Human Cell Assay Research Articles

Structurally Minimalized and Druglike TGase2 Inhibitors Based on 7-Aminoquinoline-5,8-dione Scaffolds for the Treatment of Diabetic Retinopathy.

Diabetic retinopathy is a disease that can cause vision loss leading to blindness in people with diabetes. Improved methods to treat and prevent vision loss in diabetic patients are in high demand owing to limited current treatment procedures. Herein, we report a new class of transglutaminase 2 (TGase2) inhibitors for the treatment of diabetic retinopathy based on 7-aminoquinoline-5,8-dione derivatives. 7-Amino-2-phenylquinoline-5,8-dione 11 and 7-amino-2-{4-[(1-methylpiperidin-4-yl)oxy]phenyl}quinoline-5,8-dione 23 exhibited potent inhibitory activities against TGase2 in a fibrinogen array-based on-chip TGase2 activity assay and in an in situ assay in human retinal microvascular endothelial cells, with IC50 values of 5.88 and 1.12 μM in vitro, and 0.09 and 0.07 μM in situ, respectively. Pharmacokinetically favorable 7-amino-2-{4-[(1-isopropylpiperidin-4-yl)oxy] phenyl}quinoline-5,8-dione 22 inhibited vascular leakage in the retinas of streptozotocin-induced diabetic mice via oral administration. Results from the AL5 kinetic assay and a molecular docking study suggest that the inhibitors may bind to TGase2 remote from the active site.

Cellular fate of a plant virus immunotherapy candidate

Cowpea mosaic virus (CPMV) is a plant virus that is currently being developed for intratumoral immunotherapy. CPMV relieves the immune system from tumor-induced immunosuppression; reprograms the tumor microenvironment to an activated state whereby the treated and distant tumors are recognized and eradicated. Toward translational studies, we investigated the safety of CPMV, specifically addressing whether pathogenicity would be induced in mammalian cells. We show that murine macrophage immune cells recognize CPMV; however, there is no indication of de novo viral protein synthesis or RNA replication. Furthermore, we show that CPMV does not induce hemolysis, platelet aggregation and plasma coagulation amongst other assays in human blood and immune cells. Taken together, we anticipate that these results will reinforce the development of CPMV as an immunotherapeutic platform.

Exploring the Role of miR-132 in Rat Bladders and Human Urothelial Cells during Wound Healing

Urinary bladder wound healing shares many features with skin healing, involving several molecular players, including microRNAs (miRs). This study investigated the role of miR-132 in urothelial cells. We analyzed miR-132 expression in rat bladder using in situ hybridization and conducted gain and loss of miR-132 function assays in primary human urothelial cells (HUCs). These assays included cell proliferation and migration studies. To explore the regulation of miR-132 expression, cells were treated with wound-healing-related factors such as interleukin 6 (IL-6), interleukin 10 (IL-10), and transforming growth factor beta-1 (TGF-β1). Predictive bioinformatics and a literature review identified potential miR-132 targets, which were validated through real-time polymerase chain reaction (RT-PCR) and Western blot analysis. miR-132 was found to promote cellular proliferation and migration during the early stages of urothelial wound repair. Its expression was modulated by key cytokines such as IL-6, IL-10, and TGF-β1. miR-132 played a crucial role in urothelial wound healing by enhancing cell proliferation and migration, regulated by cytokines, suggesting its action within a complex regulatory network. These findings highlight the therapeutic potential of targeting miR-132 in bladder injury repair, offering new insights into bladder repair mechanisms.

Experimental validation of a parenteral permitted daily exposure value for cleaning-induced degradants from recombinant therapeutic proteins with in vitro immunogenicity assays

