Apolipoprotein B Secretion Assay from Primary Hepatocytes.

Apolipoprotein B (APOB) is the primary structural protein of atherogenic lipoproteins, which drive atherogenesis and thereby lead to deadly cardiovascular diseases (CVDs). Plasma levels of APOB-containing lipoproteins are tightly modulated by LDL receptor-mediated endocytic trafficking and cargo receptor-initiated exocytic route; the latter is much less well understood. This protocol aims to present an uncomplicated yet effective method for detecting APOB/lipoprotein secretion. We perform primary mouse hepatocyte isolation and culture coupled with well-established techniques such as immunoblotting for highly sensitive, specific, and semi-quantitative analysis of the lipoprotein secretion process. Its inherent simplicity facilitates ease of operation, rendering it a valuable tool widely utilized to explore the intricate landscape of cellular lipid metabolism and unravel the mechanistic complexities underlying lipoprotein-related diseases. Key features • A pipeline for the isolation and subsequent culture of mouse primary hepatocytes. • A procedure for tracking the secretion of APOB-containing lipoproteins. • A rapid and sensitive assay for detecting the APOB level based on immunoblotting.

Challenges of laboratory diagnosis of Lyme disease ‒ Questionnaire survey results.

Lyme disease. Our second goal was to identify bacterial and viral co-infections occurring concurrently with Lyme disease. Furthermore, it was our intention to also analyze the correlation of laboratory testing with the occurrence of erythema migrans (EM). BACKGROUND: The accuracy in diagnostic testing for Lyme disease in the early stages of infection is an important factor necessary for delivering proper treatment to patients. METHODS: A total of 173 individuals with confirmed Lyme disease or with laboratory testing underway participated in the quantitative survey. RESULTS: ELISA was the first test conducted in 51% of the respondents, 28% of whom yielded positive findings of both IgM and IgG antibody classes. The positivity of ELISA test findings was confirmed by Western blot in 100% of results. Negative results of ELISA were consistent with Western blot only in less than half of the patients. More than half of the respondents had not been tested for any bacterial or viral co-infections. The results of serological testing were not consistent with clinical findings in all cases, including those with clinically discernible skin manifestation of erythema migrans. CONCLUSION: The comparison of results obtained by ELISA and Western blot revealed significant discrepancies. Simultaneous infections by vectors with several pathogens were detected (Tab. 3, Fig. 2, Ref. 15).

Cymbopogon proximus phytochemicals induce S-phase arrest in A549 lung cancer cell lines via CDK2/cyclin A2 inhibition: gas chromatography-mass spectrometry and molecular docking analyses.

Cymbopogon proximus comprises several phytoconstituent classes that are reported to possess anticancer activity; however, studies on the anticancer potentials of the plant are lacking. C. proximus was extracted using solvents with increasing polarity. In-vitro cytotoxic activity of C. proximus extracts was examined against liver (HepG2), lung (A549), prostate (PC3), and bone (MG63) cell lines using MTT assay in comparison to doxorubicin. Flow cytometry was used to analyze the cell cycle for identification of the phase of inhibition. Chemical composition of the most active fraction was examined using the GC/MS technique. Molecular docking was used to explore the mechanism of cytotoxicity against A549, and the results were confirmed by Western blot analysis. Petroleum ether fraction was the highly effective fraction against A549 with IC50 = 14.02 ± 2.79. GC/MS analysis of Pet.Eth led to the identification of nine compounds in unsaponifiable matter and 27 components in the saponifiable fraction. Di-N-octyl phthalate, 3-ß-hydroxylean-11.13(18)-dien-30-oic acid methyl ester, elemol hydrocarbons, linoelaidic acid and linoleic acid demonstrated the lowest docking binding scores and similar binding modes against CDK2 as compared to that attained by the native ligand R-Roscovitine "CDK2 ATP inhibitor". Western blot analysis demonstrated that CDK2/cyclinA2 protein expression has been suppressed in A549 cell lines by Pet.Eth fraction.

Identification of high-performing antibodies for the reliable detection of Tau proteoforms by Western blotting and immunohistochemistry.

