Cell Viability Growth Research Articles

Small Molecule Induces Time-Dependent Inhibition of Stat3 Dimerization and DNA-Binding Activity and Regresses Human Breast Tumor Xenografts.

Aberrantly-active signal transducer and activator of transcription (Stat)3 has a causal role in many human cancers and represents a validated anticancer drug target, though it has posed significant challenge to drug development. A new small molecule, JKB887, was identified through library screening and is predicted to interact with Lys591, Arg609 and Pro63 in the phospho-tyrosine (pTyr)-binding pocket of the Stat3 SH2 domain. JKB887 inhibited Stat3 DNA-binding activity in vitro in a time-dependent manner, with IC50 of 2.2-4.5 μM at 30-60-min incubation. It directly disrupted both the Stat3 binding to the cognate, high-affinity pTyr (pY) peptide, GpYLPQTV-NH2 in fluorescent polarization assay with IC50 of 3.5-5.5 μM at 60-90-min incubation, and to the IL-6 receptor/gp130 or Src in treated malignant cells. Treatment with JKB887 selectively blocked constitutive Stat3 phosphorylation, nuclear translocation and transcriptional activity, and Stat3-regulated gene expression, and decreased viable cell numbers, cell growth, colony formation, migration, and survival in human or mouse tumor cells. By contrast, JKB887 had minimal effects on Stat1, pErk1/2MAPK, pShc, pJAK2, or pSrc induction, or on cells that do not harbor aberrantly-active Stat3. Additionally, JKB887 inhibited growth of human breast cancer xenografts in mice. JKB887 is a Stat3-selective inhibitor with demonstrable antitumor effects against Stat3-dependent human cancers.

Evaluation of Alginate Hydrogel Microstrands for Stromal Cell Encapsulation and Maintenance.

Mesenchymal stromal cells (MSCs) have displayed potential in regenerating organ function due to their anti-fibrotic, anti-inflammatory, and regenerative properties. However, there is a need for delivery systems to enhance MSC retention while maintaining their anti-fibrotic characteristics. This study investigates the feasibility of using alginate hydrogel microstrands as a cell delivery vehicle to maintain MSC viability and phenotype. To accommodate cell implantation needs, we invented a Syringe-in-Syringe approach to reproducibly fabricate microstrands in small numbers with a diameter of around 200 µm and a porous structure, which would allow for transporting nutrients to cells by diffusion. Using murine NIH 3T3 fibroblasts and primary embryonic 16 (E16) salivary mesenchyme cells as primary stromal cell models, we assessed cell viability, growth, and expression of mesenchymal and fibrotic markers in microstrands. Cell viability remained higher than 90% for both cell types. To determine cell number within the microstrands prior to in vivo implantation, we have further optimized the alamarBlue assay to measure viable cell growth in microstrands. We have shown the effect of initial cell seeding density and culture period on cell viability and growth to accommodate future stromal cell delivery and implantation. Additionally, we confirmed homeostatic phenotype maintenance for E16 mesenchyme cells in microstrands.

Achieving High-Precision, Low-Cost Microfluidic Chip Fabrication with Flexible PCB Technology.

Soft lithography has long remained the state of the art to generate the necessary micropatterning for molded microfluidic (MF) chips. Previous attempts to use printed circuit boards (PCBs) as a cheap and accessible alternative to expensive lithographed molds for the production of PDMS MF chip prototypes have shown their limitations. A more in-depth exploration of using PCBs as a mold substrate and a novel methodology of using flexible PCBs to produce highly accurate MF chips is reported here for the first time. Cross sections highlight the improved accuracy of this method, and peel testing is performed to demonstrate suitable adhesion between the glass substrate and PDMS cast. Positive cell growth viability showcases this novel method as a high-accuracy, high-accessibility, low-cost prototyping method for microfluidic chips while still maintaining all favorable properties provided by the PDMS material.

