Regulation of the integrin αVß3- actin filaments axis in early osteogenic differentiation of human mesenchymal stem cells under cyclic tensile stress.

BACKGROUND: Integrins are closely related to mechanical conduction and play a crucial role in the osteogenesis of human mesenchymal stem cells. Here we wondered whether tensile stress could influence cell differentiation through integrin αVß3. METHODS: We inhibited the function of integrin αVß3 of human mesenchymal stem cells by treating with c(RGDyk). Using cytochalasin D and verteporfin to inhibit polymerization of microfilament and function of nuclear Yes-associated protein (YAP), respectively. For each application, mesenchymal stem cells were loaded by cyclic tensile stress of 10% at 0.5 Hz for 2 h daily. Mesenchymal stem cells were harvested on day 7 post-treatment. Western blotting and quantitative RT-PCR were used to detect the expression of alkaline phosphatase (ALP), RUNX2, ß-actin, integrin αVß3, talin-1, vinculin, FAK, and nuclear YAP. Immunofluorescence staining detected vinculin, actin filaments, and YAP nuclear localization. RESULTS: Cyclic tensile stress could increase the expression of ALP and RUNX2. Inhibition of integrin αVß3 activation led to rearrangement of actin filaments and downregulated the expression of ALP, RUNX2 and promoted YAP nuclear localization. When microfilament polymerization was inhibited, ALP, RUNX2, and nuclear YAP nuclear localization decreased. Inhibition of YAP nuclear localization could reduce the expression of ALP and RUNX2. CONCLUSIONS: Cyclic tensile stress promotes early osteogenesis of human mesenchymal stem cells via the integrin αVß3-actin filaments axis. YAP nuclear localization participates in this process of human mesenchymal stem cells. Video Abstract.

EWI2 prevents EGFR from clustering and endocytosis to reduce tumor cell movement and proliferation.

EWI2 is a transmembrane immunoglobulin superfamily (IgSF) protein that physically associates with tetraspanins and integrins. It inhibits cancer cells by influencing the interactions among membrane molecules including the tetraspanins and integrins. The present study revealed that, upon EWI2 silencing or ablation, the elevated movement and proliferation of cancer cells in vitro and increased cancer metastatic potential and malignancy in vivo are associated with (i) increases in clustering, endocytosis, and then activation of EGFR and (ii) enhancement of Erk MAP kinase signaling. These changes in signaling make cancer cells (i) undergo partial epithelial-to-mesenchymal (EMT) for more tumor progression and (ii) proliferate faster for better tumor formation. Inhibition of EGFR or Erk kinase can abrogate the cancer cell phenotypes resulting from EWI2 removal. Thus, to inhibit cancer cells, EWI2 prevents EGFR from clustering and endocytosis to restrain its activation and signaling.

Discovery of N,4-Di(1H-pyrazol-4-yl)pyrimidin-2-amine-Derived CDK2 Inhibitors as Potential Anticancer Agents: Design, Synthesis, and Evaluation.

Cyclin-dependent kinase 2 (CDK2) has been garnering considerable interest as a target to develop new cancer treatments and to ameliorate resistance to CDK4/6 inhibitors. However, a selective CDK2 inhibitor has yet to be clinically approved. With the desire to discover novel, potent, and selective CDK2 inhibitors, the phenylsulfonamide moiety of our previous lead compound 1 was bioisosterically replaced with pyrazole derivatives, affording a novel series of N,4-di(1H-pyrazol-4-yl)pyrimidin-2-amines that exhibited potent CDK2 inhibitory activity. Among them, 15 was the most potent CDK2 inhibitor (Ki = 0.005 µM) with a degree of selectivity over other CDKs tested. Meanwhile, this compound displayed sub-micromolar antiproliferative activity against a panel of 13 cancer cell lines (GI50 = 0.127-0.560 µM). Mechanistic studies in ovarian cancer cells revealed that 15 reduced the phosphorylation of retinoblastoma at Thr821, arrested cells at the S and G2/M phases, and induced apoptosis. These results accentuate the potential of the N,4-di(1H-pyrazol-4-yl)pyrimidin-2-amine scaffold to be developed into potent and selective CDK2 inhibitors for the treatment of cancer.

Ring finger protein 126 promotes breast cancer metastasis and serves as a potential target to improve the therapeutic sensitivity of ATR inhibitors.

