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To our knowledge, this study represents the first demonstration of Arthrographis kalrae biofilm formation in vitro by scanning electron microscopy and the distinct cytotoxic activity between planktonic and biofilm extracts on RAW 264.7 cell line. Higher activity was observed with biofilm. It could impact host immune response, that require furthers study.

Agradecimentos: This study was supported by Sao Paulo Research Foundation (FAPESP - grants 2017/19674-2, 2017/21801-2), National Council for Scientific and Technological Development (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Brasil (CAPES) - Finance Code 001 Abstract: Culturing bone marrow mesenchymal stem cells (BM-MSCs) is a key point in different fields of research, including tissue engineering and regenerative medicine and studies of the bone marrow microenvironment. However, isolating and expanding murine BM-MSCs in vitro has challenged researchers due to the low purity and yield of obtained cells. In this study, we aimed to evaluate five different protocols to culture murine BMMSCs in vitro. Methods: All protocols were based on the adhesion capacity of BM-MSCs to the tissue culture plastic surface and varied in the types of plate, culture media, serum, additional supplementation and initial cell density. Flow cytometry analysis was used to investigate lineage purity after expansion. Results: The expression of CD45 and CD11b was detected in the cultures generated according to all protocols, indicating low purity with the presence of hematopoietic cells and macrophages. The cellular growth rate and morphology varied between the cultures performed according to each protocol. Cells cultured according to protocol 5 (8 £ 107 cells/plate, Roswell Park Memorial Institute (RPMI) culture medium during first passage and then Iscove's Modified Delbecco's Medium (IMDM) culture medium, both supplemented with 9% fetal bovine serum, 9% horse serum, 12mM L-glutamine) presented the best performance, with a satisfactory growth rate and spindle-shape morphology. Conclusion: Our results point out that the purity and satisfactory growth rate of murine BMMSC cultures are not easily achieved and additional approaches must be tested for a proper cell expansion FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO (FAPESP) CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO (CNPQ) COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR (CAPES) Fechado

Smart biomaterials, featuring not only bioactivity, but also dynamic responsiveness to external stimuli, are desired for biomedical applications, such as regenerative medicine, and hold great potential to expand the boundaries of the modern clinical practice. Herein, a magnetically responsive three-dimensional scaffold with sandwich structure is developed by using hydroxyapatite (HA) nanowires and ferrosoferric oxide (Fe3O4) nanoparticles as building blocks. The magnetic HA/Fe3O4 scaffold is fully inorganic in nature, but shows polymeric hydrogel-like characteristics including a 3D fibrous network that is highly porous (>99.7% free volume), deformable (50% deformation) and elastic, and tunable stiffness. The magnetic HA/Fe3O4 scaffold has been shown to execute multimodal motion upon exposure to an external magnetic field including shape transformation, rolling and somersault. In addition, we have demonstrated that the magnetic scaffold can serve as a smart carrier for remotely controlled, on-demand delivery of compounds including an organic dye and a protein. Finally, the magnetic scaffold has exhibited good biocompatibility, supporting the attachment and proliferation of human mesenchymal stromal cells, thereby showing great potential as smart biomaterials for a variety of biomedical applications.

The lack of a suitable explant culture system restricts the study of factors secreted by the mouse placenta into maternal circulation. Here, we present a protocol for culturing the endocrine junctional zone of the mouse placenta, free from the decidua and labyrinthine layers in serum-free medium. We describe steps for dissecting and separating layers, dicing tissue, and culture setup. We then detail medium processing for downstream analysis. This model allows the investigation of placental signals that may regulate maternal physiology. For complete details on the use and execution of this protocol, please refer to Yung et al. (2023).1.

The purpose of this study was to research the physiological roles of ubiquitin carboxyl-terminal esterase L1 (UCHL1) in head and neck squamous cell carcinoma (HNSCC).Ten HNSCC samples and matched normal oral mucosal tissues were collected. UCHL1 expression of these tissues was detected by the immunohistochemical staining and real-time quantitative polymerase chain reaction. The human HNSCC cell line HN6 UCHL1 knockout (UCHL1 KO) cell line was constructed using CRISPR/CAS9 gene editing and verified by western blotting. Wound healing assay, cell proliferation assay, cell invasion assay, and flow cytometric analysis of the cell cycle and apoptosis were applied to research the role of UCHL1 in HNSCC. Also, an RNAseq gene expression data set and HNSCC patient survival data from The Cancer Genome Atlas were analyzed.UCHL1 was highly expressed in HNSCC tissues compared with normal oral mucosal tissues (P = .032). A decreased proliferation (P.0001), migration (P.0001), and invasion (P = .0049) ability of HN6 cells was exhibited after knockout of UCHL1. However, HN6 UCHL1 KO cells showed no significant differences in the cell cycle or apoptosis. The progression, nodal metastasis status, and stage of HNSCC had a positive correlation with the expression of UCHL1.UCHL1 plays an important role in HNSCC, and we consider that targeting UCHL1 may be a feasible therapeutic strategy for HNSCC.

The regenerative promise of nanosized extracellular vesicles (EVs) secreted by cells is widely explored. Recently, the capacity of EVs purified from blood to elicit regenerative effect has begun to be evaluated. Blood might be a readily available source of EVs, avoiding need for extensive cell culturing, but there are specific issues that complicate use of the biofluid in this area. We assess the evidence for blood containing regenerative material, progress made towards delivering blood derived EVs as regenerative therapeutics, difficulties that relate to the complexity of blood and the promise of hydrogel-based delivery of EVs.

In this study, SB216763 and cyclosporine A were identified as anti-influenza A virus (IAV) agents by transcriptome signature reversion (TSR) analysis through deep mining of the cellular transcriptome of human airway and lung cell lines infected with 3 strains of IAV and the chemical perturbations library. A synergistic effect of SB216763 and cyclosporine A against influenza A was disclosed by quantification of the network-based relationship, which was validated in vitro. Along with burgeoning omics approaches, transcriptome-based drug development is flourishing, which provides a novel insight into antivirals discovery with comprehensive cellular transcriptional information of disease and chemical perturbations in multicomponent intervention. This strategy can be applied as a new approach in discovering multitarget antiviral agents from approved drugs, clinical compounds, natural products or other known bioactive compounds.

Objective Recently, fibulin-1 (FBLN1) has been shown to be downregulated in various cancers via promoter hypermethylation. Our study aimed to determine the expression and methylation status of FBLN1 in tongue squamous cell carcinoma (TSCC) tissues and cells. Methods Methylation-specific PCR was implemented to detect the methylation status of the FBLN1 gene in TSCC tissues and Western blot analysis was used to detect the expression of FBLN1 protein. The human TSCC cell lines, CAL27 and SCC9, were cultured in vitro and treated with 5-aza-deoxycytidine (5-Aza-dC). CCK-8, colony formation, and Transwell assays were performed to test TSCC cell proliferation, migration, and invasion following 5-Aza-dC treatment or overexpression of FBLN1, which was further verified in in vivo experiments. Results FBLN1 was hypermethylated and the protein expression was reduced in TSCC tissues. After human TSCC cell lines (CAL27 and SCC9) were treated with 5-Aza-dC or overexpressed FBLN1, FBLN1 expression was upregulated and the TSCC cell proliferation, migration, and invasion abilities were suppressed. In vivo experiments further showed that demethylation or overexpression of FBLN1 slowed tumor growth in nude mice. Conclusion This study demonstrated that 5-Aza-dC treatment or overexpression of FBLN1 inhibited the growth of human TSCC.