Multiproduct manufacturing of biotherapeutic proteins generate cleaning-induced protein degradants because of extreme pH and temperature conditions during the cleaning process. Cleaning Acceptance limits are calculated based on the maximum allowable carryover (MAC) assessment of the previously manufactured active pharmaceutical ingredient (API) – or drug product – based on the permitted daily exposure (PDE) of the previously manufactured API into the dose of subsequent product. In this study, we tested a previously determined PDE value for cleaning-induced protein degradants of 650 µg/dose. A bench-scale cleaning method was used to generate cleaning induced degradants from both a half-life extension (HLE) BiTE® molecule and a mAb product. For this investigation degradants of HLE BiTE®-A and mAb-1 were characterized either alone or degradants of HLE BiTE®-A and mAb-1 spiked into mAb-1 at 650 µg. These samples were characterized by endotoxin testing, size exclusion chromatography (SEC), light obscuration by HIAC, and micro-fluidic imaging (MFI). These results suggest that significant degradation of the molecule occurs because of the cleaning procedure, and it is no longer in the intact form or active state. The biological impact was assessed using a cell line assay to assess immune activation, and a human Peripheral Blood Mononuclear Cell (PBMC) assay to assess T cell activation, T cell proliferation, and cytokine release after 20 h and 7 days. Findings from the various in vitro cell-based assays suggest that the presence of 650 µg of carryover of degradants either alone or spiked into the same or a cross-product do not increase immunogenicity risk in cell-based assays – suggesting that the current PDE of 650 µg/dose for cleaning-induced degradant carryover does not have a risk of immunogenicity in patients.

Kukoamine A alleviates nephrolithiasis by inhibiting endogenous oxalate synthesis via the IL-6/JAK/STAT3/DAO signaling pathway

BackgroundThe recurrent nature and socioeconomic burden of nephrolithiasis demand effective treatments. Delineating the crosstalk between inflammatory processes and the endogenous oxalate metabolism pathway, which underpins nephrolithiasis pathogenesis, is essential for advancing treatment strategies. PurposeWe aim to screen therapeutic Chinese herbal remedies and their bioactive constituents for kidney stone treatment using a fruit fly model, followed by efficacy and mechanism validation in a rodent nephrolithiasis model as well as in vitro human cell culture model. Study design and methodsWe developed a fruit fly model to screen for efficient traditional Chinese medicine herbs and their active compounds for kidney stone treatment. Candidate active compounds from efficient herbs were separated and identified by solid-phase chromatography coupled with LC-MS analysis. Fruit fly genetic tools were employed to manipulate the expression of related genes to explore the therapeutic mechanisms of the Lycii Cortex and kukoamine A (KuA). To confirm the therapeutic effects and mechanisms of KuA for mammalian nephrolithiasis, mouse model of glyoxylate-induced kidney stone and human renal tubular cells were used. The therapeutic role of kukoamine A in nephrolithiasis was evaluated by assessing tubular injury, crystal deposition, and adhesion. The level of expression and phosphorylation in cells and mice was assessed using RT-qPCR and western blot. ResultsOur findings indicate that Lycii Cortex potently inhibits calcium oxalate stone formation via activation of the JNK/Upd3/JAK/STAT signaling cascade, resulting in diminished endogenous oxalate synthesis by downregulating D-amino acid oxidase (DAO) gene expression, predominantly in fruit fly Malpighian tube stellate cells. KuA was identified as the principal bioactive constituent mediating these effects. Both mouse models and human cell assays confirmed KuA's efficacy in preventing calcium oxalate nephrolithiasis in mammals, through hepatic JAK/STAT3 pathway activation and upregulation of IL-6, culminating in reduced urinary crystal deposition. ConclusionOur research underscores the potential of kukoamine A as a lead compound in treating nephrolithiasis and reveals the interplay between the IL-6/JAK/STAT3 inflammatory pathway and endogenous oxalate metabolism in nephrolithiasis pathogenesis. Additionally, it highlights the utility of the fruit fly model as a powerful tool for deciphering the therapeutic mechanisms of traditional Chinese herbs.

Identification of SNHG11 as a Therapeutic Target in Pulmonary Hypertension.

Pulmonary hypertension (PH) is a life-threatening condition characterized by pulmonary vascular remodeling and endothelial dysfunction. Current therapies primarily target vasoactive imbalances but often fail to address adverse vascular remodeling. Long non-coding RNA (lncRNA), which are key regulators of various cellular processes, remain underexplored in the context of PH. To investigate the role of lncRNA in PH, we performed a comprehensive analysis using Weighted Gene Co-expression Network Analysis (WGCNA) on the GSE113439 dataset, comprising human lung tissue samples from different PH subtypes. Our analysis identified the lncRNA SNHG11 as consistently downregulated in PH. Functional assays in human pulmonary artery endothelial cells (HPAECs) demonstrated that SNHG11 plays a critical role in modulating inflammation, cell proliferation, apoptosis, and the JAK/STAT and MAPK signaling pathways. Mechanistically, SNHG11 influences the stability of PRPF8, a crucial mRNA spliceosome component, thereby affecting multiple cellular functions beyond splicing. In vivo experiments using a hypoxic rat model showed that knockdown of SNHG11 alleviates PH development and improves right ventricular function. These findings highlight SNHG11 as a key regulator in PH pathogenesis and suggest it as a potential therapeutic target.

