Counting Assay Research Articles

Gene Expression Profiling Regulated by lncRNA H19 Using Bioinformatic Analyses in Glioma Cell Lines.

Glioma, the most common type of primary brain tumor, is characterized by high malignancy, recurrence, and mortality. Long non-coding RNA (lncRNA) H19 is a potential biomarker for glioma diagnosis and treatment due to its overexpression in human glioma tissues and its involvement in cell division and metastasis regulation. This study aimed to identify potential therapeutic targets involved in glioma development by analyzing gene expression profiles regulated by H19. To elucidate the role of H19 in A172 and U87MG glioma cell lines, cell counting, colony formation, and wound healing assays were conducted. RNA-seq data analysis and bioinformatics analyses were performed to reveal the molecular interactions of H19. Cell-based experiments showed that elevated H19 levels were related to cancer cell survival, proliferation, and migration. Bioinformatics analyses identified 2,084 differentially expressed genes (DEGs) influenced by H19 which are involved in cancer progression. Specifically, ANXA5, CLEC18B, RAB42, CXCL8, OASL, USP18, and CDCP1 were positively correlated with H19 expression, while CSDC2 and FOXO4 were negatively correlated. These DEGs were predicted to function as oncogenes or tumor suppressors in gliomas, in association with H19. These findings highlight H19 and its associated genes as potential diagnostic and therapeutic targets for gliomas, emphasizing their clinical significance in patient survival.

Development of phage-containing hydrogel for treating Enterococcus faecalis-infected wounds.

Chronic wound infections caused by Enterococcus faecalis pose formidable challenges in clinical management, exacerbated by the emergence of vancomycin-resistant strains. Phage therapy offers a targeted approach but encounters delivery hurdles. Due to their biocompatibility and controlled release properties, hydrogels hold promise as carriers. This study aimed to fabricate phage-containing hydrogels using sodium alginate (SA), carboxymethyl cellulose (CMC), and hyaluronic acid (HA) to treat E. faecalis-infected wounds. We assessed the efficacy of these hydrogels both in vitro and in vivo. The hydrogel was prepared using SA-CMC-HA polymers. Phage SAM-E.f 12 was incorporated into the SA-CMC-HA hydrogel. The hydrogel's swelling index was measured after 24 h, and degradation was assessed over seven days. Surface morphology and composition were analyzed using Scanning Electron Microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR). Antibacterial activity was tested via optical density (OD) and disk diffusion assays. Phage release and stability were evaluated over a month. In vivo efficacy was tested in mice through wound healing and bacterial count assays, with histopathological analysis. Hydrogels exhibited a swelling index of 0.43, a water absorption rate of %30, and 23% degradation over seven days. FTIR confirmed successful polymer incorporation. In vitro studies demonstrated that phage-containing hydrogels significantly inhibited bacterial growth, with an OD of 0.3 compared to 1.1 for the controls. Hydrogels remained stable for four weeks. In vivo, phage-containing hydrogels reduced bacterial load and enhanced wound healing, as shown by improved epithelialization and tissue restoration. Phage-containing hydrogels effectively treat wounds infected with E. faecalis-infected wounds, promoting wound healing through controlled phage release. These hydrogels can improve clinical outcomes in the treatment of infected wounds.

Susceptibility of HPV-18 Cancer Cells to HIV Protease Inhibitors.

Cervical cancer cases continue to rise despite all the advanced screening and preventative measures put in place, which include human papillomavirus (HPV) vaccination. These soaring numbers can be attributed to the lack of effective anticancer drugs against cervical cancer; thus, repurposing the human immunodeficiency virus protease inhibitors is an attractive innovation. Therefore, this work was aimed at evaluating the potential anticancer activities of HIV-PIs against cervical cancer cells. The MTT viability assay was used to evaluate the effect of HIV protease inhibitors on the viability of cervical cancer cells (HeLa) and non-cancerous cells (HEK-293). Further confirmation of the MTT assay was performed by confirming the IC50s of these HIV protease inhibitors on cervical cancer cells and non-cancerous cells using the Muse™ Count and Viability assay. To confirm the mode of death induced by HIV protease inhibitors in the HPV-associated cervical cancer cell line, apoptosis was performed using Annexin V assay. In addition, the Muse™ Cell Cycle assay was used to check whether the HIV protease inhibitors promote or halt cell cycle progression in cervical cancer cells. HIV protease inhibitors did not affect the viability of non-cancerous cells (HEK-293), but they decreased the viability of HeLa cervical cancer cells in a dose-dependent manner. HIV protease inhibitors induced apoptosis in HPV-related cervical cancer cells. Furthermore, they also induced cell cycle arrest, thus halting cell cycle progression. Therefore, the use of HIV drugs, particularly HIV-1 protease inhibitors, as potential cancer therapeutics represents a promising strategy. This is supported by our study demonstrating their anticancer properties, notably in HPV-associated cervical cancer cell line.