Dendritic cells (DCs) control adaptive immunity and are therefore attractive for in vivo targeting to either induce immune activation or tolerance, depending on disease. Liposomes, nanoparticles comprised of a lipid bi-layer, provide a nanoplatform for loading disease-relevant antigen, adjuvant and DC-targeting molecules simultaneously. However, it is yet not fully understood how liposomal formulations affect uptake by DCs and DC function. Here, we examined monocyte-derived DC (moDC) and skin DC uptake of six different liposomal formulations, together with their DC-modulating effect. Contrary to literature, we show using imaging flow cytometry that anionic or neutral liposomes are taken up more efficiently than cationic liposomes by moDCs, or by skin DCs after intradermal injection. None of the formulations yielded significant modulation of DC function as determined by the upregulation of maturation markers and cytokine production. These results suggest that anionic liposomes would be more suitable as vaccine carriers for a dermal application.(c) 2022 The Authors. Published by Elsevier Inc. on behalf of American Pharmacists Association. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

Purpose: The goal of this study is to clarify the underlying mechanisms of metastasis suppression by CiDC (carbon-ion radiotherapy (CIRT) combined with immature dendritic cell (iDC) immunotherapy), which was previously shown to significantly suppress pulmonary metastasis in a NR-S1-bearing C3H/He mouse model. Methods and Materials: Mouse carcinoma cell lines (LLC, LM8, Colon-26 and Colon-26MGS) were grafted into the right hind paw of syngeneic mice (C57BL/6J, C3H/He and BALB/c). Seven days later, the tumors on the mice were locally irradiated with carbon-ions (290 MeV/n, 6 cm SOBP, 1 or 2 Gy). At 1.5 days after irradiation, bone marrow-derived immature dendritic cells were administrated intravenously into a subset of the mice. The number of lung metastases was evaluated within three weeks after irradiation. In vitro cultured cancer cells were irradiated with carbon-ion (290 MeV/n, mono-energy, LET approximately 70 ∼ 80 keV/µm), and then co-cultured with iDCs for three days to determine the DC maturation. Results: CiDC effectively repressed distant lung metastases in cancer cell (LLC and LM8)-bearing C57BL/6J and C3H/He mouse models. However, Colon-26 and Colon-26MGS-bearing BALB/c models did not show enhancement of metastasis suppression by combination treatment. This was further evaluated by comparing LM8-bearing C3H/He and LLC-bearing C57BL/6J models with a Colon-26-bearing BALB/c model. In vitro co-culture assays demonstrated that all irradiated cell lines were able to activate C3H/He or C57BL/6J-derived iDCs into mature DCs, but not BALB/c-derived iDCs. Conclusion: The genetic background of the host may have a strong impact on the potency of combination therapy. Future animal and clinical testing should evaluate host genetic factors when evaluating treatment efficacy.

The regeneration of the injured nerve and recovery of its function have brought attention in the medical field. Electrical stimulation (ES) can enhance the cellular biological behavior and has been widely studied in the treatment of neurological diseases. Microfluidic technology can provide a cell culture platform with the well-controlled environment. Here a novel microfluidic/microelectrode composite microdevice was developed by embedding the microelectrodes to the microfluidic platform, in which microfluidics provided a controlled cell culture platform, and ES promoted the NSCs proliferation. We performed ES on rat neural stem cells (NSCs) to observe the effect on their growth, differentiation, proliferation, and preliminary explored the ES influence on cells in vitro. The results of immunofluorescence showed that ES had no significant effect on the NSCs specific expression, and the NSCs specific expression reached 98.9% ± 0.4% after three days of ES. In addition, ES significantly promoted cell growth and the cell proliferation rate reached 49.41%. To conclude, the microfluidic/microelectrode composite microdevice can play a positive role in the nerve injury repair and fundamental research of neurological diseases.

Decreased functional β-cell mass is the hallmark of diabetes, but the cause of this metabolic defect remains elusive. Here, we show that the expression levels of the growth factor receptor-bound protein 10 (GRB10), a negative regulator of insulin and mTORC1 signaling, are markedly induced in islets of diabetic mice and high glucose-treated insulinoma cell line INS-1cells. β-cell-specific knockout of Grb10 in mice increased β-cell mass and improved β-cell function. Grb10-deficient β-cells exhibit enhanced mTORC1 signaling and reduced β-cell dedifferentiation, which could be blocked by rapamycin. On the contrary, Grb10 overexpression induced β-cell dedifferentiation in MIN6 cells. Our study identifies GRB10 as a critical regulator of β-cell dedifferentiation and β-cell mass, which exerts its effect by inhibiting mTORC1 signaling.

Introduction Tumors are usually refractory to anti-cancer therapeutics under hypoxic conditions. However, the underlying molecular mechanism remains to be elucidated. Objectives Our study intended to identify hypoxia inducible lncRNAs and their biological function in gastric cancer (GC). Methods Differentially expressed lncRNAs were determined by microarray analysis between GC cells exposed to hypoxia (1% O2) and normoxia (21% O2) for 24 h. The expression level of CBSLR was manipulated in several GC cell lines to perform molecular and biological analyses both in vitro and in vivo. Results We identified a hypoxia-induced lncRNA-CBSLR that protected GC cells from ferroptosis, leading to chem-resistance. Mechanically, CBSLR interacted with YTHDF2 to form a CBSLR/YTHDF2/CBS signaling axis that decreased the stability of CBS mRNA by enhancing the binding of YTHDF2 with the m6A-modified coding sequence (CDS) of CBS mRNA. Furthermore, under decreased CBS levels, the methylation of the ACSL4 protein was reduced, leading to protein polyubiquitination and degradation of ACSL4. This, in turn, decreased the pro-ferroptosis phosphatidylethanolamine (PE) (18:0/20:4) and PE (18:0/22:4) content and contributed to ferroptosis resistance. Notably, CBSLR is upregulated, whereas CBS is downregulated in GC tissues compared to matched normal tissues; and GC patients with high CBSLR/low CBS levels have a worse clinical outcome and a poorer response to chemotherapy. Conclusion Our study reveals a novel mechanism in how HIF1α/CBSLR modulates ferroptosis/chemoresistance in GC, illuminating potential therapeutic targets for refractory hypoxic tumors.

Calcified aortic valve disease (CAVD) is the most prevalent valve disease in the elderly. Targeted pharmacological therapies are limited since the underlying mechanisms of CAVD are not well understood. Appropriate 3D in vitro models could potentially improve our knowledge of the disease. Here, we developed a 3D in vitro aortic heart valve model that resembles the morphology of the valvular extracellular matrix and mimics the mechanical and physiological behavior of the native aortic valve fibrosa and spongiosa. We employed cryogenic electrospinning to engineer a bi-layered cryogenic electrospun scaffold (BCES) with defined morphologies that allowed valvular endothelial cell (VEC) adherence and valvular interstitial cell (VIC) ingrowth into the scaffold. Using a self-designed cell culture insert allowed us to establish the valvular co-culture simultaneously by seeding VICs on one side and VECs on the other side of the electrospun scaffold. Proof-of-principle calcification studies were successfully performed using an established osteogenic culture protocol and the here designed 3D in vitro aortic heart valve model. Statement of Significance : Three-dimensional (3D) electrospun scaffolds are widely used for soft tissue engineering since they mimic the morphology of the native extracellular matrix. Several studies have shown that cells behave more naturally on 3D materials than on the commonly used stiff two-dimensional (2D) cell culture substrates, which have no biological properties. As appropriate 3D models for the study of aortic valve diseases are limited, we developed a novel bi-layered 3D in vitro test system by using the versatile technique of cryogenic electrospinning in combination with the influence of different solvents to mimic the morphology, mechanical, and cellular distribution of a native aortic heart valve leaflet. This 3D in vitro model can be used to study valve biology and heart valve-impacting diseases such as calcification to elucidate therapeutic targets.