F13B regulates angiogenesis and tumor progression in hepatocellular carcinoma via the HIF-1α/VEGF pathway.

Hepatocellular carcinoma (HCC) is a highly aggressive malignant tumor with a poor prognosis. This research aimed to investigate the role of F13B in HCC and its underlying mechanisms. Through comprehensive bioinformatics analysis of the GSE120123 and The Cancer Genome Atlas (TCGA)-Liver hepatocellular carcinoma (LIHC) datasets, we identified 220 overlapping prognosis-related genes. Eight key genes, including the previously unreported CCDC170 and F13B in HCC, were identified through Least Absolute Shrinkage and Selection Operator (LASSO)-Cox regression analysis. F13B emerged as a significant prognostic factor in HCC, warranting further investigation in subsequent analyses. In vitro experiments showed that F13B expression was notably reduced in HCC cell lines and tissues, particularly in Huh-7 and SMMC-7721 cells. Overexpression of F13B inhibited cell invasion, migration, and proliferation, while its knockdown produced the opposite effect. A lactate dehydrogenase (LDH) activity assay in human umbilical vein endothelial cells (HUVECs) demonstrated that F13B overexpression reduced vascular endothelial growth factor (VEGF)-induced cytotoxicity, whereas knockdown increased it. Further analysis revealed that F13B negatively regulates VEGFA expression, affecting HUVEC proliferation. In HUVECs, F13B overexpression reversed VEGF-induced upregulation of key angiogenesis markers, including phospho-VEGF receptor 2 (p-VEGFR2), matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), as well as AKT/mTOR signaling proteins, phospho-Akt (p-AKT), and phospho-mTOR (p-mTOR). Additionally, F13B negatively regulated VEGFA and hypoxia-inducible factor 1 A (HIF1A) under hypoxic conditions, counteracting the hypoxia-induced increase in cell viability. These findings suggest that F13B regulates angiogenesis through the HIF-1α/VEGF pathway and plays a crucial role in HCC progression. Our results highlight the potential of F13B as a therapeutic target in HCC, providing novel insights into the molecular mechanisms of HCC and its prognostic significance.

Delineation of the substrate recognition domain of MARCHF6 E3 ubiquitin ligase in the Ac/N-degron pathway and its regulatory role in ferroptosis

Nα-terminal acetylation in eukaryotic proteins creates specific degradation signals (Ac/N-degrons) targeted for ubiquitin-mediated proteolysis via the Ac/N-degron pathway. Despite the identification of key components of the Ac/N-degron pathway over the past 15 years, the precise recognition domain (Ac/N domain) remains unclear. Here, we defined the Ac/N domain of the endoplasmic reticulum MARCHF6 E3 ubiquitin ligase through a systematic analysis of its cytosol-facing regions using alanine-stretch mutagenesis, chemical crosslinking-based co-immunoprecipitation–immunoblotting, and split-ubiquitin assays in human and yeast cells. The Ac/N domain of MARCHF6 exhibits preferential binding specificity to Nα-terminally acetylated proteins and peptides over their unacetylated counterparts, mediating the degradation of Ac/N-degron-bearing proteins, such as the G-protein regulator RGS2 and the lipid droplet protein PLIN2. Furthermore, abolishing the recognition of Ac/N-degrons by MARCHF6 stabilized RGS2 and PLIN2, thereby increasing the resistance to ferroptosis, an iron-dependent lipid peroxidation-mediated cell death. These findings provide mechanistic and functional insights into how MARCHF6 serves as a rheostatic modulator of ferroptosis by recognizing Ac/N-degron substrates via its Ac/N domain and non-Ac/N-degron substrates via distinct recognition sites.

Comprehensive analysis of the functional impact of single nucleotide variants of human CHEK2.