Exosomes with Engineered Brain Derived Neurotrophic Factor on Their Surfaces Can Proliferate Menstrual Blood Derived Mesenchymal Stem Cells: Targeted Delivery for a Protein Drug.

Despite the efficacy of brain derived neurotrophic factor (BDNF) in neuro-regenerative medicine, it can't pass the blood-brain barrier. Recently, exosomes have been harnessed for targeted delivery of therapeutics into brain. Given these facts, an engineered exosome capable of BDNF expression on the surface would be an amenable tool for drug delivery. The BDNF gene was cloned into a plex-lamp lentiviral vector and virus particles were packaged using the Torano method. HEK293T cells were transduced by the purified viruses to produce and purify recombinant exosomes displaying the fusion protein on their surfaces. Western blot, Zeta sizer, TEM, and ELISA methods were used for exosome characterization. The effect of engineered exosomes on menstrual blood-derived mesenchymal stem cells (Mens-MSCs) proliferation was evaluated by cell counting assay, MTT assay, and qPCR on the bcl2 and nestin genes. Approximately, 1.8 × 108 TdU/ml of the viral particles was purified from the transfected cells and transduced into the HEK293T. Western blot and ELISA methods confirmed the surface display of the LAMP-BDNF fusion. TEM graphs and Zeta sizer results confirmed the morphology and the size of purified exosomes. Treatment of Mens-MSCs with the targeted exosomes augmented the expression level of bcl2 and nestin genes, increased the cell proliferation, and elevated the cell number. Chimeric BDNF on the exosome surface could retain its biological activity and elevate the expression of bcl2 and nestin genes. Moreover, these exosomes are capable of elevating the Mens-MSCs proliferation.

8307 Molecularly Targeted Therapy in Oncogene-Driven Human Thyroid Cancer Cell Models: Assay Selection and Response Assessment

Abstract Disclosure: A.T. Yang: None. T.W. Laetsch: Consulting Fee; Self; Cellectis, Deciphera, GentiBio, Jazz Pharmaceuticals, Jumo Health, MassiveBio, Novartis Pharmaceuticals, Pyramid Biosciences, Y-mAbs Therapeutics, Advanced Microbubbles, AI Therapeutics, Bayer, Inc.. A.T. Franco: None. Background: The most common types of pediatric thyroid cancer (TC) demonstrate kinase-activating genomic alterations that are clinically inhibitor responsive – however, optimal integration of molecular therapy into practice remains uncertain. Human TC cell models can facilitate evaluation of combination therapies. However, prior studies vary from using traditional cell counts to newer reaction-based assays; we assessed whether a linear relationship between cell count and alternative assays exist for human TC cell models. Methods: Standardized curves for Cell TiterGlo (CTG; measures total ATP using luciferase-based luminescence) and NucBlue (DAPI; binds to DNA with nuclear fluorescence) were generated using TC cells seeded at escalating concentrations (5k to 300k cells/well). A linear equation was fitted to the CTG data and used to estimate cell count. DAPI counts were obtained with EVOS automation. Human TC cell lines TPC1 (RET fusion) / CUTC5 (BRAF V600E) were seeded at low (1.5k/2k) and high (3k/4k) cells per well, exposed for 3 days to 0-256 nM selpercatinib (RET inhibitor) or 0-192 nM dabrafenib (BRAF inhibitor), respectively, and response was measured using CTG and DAPI. All were measured in triplicate. Results: Standardized curves of CTG and DAPI demonstrated a 9-23% difference between known and measured cell counts. CTG was accurate within 20k-40k cells/well; DAPI showed linearity within 10k-80k cells/well. TC cells exhibited different IC50 depending on the assay and seeding concentration. Prior studies for selpercatinib and dabrafenib reported in vitro IC50 of 0.4-67 nM and 0.5-6 nM, respectively. In this study, IC50 for CTG/DAPI were closer at higher cell counts: TPC1 was 6.1 / 8.2 nM respectively; CUTC5 was 10.6 / 5.1 nM. Wells seeded at lower counts demonstrated larger inter-assay difference: IC50 for TPC1 was 10.1 / 5.9 nM respectively; CUTC5 was 38.4 / 5.9 nM. Conclusion: IC50 values varied by cell seeding concentrations and assay used, with a two to six fold difference at lower seeding and loss of linearity between luminescence measurements and cell count. This variability complicates evaluating the concordance among data from different laboratories, and in vitro and clinical response. Alternative assays intended to assess viability of inhibitor-exposed human TC cell models may require additional evaluation to ensure measured values demonstrate the expected relationship to the intended measure (ie cell number) and ultimately clinical outcome. Presentation: 6/3/2024