Evaluation of Probiotic Quality of Lactic Acid Bacteria Isolated from Locally Fermented Palm Wine and Kunu from Sellers in Oluku and Oba Markets in Benin City, Edo State, Nigeria

Commonly used probiotics bacteria are the lactic acid bacteria (LAB) from the gastro intestinal tract. However, other LAB from exogenous origin having similar functional properties can also confer health benefit to the host. The objective of this work is to evaluate the probiotic quality of lactic acid bacteria isolated from locally fermented palm wine and kunu from sellers in Oluku and Oba markets in Benin City, Edo State, Nigeria using standard microbiological methods. Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus casei were isolated from palm wine and kunu, and they were assessed for the following: resistance to simulated gastric and intestinal fluids, resistance to bile salt and low pH, haemolytic activity and their antimicrobial characteristic against some bacteria pathogens, mainly; Staphylococcus aureus, Klebsiella pneumonia, Pseudomonas aeruginosa and Escherichia coli. Results showed resistance/tolerance and cell viability of all LAB isolates to simulated gastric and intestinal fluids, and low pH resistance of all isolates were negative at pH 2 but positive with viable cell growth at pH 3. The L. fermentum, L. plantarum, and L. acidophilus were not haemolytic while L. casei was haemolytic (alpha haemolysis). The LAB isolates were more resistance to bile salt (with viable cell growth) at 0.1% and 0.3% concentration compared to 0.5% concentration. Antibacterial activity testing showed that L. acidophilus and L. casei had antibacterial property against Pseudomonas aeruginosa, while L. plantarum and L. fermentum had antibacterial property against Staphylococcus aureus and Klebsiella pneumonia respectively. While all lactic acid bacteria (Lactobacillus species) isolated from the locally fermented food/product samples demonstrated strong probiotic characteristic properties, further in-vivo study to establish their probiotic potential is recommended.

Sawdust-derived cellulose nanofibrils with high biosafety for potential bioprinting

Cellulose is a sustainable biopolymer with potential to reduce the adverse environmental impact of non-biodegradable plastics and promote a circular economy. Nanocellulose, a representative biobased nanomaterial, is considered as a promising material in biomedical applications due to its outstanding biocompatibility and low toxicity. However, the presence of some leachable immunogenic contaminants, such as endotoxin and (1,3)-β-D-glucan, in nanocellulose will often pose a potential risk in biomedical applications, particularly to the human body in 3D printing. In line with this challenge, a novel nanocellulose fabricating method was innovatively developed from the source of sawdust biomass to minimize the possible immunogenic contaminants presented in the cellulose nanofibrils (CNFs), which will ensure its biocompatibility of the material in applications. The resulting CNFs exhibited a very low level of leachable contaminants, recorded with endotoxin at 1.39 EU/g and (1,3)-β-D-glucan at 2.82 μg/g, respectively, which fall within the acceptable levels of Pharmacopoeia. In the following evaluation with the fabricated CNFs, the printability of the CNF hydrogel scaffolds was further optimized with hydrogel concentration at 5 %, printing pressure at 37 kPa, nozzle size setting up at 25 G, and crosslinker concentration at 200 mM, respectively. As an important property of 3D printed hydrogel scaffold, its biocompatibility was further evaluated and observed through cell culture in vitro, where the extract of hydrogel scaffold demonstrated a 36 % higher cell viability in promoting cell growth and proliferation when compared with that in pure culture medium. This exploration would suggest a possible application in the biomedical field that may not only secure a sound biocompatibility of CNFs produced from sawdust biomass, but also ensure the following 3D-printed scaffold with a viable cell growth and proliferation.