Antibodies are essential research tools whose performance directly impacts research conclusions and reproducibility. Owing to its central role in Alzheimer's disease and other dementias, hundreds of distinct antibody clones have been developed against the microtubule-associated protein Tau and its multiple proteoforms. Despite this breadth of offer, limited understanding of their performance and poor antibody selectivity have hindered research progress. Here, we validate a large panel of Tau antibodies by Western blot (79 reagents) and immunohistochemistry (35 reagents). We address the reagents' ability to detect the target proteoform, selectivity, the impact of protein phosphorylation on antibody binding and performance in human brain samples. While most antibodies detected Tau at high levels, many failed to detect it at lower, endogenous levels. By WB, non-selective binding to other proteins affected over half of the antibodies tested, with several cross-reacting with the related MAP2 protein, whereas the "oligomeric Tau" T22 antibody reacted with monomeric Tau by WB, thus calling into question its specificity to Tau oligomers. Despite the presumption that "total" Tau antibodies are agnostic to post-translational modifications, we found that phosphorylation partially inhibits binding for many such antibodies, including the popular Tau-5 clone. We further combine high-sensitivity reagents, mass-spectrometry proteomics and cDNA sequencing to demonstrate that presumptive Tau "knockout" human cells continue to express residual protein arising through exon skipping, providing evidence of previously unappreciated gene plasticity. Finally, probing of human brain samples with a large panel of antibodies revealed the presence of C-term-truncated versions of all main Tau brain isoforms in both control and tauopathy donors. Ultimately, we identify a validated panel of Tau antibodies that can be employed in Western blotting and/or immunohistochemistry to reliably detect even low levels of Tau expression with high selectivity. This work represents an extensive resource that will enable the re-interpretation of published data, improve reproducibility in Tau research, and overall accelerate scientific progress.

MiR-371-5p regulates trophoblast cell proliferation, migration, and invasion by directly targeting ZNF516.

Despite its prevalence, preeclampsia (PE) remains unclear as to its etiology. Here, we aimed to investigate the mechanisms regulating differences in the gene expression of zinc-finger protein 516 (ZNF516) in the placenta. The expression of the placental ZNF516 gene and its association with critical clinical markers were verified, and a rigorous correlation analysis was conducted. With a dual-luciferase reporter gene assay, microRNA targeting the ZNF516 gene was predicted and confirmed. Finally, the molecular processes associated with ZNF516 were explored via microarray and bioinformatic analyses. In hypoxic conditions, miR-371-5p expression was reduced, resulting in ZNF516 expression being induced. Moreover, ZNF516 was shown to hinder trophoblast cell migration and invasion while enhancing trophoblast cell death in various in vitro cellular assays, such as cell counting kit-8, colony formation, wound healing, and Transwell assays. Our findings reveal a new regulatory network facilitated by ZNF516. ZNF516 overexpression inhibits trophoblast growth, movement, and penetration, potentially causing problems with placenta formation with the help of miR-371-5p suppression.

Evaluation of the Efficacy of OSU-2S in the Treatment of Non-Small-Cell Lung Cancer and Screening of Potential Targets of Action.

(1) Background: OSU-2S is a derivative of FTY720 and exhibits significant inhibitory effects on various cancer cells. There is currently no research on the mechanism of the impact of OSU-2S on NSCLC development. We analysed and validated the hub genes and pharmacodynamic effects of OSU-2S to treat NSCLC. (2) Methods: The hub genes of OSU-2S for the treatment of NSCLC were screened in PharmMapper, genecard, and KM Plotter database by survival and expression analysis. The effect of OSU-2S on hub gene expression was verified by Western blot analysis. The ex vivo and in vivo efficacy of OSU-2S on tumour growth was verified using A549 cells and a xenografted animal model. (3) Results: A total of 7 marker genes for OSU-2S treatment of NSCLC were obtained. AURKA and S1PR1 were screened as hub genes. Significant differences in the expression of AURKA and S1PR1 between normal and lung adenocarcinoma (LUAD) tissues were found in the GEPIA2 database; Western blot showed that OSU-2S could affect p-AURKA and S1PR1 protein expression. OSU-2S significantly inhibited tumour growth in A549 cells and xenografted animal models. (4) Conclusions: Our study confirms the inhibitory effect of OSU-2S on NSCLC, screens and demonstrates its potential targets AURKA(p-AURKA) and S1PR1, and provides a research basis for treating NSCLC with OSU-2S.