A-367 Development of a Multimodal Point-of-Care Diagnostic Platform to Perform Rapid, Accurate, and Cost-Effective Molecular, Immunochemistry, Clinical Chemistry, and Cytometry Tests

Abstract Background Access to point-of-care (POC) testing can make a significant impact on the quality of care. However, because POC diagnostic platforms available today are expensive and only perform one type of test, clinicians are unable to implement all the POC tests they wish to perform. Fluxergy is developing a multimodal POC diagnostic platform that consolidates diverse diagnostic modalities into a single, compact platform to minimize the cost of POC testing and standardize testing workflow for the most common clinical chemistry, hematology, immunochemistry and molecular tests. This study describes feasibility studies performed to benchmark these assay modalities on the Fluxergy Analyzer. Methods Fluxergy Analyzer: The Fluxergy Analyzer integrates conventional detection systems—colorimetric, fluorometric, and electrochemical—into a singular POC platform. This unique design expands -omics testing capabilities, including molecular, immunochemistry, clinical chemistry, and hematology within a single instrument. Benchmarking Feasibility Studies: Molecular benchmarking was performed using a multiplex respiratory panel on contrived samples in UTM/VTM. Immunochemistry benchmarking was performed using a procalcitonin assay on spiked sodium citrate plasma samples. Cytometry benchmarking was performed using a white blood cell count (WBC) assay and testing contrived samples whole blood samples. Results For molecular testing, the Fluxergy Analyzer was able to accurately detect flu A, flu B, SARS-CoV-2, and RSV with agreements with the Luminex ARIES® Flu A/B & RSV Assay and Cepheid Xpert® SARS-CoV-2 test results. For immunochemistry testing, the Fluxergy Analyzer accurate quantified PCT levels at 0.3, 0.6, 1.25 and 2.5 ng/mL. For hematology testing, the Fluxergy Analyzer demonstrated a strong correlation across the reportable range against the standard of care HemoCue analyzer. For clinical chemistry testing, the Fluxergy Analyzer demonstrated a strong correlation against the Siemen’s epoc® Blood Analysis System. Conclusions The Fluxergy Analyzer is uniquely suited to offer a comprehensive menu of the most common POC tests on a single, cost-effective platform.

The Potential of Human Pulmonary Mesenchymal Stem Cells as Vectors for Radiosensitizing Metallic Nanoparticles: An In Vitro Study.

Metallic nanoparticles (NPs) exhibit interesting radiosensitizing effects, and finding a way to accurately deliver them appears to be crucial. Due to their tumor tropism, mesenchymal stem cells (MSCs) represent a strategic approach. Therefore, we aimed to evaluate the impact of core-shell Fe3O4@Au NPs on the functionality of human pulmonary MSCs (HPMSCs). The results showed that 100 µg/mL Fe3O4@Au NPs, accumulated in HPMSCs (revealed by Prussian blue staining), did not alter cell viability as assessed by cell counting, MTT, and LDH assays. However, caspase 9 and Bcl2 gene expression, evaluated by RT-qPCR, was regulated 72 h after exposure to the NPs. Moreover, the NPs also decreased proinflammatory cytokine/chemokine secretions, except for CXCL8 (ELISA). These modulations were associated with the downregulation of AMPK gene expression at 24 h. In contrast, the NPs did not modulate VEGF, PI3K, or PDGF gene expression. Nevertheless, a decrease in VEGF secretion was observed after 24 h of exposure to the NPs. Interestingly, the Fe3O4@Au NPs did not modulate Nrf2 gene expression, but they did regulate the expression of the genes encoding Nox4 and HMOX-1. Additionally, the NPs increased ROS production, suggesting a redox imbalance. Finally, the Fe3O4@Au NPs did not affect the HPMSCs' viability or proangiogenic/tumorigenic markers. These findings are encouraging for investigating the effects of Fe3O4@Au NPs delivered by HPMSCs to tumor sites in combination with radiation.

Bacteriophage P2-71: a promising therapeutic against multidrug-resistant Proteus mirabilis in urinary tract infections.