Pancreatic cancer (PC) is highly resistant to chemo and radiotherapy. Radiation-induced fibrosis (RIF) is a major cause of clinical concern for various malignancies, including PC. In this study, we aimed to evaluate the radiosensitizing and anti-RIF potential of fluvastatin in PC. Short-term viability and clonogenic survival assays were used to evaluate the radiosensitizing potential of fluvastatin in multiple human and murine PC cell lines. The expression of different proteins was analyzed to understand the mechanisms of fluvastatin-mediated radiosensitization of PC cells and its anti-RIF effects in both mouse and human pancreatic stellate cells (PSCs). Finally, these effects of fluvastatin and/or radiation were assessed in an immune-competent syngeneic murine model of PC. Fluvastatin radiosensitized multiple PC cell lines, as well as radioresistant cell lines in vitro, by inhibiting radiation-induced DNA damage repair response. Nonmalignant cells, such as PSCs and NIH3T3 cells, were less sensitive to fluvastatin-mediated radiosensitization than PC cells. Interestingly, fluvastatin suppressed radiation and/or TGF-β-induced activation of PSCs, as well as the fibrogenic properties of these cells in vitro. Fluvastatin considerably augmented the antitumor effect of external radiation therapy and also suppressed intra-tumor RIF in vivo. These findings suggested that along with radiation, fluvastatin co-treatment may be a potential therapeutic approach against PC.

Objective Mitochondria are considered a portal to receive, process and integrate external energy and information to maintain cellular homeostasis. We examined the effect of Chinese texts with positive and negative meaning on the growth and mitochondrial functions using a mouse kidney collecting duct cell line called M1 cells. Methods To avoid skewing the results due to differential handling of the cells or analyzing the results, we conducted experiments by keeping the texts and blanks covered in brown opaque envelopes, exposed the cells to randomly selected envelopes and examined the differences over time. All operators involved in the experiments did not know the contents of the envelopes until the end of the experiments, and all data are expressed relative to the controls. Results Cell growth rate was not affected for the group treated with positive information but was significantly reduced by 18% when treated with negative information. At the biochemical level, positive texts significantly increased whole cell adenosine triphosphate (ATP) and glutathione (GSH) by 22% and 21% respectively. Conclusions This study for the first time demonstrated the effect of written words on specific biochemical measures in cultured mammalian cells.

We identified a novel interactome, circ_0001018/miR-338-3p/SOX4, in papillary thyroid cancer (PTC), and we intended to confirm the regulatory relationship between the three and to study the effects of the three in PTC. The bioinformatics method was used to screen out the circular RNA and mRNA of interest. A cellular fractionation assay and fluorescence in situ hybridization (FISH) assay were conducted to prove that circ_0001018 and CCT4 (the host gene of circ_0001018) mRNA primarily localized in the cytoplasm of PTC cell lines. By qRT-PCR analysis, the expression of circ_0001018 and SOX4 mRNA was found upregulated while the expression of miR-338-3p was found downregulated in PTC tissues and cells. circ_0001018 silence significantly inhibited the tumor growth in xenografted nude mice. A series of cytological experiments such as a Cell Counting Kit-8 (CCK-8) assay, a 5-ethynyl-2′-deoxyuridine (EdU) assay, cell cycle profiling, wound healing, a transwell assay, and cell apoptosis were conducted and showed that circ_0001018 and SOX4 promoted cell proliferation, migration, and invasion, inhibited cell apoptosis, and reduced the cell cycle arrest at the G1 phase in PTC cells. Compared with circ_0001018 and SOX4, miR-338-3p held the opposite function. The regulatory relationship between circ_0001018 and miR-338-3p, and between miR-338-3p and SOX4 mRNA, was validated using a luciferase reporter gene assay and/or RNA immunoprecipitation (RIP assay). Our findings showed that circ_0001018 acted as the tumor promoter via sponging miR-338-3p to elevate SOX4 expression level in PTC. Importantly, this novel circ_0001018/miR-338-3p/SOX4 axis has the potential to be considered as a therapy target for PTC., Graphical Abstract, In present study, the authors’ findings showed that circ_0001018 acted as the tumor promoter via sponging miR-338-3p to elevate SOX4 expression level in papillary thyroid cancer (PTC). Importantly, this novel circ_0001018/miR-338-3p/SOX4 axis has the potential to be considered as a therapy target for PTC.

The spread of coronavirus disease 2019 (COVID-19) throughout the world has resulted in stressful healthcare burdens and global health crises. Developing an effective measure to protect people from infection is an urgent need. The blockage of interaction between angiotensin-converting enzyme 2 (ACE2) and S protein is considered an essential target for anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) drugs. A full-length ACE2 protein could be a potential drug to block early entry of SARS-CoV-2 into host cells. In this study, a therapeutic strategy was developed by using extracellular vesicles (EVs) with decoy receptor ACE2 for neutralization of SARS-CoV-2. The EVs embedded with engineered ACE2 (EVs-ACE2) were prepared; the EVs-ACE2 were derived from an engineered cell line with stable ACE2 expression. The potential effect of the EVs-ACE2 on anti-SARS-CoV-2 was demonstrated by both in vitro and in vivo neutralization experiments using the pseudovirus with the S protein (S-pseudovirus). EVs-ACE2 can inhibit the infection of S-pseudovirus in various cells, and importantly, the mice treated with intranasal administration of EVs-ACE2 can suppress the entry of S-pseudovirus into the mucosal epithelium. Therefore, the intranasal EVs-ACE2 could be a preventive medicine to protect from SARS-CoV-2 infection. This EVs-based strategy offers a potential route to COVID-19 drug development., Graphical abstract Extracellular vesicles (EVs) embedded with engineered ACE2 can inhibit the transfection of S-pseudovirus in the host cells by serving as decoy receptors and competitively binding with the virus.Image 1

The aim of this study was to investigate the role of nuclear factor erythroid 2-related factor 2 (Nrf2) and key downstream targeting molecule, malic enzyme-1 (ME-1), in the induction of 5-fluorouracil (5-FU) resistance. Resistant MKN-45 (MKN-45/DR) cell line was generated by employing IC50 concentration of the agent followed by a resting cycles. Cell viability and apoptosis were evaluated by MTT assay, DAPI staining and flow cytometry. mRNA expression levels of Nrf2 and ME-1 were measured by qRT-PCR technique. Morphology analysis and combination index calculation were assessed by ImagJ and compuSyn softwares, respectively. The value for IC50 in 5-FU resistant cells increased from 8.81 ± 0.209–142.4 ± 0.060 μM with a considerable morphological changes from round to elongated shape (p = 0.016). Nrf2 and ME-1 expression levels were decreased in resistant cells with a marked increase in MDR1 mRNA level compared to sensitive cells. Combination of Nrf2 inhibitors, luteolin and brusatol had synergistic effects on 5-FU induced cytotoxicity. Moreover, combined incubation of MKN-45/DR cells with these inhibitors, exponentially increased Nrf2 and ME-1 mRNA levels (p

Understanding pulmonary diseases requires robust culture models that are reproducible, sustainable in long-term culture, physiologically relevant, and suitable for assessment of therapeutic interventions. Primary human lung cells are physiologically relevant but cannot be cultured in vitro long term and, although engineered organoids are an attractive choice, they do not phenotypically recapitulate the lung parenchyma; overall, these models do not allow for the generation of reliable disease models. Recently, we described a new cell culture platform based on H441 cells that are grown at the air-liquid interface to produce the SALI culture model, for studying and correcting the rare interstitial lung disease surfactant protein B (SPB) deficiency. Here, we report the characterization of the effects of SALI culture conditions on the transcriptional profile of the constituent H441 cells. We further analyze the transcriptomics of the model in the context of surfactant metabolism and the disease phenotype through SFTPB knockout SALI cultures. By comparing the gene expression profile of SALI cultures with that of human lung parenchyma obtained via single-cell RNA sequencing, we found that SALI cultures are remarkably similar to human alveolar type II cells, implying clinical relevance of the SALI culture platform as a non-diseased human lung alveolar cell model., Graphical abstract, The authors investigate the transcriptome of their established human pulmonary cell culture model (SALI) that recapitulates alveolar cell markers. RNA-seq analysis reveals striking parallels between gene expression profiles of SALI culture and primary ATII cells. The findings underline the suitability of SALI culture as a clinically relevant proxy for alveolar epithelium.

