Control Cells Research Articles

PFAS-mediated disruptions on thyroid histology

Abstract Introduction/Objective Per-and polyfluroalkyl substances (PFAS), are environmental contaminants of emerging concern due to their abundance in the environment and potential adverse health impacts. PFAS are used in waterproof clothing, makeup, carpets, upholstery, cookware, and fast-food containers. These organic pollutants have been found in the blood of 98% of Americans and have been linked to disruption in thyroid hormones. During fetal life, thyroid hormones are crucial for brain development and alterations in these hormones can induce long-term impacts on intellectual and brain impairments. T3 and T4 are known to be altered by PFAS exposure, but the mechanism by which this is done remains unclear. Methods/Case Report Normal thyroid cells were treated for 72 hours with 1000 nM solution containing either GEnX, PFOA, PFOS, or 1% DMSO control. Immunofluorescence for actin, tubulin, and DAPI was performed using a Nikon spinning disk. In vivo studies where 8-week-old mice are gavaged with 7.5 mg/kg of PFOS, PFOA, and GenX or PBS control for 8 weeks. Results (if a Case Study enter NA) We hypothesized the PFAS treatment alters thyroid cells morphology and histology. Immunofluorescence staining of normal thyroid cells treated with PFAS show a decrease in actin stress fibers compared to control cells. PFAS treated mice show a decrease in follicle size compared to control (average 1017 um2 size, p=0.0483). Conclusion This is the first study to show changes in thyroid histology after 8 weeks of PFAS exposure. As structure and function are closely linked, it is likely that this reduction in follicle size and actin reorganization contribute to alterations in thyroid function. Future studies are needed to illuminate the mechanism by which PFAS alter thyroid function in vivo. Understanding the role of PFAS-mediated thyroid dysregulation will have far reaching implications on environmental regulations and considerations for limiting PFAS in our environment.

Osteoclastic ATP6AP2 maintains β-catenin levels to prevent hyper-osteoclastic activation and trabecular bone-loss.

Osteoclast (OC) formation and bone resorption are regulated by several factors, including V-ATPase, Wnt/β-Catenin, and RANKL/RANK signaling. ATP6AP2, also known as the prorenin receptor (PRR), is an accessory subunit of V-ATPase and a regulator of Wnt/β-Catenin signaling. While the V-ATPase subunit ATP6AP1 is essential for osteoclast formation and function, the role of ATP6AP2 in OC-lineage cells is less clear. Here, we provide evidence that ATP6AP2 plays a negative role in osteoclastogenesis and function, contrasting with the positive role of ATP6AP1. Mice with conditional knockout (cKO) of ATP6AP2 in OCs (Atp6ap2LysM) exhibit trabecular bone loss, likely due to the increased osteoclastogenesis and activity, since bone formation rates are comparable to control mice. In vitro assays using bone marrow macrophages (BMMs) show that Atp6ap2LysM cultures have more RANKL-induced TRAP+ OC-like cells and increased bone resorptive activity. Further studies reveal that while RANKL signaling and V-ATPase activity are normal, in ATP6AP2 KO OCs, but not BMMs, have reduced basal levels of Wnt/β-Catenin pathway proteins, such as LRP5/6 and β-Catenin, compared to controls. Wnt3A treatment induces β-Catenin and suppresses osteoclast formation in both control and ATP6AP2 KO OC-lineage cells, indicating that Wnt/β-Catenin signaling negatively regulates OC-formation and operates independently of ATP6AP2. Overall, these results suggest that ATP6AP2 is critical for maintaining basal levels of LRP5/6 receptors and β-Catenin in osteoclasts, thus acting as a negative regulator of osteoclastogenesis and activation.

Pseudomonas aeruginosa-Mannose Sensitive Hemagglutinin-based Treatment Strategy for Liver Cancer