ChatGPT's ability to generate realistic experimental images poses a new challenge to academic integrity.

The rapid advancements in large language models (LLMs) such as ChatGPT have raised concerns about their potential impact on academic integrity. While initial concerns focused on ChatGPT's writing capabilities, recent updates have integrated DALL-E 3's image generation features, extending the risks to visual evidence in biomedical research. Our tests revealed ChatGPT's nearly barrier-free image generation feature can be used to generate experimental result images, such as blood smears, Western Blot, immunofluorescence and so on. Although the current ability of ChatGPT to generate experimental images is limited, the risk of misuse is evident. This development underscores the need for immediate action. We suggest that AI providers restrict the generation of experimental image, develop tools to detect AI-generated images, and consider adding "invisible watermarks" to the generated images. By implementing these measures, we can better ensure the responsible use of AI technology in academic research and maintain the integrity of scientific evidence.

Clinical and genetic analyses of APMR4 syndrome caused by novel biallelic LSS variants.

Alopecia intellectual disability syndromes 4 (APMR4) caused by Lanosterol synthase (LSS) gene variants is a very rare autosomal recessive neuroectodermal syndrome. It is characterized by congenital alopecia and variable degrees of intellectual disability (ID), frequently associated with developmental delay (DD) and epilepsy. Currently, only three studies regarding LSS-related APMR4 have been reported, the pathogenesis of APMR4 is poorly understood. We studied one patient with LSS-related APMR4 who presented with severe intellectual disability, alopecia, early-onset epilepsy and developmental delay. She is absence of hair on the eyebrows, eyelashes, and scalp. Two novel LSS variants (c.401 T > G and c.369C > G) were detected with whole-exome sequencing (WES). Analysis via WB experiment indicated that c.369 > G reduced the protein expression level of LSS. Analysis of protein stability prediction showed a destabilizing for LSS caused by the variant c.401 T > G. This study is the first study in Asia to date. These findings expanded the variantal spectrum of LSS-related APMR4 and revealed the potential pathogenic mechanism of LSS gene variants.

Screening approach by a combination of computational and in vitro experiments: identification of fluvastatin sodium as a potential SIRT2 inhibitor.

BACKGROUND: Sirtuins (SIRTs) are NAD+-dependent deacetylases that play various roles in numerous pathophysiological processes, holding promise as therapeutic targets worthy of further investigation. Among them, the SIRT2 subtype is closely associated with tumorigenesis and malignancies. Dysregulation of SIRT2 activation can regulate the expression levels of related genes in cancer cells, leading to tumor occurrence and metastasis. METHODS: In this study, we used computer simulations to screen for novel SIRT2 inhibitors from the FDA database, based on which 10 compounds with high docking scores and good interactions were selected for in vitro anti-pancreatic cancer metastasis testing and enzyme binding inhibition experiments. The results showed that fluvastatin sodium may possess inhibitory activity against SIRT2. Subsequently, fluvastatin sodium was subjected to molecular docking experiments with various SIRT isoforms, and the combined results from Western blotting experiments indicated its potential as a SIRT2 inhibitor. Next, molecular docking, molecular dynamics (MD) simulations, and binding free energy calculations were performed, revealing the binding mode of fluvastatin sodium at the SIRT2 active site, further validating the stability and interaction of the ligand-protein complex under physiological conditions. RESULTS: Overall, this study provides a systematic virtual screening workflow for the discovery of SIRT2 activity inhibitors, identifies the potential inhibitory effect of fluvastatin sodium as a lead compound on SIRT2, and opens up a new direction for developing highly active and selectively targeted SIRT2 inhibitors.

Validation of a novel western blot assay to monitor patterns and levels of alpha dystroglycan in skeletal muscle of patients with limb girdle muscular dystrophies.

