Fetal Bovine Serum Research Articles

Curcumin alleviates the aggressiveness of breast cancer through inhibiting cell adhesion mediated by TEAD4-fibronectin axis

Breast cancer (BC) ranks first among women in the world and metastasis is the main reason of cancer death. TEA domain transcription factor 4 (TEAD4) plays an important role in TEAD family members in the Hippo signaling pathway, and its pro-cancer effect is gradually being discovered, but little is known about the mechanism of TEAD4 regulating tumor adhesion and metastasis. Curcumin is an active polyphenol compound with anti-inflammatory and anti-tumor properties. However, its effects and the underlying mechanisms on BC metastasis remain unknown. Here, we show that curcumin reduced the abilities of migration and invasion of BC cells as well as lung metastasis of BC in nude mice by TEAD4 induced. TEAD4 could regulate the transcription level of adhesion molecule Fibronectin (FN1), which is an important component of extracellular matrix participating in tumor cell adhesion and migration processes, and the binding of TEAD4 to the FN1 promoter was suppressed by curcumin. Furthermore, the metastatic mobility was significantly reduced in FN1 knockout BC cells, and FN1 knockout can reverse metastatic potential of TEAD4 overexpressed cells. Our work demonstrates that TEAD4 could promote cell adhesion and migration by binding with FN1 promoter and suggested that TEAD4-FN1 axis in tumor microenvironment is expected to become a potential target for alleviating metastasis of BC of curcumin.Abbreviations: Breast cancer (BC); Bicinchoninic acid (BCA); Bovine Serum Albumin (BSA); Curcumin at 20 μM (Cur20) and 30 μM (Cur30); Dimethyl −sulfoxide (DMSO); Dulbecco’s Modified Eagle’s Medium (DMEM); Extracellular Matrix (ECM); Fetal bovine serum (FBS); Fibronectin (FN1); Immunohistochemical (IHC); Minimum Essential Medium (MEM); The negative control with infected blank lentivirus (NC); Phenylmethanesulfonyl fluoride (PMSF); Polyvinylidene difluoride (PVDF); Real-time quantitative reverse transcription (RT-qPCR); single guide RNA (sgRNA); short hairpin RNA (shRNA); Human TEAD4 shRNA (shTEAD4); Transcriptional coactivator with PDZ-binding motif (TAZ); TEA domain transcription factor 4 (TEAD4); TEAD4 overexpressed (TEAD4-OE); Vascular endothelial growth factor (VEGF); Yes-associated protein (YAP);

The roles of regulatory-compliant media and inflammatory/oxytocin priming selection in enhancing human mesenchymal stem/stromal cell immunomodulatory properties

Osteoarthritis (OA) represents a significant global health burden without a known disease modifying agent thereby necessitating pursuit of innovative therapeutic approaches. The infrapatellar fat pad (IFP) serves as a reservoir of mesenchymal stem/stromal cells (MSC), and with adjacent synovium plays key roles in joint disease affecting local inflammatory responses. Therapeutically, IFP-MSC have garnered attention for their potential in OA treatment due to their immunomodulatory and regenerative properties. However, optimizing their therapeutic efficacy necessitates a comprehensive understanding of how growth medium and inflammatory/hormonal priming influence their behavior. In this study, we isolated and expanded IFP-MSC in three different growth media: DMEM + 10% fetal bovine serum (FBS), DMEM + 10% human platelet lysate (HPL), and xeno-/serum-free synthetic (XFSF) medium. Subsequently, cells were induced with an inflammatory/fibrotic cocktail (TIC) with or without oxytocin (OXT). We evaluated various parameters including growth kinetics, phenotype, immunomodulatory capacity, gene expression, and macrophage polarization capacity. Our results revealed significant differences in the behavior of MSC cultured in different media. IFP-MSC cultured in HPL and XFSF exhibited superior growth kinetics and colony-forming abilities compared to those cultured in FBS. Furthermore, both HPL and XFSF media enhanced the expression of MSC markers (> 90%) and potentiated their immunomodulatory properties. Notably, XFSF-conditioned IFP-MSC demonstrated the highest attenuation of peripheral blood mononuclear cell (PBMC) proliferation, indicating their robust immunosuppressive capacity. Additionally, TIC priming further augmented the immunomodulatory functionality of MSC, with IFP-MSC exhibiting enhanced suppression of PBMC proliferation upon TIC priming. Of particular interest, gene expression analysis revealed distinct patterns in TIC + OXT induced MSC compared to TIC only induced, with upregulation of genes associated with immunomodulatory and regenerative functions. Furthermore, TIC + OXT priming promoted M2 polarization in macrophages, suggesting a potential therapeutic strategy for immune-mediated inflammatory joint conditions including OA. Our findings highlight the critical influence of growth medium and inflammatory/hormonal priming on MSC behavior and therapeutic potential. XFSF and HPL media offer promising alternatives to FBS, enhancing MSC growth and immunomodulatory properties. Moreover, TIC + OXT priming represents a novel approach to augment MSC immunomodulation and promote M2 polarization, providing insights into potential therapeutic strategies for OA and other immune-mediated inflammatory conditions.