Loss of function mutations in the checkpoint kinase gene CHEK2 are associated with increased risk of breast and other cancers. Most of the 3,188 unique amino acid changes that can result from non-synonymous single nucleotide variants (SNVs) of CHEK2, however, have not been tested for their impact on the function of the CHEK2-enocded protein (CHK2). One successful approach to testing the function of variants has been to test for their ability to complement mutations in the yeast ortholog of CHEK2, RAD53. This approach has been used to provide functional information on over 100 CHEK2 SNVs and the results align with functional assays in human cells and known pathogenicity. Here we tested all but two of the 4,887 possible SNVs in the CHEK2 open reading frame for their ability to complement RAD53 mutants using a high throughput technique of deep mutational scanning (DMS). Among the non-synonymous changes, 770 were damaging to protein function while 2,417 were tolerated. The results correlate well with previous structure and function data and provide a first or additional functional assay for all the variants of uncertain significance identified in clinical databases. Combined, this approach can be used to help predict the pathogenicity of CHEK2 variants of uncertain significance that are found in susceptibility screening and could be applied to other cancer risk genes.

GelMA loaded with platelet lysate promotes skin regeneration and angiogenesis in pressure ulcers by activating STAT3

Pressure ulcers (PU) are caused by persistent long-term pressure, which compromises the integrity of the epidermis, dermis, and subcutaneous adipose tissue layer by layer, making it difficult to heal. Platelet products such as platelet lysate (PL) can promote tissue regeneration by secreting numerous growth factors based on clinical studies on skin wound healing. However, the components of PL are difficult to retain in wounds. Gelatin methacrylate (GelMA) is a photopolymerizable hydrogel that has lately emerged as a promising material for tissue engineering and regenerative medicine. The PL liquid was extracted, flow cytometrically detected for CD41a markers, and evenly dispersed in the GelMA hydrogel to produce a surplus growth factor hydrogel system (PL@GM). The microstructure of the hydrogel system was observed under a scanning electron microscope, and its sustained release efficiency and biological safety were tested in vitro. Cell viability and migration of human dermal fibroblasts, and tube formation assays of human umbilical vein endothelial cells were applied to evaluate the ability of PL to promote wound healing and regeneration in vitro. Real-time polymerase chain reaction (PCR) and western blot analyses were performed to elucidate the skin regeneration mechanism of PL. We verified PL’s therapeutic effectiveness and histological analysis on the PU model. PL promoted cell viability, migration, wound healing and angiogenesis in vitro. Real-time PCR and western blot indicated PL suppressed inflammation and promoted collagen I synthesis by activating STAT3. PL@GM hydrogel system demonstrated optimal biocompatibility and favorable effects on essential cells for wound healing. PL@GM also significantly stimulated PU healing, skin regeneration, and the formation of subcutaneous collagen and blood vessels. PL@GM could accelerate PU healing by promoting fibroblasts to migrate and secrete collagen and endothelial cells to vascularize. PL@GM promises to be an effective and convenient treatment modality for PU, like chronic wound treatment.

Exploring Mucoadhesive and Toxicological Characteristics Following Modification of Linear Polyethylenimine with Various Anhydrides.

Linear polyethylenimine (L-PEI) has numerous applications, such as in pharmaceutical formulations, gene delivery, and water treatment. However, due to the presence of secondary amine groups, L-PEI shows a relatively high toxicity and low biocompatibility. Here, various organic anhydrides were used to modify L-PEI to reduce its toxicity and enhance its functionality. We selected methacrylic anhydride, crotonic anhydride, maleic anhydride, and succinic anhydride to modify L-PEI. The structure of the resulting derivatives was characterized using 1H NMR and FTIR spectroscopies, and their behavior in aqueous solutions was studied using turbidimetric and electrophoretic mobility measurements over a broad range of pHs. A fluorescence flow through method determined the mucoadhesive properties of the polymers to the bovine palpebral conjunctiva. Methacrylated L-PEI and crotonylated L-PEI showed strong mucoadhesive properties at pH 7.4, likely due to covalent bonding with mucin thiol groups. In contrast, maleylated and succinylated L-PEI were poorly mucoadhesive as the pH was above their isoelectric point, resulting in electrostatic repulsion between the polymers and mucin. The toxicity of these polymers was evaluated using in vivo assays with planaria and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cell viability assay in human alveolar epithelial cells. Moreover, the irritancy of polymers was assessed using a slug mucosa irritation assay. The results demonstrated that anhydride modification mitigated the adverse toxicity effects seen for parent L-PEI.