Proteus mirabilis is a Gram-negative, rod-shaped bacterium widely found in natural environments. It is known for causing a range of severe illnesses in mammals, particularly urinary tract infections (UTIs). This study evaluates the therapeutic efficacy of phage P2-71 against Proteus mirabilis in vivo and in vitro environments. The in vitro therapeutic potential of bacteriophage P2-71 was assessed through the ability of phage to kill Proteus mirabilis by using a plate counting assay, and biofilm inhibition and biofilm lysis assays using a microtitre plate method. Additionally, an in vivo UTI model in C57BL/6Jmice was developed via urethral inoculation of the bacterium. Phage therapy was administered through urethral injection over a period of 5 days. Therapeutic outcomes were measured by analyzing bacterial load, phage titer, inflammatory markers, and histopathological changes in the urine, urogenital tissues, and spleen. In vitro, bacteriophage P2-71 achieved significant reductions in P. mirabilis concentrations, with log reductions of 1.537 and 0.7009 CFU/mL in laboratory and urine environments, respectively (p < 0.001). The phage also decreased biofilm formation by 34-49% and lysed 15-25% of mature biofilms at various multiplicities of infection (MOIs) (p < 0.001). In vivo, phage treatment significantly lowered bacterial concentrations in the urine on Days 1 and 3 (p < 0.0001), achieving a maximum reduction of 4.602 log₁₀ CFU/mL; however, its effectiveness diminished by Day 5 (p > 0.05). Concurrently, phage titers decreased over time. Importantly, phage treatment notably reduced bacterial load in the bladder, kidneys, and spleen (p < 0.001). Inflammatory markers such as IL-6, IL-1β, and TNF-α were significantly lower in the treatment group, especially in the bladder (p < 0.0001), indicating an effective reduction in inflammation. Histopathological analysis showed significant mitigation of tissue damage. The results demonstrated that bacteriophage P2-71 is a promising alternative therapy for UTIs caused by MDR Proteus mirabilis. This bacteriophage therapy offers a viable strategy for managing infections where traditional antimicrobials fail, highlighting its potential in clinical applications.

Compressive properties and biocompatibility of additively manufactured lattice structures by using bioactive materials

Porous bioactive materials were widely used in orthopedic implant fields because of their excellent mechanical properties and porous spaces. However, most porous types are predominantly stacked in two-dimensional configurations, which significantly limits their mechanical property range and adversely affects the modulus matching between the porous implants and surrounding bone tissues. Hence, various lattice structures were prepared using 3D printing technology with bioactive materials, and characterized by mechanical and biological tests. Numerical simulations were conducted to analyze the effect of relative density and geometric parameters on the equivalent compressive properties of the lattice structures. The results showed that the lattice structure exhibited a broad elastic modulus range, which can be adjusted to align with the mechanical properties of human cortical and cancellous bones, thereby helping to mitigate stress shielding in orthopedic implants. The biocompatibility of the 3D-printed solid materials was assessed in vitro using a cell counting assay kit-8 (CCK-8). The results indicated that poly-ether-ether-ketone (PEEK), carbon fiber reinforced PEEK (CFR/PEEK), nylon, and titanium (Ti) alloy all exhibited good biocompatibility, with no significant differences observed among the four materials. This study further enhances the understanding of bioactive lattice structures in the biomedical field and offers new possibilities for orthopedic repair.

A Facile and Green Approach for the Preparation of Silver Nanoparticles on Graphene Oxide with Favorable Antibacterial Activity.

Herein, we introduce a simple precipitation method for preparing graphene oxide-silver nanoparticle (GO/AgNP) composites, utilizing Calendula officinalis (C. officinalis) seed extract as both a reducing and stabilizing agent. Our research combines the sustainable preparation of graphene oxide (GO) with the green synthesis of silver nanoparticles (AgNPs), aiming to explore the potential of the obtained composite as a novel antibacterial material. To establish a benchmark, the synthesis was also performed using sodium citrate, a conventional reducing agent. The resultant GO/AgNP composites were characterized through several analytical techniques, including scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), energy dispersive X-ray spectroscopy (EDS), Raman spectroscopy, X-ray diffraction (XRD), infrared (IR) spectroscopy, and ultraviolet-visible (UV-vis) spectroscopy, confirming the successful functionalization of GO with AgNPs. The antibacterial effectiveness of the composites was systematically assessed against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), with nanoparticle concentrations spanning from 0 to 250 µg/mL, utilizing mostly disk diffusion and colony-forming unit (CFU) count assays. The AgNPs were characterized by a size range of 15-50 nm. Notably, the GO/AgNP composite prepared using C. officinalis seed extract demonstrated superior antibacterial activity at all tested concentrations, outperforming both pure GO and the GO/AgNP composite prepared with sodium citrate. The most pronounced antibacterial effect was observed at a concentration of 32.0 µg/mL. Therefore, this innovative synthesis approach may offer a valuable contribution to the development of new therapeutic agents to combat bacterial infections, suggesting further exploration into antibacterial coatings or potential drug development.