Realizing real-time optical molecular imaging in peripheral nerve tissue via Rhodamine B

BackgroundIatrogenic nerve injury is a consequential complication during surgery. Thus, real-time imaging of peripheral nerve (PN) possesses significant clinical implications. In recent years, the rapid advancements in optical molecular imaging (OMI) technology have provided essential technical foundations for the implementation of PN fluorescence imaging. This study aimed to realize real-time OMI of PNs via Rhodamine B.MethodsPhosphate buffered saline (PBS), normal saline (NS), 5% glucose solution (GS), and fetal bovine serum (FBS) were selected for measuring the fluorescence spectra of Rhodamine B solutions prepared in each formulation. Rhodamine B solutions, with varying doses dissolved in 100 μL of each formulation, were prepared and applied to the exposed PNs of the mice for incubation later. To ascertain the optimal formulation and dose of Rhodamine B, an analysis was performed on the signal-to-background ratio (SBR) of the nerves. Based on the experimental results, we proceeded to incubate Rhodamine B solution on the PN tissue of mice and human subjects, as well as on neuronal cells, to verify the binding sites of Rhodamine B with nerve. Subsequently, histological studies were conducted to validate the binding site between Rhodamine B and the nerves. Finally, we injected the optimal combination of Rhodamine B solution into mice via the tail vein and collected the SBR of mouse nerve tissues at different time intervals to determine the optimal pre-injection time. Fluorescence images of various tissues were collected, and Hematoxylin and Eosin (H&E) staining results were observed to determine the metabolism of Rhodamine B in mice and its toxicity.ResultsThe excitation peak of Rhodamine B in PBS, NS, 5% GS, and FBS formulations was 554 nm, and the emission peak was 576 nm. In PBS group, the maximum SBR was 15.37 ± 0.68 while the dose of Rhodamine B was 8 nmol. Through ex-vivo validation on fresh human nerve tissue and verification using mouse and human tissue sections, we observed fluorescent signals of Rhodamine Bin the regions of nerve tissue and the fluorescence signals were all concentrated on the neuronal cell membranes. After injection, the fluorescent signal in nerve tissue reached its peak at 24 hours (h), coinciding with the highest SBR (5.93 ± 0.92) in mouse nerve tissues at this time point. Additionally, the fluorescence signal could be maintained for at least 48 h. Within 24 h, lung dilation and fusion of alveoli occurred. Then these pathological manifestations gradually diminished, returning to normal at 2 weeks (w), with no significant acute or chronic adverse reactions observed in other tissues.ConclusionRhodamine B enables fluorescence imaging of PNs and has the potential for clinical translation.

In vitro Cultured Meat: Nutritional Aspects for the Health and Safety of Future Foods

In vitro cultured meat is one of the future foods that might revolutionize meat production by allowing food manufacturers to sidestep traditional animal farming. Despite being in early development, in vitro cultured meat faces challenges such as technological limitations and consumer acceptance, hindering its market integration. Beyond being an ethical alternative to conventional meat, it resembles conventionally farmed meat, making it a focus on healthcare and food safety. From a health perspective, it theoretically presents intriguing possibilities in the fields of functional foods, tailored dietary therapy, and public health. In functional foods, its unique composition allows precise nutritional manipulation for specific dietary needs, aligning with personalized nutrition. In dietary therapy, in vitro cultured meat might offer customization for individuals with dietary restrictions or health conditions, providing a novel therapeutic avenue. From a public health standpoint, it holds promise in mitigating environmental and health challenges linked to conventional meat production, aligning with sustainability and disease prevention initiatives. However, the potential health risks of in vitro cultured meat include concerns about the use of genetically modified starting cells, fetal bovine serum, growth factors, scaffolding materials, and antibiotics during the production process. These additives may have unforeseen long-term health effects if not properly regulated. Additionally, in vitro cultured meat may lack some of the natural nutrients found in conventional meat, which could lead to nutritional imbalances. There are also uncertainties about how the texture and bioavailability of nutrients in in vitro cultured meat might affect digestion and absorption in the human body. Nevertheless, specific aspects of research in the field of in vitro cultured meat demand increased focus from researchers to guarantee the optimal safety standards. An in-depth study on safety-oriented research of in vitro cultured meat should be emphasized to ensure benefits for individual and public health. HIGHLIGHTS In theory, in vitro cultured meat offers potential as a novel solution for developing functional foods by allowing precise manipulation of nutritional content, enabling the creation of health-optimized products. In vitro cultured meat could help mitigate the risk of antimicrobial resistance by minimizing the need for antimicrobial agents, reducing overall exposure and the potential for the development and spread of antimicrobial-resistant microorganisms. Starting cells derived from genetically modified cells in the in vitro cultured meat may pose risks of allergenicity and contamination, as well as potential immune responses to novel proteins. Strict safety protocols and thorough testing are crucial to address these risks and ensure the final product is safe for consumption. GRAPHICAL ABSTRACT