Background Although it does not represent a threat to healthy humans, Pseudomonas aeruginosa is a type of aerobic, gram-negative bacteria that can cause catastrophic infections in patients with immune system abnormalities and cystic fibrosis. Purpose The current study investigated the effect of Pseudomonas aeruginosa-mannose sensitive hemagglutinin (PA-MSHA) on liver cancer cell viability, colony formation, and invasion potential, and explored the underlying mechanism. Methods Proliferation potential and clonogenic survival of the cells were studied using MTT and colony formation assays, respectively. Transwell and Western blotting assays were used for the assessment of invasive potential and protein expression, respectively. Results The results revealed that exposure to PA-MSHA led to a time-dependent suppression in HepG2 and HEP3B cell proliferation. Incubation with PA-MSHA decreased HepG2 cell proliferation to 92%, 75%, 50%, 35%, and 20%, respectively, after 12, 24, 36, 48, and 72 hours. Similarly, incubation for 12, 24, 36, 48, and 72 hours reduced the proliferation of HEP3B cells to 94%, 79%, 55%, 41%, and 26%, respectively. The rate of clonogenic survival was significantly lower in HepG2 and HEP3B cells on incubation with PA-MSHA. The cell invasion potential was also significantly reduced on incubation with PA-MSHA. In HepG2 cells, incubation with PA-MSHA significantly reduced the expression of Bcl-2 (26 kDa) protein and promoted Bax (21 kDa) level. Following incubation with PA-MSHA, HepG2 cells showed higher cleaved caspase-3 level compared to the control cells. Compared to control cells, the PA-MSHA incubation of HepG2 cells resulted in a prominent reduction in Notch3 protein expression. Conclusion In summary, PA-MSHA treatment prevents liver cancer cell proliferation and targets the survival of clonogenic cells. It reduces invasiveness, targets Notch3 protein expression in liver cancer cells, and increases the level of proapoptotic and suppresses antiapoptotic protein expression. Therefore, PA-MSHA shows encouraging anticancer effects on HepG2 and HEP3B cancer cells, suggesting that it could be developed as a viable treatment option for liver cancer.

Exploring the effects of simulated microgravity on esophageal cancer cells: insights into morphological, growth behavior, adhesion, and genetic damage.

The exploration of microgravity has garnered substantial scholarly attention due to its potential to offer unique insights into the behavior of biological systems. This study presents a preliminary investigation into the effects of simulated microgravity on esophageal cancer cells, examining various aspects such as morphology, growth behavior, adhesion, inhibition rate, and DNA damage. To achieve this, a novel microgravity simulator named "Gravity Challenge" was utilized for its effectiveness in minimizing external influences that could compromise microgravity conditions. The international cell line SK-GT-4 was utilized as the focal point of this investigation. Results revealed noticeable alterations in the growth behavior of cancer cells following exposure to simulated microgravity for 24 h, characterized by a loss of adhesion properties compared to control cells. Concurrently, cell viability exhibited a decline, as evidenced by cytotoxicity testing. Furthermore, the comet assay test demonstrated that cells subjected to microgravity simulation experienced a higher incidence of DNA damage compared to their control counterparts. In conclusion, this comprehensive examination of the impact of simulated microgravity on esophageal cancer cells extends beyond morphological changes, delving into genetic implications through observed DNA damage. The diminished vitality of cells under microgravity conditions underscores the multifaceted effects on cellular behavior in response to environmental variations. These findings represent a significant step towards understanding the dynamics of cancer cells, laying the groundwork for future research aimed at identifying potential therapeutic strategies for this disease.

Integrative Omics Strategies for Understanding and Combating Brown Planthopper Virulence in Rice Production: A Review

The brown planthopper (Nilaparvata lugens, BPH) is a serious insect pest responsible for causing immense economic losses to rice growers around the globe. The development of high-throughput sequencing technologies has significantly improved the research on this pest, and its genome structure, gene expression profiles, and host–plant interactions are being unveiled. The integration of genomic sequencing, transcriptomics, proteomics, and metabolomics has greatly increased our understanding of the biological characteristics of planthoppers, which will benefit the identification of resistant rice varieties and strategies for their control. Strategies like more optimal genome assembly and single-cell RNA-seq help to update our knowledge of gene control structure and cell type-specific usage, shedding light on how planthoppers adjust as well. However, to date, a comprehensive genome-wide investigation of the genetic interactions and population dynamics of BPHs has yet to be exhaustively performed using these next-generation omics technologies. This review summarizes the recent advances and new perspectives regarding the use of omics data for the BPH, with specific emphasis on the integration of both fields to help develop more sustainable pest management strategies. These findings, in combination with those of post-transcriptional and translational modifications involving non-coding RNAs as well as epigenetic variations, further detail intricate host–brown planthopper interaction dynamics, especially regarding resistant rice varieties. Finally, the symbiogenesis of the symbiotic microbial community in a planthopper can be characterized through metagenomic approaches, and its importance in enhancing virulence traits would offer novel opportunities for plant protection by manipulating host–microbe interactions. The concerted diverse omics approaches collectively identified the holistic and complex mechanisms of virulence variation in BPHs, which enables efficient deployment into rice resistance breeding as well as sustainable pest management.