Antimicrobial properties of the edible pink oyster mushroom, Pleurotus eous: In-vivo and in-vitro studies

In recent times, there has been a notable surge in the investigation of new antibiotic substances derived from natural origins. Pleurotus eous is an edible mushroom that has various useful bioactive substances and therapeutic properties, including antimicrobial activity. The present study aims to evaluate the antimicrobial efficacy of the methanolic extract of P. eous (MEPE) through in vitro method. Notably, S. aureus demonstrated the highest susceptibility to MEPE, prompting further investigation into its antibacterial mechanisms via scanning electron microscopy (SEM), membrane integrity, and permeability assays. The in-vivo antibacterial effect of MEPE against S. aureus was also assessed, including analysis of bacterial burden in organs, hematological profiles, and cytokine profiles. Detailed phytochemical analyses of MEPE were conducted using GC-MS. Results revealed MEPE's significant (p < 0.05) efficacy against Gram-positive bacteria, particularly S. aureus (77.56 ± 0.4 μg/mL and 34 ± 6.9 μg/ml in turbidometric and viable cell count assays, respectively). Moreover, membrane permeability significantly increased in 60.32 % of S. aureus isolates following treatment with MEPE. Additionally, mice receiving MEPE exhibited decreased levels of TNF-α, IL-1β, and IL-6, suggesting its potential in combating S. aureus infection in animal models.

Biofilm Formation is Durably Prevented on Pre-Fabricated Antibiotic Cement Spacers Compared to Cobalt Chrome and Polyethylene

BackgroundA 2-stage revision remains the standard for managing chronic periprosthetic joint infection. Despite multiple spacer options, whether a particular one better resists biofilm formation remains unclear. Prefabricated polymethylmethacrylate (PMMA) articulating spacers containing antibiotics and a proprietary pore structure were developed to increase antibiotic elution characterized by a rapid burst phase for the initial one to two days and an extended slow-release phase for > 28 days. This in vitro study determined whether biofilm formation is prevented during the initial rapid burst phase and/or the slow-release phase. MethodsS. aureus-Xen36 was incubated in 1.5 mL of Luria-Bertani broth with PMMA discs with the proprietary pore structure either with or without gentamycin and vancomycin or with ‘Hoffman style’ positive-control discs (ultra-high molecular weight polyethylene or cobalt-chrome). Nonadherent bacteria were removed by three phosphate buffered saline rinses every 20 to 24 hours. Planktonic bacterial growth in the culture broth and biofilm formation on the discs were measured by colony forming unit (CFU) counting and resazurin reduction assays. Experiments were repeated > four times. ResultsNo detectable planktonic bacterial growth or biofilm formation occurred in cultures containing PMMA with antibiotics (≤ 15 CFUs/disc), whereas biofilms formed on PMMA without antibiotics, ultra-high molecular weight polyethylene, and cobalt-chrome (1 × 107 to 4 × 108 CFUs/disc, P < 0.0001). Biofilm formation was confirmed by a 100-fold decrease in sensitivity to vancomycin. To determine whether the antibiotic slow-release phase is sufficient to block biofilm formation, PMMA discs with antibiotics were preeluted for 14 days with multiple saline changes prior to bacterial inoculation. After antibiotic elution, still no detectable biofilms formed on PMMA discs with antibiotics (≤ 15 CFUs/disc, P < 0.0001). ConclusionsAntibiotic release during both the initial and slow-release phases prevented biofilm formation on PMMA with the proprietary pore structure. This may translate into improved infection eradication rates clinically.

SLC25A19 drives colorectal cancer progression by regulating p53.

