooth spherical morphology, andcompositional structure, and confirmed the conjugation of Rapamycin onto the NPs. The in vitro drug release study showed acontrolled release of the drug over time. R-Au-IONPs showed significant cytotoxicity in MCF 7 cells. Anti-proliferative assays such as trypan blue dye exclusion assay, microscopy, Fluorescent staining, and clonogenic assays were performed. NH staining, Rhodamine 123 staining, PS externalization, and the cleavage of PARP protein by western immunoblot assays confirmed the induction of apoptosis. The mechanism of R-Au-IONP-induced cell death was analyzed by flow cytometry. Our in-vitro study, on the impact of R-Au-IONPs on cell viability in the human breast adenocarcinoma cell line (MCF-7), confirms the efficacy of drug delivery using the nanoparticle system. Further results implied the induction of apoptosis. This drug delivery system using Rapamycin could be a potential candidate in the treatment of breast cancer.

Development of a hydrogel-based three-dimensional (3D) glioblastoma cell lines culture as a model system for CD73 inhibitor response study

BackgroundDespite the development of various therapeutic approaches over the past decades, the treatment of glioblastoma multiforme (GBM) remains a major challenge. The extracellular adenosine-generating enzyme, CD73, is involved in the pathogenesis and progression of GBM, and targeting CD73 may represent a novel approach to treat this cancer. In this study, three-dimensional culture systems based on three hydrogel compositions were characterized and an optimal type was selected to simulate the GBM microenvironment. In addition, the effect of a CD73 inhibitor on GBM cell aggregates and spheroids was investigated as a potential therapeutic approach for this disease.MethodsRheology measurements, Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and cell proliferation assays were performed to analyze the synthesized hydrogel and select an optimal formulation. The viability of tumor cells in the optimal hydrogel was examined histologically and by confocal microscopy. In addition, the sensitivity of the tumor cells to the CD73 inhibitor was investigated using a cell proliferation assay and real-time PCR.ResultsThe data showed that the hydrogel containing 5 wt% gelatin and 5 wt% sodium alginate had better rheological properties and higher cell viability. Therefore, it could provide a more suitable environment for GBM cells and better mimic the natural microenvironment. GBM cells treated with CD73 inhibitors significantly decreased the proliferation rate and expression of VEGF and HIF1-α in the optimal hydrogel.ConclusionOur current research demonstrates the great potential of CD73 inhibitor for clinical translation of cancer studies by analyzing the behavior and function of 3D tumor cells, and thus for more effective treatment protocols for GBM.

Microscopic phage adsorption assay: High-throughput quantification of virus particle attachment to host bacterial cells

Phages, viruses of bacteria, play a pivotal role in Earth's biosphere and hold great promise as therapeutic and diagnostic tools in combating infectious diseases. Attachment of phages to bacterial cells is a crucial initial step of the interaction. The classic assay to quantify the dynamics of phage attachment involves coculturing and enumeration of bacteria and phages, which is laborious, lengthy, hence low-throughput, and only provides ensemble estimates of model-based adsorption rate constants. Here, we utilized fluorescence microscopy and particle tracking to obtain trajectories of individual virus particles interacting with cells. The trajectory durations quantified the heterogeneity in dwell time, the time that each phage spends interacting with a bacterium. The average dwell time strongly correlated with the classically measured adsorption rate constant. We successfully applied this technique to quantify host-attachment dynamics of several phages including those targeting key bacterial pathogens. This approach should benefit the field of phage biology by providing highly quantitative, model-free readouts at single-virus resolution, helping to uncover single-virus phenomena missed by traditional measurements. Owing to significant reduction in manual effort, our method should enable rapid, high-throughput screening of a phage library against a target bacterial strain for applications such as therapy or diagnosis.

Tetrahydrofolic acid accelerates amyloid fibrillization, decreases cytotoxic oligomers and suppresses their toxicity.

Soluble cytotoxic oligomers produced during the fibrillation of both α-synuclein (αS) and amyloid-β protein (Aβ) are key pathogenic factors in Parkinson's disease (PD) and Alzheimer's disease (AD). Reducing toxic oligomers by regulating the aggregation process of αS and Aβ is an important strategy for the treatment of PD and AD. Herein, tetrahydrofolic acid (THF) is found to accelerate amyloid fibrillization, decreases cytotoxic oligomers and suppresses their toxicity. Thioflavin T and atomic force microscopy assays results showed that THF was able to accelerate the formation of dense fibrils from αS and Aβ in a dose-dependent manner. Strikingly, this was accompanied by a reduction in the abundance of toxic oligomers, and these results were confirmed by DB. Meanwhile, MTT and FDA/PI assays demonstrated that THF-induced accelerated fibril formation was accompanied by a reduction in αS- and Aβ-induced cytotoxicity. In addition, the lifespan of genetically modified αS and Aβ expressing C. elegans was extended by feeding THF, although plaque deposits of αS and Aβ increased. These findings suggest that THF enhances the conversion of αS and Aβ oligomers into less toxic fibrils and is a potential therapeutic agent for PD and AD.