Zwitterion-Conjugated Protein Coatings for Enhanced Antifouling in Complex Biofluids: Underlying Molecular Interaction Mechanisms.

Biofouling can cause severe infections, device malfunctions, and failures in diagnostics and therapeutics. Proteins such as bovine serum albumin (BSA) have recently been used as coatings to resist biofouling because they combine surface anchoring and antifouling properties. However, their antifouling effectiveness will significantly deteriorate in complex biofluids with high salinity, limiting their practical applications. In this work, we developed a zwitterion-conjugated protein with enhanced antifouling capability by grafting zwitterionic 2-methacryloyloxyethyl phosphorylcholine (MPC) onto BSA protein via a click reaction. This conjugated protein can easily anchor on various substrates, both inorganic and organic, and exhibits efficient and broad-spectrum fouling resistance to metabolites, proteins, and complex biofluids. Even in the complex fetal bovine serum with higher salinity, the BSA@MPC coating can also maintain 99% fouling resistance robustly, over 6-fold superior to native BSA-coated surfaces in antifouling capability. Direct surface forces measurement reveals that such outstanding antifouling properties of conjugated protein BSA@MPC could be attributed to the stable hydration layer on its surface and the steric repulsion from the antipolyelectrolyte behavior of zwitterionic MPC polymer in the high-salinity environment. Our findings advance the development of protein-based functional materials and provide valuable insights for designing novel antifouling surfaces for marine, food, and bioengineering applications.

Qualitative and Quantitative Luminescence Detection of 2,6-Dipicolinic Acid and Levofloxacin Based on a High-Nuclearity Eu(III) Nanomolecular Sensor.

Rapid and accurate testing of 2,6-dipicolinic acid (DPA) and levofloxacin (LFX) has been attracting much attention due to the fact that the former is an important biomarker of anthrax spores and the latter is a third-generation fluoroquinolone drug and has been recognized as an important environmental pollutant. Herein, we report the preparation of a 13-metal Eu(III) nanomolecular sensor (molecular sizes: 2.0 × 2.4 × 3.2 nm) bearing a new flexible Schiff base ligand for luminescence testing of DPA and LFX with high selectivity and sensitivity. The rapid enhancement of the Eu(III) luminescence of 1 caused by DPA and LFX is expressed by fitting equation I615nm = k × [C] + a, which can be used to quantitatively detect DPA and LFX concentrations in CH3CN, fetal calf serum (FCS), and a real drug. The response times of 1 to DPA and LFX are less than ten seconds, with detection limits as low as 3.78 × 10-2 to 2.71 nM. The test strips containing 1, as well as 1@SA films, can be used to qualitatively detect DPA and LFX by the color changes under the irradiation of UV light.

Automated Manufacturing Processes and Platforms for Large-scale Production of Clinical-grade Mesenchymal Stem/ Stromal Cells.

Mesenchymal stem/stromal cells (MSCs) hold immense potential for regenerative medicine due to their remarkable regenerative and immunomodulatory properties. However, their therapeutic application requires large-scale production under stringent regulatory standards and Good Manufacturing Practice (GMP) guidelines, presenting significant challenges. This review comprehensively evaluates automated manufacturing processes and platforms for the scalable production of clinical-grade MSCs. Various large-scale culture vessels, including multilayer flasks and bioreactors, are analyzed for their efficacy in MSCs expansion. Furthermore, automated MSCs production platforms, such as Quantum® Cell Expansion System, CliniMACS Prodigy®, NANT001/ XL, CellQualia™, Cocoon® Platform, and Xuri™ Cell Expansion System W25 are reviewed and compared as well. We also underscore the importance of optimizing culture media specifically emphasizing the shift from fetal bovine serum to humanized or serum-free alternatives to meet GMP standards. Moreover, advances in alternative cryopreservation methods and controlled-rate freezing systems, that offer promising improvements in MSCs preservation, are discussed as well. In conclusion, advancing automated manufacturing processes and platforms is essential for realizing the full potential of MSCs-based regenerative medicine and accomplishing the increasing demand for cell-based therapies. Collaborative initiatives involving industry, academia, and regulatory bodies are emphasized to accelerate the translation of MSCs-based therapies into clinical practice.