Improving the utilization efficiency of nitrogen source through co-culture of Lactobacillus strains with different nitrogen source metabolisms

The growth of lactic acid bacteria depends largely on a sufficient source of nitrogen as well as a wide variety of nitrogen sources. Nutrient dependence, especially in relation to nitrogen sources, plays a key role in controlling the positive interactions between microorganisms. However, little is known regarding the relationship between co-culture and nitrogen source dependence in current studies. This study aimed to obtain a higher proliferation of strains with the same nitrogen source content by co-cultivation of Lactobacillus. First, upon the detection of various commercial nitrogen sources, notable differences in peptide molecular weight distributions were observed. In addition, diverse Lactobacillus strains exhibit varying proficiencies in utilizing these nitrogen sources. Second, in the exploration of co-cultivation of distinct bacterial strains, three synergistic combinations were identified. These assemblies exhibited superior total viable cell counts, growth trajectories, and glucose consumption. Finally, strains possessing capsular proteinase genes adeptly hydrolyzed large peptides, yielding smaller peptides and amino acids for strains lacking analogous enzymatic and peptide transport mechanisms. This culminated in optimized utilization of the nitrogen source and provided a method for the direct production of multi-strain probiotics.

Growth capacity of hybridoma clones producing monoclonal antibody against SARS-CoV-2 in low-serum media

Monoclonal antibodies (mAbs) are biorecognition molecules in the COVID-19 detection. Previously, we used the hybridoma technique to generate anti-Spike SARS-CoV-2 monoclonal antibodies. The resulting mAbs captured the commercial Spike protein of SARS-CoV-2 on an indirect ELISA platform, and afterward, the hybridomas were adapted to live in low-serum media. However, a validated hybridoma cell and a specific mAb should be established due to its critical role in large-scale production. Hence, this study aimed to observe the hybridoma cell’s growth capacity and its mAbs production in low-serum media. Two hybridoma clones, SF2 and RF10, were grown in RPMI media supplemented with 3% FBS for 11 days. Their viable cell density, growth rate, and doubling time were measured and calculated. According to the data, the SF2 clone grew slower than the RF10 clone. However, SF2 had shown greater cell density on the logarithmic phase. This finding is linear to the ELISA result, showing that SF2 produces higher absorbance levels. These findings demonstrated that the SF2 clone had the potential to survive under low-serum circumstances and still produce mAbs. This clone might be used to produce mAbs against Spike protein SARS-CoV-2 at a low cost.

Establishment and functional characterization of immortalized rabbit dermal papilla cell lines

Hair follicle (HF) undergo periodic growth and development in mammals, which regulated by dermal papilla cells (DPCs) are reported to play an important role in HF morphogenesis and development. However, primary DPCs have low proliferative activity, age quickly, and fresh cell isolation is both time-consuming and laborious. In this study, we introduced the SV40 large T antigen (SV40T) into dissociated early passage rabbit vibrissae DPCs with lentiviral vectors and established seven immortalized DPC lines (R-1, R-2, R-3, R-4, R-5, R-6 and R-7). These cell lines displayed early passage morphology and high alkaline phosphatase activity. RT-PCR and immunofluorescence staining showed that all the immortalized cell lines expressed the DPC markers (α-SMA, IGF1, ALPL, FGF2, BMP2 and TGFβ2), but α-SMA was only expressed well in R-3, R-4, and R-7. Furthermore, it was found that R-7 was the only line to survive beyond 50 passages. Compared to melanoma cells, R-7 did not undergo malignant transformation. Karyotyping and cell growth viability analysis illustrated that the R-7 cell line preserved the basic characteristics of primary DPCs. The R-7 DPCs established have potential application for future hair research. The study provides the theoretical basis in the cell research of HF growth and development.

Structural mechanism of intracellular autoregulation of zinc uptake in ZIP transporters

Zinc is an essential micronutrient that supports all living organisms through regulating numerous biological processes. However, the mechanism of uptake regulation by intracellular Zn2+ status remains unclear. Here we report a cryo-electron microscopy structure of a ZIP-family transporter from Bordetella bronchiseptica at 3.05 Å resolution in an inward-facing, inhibited conformation. The transporter forms a homodimer, each protomer containing nine transmembrane helices and three metal ions. Two metal ions form a binuclear pore structure, and the third ion is located at an egress site facing the cytoplasm. The egress site is covered by a loop, and two histidine residues on the loop interact with the egress-site ion and regulate its release. Cell-based Zn2+ uptake and cell growth viability assays reveal a negative regulation of Zn2+ uptake through sensing intracellular Zn2+ status using a built-in sensor. These structural and biochemical analyses provide mechanistic insight into the autoregulation of zinc uptake across membranes.