Heparan sulfate-dependent phase separation of CCL5 and its chemotactic activity

Secreted chemokines form concentration gradients in target tissues to control migratory directions and patterns of immune cells in response to inflammatory stimulation; however, how the gradients are formed is much debated. Heparan sulfate (HS) binds to chemokines and modulates their activities. In this study, we investigated the roles of HS in the gradient formation and chemoattractant activity of CCL5 that is known to bind to HS. CCL5 and heparin underwent liquid–liquid phase separation and formed gradient, which was confirmed using CCL5 immobilized on heparin-beads. The biological implication of HS in CCL5 gradient formation was established in CHO-K1 (wild-type) and CHO-677 (lacking HS) cells by Transwell assay. The effect of HS on CCL5 chemoattractant activity was further proved by Transwell assay of human peripheral blood cells. Finally, peritoneal injection of the chemokines into mice showed reduced recruitment of inflammatory cells either by mutant CCL5 (lacking heparin-binding sequence) or by addition of heparin to wild-type CCL5. Our experimental data propose that co-phase separation of CCL5 with HS establishes a specific chemokine concentration gradient to trigger directional cell migration. The results warrant further investigation on other heparin-binding chemokines and allows for a more elaborate insight into disease process and new treatment strategies.

Heparan sulfate-dependent phase separation of CCL5 and its chemotactic activity.

Secreted chemokines form concentration gradients in target tissues to control migratory directions and patterns of immune cells in response to inflammatory stimulation; however, how the gradients are formed is much debated. Heparan sulfate (HS) binds to chemokines and modulates their activities. In this study, we investigated the roles of HS in the gradient formation and chemoattractant activity of CCL5 that is known to bind to HS. CCL5 and heparin underwent liquid-liquid phase separation and formed gradient, which was confirmed using CCL5 immobilized on heparin-beads. The biological implication of HS in CCL5 gradient formation was established in CHO-K1 (wild-type) and CHO-677 (lacking HS) cells by Transwell assay. The effect of HS on CCL5 chemoattractant activity was further proved by Transwell assay of human peripheral blood cells. Finally, peritoneal injection of the chemokines into mice showed reduced recruitment of inflammatory cells either by mutant CCL5 (lacking heparin-binding sequence) or by addition of heparin to wild-type CCL5. Our experimental data propose that co-phase separation of CCL5 with HS establishes a specific chemokine concentration gradient to trigger directional cell migration. The results warrant further investigation on other heparin-binding chemokines and allows for a more elaborate insight into disease process and new treatment strategies.

Discovery of new cyclopropane sulfonamide derivatives as EGFR inhibitors to overcome C797S-mediated resistance and EGFR double mutation

The C797S mutation of EGFR leads to Osimertinib resistance by blocking the covalent binding of Cys797. To develop new agents that can overcome EGFR mutation resistance, thirty seven new cyclopropane sulfonamide derivatives were synthesized and evaluated as EGFRL858R/T790M/C797S or EGFRDel19/T790M/C797S inhibitors by structure-based screening. Most of the synthesized compounds exhibit good to excellent anti proliferation activity against to BaF3-EGFR L858R/T790M/C797S and BaF3–C797S/Del19/T790M cancer cell lines. Representative compounds 8l showed inhibitory activity against the two cancer cell lines with the IC50 values of 0.0012 and 0.0013 μM, respectively. Another compound 8h, exhibited slightly lower activity (0.0042 and 0.0034 μM of the IC50 values) to both of the two tri-mutation cell lines, but excellent activities against H1975 and PC9 cells with IC50 values of 13 and 19 nM, respectively. Considering the acquired drug resistance of tumors is a gradual process, we chose 8h for further in vivo and mechanism study. 8h was demonstrated significantly inhibited tumor growth with 72.1 % of the TGI in the BaF3/EGFR-TM xenograft tumor model and 83.5 % in the H1975-DM xenograft tumor model. Compound 8h was confirmed to be safe with no significant side effects as showed by the results of in vitro assay of human normal cells and the sections of animals major organs. Mechanism studies showed that in addition to inhibiting EGFR mutations, 8h can also target the tumor microenvironment and induce tumor cell apoptosis. All these results indicate that 8h deserves further investigation as an EGFR inhibitor to overcome C797S-mediated resistance.