Virus susceptibility of a new cell line derived from the muscle of koi (Cyprinus carpio koi).

In this study, a continuous cell line (KM cells) derived from koi (Cyprinus carpio koi) muscle was established and characterized. The KM cells were subcultured for more than 70 passages and showed high viability after long-term cryopreservation. The KM cell line was optimally cultured in medium 199 containing 10% foetal bovine serum at 25°C. A chromosome analysis indicated that the cell line remained diploid, with a mean chromosome count of 100. DNA sequencing and comparative analysis of the 16S rRNA and cytochrome oxidase I gene sequences showed that the KM cell line originated from koi. In transfection experiments using the plasmid pEGFP, KM cells demonstrated a high level of transfection efficiency, suggesting their potential for use in foreign gene expression studies. Inoculation with spring viraemia of carp virus (SVCV) resulted in a substantial cytopathic effect, and the level of production of SVCV in KM cells was higher than that in the epithelioma papulosum cyprinid (EPC) cell line that is normally used to produce the virus. However, no cytopathic effect was observed when these cells were inoculated with koi herpesvirus, carp oedema virus, or grass carp reovirus. These observations suggest that the newly established KM cell line will be a valuable tool for investigating the pathogenesis of infection with spring viraemia of carp virus.

Translational implications of a novel combination of iPRF and collagen scaffold for proliferation of gingival mesenchymal stem cells

“Tissue engineering,” is a concept that involves the use of scaffolds, cells, and growth factors/signaling molecules in the field of regenerative medicine. The present study was carried out to evaluate the attachment and depth of penetration of the Gingival Mesenchymal Stem Cells (GMSCs) onto the collagen scaffold preconditioned with Fetal Bovine Serum (FBS), Injectable Platelet Rich Fibrin (i-PRF) and a combination of FBS with i-PRF using histological analysis and environmental scanning electron microscopy (ESEM) respectively. In the present study, commercially available collagen membranes were used as scaffolds and divided into 3 groups where Group I was preconditioned with FBS, Group II was preconditioned with i-PRF, and Group III with a combination of FBS and i-PRF. Scaffolds were then seeded with GMSCs and incubated in a CO2 incubator at 37 0C for 7 days. Both histological and ESEM analysis showed proliferation, attachment, and depth of penetration along with the combination of cell morphological changes in all the three groups. In group I cells showed mild depth of penetration along with a round morphological appearance. In group II the cells showed moderate depth of penetration and rounded morphology. In group III maximum depth of penetration was seen along with the round and spheroidal morphology of cells. The combination of the growth media showed the best effect on cell attachment, depth of penetration, and cell morphology. This is the first report demonstrating the effect of the combination of collagen and i-PRF supports the proliferation of GMSCs. Since FBS alone and i-PRF alone exhibited similar cell proliferation it is recommended that i-PRF could be used in clinical scenarios making it xenofree.

Enhanced stability of hairpin-functionalized DNA tetrahedral nanostructures for miRNA detection in plasma from ischemic stroke patients

The enzyme-free amplification technique using the Hybridization Chain Reaction (HCR) is gaining traction for its efficiency in miRNA analysis. Conventional HCR (C-HCR) with hairpin probes faces challenges due to enzymatic degradation in body fluids, leading to potential false-positive results. This study addresses the critical need for a more reliable method that resists enzymatic breakdown and improves diagnostic accuracy for detecting miRNA related to ischemic stroke. We have developed a novel DNA tetrahedral nanostructures-mediated HCR (DTN-HCR) platform for the precise detection of microRNA-25 (miR-25), a biomarker for ischemic stroke. Incorporating two unique DNA tetrahedral nanostructures with embedded hairpin structures (DTN-HP1 and DTN-HP2), this platform activates upon miR-25 binding, initiating a robust DTN-HCR reaction. This reaction forms extensive DNA tetrahedron clusters that significantly boost the fluorescence signal, enabling detection thresholds as low as 5.4 pM. The method showcases exceptional specificity by distinguishing target miRNA from close analogues and maintains structural integrity against DNase I and fetal bovine serum (FBS), verified through polyacrylamide gel electrophoresis (PAGE). It successfully differentiates ischemic stroke patients from healthy controls by analyzing peripheral blood-derived miRNAs. This study concludes that the DTN-HCR platform substantially enhances the specificity and stability of miRNA detection, marking a significant advancement in non-enzymatic miRNA analysis techniques. With its capability to accurately identify ischemic stroke biomarkers at very low concentrations and its resistance to enzymatic degradation, the DTN-HCR method presents a valuable diagnostic tool for ischemic stroke, potentially improving early detection and monitoring in a clinical environment.