The cell membrane protein, dystroglycan, plays a crucial role in connecting the cytoskeleton of a variety of mammalian cells to the extracellular matrix. The α-subunit of dystroglycan (αDG) is characterized by a high level of glycosylation, including a unique O-mannosyl matriglycan. This specific glycosylation is essential for binding of αDG to extracellular matrix ligands effectively. A subset of muscular dystrophies, called dystroglycanopathies, are associated with aberrant, dysfunctional glycosylation of αDG. This defect prevents myocytes from attaching to the basal membrane, leading to contraction-induced injury. Here, we describe a novel Western blot (WB) assay for determining levels of αDG glycosylation in skeletal muscle tissue. The assay described involves extracting proteins from fine needle tibialis anterior (TA) biopsies and separation using SDS-PAGE followed by WB. Glycosylated and core αDG are then detected in a multiplexed format using fluorescent antibodies. A practical application of this assay is demonstrated with samples from normal donors and patients diagnosed with LGMD2I/R9. Quantitative analysis of the WB, which employed the use of a normal TA derived calibration curve, revealed significantly reduced levels of αDG in patient biopsies relative to unaffected TA. Importantly, the assay was able to distinguish between the L276I homozygous patients and a more severe form of clinical disease observed with other FKRP variants. Data demonstrating the accuracy and reliability of the assay are also presented, which further supports the potential utility of this novel assay to monitor changes in ⍺DG of TA muscle biopsies in the evaluation of potential therapeutics.

Characterization of Bitter Taste Receptor-Dependent Autophagy in Oral Epithelial Cells.

Microbial dysbiosis is an important trigger in the development of oral diseases. Oral keratinocytes or gingival epithelial cells (GECs) offer protection against various microbial insults. Recent studies suggest that GECs expressed higher level of bitter taste receptor 14 (T2R14) compared to other taste receptors and toll-like receptors and act as innate immune sentinels. Macroautophagy or autophagy is a cellular conserved process involved in the regulation of host innate immune responses against microbial infection. Here, we describe a robust method for evaluation of T2R14-dependent autophagy flux in GECs. Autophagy flux was detected using Western blot analysis in GECs and further was confirmed using Acridine Orange-dependent flow cytometry analysis.

Downregulation of NAT1 Expression is Associated with Poor Prognosis and Immune Infiltration in COAD.

Background: An increasing corpus of evidence has identified the involvement of N-acetyltransferase 1 (NAT1), a member of the NAT family, in the progression of various cancers. However, the specific function of NAT1 in colon cancer (COAD) remains elusive. This study aims to decip her the role of NAT1 in COAD and its associated mechanisms. Methods: The Tumor Immunity Evaluation Resource (TIMER), The Cancer Genome Atlas (TCGA), and the Gene Expression Omnibus (GEO) databases were employed to assess the NAT1 expression level in COAD. The differential expression between COAD and normal colon tissue was further validated using quantitative real-time reverse-transcription PCR (RT-qPCR) and Western blot (WB) analyses. Additionally, survival analysis of NAT1 in COAD was carried out using the PrognoScan database and TCGA dataset. The functions of NAT1 were explored through gene set enrichment analysis (GSEA) and immuno-infiltration analysis. Results: There was a significant reduction in NAT1 expression in COAD samples compared to normal tissue. Notably, low NAT1 expression in COAD correlated significantly with various clinical parameters such as tumor stage (T stage, N stage, M stage, pathologic stage), primary therapy outcome, carcinoembryonic antigen (CEA) level, and lymphatic invasion. The downregulation of NAT1 was also strongly linked with poor outcomes in overall survival (OS), progression-free interval (PFI), and disease-specific survival (DSS). Cox regression analysis highlighted NAT1 as an independent prognostic indicator for overall survival in COAD patients. GSEA results revealed NAT1's involvement in multiple pathways, including the neuroactive ligand-receptor interaction, olfactory transduction, olfactory signaling, extracellular matrix receptor interaction, calcium signaling, and focal adhesion pathways. Furthermore, NAT1 expression was found to significantly correlate with infiltration levels of various immune cells. Conclusion: The findings reveal NAT1's potential as a valuable prognostic biomarker for COAD. Moreover, its associated mechanisms offer insights that might pave the way for therapeutic interventions for COAD patients.

Performance characteristics of highly automated HSV-1 and HSV-2 IgG testing.