SASH1 S519N Variant Links Skin Hyperpigmentation and Premature Hair Graying to Dysfunction of Melanocyte Lineage

A better understanding of human melanocyte (MC) and MC stem cell biology is essential for treating MC-related diseases. This study employed an inherited pigmentation disorder carrying the SASH1S519N variant in a Hispanic family to investigate SASH1 function in the MC lineage and the underlying mechanism for this disorder. We used a multidisciplinary approach, including clinical examinations, human cell assays, yeast 2-hybrid screening, and biochemical techniques. Results linked early hair graying to the SASH1S519N variant, a previously unrecognized clinical phenotype in hyperpigmentation disorders. Invitro, we identified SASH1 as a regulator in MC stem cell maintenance and discovered that TNKS2 is crucial for SASH1's role. In addition, the S519N variant is located in one of multiple tankyrase-binding motifs and alters the binding kinetics and affinity of the interaction. In summary, this disorder links both gain and loss of pigmentation in the same individual, hinting to accelerated aging in human MC stem cells. The findings offer insights into the roles of SASH1 and TNKS2 in MC stem cell maintenance and the molecular mechanisms of pigmentation disorders. We propose that a comprehensive clinical evaluation of patients with MC-related disorders should include an assessment and history of hair pigmentation loss.

Comparing the Soluble Form of Recombinant Human Insulin-like Growth Factor-1 (rhIGF-1) in Escherichia coli Using Thioredoxin as Fused and Co-expressed Protein.

Insulin-like growth factor-1 (IGF-1) is a single-chain polypeptide with various physiological functions. Escherichia coli is one of the most desirable hosts for recombinant protein production, especially for human proteins whose post-translation modifications are not essential for their bioactivity, such as hIGF-1. In this study, bacterial thioredoxin (Trx) was studied as a fused and non-fused protein to convert the insoluble form of recombinant human IGF-1 (rhIGF-1) to its soluble form in E. coli. The rhIGF-1 was expressed in the E. coli Origami strain in the form of fused-Trx. It was co-expressed with Trx and then purified and quantified. In the next step, the biological activity of rhIGF-1 was evaluated by alkaline phosphatase (ALP) activity assay in human adipose-derived stem cells (hASCs) regarding the differentiation enhancement effect of IGF-1 through the osteogenic process. Results showed that Trx in both the fused and non-fused forms had a positive effect on the production of the soluble form of rhIGF-1. A significant increase in ALP activity in hASCs after rhIGF-1 treatment was observed, confirming protein bioactivity. It was strongly suggested that the overproduction of Trx could increase the solubility of co-expressed recombinant proteins by changing the redox state in E. coli cells.

Massively parallel characterization of regulatory elements in the developing human cortex.

Nucleotide changes in gene regulatory elements are important determinants of neuronal development and diseases. Using massively parallel reporter assays in primary human cells from mid-gestation cortex and cerebral organoids, we interrogated the cis-regulatory activity of 102,767 open chromatin regions, including thousands of sequences with cell type-specific accessibility and variants associated with brain gene regulation. In primary cells, we identified 46,802 active enhancer sequences and 164 variants that alter enhancer activity. Activity was comparable in organoids and primary cells, suggesting that organoids provide an adequate model for the developing cortex. Using deep learning we decoded the sequence basis and upstream regulators of enhancer activity. This work establishes a comprehensive catalog of functional gene regulatory elements and variants in human neuronal development.

Fabrication and characterization of bifunctional PCL/chitosan scaffolds decorated with MXene nanoflakes for bone tissue engineering