Ionising and ultraviolet radiation induce the secretion of melanin in Tetrahymena pyriformis

ABSTRACT The objective of this study was to determine the effect of ionising and ultraviolet radiation on stimulating melanin secretion in Tetrahymena pyriformis. Cell cultures were exposed to different doses of radiation for 48, 72 and 96 h. After incubation, melanin was extracted and analysed. Our results showed that melanin concentration increased with increasing irradiation dose and exposure duration. Melanin was insoluble in water and organic solvents but soluble in alkaline and acidic solutions. A linear correlation between the logarithm of melanin absorbance and wavelength ranging from 400 to 600 nm was observed. The HPLC chromatograms of melanin degradation products by the alkaline hydrogen peroxide oxidation method revealed the existence of four peaks. The natural melanin standard extracted from the cuttlefish Sepia officinalis was used as a reference compound of eumelanin markers. These markers were detected in control cells, irradiated cells and black human hair but with different intensities.

Multicolor iLIFE (m-iLIFE) volume cytometry for high-throughput imaging of multiple organelles

To be able to resolve multiple organelles at high throughput is an incredible achievement. This will have immediate implications in a range of fields ranging from fundamental cell biology to translational medicine. To realize such a high-throughput multicolor interrogation modality, we have developed a light-sheet based flow imaging system that is capable of visualizing multiple sub-cellular components with organelle-level resolution. This is possible due to the unique optical design that combines an illumination system comprising two collinear light sheets illuminating the flowing cells and a dedicated dual-color 4f-detection, enabling simultaneous recording of multiple organelles. The system PSF sections up to 4 parallel microfluidic channels through which cells are flowing, and multicolor images of cell cross-sections are recorded. The data is then computationally processed (filtered using ML algorithm, shift-corrected, and merged) and combined to reconstruct the 3D multicolor volume. System testing is conducted using multicolor fluorescent nano-beads (size ∼175\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\sim 175$$\\end{document} nm) and flow-based imaging parameters (PSF size, motion-blur, flow rate, frame rate, and number of cell-sections) are determined for quality imaging. Drug treatment studies were carried out for healthy and cancerous HeLa cells to check the performance of the proposed system. The cells were treated with a drug (Vincristine, which is known to promote mitochondrial fission in cells), and the same is compared with untreated control cells. The proposed multicolor iLIFE system could screen ∼800\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\sim 800$$\\end{document} cells/min (at a flow speed of 2490μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$2490 ~\\upmu$$\\end{document}m/s), and the drug treatment studies were carried out up to 24 h. Studies showed the disintegration of mitochondrial network and dysfunctional lysosomes and their accumulation at the cell membrane, which is a clear indication of cell apoptosis. Compared to control cells (untreated), the mortality is highest at a concentration of 500 nM post 12 h of drug treatment. With the capability of multiorganelle interrogation and organelle-level resolution, the multicolor iLIFE cytometry system is suitably placed to assist optical imaging and biomedical research.

Aucubin Induces Apoptotic Cell Death Through Caspase-dependent Pathways in Human Osteosarcoma MG-63 Cells

Background Osteosarcomas (OSs) are predominantly generated by mesenchymal cells, which commonly develop in the distal and upper regions of the bones. OS has high mortality rates and treatment failures, mostly due to the presence of lung metastases. Purpose The present work aimed to scrutinize the anticancer potentials of aucubin against OS human osteosarcoma (MG-63) cells. Methods The proliferation of the control and aucubin-treated MG-63 cells was studied by an XTT assay. Various fluorescent staining assays were done to assess the endogenous reactive oxygen species (ROS) accumulation and apoptosis in the aucubin-treated MG-63 cells. The caspase-3, -8, and -9 activities were investigated using the assay kits. Results The XTT assay results confirmed that the aucubin treatment considerably reduced the MG-63 cells. The findings of the various fluorescent staining assays evidenced that the aucubin treatment remarkably promoted endogenous ROS accumulation and apoptosis in the OS cells. The caspase activities were also improved in the OS cells after the aucubin treatment. Conclusion The results of this work show that aucubin treatments inhibit cell proliferation and trigger apoptosis in MG-63 cells, suggesting its potential as a promising anticancer agent. These findings will facilitate the future development of aucubin as a talented anticancer agent to treat OS.