BACKGROUND/AIMS: This study explores the relationship between the E3 ubiquitin ligase Ring finger protein 126 (RNF126) and early breast cancer metastasis and tests the hypothesis that RNF126 determines the efficacy of inhibitors targeting Ataxia telangiectasia mutated and Rad3-related kinase (ATR). METHODS: Various metastasis-related genes were identified by univariable Cox proportional hazards regression analysis based on the GSE11121 dataset. The RNF126-related network modules were identified by WGCNA, whereas cell viability, invasion, and migration assays were performed to evaluate the biological characteristics of breast cancer cells with or without RNF126 knockdown. MTT, immunoblotting, immunofluorescence, and DNA fiber assays were conducted to determine the efficiency of ATR inhibitor in cells with or without RNF126 knockdown. RESULTS: RNF126 was associated with early breast cancer metastasis. RNF126 promoted breast cancer cell proliferation, growth, migration, and invasion. ATR inhibitors were more effective at killing breast cancer cells with intact RNF126 due to replication stress compared with the corresponding cells with RNF126 knockdown. Cyclin-dependent kinase 2 (CDK2) was involved in regulating replication stress in breast cancer cells with intact RNF126. CONCLUSION: A high level of expression of RNF126 in early breast cancer patients without lymph node metastases may indicate a high-risk type of metastatic disease, possibly due to RNF126, which may increase breast cancer cell proliferation and invasion. RNF126-expressing breast cancer cells exhibit CDK2-mediated replication stress that makes them potential targets for ATR inhibitors.

Single-cell transcriptomic sequencing analyses of cell heterogeneity during osteogenesis of human adipose-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are known for their multilineage differentiation potential with immune-modulatory properties. The molecular underpinnings of differentiation remain largely undefined. In this study, we investigated the cellular and molecular features of chemically induced osteogenesis from MSC isolated from human adipose tissue (human adipose MSCs, hAMSCs) using single-cell RNA-sequencing (scRNA-seq). We found that a near complete differentiation of osteogenic clusters from hAMSCs under a directional induction. Both groups of cells are heterogeneous, and some of the hAMSCs cells are intrinsically prepared for osteogenesis, while variant OS clusters seems in cooperation with a due division of the general function. We identified a set of genes related to cell stress response highly expressed during the differentiation. We also characterized a series of transitional transcriptional waves throughout the process from hAMSCs to osteoblast and specified the unique gene networks and epigenetic status as key markers of osteogenesis.

BCR/ABL1ΔE7-8-9 isoform contributes to tyrosine kinase inhibitor resistance in chronic myeloid leukemia.

In chronic myeloid leukemia (CML) patients, the involvement of the BCR/ABL1 isoform in tyrosine kinase inhibitors (TKIs) resistance has attracted lots of attention. In this work, a novel isoform that encoded truncated protein due to the deletion of ABL1 exon7, 8, and 9 was reported and named BCR/ABL1ΔE7-8-9 here. This isoform was detected only in 10.2% of CML patients with inadequate responses to TKIs. BCR/ABL1Δexon7-8-9 isoform promoted S phase cell proliferation and reduced the expression of fusion gene and ABL1 phosphorylation level more slowly than that of control cells after TKIs treatment. The novel isoform has the qualities of a functional tyrosine kinase, localized in the cytoplasm, and could not be imported into the nucleus by TKIs. These results indicated that BCR/ABL1Δexon7-8-9 showed poorer sensitivity to imatinib and nilotinib than wild-type BCR/ABL1. According to molecular docking studies, nilotinib and imatinib present different binding sites and have a lower binding capacity with BCR/ABL1ΔE7-8-9 protein than the wild type. Our findings suggested that the novel isoform BCR/ABL1ΔE7-8-9 may contribute to TKIs resistance in CML due to its weakened TKIs binding ability. It enriched the mechanism of spliceosome involved in TKIs resistance. Monitoring the expression of BCR/ABL1ΔE7-8-9 helps guide the treatment of CML patients in the clinic.

Spatial organization and crosstalk of vimentin and actin stress fibers regulate the osteogenic differentiation of human adipose-derived stem cells.

Human adipose-derived stem cells (hASCs) are ideal seed cells for tissue engineering due to their multidirectional differentiation potential. Microfilaments, microtubules, and intermediate filaments are responsible for supporting the intracellular space. Vimentin, a type III intermediate filament protein that is specifically expressed in cells of mesenchymal origin, can function as a scaffold and endow cells with tension and shear stress resistance. Actin stress fibers (ASF) act as an important physical device in stress signal transduction, providing stiffness for cells, and promoting osteogenesis. Through direct physical contact, cross-linkers, and spatial interactions, vimentin and actin networks exist as intersecting entities. Spatial interactions occur in the overlapping area of cytoskeleton subsystems, which could affect cell morphology, cell mechanics, and cell fate. However, how does the spatial organization between the cytoskeletal subsystems changed during osteogenesis, especially between vimentin and ASF, is still not understood, and its mechanism effect on cell fate remains unclear. In our study, WB experiment was used to detect the expression changes in Vimentin, ASF, and other proteins. Cells were reconstructed by three-dimensional scanning with fluorescence microscope, and the spatial thickness of vimentin and ASF cytoskeletons and the thickness of the overlapping area between them were calculated, respectively, so as to observe the spatial reorganization of vimentin and ASF in cells. Cytochalasin D (an inhibitor of actin polymerization) and vimentin upregulated/downregulated cells were used to verify the change in the spatial organization between vimentin and ASF and its influence on osteogenesis. Then, heat shock protein 27 (HSP27) was downregulated to illuminate the regulatory mechanisms of spatial organization between vimentin and ASF during osteogenesis. The amounts and the spatial positions of vimentin and actin stress fiber exhibited opposite trends during osteogenesis. Through controlling the anchor sites on the nucleus, intermediate filaments vimentin can reduce the spatial proportion of actin stress fibers, which can be regulated by HSP27. In addition, depolymerization of actin stress fibers lead to lower osteogenic differentiation ability, resulting in osteogenesis and lipogenesis existed simultaneously, that can be resisted by vimentin. Our data indicate that the spatial reorganization of vimentin and actin stress fibers is a key factor in the regulation of the differentiation state of hASCs. And their spatial overlapping area is detrimental to hASCs osteogenesis, providing a new perspective for further exploring the mechanism underlying hASCs osteogenesis.