Magnetic graphene oxide increases the biocompatibility and nuclear factor erythroid 2-related factor 2 antioxidant of human cumulus cells: A lab-trial study.

Although assisted reproductive technology has been improved, the success rate is only 30%. Since the interaction between oocytes and cumulus cells (CCs) is necessary for the formation of a fertile oocyte, increasing the survival rate of CCs can improve the function of oocytes in infertile women. This study aimed to investigate the effects of magnetic graphene oxide (MGO) nanocomposite on the biocompatibility and antioxidant activity of human CCs. In this lab-trial study, from July 2021-2023 human CCs were collected from 37 women aged 20-37 yr and cultured in a medium containing Dulbecco's Modified Eagle's/F12, fetal bovine serum (10%), and penicillin-streptomycin (1%). Then CCs were treated with increasing concentrations of nano-MGO for 24, 48, and 72 hr (3[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyltetrazolium bromide) assay and flow cytometry technique were used to compare the survival rate and apoptosis of CCs before and after treatment. Western blot test was used for expressing nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant in 2 groups. The results of the present study showed that treatment with MGO increased the viability of CCs at a concentration of 50 µg/ml after 48 hr (p 0.01). At higher doses (100 µg/ml) MGO decreased the survival rate of CCs (p 0.05). Also, treatment with MGO at a concentration of 50 µg/ml increased the expression level of antioxidant protein Nrf2 in human CCs. Our results highlight the use of MGO in a new strategy that improves CCs viability and secretion of antioxidant protein Nrf2, thereby potentially increasing in vitro fertilization outcomes.

Mouse liver blood flow is regulated by hepatic stellate cells in response to the sympathetic neurotransmitter norepinephrine

BackgroundThere is no clear information on the regulation of liver blood flow by the autonomic nervous system. We conducted this study to investigate whether quiescent hepatic stellate cells (qHSCs) regulate liver blood flow in response to the sympathetic neurotransmitter norepinephrine (NE). MethodsqHSCs isolated from mice were cultured in Dulbecco's modified Eagle medium without fetal bovine serum for 1 day on collagen gel. NE-induced qHSC contraction was evaluated using the quantitative single-cell contraction measurement method that we had developed previously. For the measurement of liver perfusion pressure in situ, a buffer solution was perfused from the portal vein in mice. ResultsNE-induced a reversible contraction of qHSCs. This contraction was suppressed by the nonmuscle myosin II inhibitor blebbistatin, the myosin light chain kinase inhibitor ML-9, the Rho kinase inhibitor H-1152, the calmodulin inhibitor W-7, the store-operated calcium channel inhibitor YM-58483, and the IP3 receptor inhibitor xestospongin C. In contrast, the transient receptor potential C channel inhibitor SKF96365 did not affect the NE-induced contraction. ConclusionThese results suggest that qHSCs contract in response to NE. New & noteworthyThe present study provides direct evidence for the first time that norepinephrine (NE) induces a reversible contraction of isolated single quiescent hepatic stellate cells (qHSCs) and further suggests that the NE-mediated qHSC contraction participates in the regulation of liver blood flow in vivo.

Beyond a ferroptosis inducer: erastin can suppress nutrient deprivation induced cell death in the intervertebral disc