COL8A2 activation enhances function of corneal endothelial cells through HIPPO signaling/mitochondria pathway

Corneal endothelial cells (CECs) are essential for maintaining corneal transparency and hydration through their barrier and pump functions. The COL8A2 gene encodes a component of the extracellular matrix of the cornea, which is crucial for the normal functioning of these cells. Mutations in COL8A2 are linked to corneal dystrophies, emphasizing the gene's importance in corneal health. The purpose of this research is to explore the effects of COL8A2 activation within CECs, to understand its contribution to cellular behavior and health. COL8A2 CRISPR/dCas9 activation system (aCOL8A2) was used to activate the COL8A2. In rats, wound healing and mitochondrial function were assessed after COL8A2 activation. As a result, aCOL8A2 promoted wound healing of rat corneal endothelium by increasing mitochondrial membrane potential. In cultured human CECs, proteomic analysis was performed to screen and identify the differential protein profiles between control and aCOL8A2 cells. Western blot was used to validate the differential proteins from both cells. Mitochondrial function and intracellular distribution were assessed by measuring ATP production and mitochondrial membrane potential. In cultured human CECs, aCOL8A2 increased COL8A2 and phospho-YAP levels. Transendothelial electrical resistance (TEER) was increased and actin cytoskeleton was attenuated by aCOL8A2. Gene ontology analysis revealed that the proteins were mainly involved in the regulation of folate biosynthesis, ECM-receptor interaction, cell differentiation, NADP activity and cytoskeleton. ATP production was increased, mitochondrial membrane potential was polarized and mitochondrial distribution was widespread in the aCOL8A2 group. In conclusion, aCOL8A2 induces a regulatory cascade affecting mitochondrial positioning and efficiency, mediated by alterations in the cytoskeletal architecture and the YAP signaling pathway. This sequence of events serves to bolster the functional capacities of corneal endothelial cells, including their pump and barrier functions, essential for corneal health and transparency.

SRSF1 Is Crucial for Maintaining Satellite Cell Homeostasis During Skeletal Muscle Growth and Regeneration.

The splicing factor SRSF1 emerges as a mater regulator of cell proliferation, displaying high expression in actively proliferative satellite cells (SCs). In SRSF1 knockout mice (KO) generated via MyoD-Cre, early mortality and muscle atrophy are observed during postnatal muscle growth. Despite these findings, the precise mechanisms through which SRSF1 loss influences SCs' functions and its role in muscle regeneration remain to be elucidated. To unravel the exact mechanisms underlying the impact of SRSF1 deficiency SC functions, we employed single-cell RNA sequencing (scRNA-seq) on a mononuclear cell suspension isolated from the newborn diaphragm of KO and control mice. Concurrently, we subjected diaphragm muscles to RNA-seq analysis to identify dysregulated splicing events associated with SRSF1 deletion. For the analysis of the effect of SRSF1 deletion on muscle regeneration, we generated mice with inducible SC-specific Srsf1 ablation through Pax7-CreER. SRSF1 ablation was induced by intraperitoneal injection of tamoxifen. Using cardiotoxin-induced muscle injury, we examined the consequences of SRSF1 depletion on SC function through HE staining, immunostaining and EdU incorporation assay. C2C12 myoblasts and isolated myoblasts were employed to assess stem cell function and senescence. Utilizing scRNA-seq analysis, we observed a noteworthy increase in activated and proliferating myoblasts when SRSF1 was absent. This increase was substantial, with the proportion rising from 28.68% in the control group to 77.06% in the knockout group. However, these myoblasts experienced mitotic abnormalities in the absence of SRSF1, resulting in cell cycle arrest and the onset of cellular senescence. In the knockout mice, the proportion of Pax7+ cells within improper niche positioning increased significantly to 25% compared to 12% in the control cells (n ≥ 10, p < 0.001). Furthermore, there was an observation of persistent cell cycle exit specifically in the Pax7+ cells deficient in SRSF1 (n = 6, p < 0.001). SRSF1 plays a pivotal role in regulating the splicing of Fgfr1op2, favouring the full-length isoform crucial for mitotic spindle organization. Disrupting SRSF1 in C2C12 and primary myoblasts results in multipolar spindle formation (p < 0.001) and dysregulated splicing of Fgfr1op2 and triggers cellular senescence. Consequently, adult SCs lacking SRSF1 initially activate upon injury but face substantial challenge in proliferation (n = 4, p < 0.001), leading to a failure in muscle regeneration. SRSF1 plays a critical role in SCs by ensuring proper splicing, maintaining mitotic progression and preventing premature senescence. These findings underscore the significant role of SRSF1 in controlling SC proliferation during skeletal muscle growth and regeneration.