Investigating the molecular mechanism of colorectal cancer (CRC), a common lethal malignancies worldwide, is of great clinical significance. Solute carrier family 25 member 19 (SLC25A19) is a member of the solute carrier family that contribute to cellular functions, including tumor biology. Recently, many studies have attention on uncovering the relationship of SLC25A19 with malignant cancers, but its precise involvement in the regulation of CRC has not been thoroughly understood. This study sought to uncover the role and mechanism of SLC25A19 in CRC development. The GEPIA database and immunohistochemical staining were utilized to detect the expression of SLC25A19 in CRC tissues. The functional influences of SLC25A19 on CRC cell phenotypes were evaluated through a series of assays including celigo cell count, colony formation, CCK-8, flow cytometry, wound healing, and transwell assays following knocking down SLC25A19. Subsequently, the xenograft tumor model was constructed to evaluate the effect of SLC25A19 on tumor growth invivo. The underlying mechanisms of SLC25A19 silencing were investigated using the human phospho-kinase array. This study demonstrated the upregulation of SLC25A19 in CRC and its significant correlation with unfavorable prognosis in CRC patients. Suppression of SLC25A19 resulted in significant inhibition of cell proliferation, colony formation, and cell migration, alongside a boost in cell apoptosis. Invivo experiments revealed that silenced SLC25A19 displayed reduced growth rates and formed smaller xenografts. Mechanistically, the p53 pathway was found to be upregulated by SLC25A19 knockdown and mediated the function of SLC25A19. Consequently, SLC25A19 was identified as a novel molecule with key regulatory ability in CRC development.

Inhibitory Effects of Lactobionic Acid on Biofilm Formation and Virulence of Staphylococcus aureus.

Staphylococcus aureus biofilm is a common bio-contaminant source that leads to food cross-contamination and foodborne disease outbreaks. Hence, there is a need for searching novel antibiofilm agents with potential anti-virulence properties to control S. aureus contamination and infections in food systems. In this study, the antibiofilm effects of lactobionic acid (LBA) against S. aureus and its influence on virulence were explored. The minimum inhibition concentration of LBA on S. aureus was 8 mg/mL. Viable count and crystal violet assays revealed that LBA inhibited and inactivated S. aureus biofilms. Microscopic observations further confirmed the antibiofilm activity of LBA on S. aureus that disrupted the biofilm architecture and inactivated the viable cells in biofilms. Moreover, LBA decreased the release of extracellular DNA (eDNA) and extracellular polysaccharide (EPS) in S. aureus biofilms. LBA suppressed biofilm formation by intervening metabolic activity and reduced virulence secretion by repressing the hemolytic activity of S. aureus. Furthermore, LBA altered the expressions of biofilm- and virulence-related genes in S. aureus, further confirming that LBA suppressed biofilm formation and reduced the virulence secretion of S. aureus. The results suggest that LBA might be useful in preventing and controlling biofilm formation and the virulence of S. aureus to ensure food safety.

Targeting NUF2 suppresses gastric cancer progression through G2/M phase arrest and apoptosis induction.

Gastric cancer (GC), a malignant tumor with poor prognosis, is one of the leading causes of cancer-related deaths worldwide; consequently, identifying novel therapeutic targets is crucial for its corresponding treatment. NUF2 , a component of the NDC80 kinetochore complex, promotes cancer progression in multiple malignancies. Therefore, this study aimed to explore the potential of NUF2 as a therapeutic target to inhibit GC progression. Clinical samples were obtained from patients who underwent radical resection of GC at Lanzhou University Second Hospital from 2016 to 2021. Cell count assays, colony formation assays, and cell-derived xenotransplantation (CDX) models were used to determine the effects of NUF2 on GC progression. Flow cytometry was used to detect the effect of NUF2 or quercetin on cell cycle progression and apoptosis. A live-cell time-lapse imaging assay was performed to determine the effect of NUF2 on the regulation of mitotic progression. Transcriptomics was used to investigate the NUF2 -associated molecular mechanisms. Virtual docking and microscale thermophoresis were used to identify NUF2 inhibitors. Finally, CDX, organoid, and patient-derived xenograft (PDX) models were used to examine the efficacy of the NUF2 inhibitor in GC. NUF2 expression was significantly increased in GC and was negatively correlated with prognosis. The deletion of NUF2 suppressed GC progression both in vivo and in vitro . NUF2 significantly regulated the mitogen-activated protein kinase (MAPK) pathway, promoted G2/M phase transition, and inhibited apoptosis in GC cells. Additionally, quercetin was identified as a selective NUF2 inhibitor with low toxicity that significantly suppressed tumor growth in GC cells, organoids, CDX, and PDX models. Collectively, NUF2 -mediated G2/M phase transition and apoptosis inhibition promoted GC progression; additionally, NUF2 inhibitors exhibited potent anti-GC activity. This study provides a new strategy for targeting NUF2 to suppress GC progression in clinical settings.