Comparison of fine-scale malaria strata derived from population survey data collected using RDTs, microscopy and qPCR in South-Eastern Tanzania

BackgroundMalaria-endemic countries are increasingly adopting data-driven risk stratification, often at district or higher regional levels, to guide their intervention strategies. The data typically comes from population-level surveys collected by rapid diagnostic tests (RDTs), which unfortunately perform poorly in low transmission settings. Here, a high-resolution survey of Plasmodium falciparum prevalence rate (PfPR) was conducted in two Tanzanian districts using rapid diagnostic tests (RDTs), microscopy, and quantitative polymerase chain reaction (qPCR) assays, enabling the comparison of fine-scale strata derived from these different diagnostic methods.MethodsA cross-sectional survey was conducted in 35 villages in Ulanga and Kilombero districts, south-eastern Tanzania between 2022 and 2023. A total of 7,628 individuals were screened using RDTs (SD-BIOLINE) and microscopy, with two thirds of the samples further analysed by qPCR. The data was used to categorize each district and village as having very low (PfPR < 1%), low (1%≤PfPR < 5%), moderate (5%≤PfPR < 30%), or high (PfPR ≥ 30%) parasite prevalence. A generalized linear mixed model was used to analyse infection risk factors. Other metrics, including positive predictive value (PPV), sensitivity, specificity, parasite densities, and Kappa statistics were computed for RDTs or microscopy and compared to qPCR as reference.ResultsSignificant fine-scale variations in malaria risk were observed within and between the districts, with village prevalence ranging from 0% to > 50%. Prevalence varied by testing method: Kilombero was low risk by RDTs (PfPR = 3%) and microscopy (PfPR = 2%) but moderate by qPCR (PfPR = 9%); Ulanga was high risk by RDTs (PfPR = 39%) and qPCR (PfPR = 54%) but moderate by microscopy (PfPR = 26%). RDTs and microscopy classified majority of the 35 villages as very low to low risk (18–21 villages). In contrast, qPCR classified most villages as moderate to high risk (29 villages). Using qPCR as the reference, PPV for RDTs and microscopy ranged from as low as < 20% in very low transmission villages to > 80% in moderate and high transmission villages. Sensitivity was 62% for RDTs and 41% for microscopy; specificity was 93% and 96%, respectively. Kappa values were 0.7 for RDTs and 0.5 for microscopy. School-age children (5–15 years) had higher malaria prevalence and parasite densities than adults (P < 0.001). High-prevalence villages also had higher parasite densities (Spearman r = 0.77, P < 0.001 for qPCR; r = 0.55, P = 0.003 for microscopy).ConclusionThis study highlights significant fine-scale variability in malaria burden within and between the study districts and emphasizes the variable performance of the testing methods when stratifying risk at local scales. While RDTs and microscopy were effective in high-transmission areas, they performed poorly in low-transmission settings; and classified most villages as very low or low risk. In contrast, qPCR classified most villages as moderate or high risk. The findings emphasize that, where precise mapping and effective targeting of malaria are required in localized settings, tests must be both operationally feasible and highly sensitive. Furthermore, when planning microstratification efforts to guide local control measures, it is crucial to carefully consider both the strengths and limitations of the available data and the testing methods employed.

Novel homozygous SPAG17 variants cause human male infertility through multiple morphological abnormalities of spermatozoal flagella related to axonemal microtubule doublets.

Male infertility can result from impaired sperm motility caused by multiple morphological abnormalities of the flagella (MMAF). Distinct projections encircling the central microtubules of the spermatozoal axoneme play pivotal roles in flagellar bending and spermatozoal movement. Mammalian sperm-associated antigen 17 (SPAG17) encodes a conserved axonemal protein of cilia and flagella, forming part of the C1a projection of the central apparatus, with functions related to ciliary/flagellar motility, skeletal growth, and male fertility. This study investigated two novel homozygous SPAG17 mutations (M1: NM_206996.2, c.829+1G>T, p.Asp212_Glu276del; and M2: c.2120del, p.Leu707*) identified in four infertile patients from two consanguineous Pakistani families. These patients displayed the MMAF phenotype confirmed by Papanicolaou staining and scanning electron microscopy assays of spermatozoa. Quantitative real-time polymerase chain reaction (PCR) of patients' spermatozoa also revealed a significant decrease in SPAG17 mRNA expression, and immunofluorescence staining showed the absence of SPAG17 protein signals along the flagella. However, no apparent ciliary-related symptoms or skeletal malformations were observed in the chest X-rays of any of the patients. Transmission electron microscopy of axoneme cross-sections from the patients showed incomplete C1a projection and a higher frequency of missing microtubule doublets 1 and 9 compared with those from fertile controls. Immunofluorescence staining and Western blot analyses of spermatogenesis-associated protein 17 (SPATA17), a component of the C1a projection, and sperm-associated antigen 6 (SPAG6), a marker of the spring layer, revealed disrupted expression of both proteins in the patients' spermatozoa. Altogether, these findings demonstrated that SPAG17 maintains the integrity of spermatozoal flagellar axoneme, expanding the phenotypic spectrum of SPAG17 mutations in humans.