B Cell and Antibody Responses in Bovine Tuberculosis.

The development of vaccines and effective diagnostic methods for bovine tuberculosis requires an understanding of the immune response against its causative agent, Mycobacterium bovis. Although this disease is primarily investigated and diagnosed through the assessment of cell-mediated immunity, the role of B cells and antibodies in bovine tuberculosis has been relatively undervalued and understudied. Current evidence indicates that circulating M. bovis-specific antibodies are not effective in controlling the disease. However, local humoral immune responses may contribute to either defence or pathology. Recent studies in animal models and cattle vaccine trials suggest a potential beneficial role of B cells in tuberculosis control. This review discusses the role of B cells and antibodies in bovine tuberculosis and explores antibody-based diagnostics for the disease, including traditional techniques, such as different ELISA, new platforms based on multiple antigens and point-of-care technologies. The high specificity and sensitivity values achieved by numerous antibody-based tests support their use as complementary tests for the diagnosis of bovine tuberculosis, especially for identifying infected animals that may be missed by the official tests.

Proanthocyanidin Regulates NETosis and Inhibits the Growth and Proliferation of Liver Cancer Cells - In Vivo, In Vitro and In Silico Investigation.

Liver cancer ranks third in global cancer-related mortality, with about 700,000 deaths recorded yearly, making it one of the most common cancers worldwide. Even though prognoses differ according to the severity of the diseases, many patients now exhibit an increased life cycle since the implementation of chemotherapy. In the current study, we investigated the effect of proanthocyanidin ‒a polyphenol molecule found in many plants‒ on the proliferation and invasion of liver cancer cells. In particular, we determined the effect of proanthocyanidin on the serum levels of four strategic liver cancer target, TNFα, IL-6, cfDNA, and IL-1β. Further molecular insight on the inhibitory mechanism of proanthocyanidin against TNFα, IL-6, and IL-1β was obtained via molecular docking, molecular dynamics simulations and binding free energy calculations. Results showed that proanthocyanidin inhibited the growth of HepG2 and HEP3B cells, and effectively reduced clonogenic survival and invasion potential when compared to control cells. Proanthocyanidin was also found to suppress the expression of Bcl-2 (26 kDa) protein in HepG2 cells, while increasing the expression of Bax (21 kDa). Molecular dynamics (MD) and thermodynamic binding free energy calculations showed that proanthocyanidin maintained stable binding within the active site of target proteins across the entire 100 ns MD simulation period, and its binding affinity outscored respective control molecules.In conclusion, the multifaceted analysis showcased in this study demonstrated promising anti-cancer effect of proanthocyanidin on HepG2 and HEP3B cancer cells, highlighting its potential as a viable liver cancer therapeutic alternative.

A Copper-Based Coating for the Control of Airborne Viable Bacteria in a Prison Environment

Infections in confined environments can spread by direct contact, contaminated surfaces, and airborne transmission. This is critical in prison facilities, where cleaning and sanitary conditions are inadequate. An alternative is the development of antimicrobial surfaces. A new antimicrobial coating was developed by incorporating copper microparticles into a standard commercial paint, aiming to reduce the concentration of bacteria on surfaces by granting antimicrobial properties to surfaces. The copper additive comprised Cu2Cl(OH)3 deposited on polyhedral zeolite. The efficacy of this coating was evaluated in detention cells in a police station, which are temporary prisons and inherently dirty environments. The experiment compared a cell painted with the copper additive coating and a control cell with the standard paint. Viable coliforms were measured on different surfaces and in the air for five months under normal usage. Bacterial load was reduced by ca. 68% by the copper-amended paint on cement surfaces. Surprisingly, airborne viable coliforms were reduced by ca. 87% in the detention cell treated with the copper coating. This research highlights the potential of antimicrobial coatings in controlling the spread of infections through contact with contaminated surfaces and emphasizes the significant reduction in airborne bacterial load. It is especially relevant for controlling infections where sanitization is limited but can be extended to other built environments, such as healthcare facilities.