Management of Heparin-Induced Thrombocytopenia: A Contemporary Review.

Heparin-induced thrombocytopenia (HIT) is a life- and limb-threatening immune-mediated emergency classically associated with heparin therapy. This review focuses on type II HIT, characterized by the development of antibodies against platelet-factor 4 (PF4) bound to heparin after exposure, causing life-threatening thrombocytopenia, arterial thrombosis, and/or venous thrombosis. The high morbidity and mortality rates emphasize the need for early recognition and urgent intervention with discontinuation of heparin and initiation of non-heparin anticoagulation. We discuss the management of HIT with an emphasis on recent developments: (i) incorporating the phases of HIT (i.e., suspected, acute, subacute A and B, and remote) into its management, categorized according to platelet count, immunoassay, and functional assay results and (ii) direct-acting oral anticoagulants (DOACs), which are increasingly used in appropriate cases of acute HIT (off-label). In comparison to parenteral options (e.g., bivalirudin and danaparoid), they are easier to administer, are more cost-effective, and obviate the need for transition to an oral anticoagulant after platelet recovery. We also identify the knowledge gaps and suggest areas for future research.

Heraclenin promotes the osteogenic differentiation of bone marrow stromal cells by activating the RhoA/ROCK pathway.

Osteoporosis is a devastating skeletal disease, the pathogenesis of which is related to abnormal bone metabolism, featured by the imbalance between osteoblastic bone formation and osteoclastic bone resorption. Stem cell-based therapies have been demonstrated to improve osteoporosis treatment. Previously, the linear furanocoumarin heraclenin was reported to enhance osteoblast differentiation and mineralization in mouse mesenchymal stem cells (MSCs), suggesting its potential for osteogenic differentiation and bone regeneration. Our study was designed to confirm the promotive role of heraclenin on osteogenic differentiation of human bone MSCs (BMSCs) and explore the underlying mechanisms. Human BMSCs were treated for 24, 48, and 72h with heraclenin (5, 10, 20, 40, and 80 μM), and cell viability was determined by Cell Counting Kit-8 (CCK-8) assay. To further evaluate the cytotoxicity of heraclenin, cell suspension obtained from BMSCs treated with heraclenin (5, 10, and 20 μM) for 72h was subjected to a MUSE™ cell analyzer for cell viability and count assay. BMSCs were incubated in osteogenic induction medium for 7 days. Then, osteogenic differentiation and mineralization of BMSCs were assessed through alkaline phosphatase (ALP) and Alizarin Red S staining. The expression of osteogenesis markers including ALP, osteocalcin (OCN), osterix (OSX), and runt-related transcription factor 2 (RUNX2) was detected via reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting. The effects of heraclenin on the RhoA/ROCK pathway were estimated through western blotting. Y-27632, the ROCK inhibitor, was used to confirm the role of the RhoA/ROCK pathway in heraclenin-mediated osteogenic differentiation of BMSCs. Heraclenin (5-80 μM) was non-toxic on human BMSCs. Heraclenin treatment (5-20 μM) dose-dependently enhanced ALP activity and calcium deposition. Furthermore, heraclenin promoted ALP, OCN, OSX, and RUNX2 mRNA and protein expression. Mechanically, heraclenin treatment increased RhoA and ROCK1 mRNA expression, stimulated the translocation of ROCK from the cytosolic to the membrane fraction, and elevated the protein levels of phosphorylated cofilin (p-cofilin) and active RhoA. Additionally, treatment with Y-27632 overturned the promotion of heraclenin on ALP activity, calcium deposition, the expression of osteogenesis markers, and the RhoA/ROCK signaling pathway. Heraclenin facilitates the osteogenic differentiation of human BMSCs through the activation of the RhoA/ROCK pathway.

Rutin alleviates psoriasis-related inflammation in keratinocytes by regulating the JAK2/STAT3 signaling.