Iron dyshomeostasis is associated with hepatocellular carcinoma (HCC) development. However, the role of iron in HCC metastasis is unknown. This study aimed to elucidate the underlying mechanisms of iron’s enhancement activity on HCC metastasis. In addition to the HCC cell lines and clinical samples in vitro, iron deficient (ID) mouse models were generated using iron-free diet and transferrin receptor protein knock out, followed by administration of HCC tumors through either orthotopic or ectopic route. Clinical metastatic HCC samples showed significant ID status, accompanied by overexpression of sphingosine-1-phosphate transporter spinster homologue 2 (SPNS2). Mechanistically, ID increased SPNS2 expression, leading to HCC metastasis in both cell cultures and mouse models. ID not only altered the anti-tumor immunity, which was indicated by phenotypes of lymphatic subsets in the liver and lung of tumor-bearing mice, but also promoted HCC metastasis in a cancer cell autonomous manner through the SPNS2. Since germline knockout of globe SPNS2 showed significantly reduced HCC metastasis, we further developed hepatic-targeting recombinant adeno-associated virus vectors to knockdown SPNS2 expression and to inhibit iron-regulated HCC metastasis. Our observation indicates the role of iron in HCC pulmonary metastasis and suggests SPNS2 as a potential therapeutic target for the prevention of HCC pulmonary metastasis.

Graphical abstract, Highlights • Virtual screening based on machine learning with multiple proteins was developed. • Discovery of a novel PI3Kγ inhibitor integrating virtual screening and bio-assays. • JN-KI3 selective inhibit PI3Kγ enzymatic activity and hematologic malignancies. • The selective γ-inhibition mechanism of JN-KI3 was highlighted using MD simulation., Introduction Phosphoinositide 3-kinase gamma (PI3Kγ) has been regarded as a promising drug target for the treatment of various diseases, and the diverse physiological roles of class I PI3K isoforms (α, β, δ, and γ) highlight the importance of isoform selectivity in the development of PI3Kγ inhibitors. However, the high structural conservation among the PI3K family makes it a big challenge to develop selective PI3Kγ inhibitors. Objectives A novel machine learning-based virtual screening with multiple PI3Kγ protein structures was developed to discover novel PI3Kγ inhibitors. Methods A large chemical database was screened using the virtual screening model, the top-ranked compounds were then subjected to a series of bio-evaluations, which led to the discovery of JN-KI3. The selective inhibition mechanism of JN-KI3 against PI3Kγ was uncovered by a theoretical study. Results 49 hits were identified through virtual screening, and the cell-free enzymatic studies found that JN-KI3 selectively inhibited PI3Kγ at a concentration as low as 3,873 nM but had no inhibitory effect on Class IA PI3Ks, leading to the selective cytotoxicity on hematologic cancer cells. Meanwhile, JN-KI3 potently blocked the PI3K signaling, finally led to distinct apoptosis of hematologic cell lines at a low concentration. Lastly, the key residues of PI3Kγ and the structural characteristics of JN-KI3, which both would influence γ isoform-selective inhibition, were highlighted by systematic theoretical studies. Conclusion The developed virtual screening model strongly manifests the robustness to find novel PI3Kγ inhibitors. JN-KI3 displays a specific cytotoxicity on hematologic tumor cells, and significantly promotes apoptosis associated with the inhibition of the PI3K signaling, which depicts PI3Kγ as a potential target for the hematologic tumor therapy. The theoretical results reveal that those key residues interacting with JN-KI3 are less common compared to most of the reported PI3Kγ inhibitors, indicating that JN-KI3 has novel structural characteristics as a selective PIK3γ inhibitor.

CD19-directed chimeric antigen receptor (CAR) T cells have yielded impressive response rates in refractory/relapse B cell acute lymphoblastic leukemia (B-ALL); however, most patients ultimately relapse due to poor CAR T cell persistence or resistance of either CD19(+) or CD19(-) B-ALL clones. CD22 is a pan-B marker whose expression is maintained in both CD19(+) and CD19(-) relapses. CD22-CAR T cells have been clinically used in B-ALL patients, although relapse also occurs. T cells engineered with a tandem CAR (Tan-CAR) containing in a single construct both CD19 and CD22 scFvs may be advantageous in achieving higher remission rates and/or preventing antigen loss. We have generated and functionally validated using cutting-edge assays a 4-1BB-based CD22/CD19 Tan-CAR using in-house-developed novel CD19 and CD22 scFvs. Tan-CAR-expressing T cells showed similar in vitro expansion to CD19-CAR T cells with no increase in tonic signaling. CRISPR-Cas9-edited B-ALL cells confirmed the bispecificity of the Tan-CAR. Tan-CAR was as efficient as CD19-CAR in vitro and in vivo using B-ALL cell lines, patient samples, and patient-derived xenografts (PDXs). Strikingly, the robust antileukemic activity of the Tan-CAR was slightly more effective in controlling the disease in long-term follow-up PDX models. This Tan-CAR construct warrants a clinical appraisal to test whether simultaneous targeting of CD19 and CD22 enhances leukemia eradication and reduces/delays relapse rates and antigen loss.

The phenotype of glioma-initiating cells (GIC) is modulated by cell-intrinsic and cell-extrinsic factors. Phenotypic heterogeneity and plasticity of GIC is an important limitation to therapeutic approaches targeting cancer stem cells. Plasticity also presents a challenge to the identification, isolation, and propagation of purified cancer stem cells. Here we use a barcode labelling approach of GIC to generate clonal populations over a number of passages, in combination with phenotyping using the established stem cell markers CD133, CD15, CD44, and A2B5. Using two cell lines derived from isocitrate dehydrogenase (IDH)-wildtype glioblastoma, we identify a remarkable heterogeneity of the phenotypes between the cell lines. During passaging, clonal expansion manifests as the emergence of a limited number of barcoded clones and a decrease in the overall number of clones. Dual-labelled GIC are capable of forming traceable clonal populations which emerge after as few as two passages from mixed cultures and through analyses of similarity of relative proportions of 16 surface markers we were able to pinpoint the fate of such populations. By generating tumour organoids we observed a remarkable persistence of dominant clones but also a significant plasticity of stemness marker expression. Our study presents an experimental approach to simultaneously barcode and phenotype glioma-initiating cells to assess their functional properties, for example to screen newly established GIC for tumour-specific therapeutic vulnerabilities., The authors barcoded glioma-initiating cells (GIC) using combinations of virally encoded fluorophores. GIC show a strong tendency to form clonal populations over as few as two passages. Combined with stem cell marker phenotyping and computational analysis, they could trace the fate of such populations. This model presents an approach for rapid assessment of newly established GIC to assess tumour-specific vulnerabilities.

We report a novel topoisomerase IIα inhibitor, mercaptopyridine oxide (MPO), which induces G2/M arrest and senescence with distinctly different cell cycle regulators (p21 or p14ARF) in HCT116p 53WT and HCT116 p53−/− cells, respectively. MPO treatment induced defective topoisomerase IIα-mediated decatenation process and inhibition of the enzyme's catalytic activity that stalled M phase progression. Topoisomerase IIα inhibition was associated with ROS-mediated activation of ATM-Chk2 kinase axis in HCT116 p53WT cells, but not in HCT116 p53−/− cells displaying early Chk1 activation. Results suggest that E2F1 stabilization might link MPO-induced p53 phospho-activation in HCT116 p53WT cells or p14ARF induction in HCT116 p53−/− cells. Also, interaction between topoisomerase IIα and Chk1 was induced in both cell lines, which could be important for decatenation checkpoint activation, even upon p53 ablation. Notably, TCGA dataset analyses revealed topoisomerase IIα upregulation across a wide array of cancers, which was associated with lower overall survival. Corroborating that increased topoisomerase IIα expression might offer susceptibility to the novel inhibitor, MPO (5 μM) induced strong inhibition in colony forming ability of pancreatic and hepatocellular cancer cell lines. These data highlight a novel topoisomerase IIα inhibitor and provide proof-of-concept for its therapeutic potential against cancers even with loss-of-function of p53.