Silmitasertib in Combination With Cabozantinib Impairs Liver Cancer Cell Cycle Progression, Induces Apoptosis, and Delays Tumor Growth in a Preclinical Model.

The rising incidence of hepatocellular carcinoma (HCC) is a global problem. Several approved treatments, including immune therapy and multi-tyrosine kinase inhibitors, are used for treatment, although the results are not optimum. There is an unmet need to develop highly effective chemotherapies for HCC. Targeting multiple pathways to attack cancer cells is beneficial. Cabozantinib is an orally available bioactive multikinase inhibitor and has a modest effect on HCC treatment. Silmitasertib is an orally bioavailable, potent CK2 inhibitor with a direct role in DNA damage repair and is in clinical trials for other cancers. In this study, we planned to repurpose these existing drugs on HCC treatment. We observed a stronger antiproliferative effect of these two combined drugs on HCC cells generated from different etiologies as compared to the single treatment. Global RNA-seq analyses revealed a decrease in the expression of G2/M cell cycle transition genes in HCC cells following combination treatment, suggesting G2 phase cell arrest. We observed G2/M cell cycle phase arrest in HCC cells upon combination treatment compared to the single-treated or vehicle-treated control cells. The downregulation of CCNA2 and CDC25C following combination therapy further supported the observation. Subsequent analyses demonstrated that combination treatment inhibited 70 kDa ribosomal protein S6 kinase (p70S6K) phosphorylation, and increased Bim expression. Apoptosis of HCC cells were accompanied by increased poly (ADP-ribose) polymerase cleavage and caspase-9 activation. Next, we observed that a combination therapy significantly delayed the progression of HCC xenograft growth as compared to vehicle control. Together, our results suggested combining cabozantinib and silmitasertib would be a promising treatment option for HCC.

NRF2 and Thioredoxin Reductase 1 as Modulators of Interactions between Zinc and Selenium.

Selenium and zinc are essential trace elements known to regulate cellular processes including redox homeostasis. During inflammation, circulating selenium and zinc concentrations are reduced in parallel, but underlying mechanisms are unknown. Accordingly, we modulated the zinc and selenium supply of HepG2 cells to study their relationship. HepG2 cells were supplied with selenite in combination with a short- or long-term zinc treatment to investigate intracellular concentrations of selenium and zinc together with biomarkers describing their status. In addition, the activation of the redox-sensitive transcription factor NRF2 was analyzed. Zinc not only increased the nuclear translocation of NRF2 after 2 to 6 h but also enhanced the intracellular selenium content after 72 h, when the cells were exposed to both trace elements. In parallel, the activity and expression of the selenoprotein thioredoxin reductase 1 (TXNRD1) increased, while the gene expression of other selenoproteins remained unaffected or was even downregulated. The zinc effects on the selenium concentration and TXNRD activity were reduced in cells with stable NRF2 knockdown in comparison to control cells. This indicates a functional role of NRF2 in mediating the zinc/selenium crosstalk and provides an explanation for the observed unidirectional behavior of selenium and zinc.

Chemical synthesis, in vitro testing and in silico Nampt-based molecular docking of novel aniline aromatic ring-substituted 2-Aminothiazole analogues.

The heterocyclic 2-Aminothiazoles scaffolds are used in a wide range of therapeutic applications against various diseases for its antioxidant, anti-inflammatory, antimicrobial and anticancer actions. In the present study, we synthesized novel aniline aromatic ring-substituted 2-Aminothiazole derivatives. Molecular docking was performed using Glide module of the Schrödinger Suite to fit compounds JG-49, JG-62, and KBA-18 against the Nicotinamide phosphoribosyl transferase (Nampt) enzyme, an intracellular regulator of Nicotinamide adenine dinucleotide (NAD) redox cofactor involved in energy metabolism and epigenetics and are implicated in aging and metabolic diseases. The three compounds viz. JG-49, JG-62, and KBA-18 showed an increase in Nampt enzymatic activity in vitro. All three substituted derivatives of 2-Aminothiazole showed no cytotoxicity with the mouse C2C12 myoblasts cultures assessed with the MTT cell viability assay. Moreover, the wound closure of the mouse C2C12 myoblasts in vitro displayed no significant difference between the treatment groups of the 2-Aminothiazole derivatives compared with the control naïve and DMSO treated myoblasts cultures, except for the 2-Aminothiazole substituted derivatives JG-62 and KBA-18, which showed a significant increase in the wound closure compared with the control cells at different concentrations. Taken together, we demonstrated that 2-Aminothiazole substituted derivatives provide enhanced Nampt activity, wound closure, and no cytotoxic effects in vitro. Further studies will allow to improve the substitution of 2-Aminothiazole derivatives and test their potential therapeutic applications.