The implantation of bioresorbable compositions has emerged as a promising therapeutic strategy to treat damaged bone tissues. To develop versatile bone scaffolds, polycaprolactone (PCL) is known as a dominant polymeric material regarding its unique biocompatibility and biodegradability. However, using PCL in a single structure reveals insufficient mechanical properties, in tandem with poor biological activity. To modulate these features, the combination of PCL and chitosan has been broadly proposed due to the proper bioactivity behavior of the chitosan component. It is also declared that the presence of MXene elements in the bone scaffolds could enhance the osteogenic impact and modify the mechanical behavior to match the surrounding bone tissue. To explore the synergetic effect of chitosan and MXene in enhancing the features of PCL structure, the PCL/chitosan/MXene ternary system was designed in this study. Accordingly, MXene nanosheets were synthesized and incorporated with various filler contents of 1, 3, and 5 % into PCL/chitosan to boost the overall characteristics. The obtained data exhibited a rise in porosity ratio from 22 to 30 % and a decrease in contact angle from 95.45 to 79.86⁰ by embedding MXene up to 3 %. Young's modulus was enhanced from 0.4 to 1.2 MPa, corroborating the generation of scaffolds with superior mechanical strength and rigidity. The compositions containing MXene up to 3 % revealed no cytotoxicity and human osteoblast cell assay approved the cell adhesion affinity of the designed architectures. Furthermore, the presence of MXene nanosheets resulted in the promoting of antimicrobial features in the membranes as a bifunctional behavior. Correspondingly, embedding an optimized ratio of MXene nanoparticles into the PCL/chitosan scaffold could create a suitable scaffolding architecture toward the generation of new bone tissues, benefiting appropriate cell attachment, proliferation, and differentiation.

Towards optimized tissue regeneration: a new 3D printable bioink of alginate/cellulose hydrogel loaded with thrombocyte concentrate.

Autologous platelet concentrate (APC) are pro-angiogenic and can promote wound healing and tissue repair, also in combination with other biomaterials. However, challenging defect situations remain demanding. 3D bioprinting of an APC based bioink encapsulated in a hydrogel could overcome this limitation with enhanced physio-mechanical interface, growth factor retention/secretion and defect-personalized shape to ultimately enhance regeneration. This study used extrusion-based bioprinting to create a novel bioink of alginate/cellulose hydrogel loaded with thrombocyte concentrate. Chemico-physical testing exhibited an amorphous structure characterized by high shape fidelity. Cytotoxicity assay and incubation of human osteogenic sarcoma cells (SaOs2) exposed excellent biocompatibility. enzyme-linked immunosorbent assay analysis confirmed pro-angiogenic growth factor release of the printed constructs, and co-incubation with HUVECS displayed proper cell viability and proliferation. Chorioallantoic membrane (CAM) assay explored the pro-angiogenic potential of the prints in vivo. Detailed proteome and secretome analysis revealed a substantial amount and homologous presence of pro-angiogenic proteins in the 3D construct. This study demonstrated a 3D bioprinting approach to fabricate a novel bioink of alginate/cellulose hydrogel loaded with thrombocyte concentrate with high shape fidelity, biocompatibility, and substantial pro-angiogenic properties. This approach may be suitable for challenging physiological and anatomical defect situations when translated into clinical use.

RFC3 drives the proliferation, migration, invasion and angiogenesis of colorectal cancer cells by binding KIF14.

Colorectal cancer (CRC) is a deadly and aggressive type of cancer that has a high fatality rate. The expression levels of replication factor C subunit 3 (RFC3) and kinesin family member 14 (KIF14) have been reported to be increased in CRC. The current study aimed to explore the effects of RFC3 on the malignant behaviors of CRC cells and its possible underlying mechanism involving KIF14. RFC3 and KIF14 expression levels in CRC tissues were analyzed using TNMplot database and Gene Expression Profiling Interactive Analysis database bioinformatics tools. RFC3 and KIF14 levels in CRC cells were examined using reverse transcription-quantitative PCR and western blotting. Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine assays were performed to assess cell proliferation. Cell apoptosis was determined using flow cytometric analysis. Wound healing and Transwell assays were adopted for the evaluation of cell migration and invasion. Tube formation assay in human umbilical vein endothelial cells was used to measure angiogenesis. Western blotting analysis was performed to determine the expression of apoptosis-, migration- and angiogenesis-associated proteins. Additionally, bioinformatics tools predicted the co-expression and interaction of RFC3 and KIF14, which was verified by a co-immunoprecipitation assay. RFC3 displayed elevated expression in CRC tissues and cells, and depletion of RFC3 halted the proliferation, migration, invasion and angiogenesis, while increasing the apoptosis of CRC cells; this was accompanied by changes in the expression levels of related proteins. In addition, RFC3 bound to KIF14 and interference with RFC3 reduced KIF14 expression. Moreover, KIF14 upregulation reversed the effects of RFC3 depletion on the aggressive cellular behaviors in CRC. In conclusion, RFC3 might interact with KIF14 to function as a contributor to the malignant development of CRC.