Objectives The detection of SARS-CoV-2 RNA in blood and platelet concentrates from asymptomatic donors, and the detection of viral particles on the surface and inside platelets during in vitro experiments, raised concerns over the potential risk for transfusion-transmitted-infection (TTI). The objective of this study was to assess the efficacy of the amotosalen/UVA pathogen reduction technology for SARS-CoV-2 in human platelet concentrates to mitigate such potential risk. Material and methods Five apheresis platelet units in 100% plasma were spiked with a clinical SARS-CoV-2 isolate followed by treatment with amotosalen/UVA (INTERCEPT Blood System), pre- and posttreatment samples were collected as well as untreated positive and negative controls. The infectious viral titer was assessed by plaque assay and the genomic titer by quantitative RT-PCR. To exclude the presence of infectious particles post-pathogen reduction treatment below the limit of detection, three consecutive rounds of passaging on permissive cell lines were conducted. Results SARS-CoV-2 in platelet concentrates was inactivated with amotosalen/UVA below the limit of detection with a mean log reduction of > 3.31 ± 0.23. During three consecutive rounds of passaging, no viral replication was detected. Pathogen reduction treatment also inhibited nucleic acid detection with a log reduction of > 4.46 ± 0.51 PFU equivalents. Conclusion SARS-CoV-2 was efficiently inactivated in platelet concentrates by amotosalen/UVA treatment. These results are in line with previous inactivation data for SARS-CoV-2 in plasma as well as MERS-CoV and SARS-CoV-1 in platelets and plasma, demonstrating efficient inactivation of human coronaviruses.

Organ-on-a-chip technology, a promising three-dimensional (3D) dynamic culture method, ensures accurate and efficient cell culture and has great potential for replacing animal models in preclinical testing. The circulatory system, the most abundant organ in the human body, plays a crucial role in oxygen exchange and mass transfer, which is the determining factor for the survival of tissues and organs. Thus, it is essential to integrate the circulatory system into an organ-on-a-chip to recreate tissue and organ microenvironments and physiological functions. This review discusses the synergy between the vasculature and the emerging organ-on-a-chip technology, which offers even better possibilities of duplicating physiology and disease characteristics. In addition, we review the different steps of a vascularized organ-on-a-chip fabrication process, including structure fabrication and tissue construction using different biofabrication strategies. Finally, we outline the applicability of this technology in the fascinating and fast-developing field of organ and tumor culture.

The molecular and cellular mechanism of microgravity-induced osteoporosis is unclear. Our previous study showed that it may be due to the decreased mechanosensitivity of osteocytes. Some research indicated that endoplasmic reticulum stress (ERS) could affect the mechanical response of cells. This study investigated the role of ERS in the altered mechanosensitivity of osteocytes under simulated microgravity (SMG). MLO-Y4 cells (osteocyte-like cell line) were cultured under SMG for 48 h and then applied 15 dyn/cm2 fluid shear stress (FSS) in the flow chamber for 1 h. In addition, melatonin (MT, 200 nmol/L), an ERS antagonist, was added to inhibit ERS. The morphology of endoplasmic reticulum was examined by immunofluorescence, ERS marker (GRP78) by Western blot and qPCR, unfolded protein pathway (UPR) related factors (IRE1, XBP1s, PERK, ATF4, ATF6) and apoptosis-related factor (CHOP) by qPCR and the mechanical sensitive factors (NO, PGE2 and ATP) by ELISA were determined. The results showed that under SMG, the endoplasmic reticulum enlarged as the endoplasmic reticulum/nucleus area ratio increased significantly and the morphology was irregular; the expression of GRP78 and UPR related factors increased significantly; NO, PGE2 and ATP induced by FSS decreased. Furthermore, MT could counter above SMG-induced effect. ERS may play an important role in the mechanosensitivity alteration in osteocytes under microgravity.

Micro-fabrication and nano-fabrication provide useful approaches to address fundamental biological questions by mimicking the physiological microenvironment in which cells carry out their functions. In particular, 2D patterns and 3D scaffolds obtained via lithography, direct laser writing, and other techniques allow for shaping hydrogels, synthetic polymers and biologically derived materials to create structures for (single) cell culture. Applications of micro-scaffolds mimicking cell niches include stem cell self-renewal, differentiation, and lineage specification. This review moves from technological aspects of scaffold microfabrication for cell biological applications to a broad overview of advances in (stem) cell research: achievements for embryonic, induced pluripotent, mesenchymal, and neural stem cells are treated in detail, while a particular section is dedicated to micro-scaffolds used to study single cells in basic cell biology.

Gliomas are the most common and recalcitrant intracranial tumors, approximately a quarter of which are classified as lower-grade gliomas (WHO II–III). Although the prognosis of lower-grade gliomas (LGGs) is significantly better than that of higher-grade gliomas, as a highly heterogeneous tumor type, the prognosis of LGGs varies greatly based on the molecular diagnosis. IDH wild-type used to be regarded as a dismal prognostic biomarker in LGGs; however, several studies revealed that IDH wild-type LGGs might not always be equivalent to glioblastoma (WHO IV). Hence, we hypothesize that underlying biological events in LGGs can result in different prognosis. In our study, transcriptome profiling was performed in 24 samples of LGG, and the results showed that the expression of phospholipase Cγ1 (PLCG1) was significantly correlated with IDH1/2 status and patients’ clinical outcome. Furthermore, the cancer genome atlas (TCGA) and the Chinese glioma genome atlas (CGGA) databases verified that elevated PLCG1 expression was associated with tumor progression and poor survival in LGG patients. Moreover, PLCG1-targeted siRNA dramatically affected the growth, migration and invasiveness of IDH wild-type LGG cell lines. In in vitro and in vivo experiments, the PLC-targeted drug significantly suppressed the tumor growth of IDH wild-type LGG cell lines in vitro and tumors in mouse models. Taken together, our results demonstrated that higher PLCG1 expression was associated with tumor growth and worse prognosis in IDH wild-type LGGs and PLCG1 could serve as a potential therapeutic target for IDH wild-type LGG patients., The mRNA expression levels of phospholipase Cγ1 (PLCG1) are much higher in IDH wild-type (IDHwt) lower-grade gliomas (LGGs) than in that of IDH mutant (IDHmut) LGGs. Higher PLCG1expression in IDHwt LGGs indicates poor clinical outcome. PLCG1 amplification may act as the key mechanism of PLCG1 upregulation. Depletion of PLCG1 expression can inhibits proliferation and invasion of IDHwt LGG cell lines in vitro and in vivo. The PLC inhibitor U73122 may therefore be a potential therapeutical agent for IDHwt LGGs.

All active natural molecules are not fully exploited as therapeutic agents, causing delays in the advancement of anticancer drug discovery. Viridiflorol is a natural volatile element that may work as anti-cancer compound. We tested the anticancer properties of viridiflorol at different concentrations ranging from 0.03 to 300 μM in vitro on three cancer cells including breast (MCF-7), lung (A549) and brain (Daoy). The cancer cells responses were documented after treatment using MTT and Annexin V assays. Viridiflorol showed cytotoxic effects against all tested cell lines, reducing cell viability in a concentration-dependent manner with variable IC50 values. Daoy and A549 cell lines were more sensitive to viridiflorol when compared with temozolomide and doxorubicin, respectively. Viridiflorol demonstrated the highest anticancer activity against the Daoy cells with an estimated IC50 of 0.1 µM followed by MCF-7 at 10 µM, and A549 at 30 µM. In addition, upon exposure to concentrations ranging from 30 µM to 300 µM of viridiflorol, early and late apoptotic cell death was induced in a concentration dependent manner in Daoy (55.8%-72.1%), MCF-7 (36.2%-72.7%) and A459 (35%-98.9%) cell lines, respectively. In conclusion, viridiflorol demonstrates cytotoxic and apoptotic ability in three different cancer cell lines (brain, breast and lung).