Herpes simplex virus (HSV) infections are one of the most common and stigmatized infections of humankind, affecting more than 4 billion people around the world and more than 100 million Americans. Yet, most people do not know their infection status, and antibody testing is not recommended, partly due to poor test performance. Here, we compared the test performance of the Roche Elecsys HSV-1 IgG and HSV-2 IgG, DiaSorin LIAISON HSV-1/2 IgG, and Bio-Rad BioPlex 2200 HSV-1 and HSV-2 IgG assays with the gold-standard HSV western blot in 1,994 persons, including 1,017 persons with PCR or culture-confirmed HSV-1 and/or HSV-2 infection. Across all samples, the Bio-Rad and Roche assays had similar performance metrics with low sensitivity (<85%) but high specificity (>97%) for detecting HSV-1 IgG and both high sensitivity (>97%) and high specificity (>98%) for detecting HSV-2 IgG. The DiaSorin assay had a higher sensitivity (92.1%) but much lower specificity (88.7%) for detecting HSV-1 IgG and comparatively poor sensitivity (94.5%) and specificity (94.2%) for detecting HSV-2 IgG. The DiaSorin assay performed poorly at low-positive index values with 60.9% of DiaSorin HSV-1 results and 20.8% of DiaSorin HSV-2 results with positive index values <3.0 yielding false positive results. Based on an estimated HSV-2 seroprevalence of 12% in the United States, positive predictive values for HSV-2 IgG were 96.1% for Roche, 87.4% for Bio-Rad, and 69.0% for DiaSorin, meaning nearly one of every three positive DiaSorin HSV-2 IgG results would be falsely positive. Further development in HSV antibody diagnostics is needed to provide appropriate patient care.IMPORTANCESerological screening for HSV infections is currently not recommended in part due to the poor performance metrics of widely used commercial HSV-1 and HSV-2 IgG assays. Here, we compare three Food and Drug Administration (FDA)-cleared automated HSV-1 and HSV-2 IgG assays to the gold-standard western blot across nearly 2,000 samples. We find that not all commercially available HSV assays are created equal, with comparably low sensitivities for HSV-1 IgG across platforms and high false positivity rates for DiaSorin on HSV-2 IgG. This study is the first large-scale comparison of performance metrics for the Bio-Rad and Roche assays in over 10 years. Our study confirms that there remains room for improvement in HSV serological diagnostic testing-especially in regard to low sensitivities for HSV-1 IgG detection-and highlights that some previously less-studied assays may have better performance metrics than previously considered typical of commercially available HSV-2 IgG assays.

Synthesis, characterization and irradiation enhances anticancer activity of liposome-loaded iridium(III) complexes.

Herein, we synthesized and characterized two novel iridium (III) complexes: [Ir(bzq)2(PPD)](PF6) (4a, with bzq = deprotonated benzo[h]quinoline and PPD = pteridino[6,7-f][1,10]phenanthroline-11,13-diamine) and [Ir(piq)2(PPD)](PF6) (4b, with piq = deprotonated 1-phenylisoquinoline). The anticancer efficacy of these complexes, 4a and 4b, was investigated using 3-(4,5-dimethylthiazole)-2,5-diphenltetraazolium bromide (MTT). Complex 4a exhibited no cytotoxic activity, while 4b demonstrated moderate efficacy against SGC-7901, A549, and HepG2 cancer cells. To enhance their anticancer potential, we explored two strategies: (I) light irradiation and (II) encapsulation of the complexes in liposomes, resulting in the formation of 4alip and 4blip. Both strategies significantly increased the ability of 4a, 4b to kill cancer cells. The cellular studies indicated that both the free complexes 4a, 4b and their liposomal forms 4alip and 4blip effectively inhibited cell proliferation. The cell cycle arrest analysis uncovered 4alip and 4blip arresting cell growth in the S period. Additionally, we investigated apoptosis and ferroptosis pathways, observing an increase in malondialdehyde (MDA) levels, a reduction of glutathione (GSH), a down-regulation of GPX4 (glutathione peroxidase) expression, and lipid peroxidation. The effects on mitochondrial membrane potential and intracellular Ca2+ concentrations were also examined, revealing that both light-activated and liposomal forms of 4alip and 4blip caused a decline in mitochondrial membrane potential and an enhancement in intracellular Ca2+ levels. In conclusion, these complexes and them encapsulated liposomes induce cell death through apoptosis and ferroptosis.