Viability of Clostridium difficile in mortadella with added essential oils and reduced sodium nitrite content

The effects of additioning essential oil mixtures and reducing the sodium nitrite content (75 ppm) on Clostridium difficile inoculated in mortadellas and on the product’s technological characteristics were evaluated. 15 essential oils were tested preliminarily, but only four were selected. The minimum bactericidal concentrations found were 1.2 % for Ocimum basilicum, 0.3 % for Origanum vulgare and for Thymus vulgaris and 0.15 % for Litsea cubeba. There wasn`t significant difference in viable cell growth between treatments and the control; however, there was an increase in viable cells of approximately 2.5 log10 MPN/g after the fifth day. Combinations of oils didn`t prevent sporulation, however a synergistic effect was observed in vitro. There was a reduction in pH during storage and a greater amount of residual nitrite for the F1 treatment (Origanum vulgare (0.2 %); Thymus vulgaris (0.05 %) and Litsea cubeba (0.025 %), with 10.23 ppm, and reduction of residual nitrite during the storage period from 18.75 ppm on the first day of storage to 5.11 ppm on the last. No significant changes were observed in the technological characteristics of the product with the analyzed treatments.

Optimization of Cocktail Composition and Up-scale Fermentation Process Development of Four Skin Commensal Bacterial Strains

Analyzing the actual microbial composition in the skin microbiome is riveting to create a potential sourceof active substances in skincare products. Our previous study isolated four bacterial strains from Javanese male and female skin samples, Staphylococcus hominis MBF12–19J, Staphylococcus warneri MBF02–19J, Bacillus subtilis MBF10–19J, and Micrococcus luteus MBF05–19J. This study aimed to determine the composition of those strains in a bacterial cocktail and optimize the condition for up-scale laboratory production of the cocktail. To assess the ability of bacteria to coexist and live in cocktail communities, simultaneous monitoring was performed using the Deferred Growth Inhibition Assay (DGIA) method and real-time PCR for DNA copy number measurement. Results showed the best composition based on even distribution and cell growth viability was at the ratio 1.5:1:0.5:0.5 of Micrococcus luteus MBF05-19J, Bacillus subtilis MBF10-19J, Staphylococcus warneri MBF02-19J, Staphylococcus hominis MBF12-19J, which is equivalent to DNA copy number/mL 1.209 x 1024 CFU : 2.484 x 1041 CFU : 2.645 x 1041 CFU : 9.041 x 1035 CFU, respectively. The optimum growth incubation time of individual bacterial cultures for an up-scale 2-L bio-fermentor mixture was as follows; Micrococcus luteus MBF05-19J = 21 hrs; Bacillus subtilis MBF10-19J = 7 hrs; Staphylococcus warneri MBF02-19J = 17 hrs; Staphylococcus hominis MBF12-19 = 15 hrs. For up-scale conditions, the fermentation incubation time was 3 hours at 37oC, agitation 50 RPM, and aeration 5% dissolved oxygen.

Mechanical and histological properties of an electrospun scaffold with a modified surface by plasma polymerization implanted in an in vivo model

This article presents the construction of scaffolds composed of polylactic acid (PLA) with different concentrations of hydroxyapatite (HA) by electrospinning, which were superficially modified with polypyrrole (PPy/I) by plasma polymerization. A preliminary study was conducted of the biological and mechanical behavior of the scaffolds when they were implanted in the back of rabbits for 30 days; bone cells differentiated from mesenchymal stem cells (MSCs) were used. The bone cell and scaffold structures were characterized by histological, immunohistochemical, and mechanical stress tests. Hematoxylin–eosin staining showed good tissue conformation. The immunohistochemical tests highlighted the presence of the main bone tissue proteins, such as collagen, osteocalcin, and osteopontin. The PLA/HA scaffolds were observed to exhibit cell adhesion and proliferation properties; however, the response was much better in the scaffolds that had a higher concentration of HA and that were coated with PPy/I. The results of the mechanical tests of the scaffolds indicated that the plasma treatment improved the adhesion and cell proliferation properties and contributed to the mechanical support, allowing the formation of neotissues with good viability of cell growth.