Psoriasis is a chronic inflammatory skin disease that can cause systemic inflammation in various organs. Rutin has been suggested to fight psoriasis, but the signaling pathways by which it works need to be explored. HaCaT cells co-stimulated with interleukin (IL)-17, IL-22, tumor necrosis factor-alpha (TNF-α), IL-1α, and oncostatin M (M5) were used as an in vitro cell model of psoriasis. The proliferation and viability of HaCaT cells were determined by 5-ethynyl-2'-deoxyuridine and cell counting assays. Relative mRNA levels of IL-6, TNF-α, chemokines (CXCL1 and CXCL2), and anti-microbial peptides (S100A7 and S100A8) were detected by reverse transcriptase-quantitative PCR. Release of IL-6 and TNF-α from HaCaT cells was measured by enzyme-linked immunosorbent assay. Keratin1, Keratin5, p-JAK2, and p-STAT3 protein levels were estimated with western blotting. Molecular docking predicted binding sites for Rutin and STAT3. Rutin treatment undercut M5-urged viability increase and proliferation boost in HaCaT cells. Moreover, M5 stimulation mediated upregulation of IL-6, TNF-α, CXCL1, CXCL2, S100A7, and S100A8 was partially reversed after Rutin treatment. In addition, M5 stimulation induced downregulation of Keratin1 and Keratin5 proteins as well as upregulation of p-JAK2 and p-STAT3 proteins were attenuated in response to Rutin treatment, manifesting that Rutin treatment inhibited M5-promoted aberrant differentiation and impaired M5-mediated activation of the JAK2/STAT3 signaling in HaCaT cells. Molecular docking discovered that residues GLN326 and ASP334 in STAT3 might bind to Rutin. Rutin treatment blocked the JAK2/STAT3 signaling, thus attenuating psoriasis-related inflammation and anomalous differentiation in keratinocytes.

GLP-1 analogue semaglutide regulates pancreatic beta-cell proliferation via PDX-1 expression control

Semaglutide as an agonist of glucagon-like peptide-1 receptor was shown to potentiate insulin release and suppress food motivation targeting pancreatic islet beta cells and brain region including subfornical organ and hypothalamus, which effectively treats type 2 diabetes and obesity. The investigation of prolonged response with semaglutide on beta cells triggering proliferation and apoptotic resistance is yet to be confirmed. Previous research findings have shown that glucagon-like peptide-1 and liraglutide as agonists for glucagon-like peptide-1 receptors on beta cells result in proliferation through upregulation of PDX-1 transcription factor. Semaglutide is investigated to show whether long-term beta cell survival regulation is achieved through the same downstream signalling pathways. Both in vivo and in vitro methods were designed to show the proliferation effect of the semaglutide in C57BL/6 mice using tissue imaging, cell counting and immunosorbent assay for quantitative analysis of PDX-1 expression. The proliferation effect would broaden the application of semaglutide from insulin augmentation to the sustained benefit of beta cell survival.

Antimicrobial impact of a propolis/PVA/chitosan composite and its prospective application against methicillin resistance bacterial infection

Seriously damaged skin could be infected by methicillin-resistant bacteria, which delays restoration. Propolis has bioactivity linked with its minor components, such as antimicrobials and antioxidants. Active sites in polyvinyl alcohol (PVA) and chitosan (CS) can enhance the nano-loading of natural extracts with activity amelioration. Korean propolis extract (KPE) loading to a nanocomposite possibly enhances its antimicrobial and anti-inflammatory potency. Composites were formed using two PVA/CS structures (1:1; 2:1), and their skin-application appropriateness was determined by mechanical properties, moisture content, water activity, and color. The composite of PVA/CS (1:1) was more practicable for KPE-loading. Increasing KPE concentrations (50, 100, 150, and 200 ng/mL) alters composite bioactivity measured by Fourier transmission infrared (FT-IR). Antibacterial potency of 200 ng KPE/mL was the most effective concentration, followed by 150 ng KPE/mL, against Staphylococcus aureus (MRSA) and Clostridium perfringens. The composite activity was measured as minimum inhibition (MIC) and minimum bacterial concentrations (MBC). At 200 ng KPE/mL, MIC and MBC against MRSA were 14.93 ± 1.21 and 20.21 ± 1.97 mg composite/mL, respectively. Significant inhibition was also recorded for antibiofilm formation, where MRSA growth was not detected after 4 hours of time intervals to the stainless-steel coupon. Compared to planktonic bacteria, the formed barrier of PVA/CS restrained the biofilm matrix formation and supported KPE antimicrobial. The impact of inhibition against biofilm formation depends on two parallel mechanisms (PVA barrier with hydrogen bonds, besides nano-KPE particle penetration into bacterial cells). The KPE-composite application to rats’ wounds shows significantly reduced MRSA infection. The results demonstrate the capability of KPE composite in reducing infection, healing correctly, and restoring hair. The wound swabbed test emphasizes this capacity, in which bacterial growth rate restriction was evaluated using a plate count assay. The results recommended 150 ng KPE/mL loading into CS/PVA (1:1) as an effective anti-pathogenic treatment, particularly against the MRSA infection of wounds.