Advanced three dimensional cell culture techniques have been adopted in many laboratories to better model in vivo tissue by recapitulating multi-cellular architecture and the presence of extracellular matrix features. We describe here a 3D cell culture platform in a small molecule screening workflow that uses traditional biomarker and intracellular kinase end point assay readouts. By combining the high throughput bioprinter Rastrum with the high throughput screening assay AlphaLISA, we demonstrate the utility of the protocol in 3D synthetic hydrogel cultures with breast cancer (MDA-MB-231 and MCF-7) and fibroblast cells. To establish and validate the workflow, we treated the breast cancer cultures with doxorubicin, while fibroblast cultures were stimulated with the pro-inflammatory lipopolysaccharide. 3D and 2D MDA-MB-231 cultures were equally susceptible to doxorubicin treatment, while showing opposite ERK phosphorylation changes. Doxorubicin readily entered embedded MCF-7 spheroids and markedly reduced intracellular GSK3β phosphorylation. Furthermore, quantifying extracellular interleukin 6 levels showed a very similar activation profile for fibroblasts in 2D and 3D cultures, with 3D fibroblast networks being more resistant against the immune challenge. Through these validation experiments we demonstrate the full compatibility of the bioprinted 3D cell cultures with several widely-used 2D culture assays. The efficiency of the workflow, minimal culture handling, and applicability of traditional screening assays, demonstrates that advanced encapsulated 3D cell cultures can be used in 2D cell culture screening workflows, while providing a more holistic view on cell biology to increase the predictability to in vivo drug response.

Chinese Hamster Ovary (CHO) cells are the most frequently used biopharmaceutical production hosts, although industry is presently suffering from their variable recombinant product quality, insufficient long-term stability and low productivity. Here, we present an effort to address overall cell line engineering by a novel bottom-up microRNA (miRNA) screening approach. miRNAs are small non-coding RNAs known to regulate global gene expression at the post-transcriptional level and have proved to serve as promising tools for cell line engineering for over a decade. Here the miRNome of plasma cells (PCs) has been analyzed as the natural blueprint for optimized production and secretion of antibodies. Performing comparative miRNome cross-species expression analysis of four murine/human PC-derived (PCD) and two CHO cell lines showed 147 conserved miRNAs to be differentially expressed between PCDs and CHOs. Conducting a targeted miRNA screen of this PC-specific miRNA subset revealed 14 miRNAs to improve bioprocess relevant parameters in CHO cells, among them the PC-characteristic miR-183 cluster. Finally, miRNA target prediction tools and transcriptome analysis were combined to elucidate differentially regulated lysine degradation and fatty acid metabolism pathways in monoclonal antibody (mAb) expressing CHO-DG44 and CHO-K1 cells, respectively. Thus, substantial new insights into molecular and cellular mechanisms of biopharmaceutical production cell lines can be gained by targeted bottom-up miRNA screenings.

Cancer occurs when cells begin to grow abnormally and abruptly in the body. Cancer is one of the dangerous diseases due to which many people die in the world. Many kinds of cancers occur in the human body in which ovarian cancer is also one of the most fatal cancers found among women. It causes many deaths in women. Ovarian cancer generally occurs because of hereditary. Both types of molecules can be used in medicines; i.e., synthesized by man in the laboratories and naturally occurring molecules. Phytochemicals are the naturally occurring molecules found in plants. Phytochemicals are of great importance nowadays because our research is more focusing on plant-based molecules which can be helpful in the treatment of any kind of disease and have fewer side effects. In this review article, we examined how various phytochemicals are utilized as inhibitors in the therapy of ovarian malignancy. Phytochemicals including flavonoids, terpenes, and organo-Sulphur compounds have antioxidant, anti-inflammatory, antitumor, antiviral, apoptosis induction, anti-proliferation, and cell cycle arrest induction properties that help in the inhibition of CAOV3, OVCAR3, OVCAR5, TOV112D, and A2780 ovarian cancer cells. Also helps in suppressing the expression of c-Myc (cellular-Myc: An oncogene on chromosome 8q24 of cellular), VEGF (Vascular endothelial growth factor), and NF-κB (Nuclear factor-kappa B) in ovarian cancer cell lines.

As a newly developed cadmium-free quantum dot (QD), CuInS2/ZnS has great application potential in many fields, but its biological safety has not been fully understood. In this study, the in vitro toxicity of CuInS2/ZnS QDs on U87 human glioma cell line was explored. The cells were treated with different concentrations of QDs (12.5, 25, 50 and 100 μg/mL), and the uptake of QDs by the U87 cells was detected by fluorescence imaging and flow cytometry. The cell viability was observed by MTT assay, and the gene expression profile was analyzed by transcriptome sequencing. These results showed that QDs could enter the cells and mainly located in the cytoplasm. The uptake rate was over 90 % when the concentration of QDs reached 25 μg/mL. The cell viability (50 and 100 μg/mL) increased at 24 h (P < 0.05), but no significant difference after 48 h and 72 h treatment. The results of differential transcription showed that coding RNA accounted for the largest proportion (62.15 %), followed by long non-coding RNA (18.65 %). Total 220 genes were up-regulated and 1515 genes were down-regulated, and significantly altered gene functions included nucleosome, chromosome-DNA binding, and chromosome assembly. In conclusion, CuInS2/ZnS QDs could enter U87 cells, did not reduce the cell viability, but would obviously alter the gene expression profile. These findings provide valuable information for a proper understanding of the toxicity risk of CuInS2/ZnS QD and promote the rational utilization of QDs in the future.

Advances in anticancer drug discovery are focusing on the invention of drugs that specifically prompt cell death and pose lesser toxicity to the customary cells. The proposal aims at evaluating the cytotoxic potential of the non-metallic carbon dot (CDs) nanoparticles as a selective anticancer drug against targeted human carcinoma cell lines of liver (HepG2) and breast (MCF-7) in vitro. CDs were synthesized by a chemogenic method from three different natural carbohydrate derivatives glucose (GCD), sucrose (SCD), and fructose (FCD) and were characterized by UV–Visible, Fluorescence, HRTEM, ATR-IR and Raman spectroscopic techniques. Further compared for their anticancer activity against above mentioned cells. The percent cell viability investigations showed a suitability of CDs for cytotoxic studies. The IC50 of the CDs nanoparticles (SCD, GCD, FCD) on HepG2 cells were

Background and purpose Inhibitors of DNA-dependent protein kinase (DNA-PK) are effective radiation sensitisers in preclinical tumours, but little is known about risks of normal tissue radiosensitisation. Here, we evaluate radiosensitisation of head and neck squamous cell carcinoma (HNSCC) cells by DNA-PK inhibitor AZD7648 under oxia and anoxia in vitro, and tumour (SCCVII), oral mucosa and small intestine in mice. Materials and methods Radiosensitisation of human (UT-SCC-54C) and murine (SCCVII) HNSCC cells by AZD7648 under oxia and anoxia was evaluated by clonogenic assay. Radiosensitisation of SCCVII tumours in C3H mice by oral AZD7648 (75 mg/kg) was determined by ex vivo clonogenic assay 3.5 days post-irradiation, with evaluation of normal tissue surrogate endpoints using 5-ethynyl-2’-deoxyuridine to facilitate detection of regenerating crypts in the ileum and repopulating S-phase cells in the ileum and oral mucosa of the same animals. Results AZD7648 potently radiosensitised both cell lines, with similar sensitiser enhancement ratios for 10% survival (SER10) under oxia and anoxia. AZD7648 diffused rapidly through multicellular layers, suggesting rapid equilibration between plasma and hypoxic zones in tumours. SCCVII tumours were radiosensitised by AZD7648 (SER10 2.5). AZD7648 also enhanced radiation-induced body weight loss and suppressed regenerating intestinal crypts and repopulating S-phase cells in the ileum and tongue epithelium with SER values similar to SCCVII tumours. Conclusion AZD7648 is a potent radiation sensitiser of both oxic and anoxic tumour cells, but also markedly radiosensitises stem cells in the small intestine and oral mucosa.