Development and Characterization of A Novel SpyTagged Modular Nanobody as A Detection Platform for CD22-Positive Cells.

CD22, as a surface protein of B cells, is used in the diagnosis and target-specific immunotherapy of B-cell malignancies. SpyTag and SpyCatcher, on the other hand, are two covalently coupled proteins capable of developing a bi- or multi-specific modular protein. The aim of this study was to develop FITC-conjugated SpyCatcher-SpyTagged anti-CD22 Nanobody (FITC-SpyC-SpyT-CD22Nb) to recognize CD22 on the surface of malignant B cells. In this experimental study, the SpyTag-CD22Nb construct was subcloned into a pET22 vector and expressed in E. coli BL21 (DE3). After purification using His-tag affinity chromatography, the size of the eluted protein was confirmed on a Western blot. In addition, the SpyCatcher protein, subcloned into pET28, was expressed in E. coli BL21 (DE3), purified by His-tag affinity chromatography and subjected to FITC labeling. FITC-SpyCatcher and SpyTag-CD22Nb were coupled in a 1:1 molar ratio. The specific binding of the produced FITC-SpyC-SpyT-CD22Nb was tested using CD22+ Raji and CD22- K562 cell lines and was evaluated by flow cytometry. SpyTag-CD22Nb and SpyCatcher were successfully expressed in E. coli BL21 (DE3). The 1:1 molar ratio of SpyTag-CD22Nb and FITC-SpyCatcher successfully formed FITC-SpyC-SpyT-CD22Nb at room temperature. The flow cytometry results showed that FITC-SpyC-SpyT-CD22Nb specifically binds to the CD22+ Raji cells, while there is no binding to the CD22- K562 control cells. The novel FITC-SpyC-SpyT-CD22Nb produced in the present study is capable of detecting the surficial expression of CD22. According to our findings, FITC-SpyC-SpyT-CD22Nb is applicable for specific targeting of CD22 in a therapeutic manner, i.e., chimeric antigen receptor (CAR)-T cell therapy and antibody drug conjugates (ADCs).

Transgenic overexpression of the miR-200b/200a/429 cluster prevents mammary tumor initiation in Neu/Erbb2 transgenic mice.

Although significant progress in the treatment of breast cancer has been achieved, toxic therapies would not be required if breast cancer could be prevented from developing in the first place. While breast cancer prevention is difficult to study in humans due to long disease latency and stochastic cancer development, transgenic mouse models with 100% incidence and defined mammary tumor onset, provide excellent models for tumor prevention studies. In this study, we used Neu/Erbb2 transgenic mice (MTB-TAN) as a model of human HER2+ breast cancer to investigate whether a family of microRNAs, known as the miR-200 family, can prevent mammary tumor development. Overexpression of Neu induced palpable mammary tumors in 100% of the mice within 38 days of Neu overexpression. When the miR-200b/200a/429 cluster was co-overexpressed with Neu in the same mammary epithelial cells (MTB-TANba429 mice), the miR-200b/200a/429 cluster prevented Neu from inducing mammary epithelial hyperplasia and mammary tumor development. RNA sequencing revealed alterations in the extracellular matrix of the mammary gland and a decrease in stromal cells including myoepithelial cells in Neu transgenic mice. Immunohistochemistry for smooth muscle actin confirmed that mammary epithelial cells in control and MTB-TANba429 mice were surrounded by a layer of myoepithelial cells and these myoepithelial cells were lost in MTB-TAN mice with hyperplasia. Thus, we have shown for the first time that elevated expression of miR-200 family members in mammary epithelial cells can completely prevent mammary tumor development in Neu transgenic mice possibly through regulating myoepithelial cells.