Background: Erastin has been found to induce ferroptosis; however, whether erastin may have roles other than ferroptosis inducer in cells is unknown. Nutrient deficiency is one of the major causes of many diseases including intervertebral disc (IVD) degeneration. Purpose: The current study investigates the effect of erastin in nucleus pulposus cells under nutrient deprivation condition. Study Design: Experiment in vitro and ex vivo. Methods: The effect of erastin on the cell survival of nucleus pulposus cells was evaluated in fetal bovine serum (FBS) and glucose deprivation condition. RSL3 and ferrostatin-1 were applied to illustrate whether the effect of erastin is ferroptosis dependent. The involvement of solute carrier family 7, membrane 11(SLC7A11), autophagy as well as mechanistic target of rapamycin kinase complex 1(mTORC1) and transcription factor EB (TFEB) were assessed to demonstrate the working mechanism of erastin. Results: Erastin may induce cell death at the concentration of ≥ 5μM; however, it may protect nucleus pulposus cells against nutrient deprivation induced cell death at lower concentration (0.25-1μM) and the effect of erastin is ferroptosis independent. The mechanism study showed that the effect of erastin may relate to its SCL7A11 regulation, as SCL7A11 knock-down may have the similar effect as erastin. Furthermore, it was also demonstrated that mTORC1-TFEB mediated autophagy was involved in protective effect of erastin. Conclusions: Low dose erastin may promote cell survival under nutrient deprivation condition, and its effect is ferroptosis independent; erastin may exert its protective effect through mTORC1-TFEB mediated autophagy regulation. Clinical Significance: Nutrient deprivation is a major contributor to intervertebral disc degeneration. Our in vitro and ex vivo study showed that low dose of erastin may suppress nutrient deprivation induced cell death in IVD degeneration. Although it was not validated in vivo model due to lack of in vivo nutrient deprivation induced IVD degeneration model currently, this study may still provide a potential therapeutic option for IVD degeneration, which of cause need further validation.

Design of Experiments to Tailor the Potential of BSA-Coated Peptide Nanocomplexes for Temozolomide/p53 Gene Co-Delivery.

Background: Gene therapy can be viewed as a promising/valuable therapeutic approach directed to cancer treatment, including glioblastoma. Concretely, the combination of gene therapy with chemotherapy could increase its therapeutic index due to a synergistic effect. In this context, bovine serum albumin (BSA)-coated temozolomide (TMZ)-peptide (WRAP5)/p53 gene-based plasmid DNA complexes were developed to promote payload co-delivery. Methods: Design of experiments (DoE) was employed to unravel the BSA-coated TMZ-WRAP5/p53 nanocomplexes with the highest potential by considering the nitrogen to phosphate groups ratio (N/P), and the BSA concentration as inputs and the size, polydispersity index, surface charge and p53-based plasmid complexation capacity (CC) as DoE outputs. Results: The obtained quadratic models were statistically significant (p-value < 0.05) with an adequate coefficient of determination, and the correspondent optimal points were successfully validated. The optimal complex formulation had N/P of 1.03, a BSA concentration of 0.08%, a size of approximately 182 nm, a zeta potential of +9.8 mV, and a pDNA CC of 96.5%. The optimal nanocomplexes are approximately spherical. A cytotoxicity assay showed that these BSA-coated TMZ-WRAP5/p53 complexes did not elicit toxicity in normal brain cells, and a hemolysis study demonstrated the hemocompatibility of the complexes. The complexes were stable in cell culture medium and fetal bovine serum and assured pDNA protection and release. Moreover, the optimal BSA-coated complexes were able of gene transcription and promoted a significant inhibition of glioblastoma cell viability. Conclusions: The reported findings instigate the development of future research to evaluate their potential utility to TMZ/p53 co-delivery. The DoE tool proved to be a powerful approach to explore and tailor the composition of BSA-coated TMZ-WRAP5/p53 complexes, which are expected to contribute to the progress toward a more efficient therapy against cancer and, more specifically, against glioblastoma.

A dual-template molecularly imprinted electrochemical sensor assisted with BO-XGBoost algorithm for simultaneous determination of dopamine and acetaminophen

The simultaneous detection of dopamine (DA) and acetaminophen (AP) is crucial for diagnosing and treating related mental disorders. However, accurately determining DA and AP in biological samples remains challenging due to the interference from other biomolecules, such as ascorbic acid (AA), uric acid (UA), epinephrine (EP), etc. Here, we present a dual-template molecularly imprinted electrochemical sensor for the selective recognition of DA and AP. Reduced graphene(rGO)-gold nanoparticles(AuNPs) composite were utilized to enhance the effective area and increase the electron transfer rate of glassy carbon electrode(GCE), thus the electrochemical response signals were amplified. This sensor combined the advantages of nanocomposites and molecularly imprinted polymer (MIP) to achieve highly sensitive and selective detection of DA and AP. However, decreases in the current response of some analytes and variations in relationships of the peak current versus analyte concentration will occur due to adsorption competition on active sites in multianalyte mixtures, rendering the detection range constrained and traditional linear fitting methods inaccurate for predicting analyte concentrations. Therefore, a concentration prediction model based on XGBoost was developed for the sensor to achieve the reliable multi-component determination of DA and AP within the identical measurement range as individual detection. In this model, nine characteristic parameters were extracted from the differential pulse voltammetry (DPV) response curve and Bayesian optimization (BO) was adopted for automatic hyperparameter search, thereby the prediction errors were reduced and the generalization ability was improved. Experiments indicated that the MIP/AuNPs/rGO/GCE exhibited a wide detection range of 2–240 μM and 3–240 μM for DA and AP, with detection limits down to 0.26 μM and 0.33 μM, respectively. In addition, the intelligent MIP/AuNPs/rGO/GCE sensor based on BO-XGBoost model provides more accurate prediction results for untrained concentration combinations obtained from fetal bovine serum samples, indicating that the proposed detection scheme holds potential in future clinical diagnosis.