TSC renal cystic disease is poorly understood and has no approved treatment. In a new principal cell-targeted murine model of Tsc cystic disease, the renal cystic epithelium is mostly composed of type A intercalated cells with an intact Tsc2 gene confirmed by sequencing, although these cells exhibit a Tsc-mutant disease phenotype. We used a newly derived targeted murine model in lineage tracing and extracellular vesicle (EV) characterization experiments and a cell culture model in EV characterization and cellular induction experiments to understand TSC cystogenesis. Using lineage tracing experiments, we found principal cells undergo clonal expansion but contribute very few cells to the cyst. We determined that cystic kidneys contain more interstitial EVs than noncystic kidneys, excrete fewer EVs in urine, and contain EVs in cyst fluid. Moreover, the loss of the Tsc2 gene in EV-producing cells greatly changes the effect of EVs on renal tubular epithelium, such that the epithelium develops increased secretory and proliferative pathway activity. We demonstate that the mTORC1 pathway activity is independent form the EV production, and that the EV effects for a single cell line can vary significantly. TSC cystogenesis involves significant contribution from genetically intact cells conscripted to the mutant phenotype by mutant cell derived EVs.

Objective The role of circadian clock in cementogenesis is unclear. This study examines the role of REV-ERBs, one of circadian clock proteins, in proliferation, migration and mineralization of cementoblasts to fill the gap in knowledge. Methods Expression pattern of REV-ERBα in cementoblasts was investigated in vivo and in vitro. CCK-8 assay, scratch wound healing assay, alkaline phosphatase (ALP) and alizarin red S (ARS) staining were performed to evaluate the effects of REV-ERBs activation by SR9009 on proliferation, migration and mineralization of OCCM-30, an immortalized cementoblast cell line. Furthermore, mineralization related markers including osterix (OSX), ALP, bone sialoprotein (BSP) and osteocalcin (OCN) were evaluated. Results Strong expression of REV-ERBα was found in cellular cementum around tooth apex. Rev-erbα mRNA oscillated periodically in OCCM-30 and declined after mineralization induction. REV-ERBs activation by SR9009 inhibited proliferation but promoted migration of OCCM-30 in vitro. Results of ALP and ARS staining suggested that REV-ERBs activation negatively regulated mineralization of OCCM-30. Mechanically, REV-ERBs activation attenuated the expression of OSX and its downstream targets including ALP, BSP and OCN. Conclusions REV-ERBs are involved in cementogenesis and negatively regulate mineralization of cementoblasts via inhibiting OSX expression. Our study provides a potential target regarding periodontal and cementum regeneration.

Molecular biomarkers play an important guidance role in the diagnosis and treatment of glioma. It has been found that TRAM2 (translocation associated membrane protein 2) drives human cancers development. Here we report that TRAM2 activity is required for malignancy properties of glioma. In this study, we demonstrated that TRAM2 is over-expressed in glioma and cell lines, particularly in the mesenchymal subtype, and glioma patients with high expression of TRAM2 is associated with poorer survival. Silencing of TRAM2 significantly suppresses glioma cell proliferation, invasion, migration and EMT in vitro, and inhibits tumorigenicity of glioma cell in vivo. We further identify that TRAM2 is positively associated with activation of the PI3K/AKT/mTOR signaling in glioma. 740Y-P, a PI3K activator, reversed the effects of TRAM2 silencing on glioma cell proliferation, invasion, migration and EMT process. Taken together, these findings establish that TRAM2/PI3K/AKT/mTOR signaling drives malignancy properties of glioma and indicate that TRAM2 may act as a potential therapeutic target for glioma.

The protein-coding ability of circular RNAs (circRNAs) has recently been a hot topic, but the expression and roles of protein-coding circRNAs in triple-negative breast cancer (TNBC) remain uncertain. By intersecting circRNA sequencing data from clinical samples and cell lines, we identified a circRNA, termed circ-EIF6, which predicted a poorer prognosis and correlated with clinicopathological characteristics in a cohort of TNBC patients. Functionally, we showed that circ-EIF6 promoted the proliferation and metastasis of TNBC cells in vitro and in vivo. Mechanistically, we found that circ-EIF6 contains a 675-nucleotide (nt) open reading frame (ORF) and that the -150-bp sequence from ATG functioned as an internal ribosome entry site (IRES), which is required for translation initiation in 5' cap-independent coding RNAs. circ-EIF6 encodes a novel peptide, termed EIF6-224 amino acid (aa), which is responsible for the oncogenic effects of circ-EIF6. The endogenous expression of EIF6-224aa was further examined in TNBC cells and tissues by specific antibody. Moreover, EIF6-224aa directly interacted with MYH9, an oncogene in breast cancer, and decreased MYH9 degradation by inhibiting the ubiquitin-proteasome pathway and subsequently activating the Wnt/beta-catenin pathway. Our study provided novel insights into the roles of protein-coding circRNAs and supported circ-EIF6/EIF6-224aa as a novel promising prognostic and therapeutic target for tailored therapy in TNBC patients.

Nuclear pore complexes (NPC) regulate molecular traffics on nuclear envelope, which plays crucial roles during cell fate specification and diseases. The viral accessory protein NSP9 of SARS-CoV-2 is reported to interact with nucleoporin 62 (NUP62), a structural component of the NPC, but its biological impact on the host cell remain obscure. Here, we established new cell line models with ectopic NSP9 expression and determined the subcellular destination and biological functions of NSP9. Confocal imaging identified NSP9 to be largely localized in close proximity to the endoplasmic reticulum. In agreement with the subcellular distribution of NSP9, association of NSP9 with NUP62 was observed in cytoplasm. Furthermore, the overexpression of NSP9 correlated with a reduction of NUP62 expression on the nuclear envelope, suggesting that attenuating NUP62 expression might have contributed to defective NPC formation. Importantly, the loss of NUP62 impaired translocation of p65, a subunit of NF-κB, upon TNF-α stimulation. Concordantly, NSP9 over-expression blocked p65 nuclear transport. Taken together, these data shed light on the molecular mechanisms underlying the modulation of host cells during SARS-CoV-2 infection.

Anaplastic thyroid cancer is a rare but devastating malignancy. Anaplastic thyroid cancer cells exhibit the Warburg effect by preferentially undergoing glycolysis even in aerobic conditions, leading to high glucose use. Here we assess if targeted inhibition of glycolysis can diminish anaplastic thyroid cancer growth and improve outcomes.Human anaplastic thyroid cancer cell line 8505C was grown in medium containing high (25 mmol/L) or low (3 mmol/L) glucose concentration and hexokinase II inhibitor 3-bromopyruvate (200 μM). Cellular proliferation, migration, and invasion were measured. An orthotopic xenograft model of anaplastic thyroid cancer was generated in nude mice using 8505C cells. Animals were provided standard chow or a ketogenic diet and treated with 3-bromopyruvate (1.8 mg/kg). Overall survival time was monitored. Necropsies were performed to harvest tumors for analysis.Growth of 8505C in low-glucose medium with 3-bromopyruvate decreased cell proliferation by 89%, migration by 44%, and invasion by 73% (P.001 for all) compared with high glucose. Animals concomitantly receiving a ketogenic diet and 3-bromopyruvate exhibited smaller tumor volumes (P = .03), slower tumor growth rates (P = .01), and improved overall survival (P = .006) compared with standard-diet control subjects. Monotherapy with a ketogenic diet or 3-bromopyruvate alone did not reduce tumor size or increase survival over the standard-diet control group.Glycolytic inhibition with 3-bromopyruvate inhibits tumor growth and extends survival in a murine model of anaplastic thyroid cancer when combined with the ketogenic diet. Thus, targeted glycolytic inhibition of anaplastic thyroid cancer exhibits context-specific utility and may only be effective during ketosis induced by dietary restriction of glycolytic inputs.