Assessing Protein Expression in Patient-Derived Xenografts Using Western Blotting.

The use of patient-derived xenografts (PDXs) in cancer research is increasing due to their ability to closely mimic the features of patient tumors. The ability to quickly and robustly measure protein expression levels in these tissues is a key methodology required in a broad range of experimental designs. Western blotting (WB) is a cost effective and simple tool that is highly specific and sensitive for detecting and quantifying individual proteins, posttranslational modifications and aberrant signaling pathways. Here, we described a method to assess protein expression in PDX tissues using WB to detect proteins involved in cell growth signaling pathways.

Iron Oxide Nanoparticles: Selectively Targeting Melanoma Cells In Vitro by Inducing DNA Damage via H2AX Phosphorylation and Hindering Proliferation through ERK Dephosphorylation.

This study investigates the distinctive characteristics of iron oxide magnetic nanoparticles (mNPs) and their potential application in cancer therapy, focusing on melanoma. Three types of mNPs, pre-validated for safety, underwent molecular analysis to uncover the activated signaling pathways in melanoma cells. Using the Western blot technique, the study revealed that mNPs induce cytotoxicity, hinder proliferation through ERK1/2 dephosphorylation, and prompt proapoptotic effects, including DNA damage by inducing H2AX phosphorylation. Additionally, in vitro magnetic hyperthermia notably enhanced cellular damage in melanoma cells. Moreover, the quantification of intracellular iron levels through Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis unveils the precise dosage required to induce cellular damage effectively. These compelling findings not only shed light on the therapeutic potential of mNPs in melanoma treatment but also open exciting avenues for future research, heralding a new era in the development of targeted and effective cancer therapies. Indeed, by discerning the effective dose, our approach becomes instrumental in optimizing the therapeutic utilization of iron oxide magnetic nanoparticles, enabling the induction of precisely targeted and controlled cellular responses.

New biflavonoids isolated from Xylia kerrii leaves extract with selective cytotoxicity against MH7A human rheumatoid arthritis synovial fibroblast cells.

Three new biflavonoids (1-3) and two known flavonoids (4, 5) were isolated from Xylia kerrii collected in Thailand. Compounds 1-5 showed selective cytotoxicity against the rheumatoid fibroblast-like synovial MH7A cell line, and these compounds showed weak cytotoxicity against the human lung synovial fibroblast WI-38 VA13 sub 2 RA cell line. Notably, compound 1 was highly selective toward MH7A cells with an IC50 value of 6.9 µM, whereas the IC50 value for WI-38 VA13 sub 2 RA cells was > 100 µM. The western blotting analysis of MH7A cells treated with compound 1 showed increased CDKN2A /p16INK4A and caspase-8 levels.

Design, synthesis, in silico and biological evaluation of new indole based oxadiazole derivatives targeting estrogen receptor alpha.

A series of new indole-oxadiazole derivatives was designed and synthesized to develop potential anti-breast cancer agents. The compounds exhibited significant inhibitory activity with IC50 values ranging from 1.78 to 19.74 µM against ER-positive human breast cancer (BC) cell lines T-47D and MCF-7. Among them, compounds (5a, 5c, 5e-5h, 5j-5o) displayed superior activity against ER-α dominant (ratio of ER-α/ER-ß is 9/1) T-47D cells compared to the standard drug bazedoxifene (IC50 = 12.78 ± 0.92 µM). Compounds 5c and 5o exhibited remarkable anti-proliferative activity with IC50 values of 3.24 ± 0.46 and 1.72 ± 1.67 µM against T-47D cells, respectively. Further, compound 5o manifested 1589-fold higher ER-α binding affinity (213.4 pM) relative to bazedoxifene (339.2 nM) in a competitive ER-α binding assay, while compound 5c showed a binding affinity of 446.6 nM. The Western blot analysis proved that both compounds influenced the ER-α protein's expression, impeding its subsequent transactivation and signalling pathway within T-47D cells. Additionally, a molecular docking study suggests that compounds 5c and 5o bind in such a fashion that induces conformational changes in the protein, culminating in their antagonistic effect. Also, pharmacokinetic profiles showed that all compounds have drug-like properties. Further, molecular dynamic (MD) simulations and density functional theory (DFT) analysis confirmed the stability, conformational behaviour, reactivity, and biological feasibility of compounds 5c and 5o. In conclusion, based on our findings, compounds 5c and 5o, which exhibit significant ER-α antagonistic activity, can act as potential lead compounds for developing anti-breast cancer agents.