Reducing the danger of phytopathogen toxins by using an organomineral composition

Relevance. Contamination by toxins of phytopathogens or mycotoxins of food and feed raw materials of plant origin poses a serious threat to the production of high-quality and safe agricultural products. Reducing the risks of contamination of plant raw materials with mycotoxins depends on the effective use of biological products to reduce the toxicity of mycotoxins.Methods. Experiments were carried out on primary rat liver cells. Primary liver cells were cultured in DMEM medium in the presence of 10% fetal calf serum at 37OC and 5% CO2. Zearalenone and T-2 toxin were dissolved in a mixture of DMSO and 96% alcohol in a ratio 1:1. Zearalenone T-2 toxin and protective compositions were mixed and aged together for 6 hours and after exposure added to a medium with a cellular monolayer. The concentration of bacterial-based compositions KMBI-3 and KMCI-3 was used for research in three variants: 0.4 mg/ml, 2 mg/ml, 4 mg/ml. After 24 hours of cultivation, the cell layer was evaluated using an inverted microscope according to the following parameters: percentage of surface coverage, cell shape, number of cell aggregates, number of floating cells.Results. A dose-dependent decrease in cell viability was revealed when exposed to zearalenone and T-2 toxin, the most toxic effect was observed at doses from 0.5 х 10-4 and 8.6 х 10-8 M and 2.14 х 10-7 M, respectively. When using the protective composition KMBI-3 at a dose of 4 mg/ml, the least negative effect of zearalenone and T-2 toxin on cell culture was observed. The use of compositions of organomineral origin KMBI-3 helps to increase cell viability when exposed to the toxins zearalenone and T-2 toxin, which indicates the activation of proliferative processes in comparison with the group without the use of drugs. The use of the biopreparation KMBI-3 reduces the pathogenic effect of zearalenone and T-2 toxin on cell culture, increases the resistance of liver cells to the effects of phytopathogen toxins.

Regulation of hepatic transporters OATP1A2 and OATP1B1 by the action of nitric oxide (II)

Nitric oxide II (NO) is a signaling molecule that has a wide range of physiological effects, including the regulation of gastrointestinal processes. The liver actively expresses the clinically significant transporters OATP1A2 and OATP1B1, which are involved in the influx of biologically active and medicinal substances. That is why it seems relevant to determine the pathways of regulation of hepatic transporters under the influence of NO. Aim. To study the effect of NO on the relative amount and expression of the transporters OATP1A2 and OATP1B1 in vitro in HepG2 cells. Materials and methods. The study was performed on a culture of HepG2 cells, which were cultured in 6-well plates at 37 °C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) with a high glucose content (4500 mg/l) containing L-glutamine (4 mM), 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin (all components from Sigma-Aldrich, Germany). S-nitrosoglutathione (Sigma-Aldrich, Germany) was added to the culture medium at concentrations of 1, 10, 50, 100 and 500 µM, incubated for 24 and 72 hours. Water for injection (solvent) was added to control cells in an equivalent volume S-nitrosoglutathione). The relative amounts of OATP1A2 and OATP1B1 proteins were assessed by Western blot, and the expression of SLCO1A2 and SLCO1B1 by real-time PCR. The results of the study. In the course of this study, it was shown that the addition of S-nitrosoglutathione in the concentration range of 10-500 μM and exposure duration of 24 and 72 hours causes an increase in the intracellular level of nitric oxide metabolites, which indicates the adequacy of the use of this NO donor. At the same time, under the influence of NO, there was an increase in the relative amount of the studied transporters - OATP1A2 at an exposure period of 24 hours and S-nitrosoglutathione concentrations of 50 and 100 μM, OATP1B1-24 and 72 hours, at concentrations of 10-500 μM, a similar trend was noted for the expression genes SLCO1A2 and SLCO1B1. Conclusion. The NO donor - S-nitrosoglutathione causes an increase in the relative amount of OATP family transporters - OATP1A2 and OATP1B1, due to increased expression of the SLCO1A2 and SLCO1B1 genes, in vitro in HepG2 cells.