Centromere protein F promotes progression of hepatocellular carcinoma through ERK and cell cycle-associated pathways.

Hepatocellular carcinoma (HCC) is one of the deadliest cancer types worldwide. The centromere proteins (CENPs) are critical for the mitosis-related protein complex and are involved in kinetochore assembly and spindle checkpoint signaling during mitosis. However, the clinical significance of CENPs in the recurrence and progression of HCC remains poorly understood. Here, we examined the expression of all CENPs and their association with recurrence and survival of HCC patients using the global gene expression profile dataset established in our laboratory. The effect of silencing CENPF on cell viability, migration, and epithelial-mesenchymal transition (EMT) were detected using CCK-8, transwell, and western blot, respectively. RT-qPCR and western blot were performed to confirm the silencing of CENPF and the relationship between STAT5A and CENPF, while tumorigenesis was tested using the HCC Huh7 xenograft mouse model. Most of the CENPs is overexpressed in HCC, and overexpression of CENPF was significantly associated with the poor survival of HCC patients. CENPF promoted HCC cell lines migration and EMT progression. Knockdown CENPF inhibited cell growth activity against human HCC cells in vitro and xenograft tumors in vivo. Bioinformatics analysis revealed that CENPF genes are enriched in the cell cycle. Silencing CENPF arrested cell cycle at the G2/M phase and inhibited Cyclin B1 and Cyclin E1 expressions. Meanwhile, silencing CENPF prohibited phosphorylation of ERK and the expression of NEK2. Additionally, we found that STAT5A down-regulated CENPF expression and inhibited cancer cell growth viability. In conclusion, our data suggested that CENPF could be potentially developed into a theranostic biomarker to tackle HCC progression.

Regulation of Apelin-13 on Bcl-2 and Caspase-3 and Its Effects on Adipocyte Apoptosis.

Objective The effects of apelin-13 on the expression of Bcl-2 and caspase-3 factors and the apoptosis of adipocytes were studied at the cellular and animal levels. Methods 3T3-L1 preadipocytes were cultured and grouped. The third-generation cells were added to the control DMSO solvent and amidation-modified apelin-13. The expression of Bcl-2 and caspase-3 were detected. The cell growth viability and cell apoptosis were detected. DOI model rats were established. The effects of apelin-13 on DOI rat biochemical indicators, the expression of Bcl-2, caspase-3, and cell apoptosis were investigated by injecting amidation-modified apelin-13 through the tail vein. Result In in vitro experiments, amidation-modified apelin-13 can significantly reduce the growth viability of adipocytes and the expression of Bcl-2, increase the expression of caspase-3, and promote the apoptosis of adipocytes. Animal experiments also show that apelin-13 modified by amidation can adjust the abnormal biochemical indicators of DOI rats, decrease the expression of Bcl-2 in adipose tissue, increase the expression of caspase-3, and promote the apoptosis of adipocytes. Conclusion Amidation of apelin-13 can promote fat cell apoptosis and reduce the incidence of obesity. The mechanism may be accomplished by inhibiting Bcl-2 and caspase-3 factors. This study helps us understand the effect of apelin-13 on fat cell apoptosis and hopes to provide a basis for the development of antiobesity drugs.

Fructose-1,6-bisphosphatase 2 represses cervical cancer progression via inhibiting aerobic glycolysis through promoting pyruvate kinase isozyme type M2 ubiquitination.