Purpose Previously, we showed that Acyl-CoA wax-alcohol acyltransferase 2 (AWAT2), an essential enzyme required for meibum wax ester synthesis, was not expressed by immortalized human meibomian gland epithelial cells (hMGEC) in culture. To begin to understand the mechanisms controlling AWAT2 expression, we have analyzed its expression in human and rabbit meibomian glands and cultured meibocytes. Methods Rabbit meibocyte progenitor cells (rMPC) were first grown in Cnt-BM.1 basal medium (Cellntec) supplemented with rhEGF, FGF10, and ROCK inhibitor (Y-27632 dihydrochloride), and then passed at 70–80% confluency with Accutase. Differentiation of rMPC to meibocytes (rMC) was induced by removal of Y-27632 and addition of 1 mM calcium with and without PPARγ agonists. RNA from the tissue, primary, passaged rMPC and differentiated rMC were obtained for AWAT2 qPCR analysis. Proteins and cells were evaluated for western blotting and neutral lipid synthesis, respectively. For comparison, human meibomian glands were separated for RNA and protein analysis. hMGEC was cultured to collect RNA and protein. Results Rabbit rMPCs were successfully grown, passaged, and differentiated, showing a significant increase in lipid droplet accumulation. AWAT2 RNA was highly expressed in tissue but showed a −16.9 log2 fold decrease in primary and passaged rMPCs and was not induced by differentiation to rMC. By comparison, human meibomian glands showed high expression of AWAT2, and hMGEC expressed non-detectable levels of AWAT2 transcripts or protein. Conclusions AWAT2 expression is lost in cultured rMPC and rMC suggesting that cells in culture do not undergo complete meibocyte differentiation and require yet to be identified culture conditions.

Objective Oral squamous cell carcinoma (OSCC) is a malignant tumor. This study aimed to investigate the role of a long noncoding RNA (lncRNA), LINC01123, in OSCC prognosis and progression and to explore the underlying mechanisms. Study Design OSCC tissues were collected from 102 patients, and 4 OSCC cell lines were analyzed. The expression levels of LINC01123 and miR-34a-5p were estimated using quantitative real-time polymerase chain reaction (qRT-PCR). Cell counting kit-8 (CCK-8) and Transwell assays were used to assess the proliferation, migration, and invasion of OSCC cells. Kaplan-Meier survival analysis was used to analyze the prognostic value of LINC01123 in OSCC. Results The analysis results showed that LINC01123 was overexpressed in OSCC tumor tissues; also, the prognosis of patients with OSCC with high LINC01123 expression levels was poor. The knockdown of LINC01123 inhibited the proliferation, migration, and invasion of OCSS cells. MiR-34a-5p was a target of LINC01123, and its inhibitor could reverse the effect of silenced LINC01123 on the progression of OSCC. Conclusions Highly expressed LINC01123 was associated with poor prognosis of OSCC and regulated OSCC cell proliferation, invasion, and migration by sponging miR-34a-5p. Therefore, the LINC01123/miR-34a-5p axis may provide new ideas for the prognosis and treatment of OSCC.

Acute myeloid leukemia (AML) remains a clinical challenge. Venetoclax is an effective Bcl-2 selective inhibitor approved by the U.S. Food and Drug Administration (FDA) for treatment of AML in patients who are 75 years and older or who have comorbidities. However, resistance to venetoclax limits its clinical efficacy. Mcl-1 has been identified as one determinant of resistance to venetoclax treatment. In this study, we investigate the Mcl-1 inhibitor S63845 in combination with venetoclax in AML cells. We found that S63845 synergizes with venetoclax in AML cell lines and primary patient samples. Bak/Bax double knockdown and treatment with the pan-caspase inhibitor Z-VAD-FMK revealed that the combination induces intrinsic apoptosis in AML cells. Inhibition of Mcl-1 using another Mcl-1 selective inhibitor, AZD5991, also synergistically enhanced apoptosis induced by venetoclax in a caspase-dependent manner. Importantly, S63845 in combination with venetoclax can effectively combat AML cells with acquired resistance to the standard chemotherapy drug cytarabine. In light of these facts, the combined inhibition of Mcl-1 and Bcl-2 shows promise against AML cells, including relapse/refractory AML.

Introduction The treatment of such primary brain tumours such as glioblastoma multiforme (GBM) presents numerous difficulties, considerably impacting patient survival rates. The search for new compounds with therapeutic potential focuses on the identification of molecules that can be produced through chemical and enzymatic processes and that present anticancer effects in different types of tumours. Methods Given the need for new treatments for GBM, we conducted a study to synthesise ortho-coumaric acid alkyl esters (OCAAE) and to evaluate them in an appropriate study model, a three-dimensional (3D) culture using the Matrigel extracellular matrix. We tested cytotoxicity with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) technique and by determining phosphatidylserine externalisation with annexin V and active caspase-3 expression as markers of apoptosis, and the subsequent degradation of DNA as a marker of cell death. Results Medium-chain OCAAEs, such as butyl-o-coumarate (BOC) and its isomer, showed the greatest effect on most of the parameters evaluated in 3D cultures of GBM, followed by methyl-, propyl-, and ethyl-o-coumarate (MOC, POC and EOC); these compounds showed effects both in reducing cell viability and in increasing the expression of active caspase-3 and annexin V and the degradation of DNA, in comparison with the first-line treatment for GBM, temozolomide. Conclusion These results, together with those of previous researchers, show that the different OCAAEs have a potential therapeutic effect on GBM, and that 3D culture of GBM cells is an appropriate model for the study of new antitumour molecules.

Background It is well accepted that the bidirectional crosstalk between platelets and cancer cells promote tumorigenesis and metastasis. In an early step cancer cells trigger platelet granule and extracellular vesicle release that is needed to facilitate cancer cells survival in circulation. Objectives To discover the early crosstalk of cancer cells and platelets. Methods Cancer cells were incubated with freshly isolated and stained human platelets. Confocal laser scanning microscopy and flow cytometry was used to visualize and to quantify platelet uptake and the membrane presence of CD42 on cancer cells. Dyngo4a was used to test if platelet uptake is a dynamin-dependent process. Results We found a dynamin dependent uptake of platelets by cancer cells. This is followed by the recycling of the platelet-specific protein CD42a and its incorporation into cancer cell`s plasma membrane which is not a result of platelet RNA transfer by platelet derives microparticles and exosomes. Time course of platelet uptake follows a sigmoid function revealing that 50% of the cancer cells are positive for platelets after approx. 38min. Platelet uptake was observed for the tested cancerous cells (A549, MCF-7 and MV3) but not for the non-cancerous cell line 16HBE14o-. Conclusions Our results demonstrate that cancer cells hijack platelets by phagocytosis and recycling of platelet membrane proteins. The uptake of platelets has additional advantages for cancer cells: Access to the entire and undiluted platelet proteome, transcriptome and secretome. These novel findings will allow further mechanistic elucidation and thus gain deeper insights into platelet-assisted hematogenous metastasis.