Carbon cloth surface engineering for simultaneous detection of ascorbic acid, dopamine, and uric acid in fetal bovine serum

Carbon cloth (CC) was electrochemically activated using three electrochemical methods in different electrolytes. The gases released during the activation process etched the surface of the CC, thereby increasing its surface area of the activated CC (FCC). Moreover, functional groups such as oxygen-containing groups and N-doping have been introduced. The quantity and type of functional groups introduced during the activation process were related to the anions in the solution. FCC electrodes were used to construct electrochemical sensors for the simultaneous determination of ascorbic acid (AA), dopamine (DA), and uric acid (UA). The PFCC electrode activated by advanced cyclic voltammetry in (NH4)2HPO4 showed linear ranges for AA, DA, and UA concentrations of 0.1 to 2.1 mM, 0.5 to 11 µM, and 0.5 to 11 µM, respectively. The detection limits are 72.93, 0.22, and 0.42 µM, respectively. The good flexibility the PFCC electrode made it suitable for the preparation of flexible sensors. The simultaneous determination of AA, DA, and UA in fetal bovine serum showed a reliable recovery ratio. This study provides a simple and green approach for activating carbon cloth and constructing flexible electrochemical sensors with the potential to simultaneously detect AA, DA, and UA.

The Biological Effect of Enriching the Plasma Content in Platelet-Rich Plasma: An In Vitro Study.

Platelet-rich plasma (PRP) formulations have become valuable therapeutic tools in regenerative medicine. In addition, these blood derivates have been successfully included in cell therapy as fetal bovine serum substitutes, due to the real need to avoid the risk of host immunologic reactions and the animal disease transmission associated with reagents from animal origin. However, the protocols for obtaining them should be optimized to improve their biological potential. PRP-derived preparations with different concentrations of the platelet and plasma components were obtained from the blood of five donors by freeze-drying. Measurements of the pH, protein, and growth factor concentration were performed. Moreover, their biological effects on cell proliferation and migration and their angiogenic potential were assessed. An increased plasma component concentration resulted in an augmented quantity of the total protein content, a significative variation in the hepatocyte growth factor concentration, and an experimental but clinically irrelevant alteration of the pH value. No significant changes were induced in their potential to enhance proliferative and migratory responses in epithelial cells, with the latter being reduced for dermal fibroblasts. The endothelial cell capacity for tube formation was significatively reduced. An increased blood plasma content did not improve the biological potential of the formulations. However, they have emerged as a promising approach for regenerative therapies where neovascularization must be avoided.

Lysate of bovine adipose-derived stem cells accelerates in-vitro development and increases cryotolerance through reduced content of lipid in the in vitro fertilized embryos

Mesenchymal stem cells such as adipose-derived stem cells (ADSCs) are known to secrete factors that stimulate cell division and promote regeneration in neighboring cells. While conditioned medium from stem cells has been used in blastocyst production, no studies have examined the use of cell lysates. In this study we investigated the effects of adding ADSC lysate to in vitro culture (IVC) medium. ADSCs and fibroblasts were isolated from bovine adipose tissue and auricular tissue, respectively, and their lysates were prepared by freeze-thaw disruption. ADSC lysate was added to synthetic oviductal fluid medium. The effects on cleavage, blastocyst development rates, cell numbers, cryotolerance, gene expression (POU5F1, BAX, IGF1R, IGF2R, SOD2), lipid content, and membrane integrity were evaluated according to the experimental design. In Expt. 1, the comparison involved adding ADSC or fibroblast lysate alongside the control group. The total blastocyst rate increased when ADSC lysate was introduced (ADSCs: 40.1 %, fibroblasts: 33.1 %, control: 27.3 %). However, there were no significant differences in the number of trophoblast cells or in the inner cell mass. Experiment 2 confirmed that this increase in blastocyst development was not due to the solvent, PBS(−). In Expt. 3, addition of 10 % fetal calf serum (FCS) or ADSC lysate increased the total blastocyst rate compared to the control (control, 26.2 %; 10 % FCS, 43.4 %; 1 % ADSC lysate, 34.2 %; 10 % ADSC lysate, 48.1 %). After freezing and thawing, the survival and hatching rates of embryos with FCS were significantly lower than those of the control as well as those with added ADSC lysate. In Expt. 4, the addition of ADSC lysate or FCS had no significant effect on gene expression in blastocysts compared to control. However, the addition of FCS significantly increased the gray intensity, indicating higher lipid content compared to the control, with a significant increase in the number of dead cells in the blastocyst. These results indicate that the addition of ADSC lysate to the IVC medium accelerates bovine blastocyst development and that its 10 % addition, corresponding to 1 × 105 cells/mL, is as effective as 10 % FCS without a decrease in cryotolerance due to the increased lipid content.