Growing evidence has shown that aerobic glycolysis, as a hallmark of cancer cells, plays a crucial role in cervical cancer. The aim of the study is to uncover whether fructose-1,6-bisphosphatase 2 (FBP2) is involved in cervical cancer progression via the aerobic glycolysis pathway. FBP2 levels were determined by quantitative PCR (qPCR) and western blotting. Cell growth viability and apoptosis were tested by cell counting kit-8 (CCK-8) and flow cytometry assays. Immunoprecipitation assay was applied for the detection of the FBP2 effect on pyruvate kinase isozyme type M2 (PKM2) ubiquitination. FBP2 level was decreased in cervical cancer, which is closely linked to shorter overall survival. FBP2 decreased cell growth and aerobic glycolysis and increased cell apoptosis, as well as decreased PKM2 expression and increased its ubiquitination level. The above-mentioned roles of FBP2 were weakened followed by PKM2 overexpression. FBP2 inhibited cervical cancer cell growth via inhibiting aerobic glycolysis by inducing PKM2 ubiquitination.

A Novel Mutant Wasp in X-Linked Neutropenia Partially Recapitulates Defective Neutrophil Production through Its Expression in WASP-Deficient NB4 Cells

BackgroundSevere congenital neutropenia (SCN) is characterized by prolonged severe neutropenia (absolute neutrophil count < 500/µL) commencing in early infancy, and recurrent bacterial and/or fungal infections. X-linked neutropenia (XLN) is a very rare type of SCN caused by a gain-of-function mutation in the Wiskott-Aldrich syndrome gene (WAS) located on the short arm of the X chromosome. To the best of our knowledge, only three WAS protein (WASp) mutations (L270P, S272P, and I294T) have been described to date. Herein, we report an identification and functional analysis of novel WASP mutation (I290T), which was identified in the first Asian XLN case by phenotype-based gene analysis of next-generation sequencing (NGS) data.CasePatient was 29-year-old male who had been clinically diagnosed with SCN at 6 years of age, however the cause of SCN remained unknown. We performed targeted exome sequencing of 4813 disease-relevant genes using a TruSight One Sequencing Panel (Illumina, San Diego, CA). The sequencing data were analyzed in terms of somatic mutations using our in-house pipeline, “Genomon” (http://genomon.hgc.jp/exome/en/). The resulting gene list, including 925 single nucleotide variants (SNVs), was subjected to examination by Phenolyzer (http://phenolyzer.usc.edu/), a web-based computational tool that analyzes clinical phenotypes on the basis of free-text term(s) and predicts the most likely candidate gene(s) by integrating information from multiple databases. The terms entered were ‘Congenital neutropenia’, ‘Familial’, ‘Myelodysplastic syndrome’, and ‘Autosomal’. Phenolyzer prioritized the WAS gene as the most probable candidate. Additionally, we identified a missense mutation in exon 9 of the patient's WAS gene (c.T869C, p.I290T) using the Integrative Genomics Viewer (IGV).Materials and MethodsTo investigate the molecular mechanisms of WASp-I290T mutation in XLN, we cloned WASp to a human cell line that can differentiate to neutrophils (NB4). Endogenous WASp in NB4 cells was fully knocked out by utilizing CRISPR/Cas9 system. We constructed a lentiviral AcGFP-tagged WASp overexpression plasmid controlled by CMV promoter, and AcGFP-tagged WASp was induced to NB4 cells. Cell viability evaluation, immunofluorescence imaging, and gene expression analysis were performed to compare NB4 cells treated with/without ATRA harboring wild type and mutant WASp.ResultsWestern blot analysis of AcGFP-WASp clearly showed a molecular mass of 84 kD for both wild type and mutant proteins. The viable cell growth assay showed no significant differences between mutant and wild type WASp-transduced NB4ΔWASPcells (NB4WASp-WT and NB4WASp-I290T cells, respectively) (Figure 1A). In contrast, upon ATRA-induced neutrophilic differentiation, the viable cell number significantly decreased in NB4WASp-I290T cells after 72 hours, compared to NB4, NB4ΔWASP and NB4WASp-WT cells. This result was further confirmed by FACS analysis, whereas CD11b expression was induced in surviving NB4WASp-I290T cells, comparably to other sublines of NB4 cells. Confocal laser microscopy revealed that WASp-I290T preferentially localized in nucleus rather than cytoplasm, compared to wild-type WASP, which was compatible with the previous findings reported for other WASp mutants in XLN. Expression of myeloid differentiation-related genes including C/EBP-alpha, -beta, -epsilon, CSF3R and PU.1 was examined at regular intervals by quantitative RT-PCR, indicating that C/EBP-epsilon transcripts were upregulated in WASp-I290T cells to higher extent than other sublines of NB4 cells after 24 hours of ATRA treatment, and that this difference was more prominent after 72 hours (Figure 1B).DiscussionIn this study, a novel WASp-I290T mutant identified in an adult XLN patient partially recapitulated defective neutrophil production through its expression in ATRA-treated NB4 cells lacking endogenous WASp. Our results suggest that premature upregulation of C/EBP-epsilon may be involved in impaired growth of ATRA-treated NB4WASp-I290T cells. A number of reports suggest the putative role of nuclear WASp in transcriptional regulation. Considering the preferential nuclear localization of WASp-I290T, further investigation of its interaction with nuclear proteins such as histone modifying enzymes is required. [Display omitted] DisclosuresTojo:AMED grant: Research Funding.