Eco-friendly green synthesis of N­pyrazole amino chitosan using PEG-400 as an anticancer agent against gastric cancer cells via inhibiting EGFR.

The present study was conducted to develop a green process that provides access to the development of Schiff base derivatives of chitosan with the heterocyclic moiety as a novel class of anti-gastric cancer agent. In the present study, we have synthesized these derivatives by reacting various pyrazoles with chitosan using CAN in PEG400. The compounds were synthesized in 20 min in excellent yield by using CAN at 5% in PEG400 at 80°C in the shortest reaction time of 20 min. The PEG400 could be efficiently recycled for the three consecutive runs. The developed compounds were tested for EGFR-TK inhibition using a Kinase-Glo Plus luminescence kinase assay kit where they exhibited significant activity revealing compound 2d as the most potent analog, while other compounds showed mild to moderate inhibitory activity. MTT assay was conducted to determine the effect of the three most potent EGFR inhibitors (2b, 2c, and 2d) on the proliferation of gastric cancer cells (SGC-7901). The results showed compound 2d as the most potent anticancer agent against SGC7901 cells. The effect of compound 2d was also quantified on the apoptosis and cell phase of SGC7901 cells using flow cytometry assay at various concentrations ranging from 0, 10, 20, and 30 µM. Results suggest that compound 2d showed significant inhibition of SGC-7901 by inducing apoptosis and arresting G0/G1 cell phase. The western blot analysis also revealed that compound 2d significantly inhibited the overexpression of EGFR in SGC-7901 cells. The study successfully demonstrated the development of N­pyrazole amino chitosan as a novel class of agent against gastric cancer via inhibition of EGFR.

Kaempferol as an Alternative Cryosupplement for Bovine Spermatozoa: Cytoprotective and Membrane-Stabilizing Effects.

Kaempferol (KAE) is a natural flavonoid with powerful reactive oxygen species (ROS) scavenging properties and beneficial effects on ex vivo sperm functionality. In this paper, we studied the ability of KAE to prevent or ameliorate structural, functional or oxidative damage to frozen-thawed bovine spermatozoa. The analysis focused on conventional sperm quality characteristics prior to or following thermoresistance tests, namely the oxidative profile of semen alongside sperm capacitation patterns, and the levels of key proteins involved in capacitation signaling. Semen samples obtained from 30 stud bulls were frozen in the presence of 12.5, 25 or 50 µM KAE and compared to native ejaculates (negative control-CtrlN) as well as semen samples cryopreserved in the absence of KAE (positive control-CtrlC). A significant post-thermoresistance test maintenance of the sperm motility (p < 0.001), membrane (p < 0.001) and acrosome integrity (p < 0.001), mitochondrial activity (p < 0.001) and DNA integrity (p < 0.001) was observed following supplementation with all KAE doses in comparison to CtrlC. Experimental groups supplemented with all KAE doses presented a significantly lower proportion of prematurely capacitated spermatozoa (p < 0.001) when compared with CtrlC. A significant decrease in the levels of the superoxide radical was recorded following administration of 12.5 (p < 0.05) and 25 µM KAE (p < 0.01). At the same time, supplementation with 25 µM KAE in the cryopreservation medium led to a significant stabilization of the activity of Mg2+-ATPase (p < 0.05) and Na+/K+-ATPase (p < 0.0001) in comparison to CtrlC. Western blot analysis revealed that supplementation with 25 µM KAE in the cryopreservation medium prevented the loss of the protein kinase A (PKA) and protein kinase C (PKC), which are intricately involved in the process of sperm activation. In conclusion, we may speculate that KAE is particularly efficient in the protection of sperm metabolism during the cryopreservation process through its ability to promote energy synthesis while quenching excessive ROS and to protect enzymes involved in the process of sperm capacitation and hyperactivation. These properties may provide supplementary protection to spermatozoa undergoing the freeze-thaw process.