7971 The Role of PDX1 In Beta Cell Response To GLP1

Abstract Disclosure: C. Bheeman: None. K. Vann: None. O. Astapova: None. Mature Onset of Diabetes of the Young Type 4 is a rare form of diabetes mellitus caused by a defect in the Pancreatic and Duodenal homeobox 1 (PDX-1) gene, which is predominantly expressed in β-cells and is an important factor in insulin gene transcription. We aim to study the impact that a PDX1 mutation (modeled after a patient) has on the beta cell response to GLP1. This was a 33-year-old nonobese man who presented to the clinic with new onset diabetes without ketosis. He was treated with insulin and metformin and his hemoglobin A1c decreased from 16% to 6.1% in six months. Other labs showed c-peptide of 8.9 ng/mL in setting of a blood glucose of 231 mg/dL, negative islet cell antibodies and glutamic acid antibodies. Of note, patient also had significant family history of diabetes. Due to high probability of MODY, genetic testing was obtained. He was found to have a heterozygous frameshift mutation in codons 228 -233 leading to a 17 base pair deletion in the PDX1 gene causing replacement of the 56 C- terminal amino acids with a shorter, missense C-terminal tail. Patient was started on dulaglutide 0.75 mg weekly but was hypoglycemic on the smallest dose so he was then switched to Semaglutide and has been maintained on less than 0.25 mg weekly. The clinical question we aim to answer was why was this patient so sensitive to GLP1 agonist activity. To study how the mutation affects response to GLP1 we are using mouse beta-TC-6 cell as they are insulin secreting, and regulated by glucose. The PDX-1 gene is highly conserved among species and the mouse PDX gene is comparable to the human gene. We used CRISPR-Cas9 to introduce the same 17 base-pair deletion into the mouse PDX-1 gene in beta-TC-6 cells. We first measured the glucose stimulated insulin secretion in control cells to examine first and second phases of insulin secretion, determining that the first phase of insulin occurs in twenty minutes and the second phase starts at about two hours. We also evaluated if these cells responded GLP-1 by increasing insulin secretion. We found that these cells respond to glucose and GLP1 in an additive manner. Insulin was measured by enzyme-linked immunosorbent assay (ELIZA). In the next phase of the experiment we aim to determine the effect of the GLP-1 in cell lines with the PDX1 deletion mutation as compared to control cell line. We will measure insulin secretion by ELISA in both mutant and the control cells treated with and without glucose and GLP1. We will measure insulin at 0 minutes and 30 minutes and insulin mRNA at 0 minutes and 6 hours to determine the first and second phases of insulin production. Our results will help guide personalized treatment for patients with MODY type 4 and improve our understanding of beta cell response to GLP1. Presentation: 6/2/2024

Proteomic analysis of HeLa cells after stable transfection with the Chlamydia trachomatis CT143 gene

BackgroundThe CT143 protein of Chlamydia trachomatis (Ct) is a key immunodominant antigen and candidate type-III secretion substrate. Although CT143 expression has not been detected in the cytosol of infected cells, it is known to interfere with the physiological behavior of HeLa cells. This study aims to investigate how the CT143 protein affects the protein expression profile of HeLa cells, providing a basis for further research into Ct’s pathogenic mechanisms. MethodsWe constructed a stably transfected HeLa cell line, pCD513B-1-CT143-HeLa, and a control cell line, pCD513B-1-HeLa. Protein expression profiles of these cell lines were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins were identified, constructed into a database, and verified using parallel reaction monitoring (PRM). Bioinformatics software facilitated the preliminary analysis of the biological functions of these differential proteins. ResultsA total of 221 host proteins were differentially expressed, with 68 upregulated and 153 downregulated. These variations influence the regulation of peptidase activity and are crucial in biological processes such as cell secretion and protease activity. Significant changes were noted in protein processing, alcohol dehydrogenase activity, Aldo-Keto reductase activity, and peptidase regulator activity. Furthermore, alterations were observed in cellular components like the plasma membrane and cell periphery. Pathways involving the hematopoietic system, glycosaminoglycan degradation, retinol metabolism, and cytochrome P450-mediated exogenous drug metabolism were notably affected. Indirect interactions among differentially expressed proteins included three key nodal proteins: C3, IFIT3, and IFIT1. ConclusionThe successful construction of a host differential protein expression profile was achieved through stable transfection of HeLa cells with the CT143 gene. The differential proteins identified are implicated in regulating various biological processes such as intracellular signal transduction, cell secretion, protein processing, hydrolysis, and enzyme activity. These findings suggest that the CT143 protein may influence the host cell’s biological behavior by altering host protein expression, potentially hindering Ct growth and development.