Knockdown of lncRNA TUG1 attenuates cerebral ischemia/reperfusion injury through regulating miR-142-3p.

Cerebral ischemia-reperfusion injury (CI/RI) is one of the most common diseases of the central nervous system. At present, there is no specific treatment for CI/RI. It is necessary to explore the mechanism of CI/RI and find new ways to prevent and treat CI/RI. An oxygen and glucose deprivation/recovery (OGD/R) model was established to evaluate the effects of mouse astrocytes (MA-C) cell viability and apoptosis of stepwise exposure to oxygen and glucose deprivation followed by their replenishment. This assessment included using taurine upregulated gene 1-small interfering RNAs (TUG1-siRNA) transfection to determine the effects of TUG1 knockdown on MA-C survival and apoptosis. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to evaluate TUG1 and miR-142-3p expression levels. The luciferase gene reporter assay was performed to validate that miR-142-3p is a TUG1 target. Accordingly, the effects of miR-142-3p knockdown on TUG1-induced MA-C apoptosis were determined using flow cytometry. Methyl thiazolyl tetrazolium (MTT) method was used to detect cell growth viability. Western blotting analysis was performed to detect the expression levels of apoptosis-related proteins. TUG1 was upregulated, while miR-142-3p was downregulated in the OGD/R model of MA-C cells. Inhibiting the expression of TUG1 could protect MA-C cells and reverse the decrease in growth viability and increasing apoptosis of MA-C cells caused by OGD/R stimulation. On the other hand, the inhibition of miR-142-3p offset the effect of TUG1 knockdown on cell viability and apoptosis. Inhibition of OGD/R-induced increases in TUG1 expression that in turn reduces miR-142-3p upregulation may suppress reperfusion-induced losses in cell viability.

Anti-inflammatory and cytoprotective potentials of Meloxicam solid dispersions prepared by different techniques on lipopolysaccharide-stimulated RAW 264.7 macrophages

The present study investigates the anti-inflammatory and cytoprotective potentials of a selective COX-II inhibitor, Meloxicam (MELOX) from solid-dispersions (SDs), using lipopolysaccharide (LPS)–stimulated RAW 264.7 macrophages cell line. MELOX/SDs were prepared by fusion (FM) & hot-melt-extrusion (HME) techniques using Soluplus/Lutrol F127 as mixed-polymers. SDs were physicochemically evaluated by SEM, XRD and solubility-testing. Viability of cell-growth was determined by MTT assay. Evaluation of inflammation was based on production of nitric oxide (NO), tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2). MELOX Solubility increased by 3- and 4- folds from SDs prepared by FM and HME, respectively, compared to pure drug. XRD/SEM analyses confirmed drug-dispersion within mixed-polymers. Exposure of normal-cells to different MELOX-treatments maintained the integrity of cell-growth. LPS-stimulated cells treated with MELOX/SDs exhibited higher viabilities compared to cells treated with pure MELOX. The prepared MELOX/SDs showed statistically stronger inhibition of inflammatory-mediators with respect to pure drug. Only, MELOX/SDs prepared by HME, successfully restored LPS-induced TNF-α level to that of normal-cells. SDs prepared by HME provided superior reduction of NO, TNF-α and PGE2, while maintaining normal cell-growth. The present study highlights the effect of SDs preparation techniques, on MELOX treatment performance.

Hypoxia-induced cofilin 1 promotes hepatocellular carcinoma progression by regulating the PLD1/AKT pathway.

BackgroundHepatocellular carcinoma (HCC) is the fourth fatal malignant tumour type worldwide. However, the exact molecular mechanism involved in HCC progression remains unclear.MethodsThree pairs of HCC and matched portal vein tumour thrombus (PVTT) tissue samples were analysed by isobaric tags for relative and absolute quantification (iTRAQ) assay to investigate the differentially expressed proteins. Real‐time quantitative PCR, immunostaining, and immunoblotting were performed to detect cofilin 1 (CFL1) in HCC and non‐tumour tissues. CCK8 and EdU, and Transwell assays, respectively, determined cell proliferation, migration, and invasion of HCC cells. Further, subcutaneous and tail vein injection were performed in nude mice for investigating HCC growth and lung metastasis in vivo. Regulatory effect of hypoxia‐inducible factor‐1α (HIF‐1α) on CFL1 was confirmed by chromatin immunoprecipitation (ChIP) assay. Finally, interaction between CFL1 and phospholipase D1 (PLD1) was studied using immunoprecipitation (IP) assay.ResultsThe iTRAQ analysis identified expression of CFL1 to be significantly upregulated in PVTT than in HCC tissues. Increased expression of CFL1 was closely associated with unfavourable clinical features, and was an independent risk predictor of overall survival in HCC patients. The knockdown of CFL1 inhibited cell growth viability, invasiveness, and epithelial‐mesenchymal transformation (EMT) in HCC cells. Furthermore, CFL1 silencing significantly suppressed the growth and lung metastasis of HCC cells in nude mice. Next, HIF‐1α directly regulated CFL1 transcription by binding to the hypoxia‐responsive element (HRE) in the promoter. Moreover, we disclosed the interaction between CFL1 and PLD1 in HCC cells using IP assay. Mechanistically, CFL1 maintained PLD1 expression by repressing ubiquitin‐mediated protein degradation, thereby activating AKT signalling in HCC cells. Notably, the CFL1/PLD1 axis was found mediating the hypoxia‐induced activation of the AKT pathway and EMT.ConclusionThe analysis suggests that hypoxia‐induced CFL1 increases the proliferation, migration, invasion, and EMT in HCC by activating the PLD1/AKT pathway.

Two-Step Laser Post-Processing for the Surface Functionalization of Additively Manufactured Ti-6Al-4V Parts.

Laser powder bed fusion (LPBF) is one of the additive manufacturing methods used to build metallic parts. To achieve the design requirements, the LPBF process chain can become long and complex. This work aimed to use different laser techniques as alternatives to traditional post-processes, in order to add value and new perspectives on applications, while also simplifying the process chain. Laser polishing (LP) with a continuous wave laser was used for improving the surface quality of the parts, and an ultrashort pulse laser was applied to functionalize it. Each technique, individually and combined, was performed following distinct stages of the process chain. In addition to removing asperities, the samples after LP had contact angles within the hydrophilic range. In contrast, all functionalized surfaces presented hydrophobicity. Oxides were predominant on these samples, while prior to the second laser processing step, the presence of TiN and TiC was also observed. The cell growth viability study indicated that any post-process applied did not negatively affect the biocompatibility of the parts. The presented approach was considered a suitable post-process option for achieving different functionalities in localized areas of the parts, for replacing certain steps of the process chain, or a combination of both.