An overview of CDK3 in cancer: clinical significance and pharmacological implications.

Cyclin-dependent kinase 3 (CDK3) is a major player driving retinoblastoma (Rb) phosphorylation during the G0/G1 transition and in the early G1 phase of the cell cycle, preceding the effects of CDK4/cyclin D, CDK6/cyclin D, and CDK2/cyclin E. CDK3 can also directly regulate the activity of E2 factor (E2F) by skipping the role of Rb in late G1, potentially via the phosphorylation of the E2F1 partner DP1. Beyond the cell cycle, CDK3 interacts with various transcription factors involved in cell proliferation, differentiation, and transformation driven by the epidermal growth factor receptor (EGFR)/rat sarcoma virus (Ras) signaling pathway. The expression of CDK3 is extremely low in normal human tissue but upregulated in many cancers, implying a profound role in oncogenesis. Further evaluation of this role has been hampered by the lack of selective pharmacological inhibitors. Herein, we provide a comprehensive overview about the therapeutic potential of targeting CDK3 in cancer.

Roadmap on emerging hardware and technology for machine learning.

Recent progress in artificial intelligence is largely attributed to the rapid development of machine learning, especially in the algorithm and neural network models. However, it is the performance of the hardware, in particular the energy efficiency of a computing system that sets the fundamental limit of the capability of machine learning. Data-centric computing requires a revolution in hardware systems, since traditional digital computers based on transistors and the von Neumann architecture were not purposely designed for neuromorphic computing. A hardware platform based on emerging devices and new architecture is the hope for future computing with dramatically improved throughput and energy efficiency. Building such a system, nevertheless, faces a number of challenges, ranging from materials selection, device optimization, circuit fabrication and system integration, to name a few. The aim of this Roadmap is to present a snapshot of emerging hardware technologies that are potentially beneficial for machine learning, providing the Nanotechnology readers with a perspective of challenges and opportunities in this burgeoning field.

Actin polymerization state regulates osteogenic differentiation in human adipose-derived stem cells.

BACKGROUND: Actin is an essential cellular protein that assembles into microfilaments and regulates numerous processes such as cell migration, maintenance of cell shape, and material transport. METHODS: In this study, we explored the effect of actin polymerization state on the osteogenic differentiation of human adipose-derived stem cells (hASCs). The hASCs were treated for 7 days with different concentrations (0, 1, 5, 10, 20, and 50 nM) of jasplakinolide (JAS), a reagent that directly polymerizes F-actin. The effects of the actin polymerization state on cell proliferation, apoptosis, migration, and the maturity of focal adhesion-related proteins were assessed. In addition, western blotting and alizarin red staining assays were performed to assess osteogenic differentiation. RESULTS: Cell proliferation and migration in the JAS (0, 1, 5, 10, and 20 nM) groups were higher than in the control group and the JAS (50 nM) group. The FAK, vinculin, paxillin, and talin protein expression levels were highest in the JAS (20 nM) group, while zyxin expression was highest in the JAS (50 nM) group. Western blotting showed that osteogenic differentiation in the JAS (0, 1, 5, 10, 20, and 50 nM) group was enhanced compared with that in the control group, and was strongest in the JAS (50 nM) group. CONCLUSIONS: In summary, our data suggest that the actin polymerization state may promote the osteogenic differentiation of hASCs by regulating the protein expression of focal adhesion-associated proteins in a concentration-dependent manner. Our findings provide valuable information for exploring the mechanism of osteogenic differentiation in hASCs.

Bioinformatics analysis of the biological changes involved in the osteogenic differentiation of human mesenchymal stem cells.

The mechanisms underlying the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) remain unclear. In the present study, we aimed to identify the key biological processes during osteogenic differentiation. To this end, we downloaded three microarray data sets from the Gene Expression Omnibus (GEO) database: GSE12266, GSE18043 and GSE37558. Differentially expressed genes (DEGs) were screened using the limma package, and enrichment analysis was performed. Protein-protein interaction network (PPI) analysis and visualization analysis were performed with STRING and Cytoscape. A total of 240 DEGs were identified, including 147 up-regulated genes and 93 down-regulated genes. Functional enrichment and pathways of the present DEGs include extracellular matrix organization, ossification, cell division, spindle and microtubule. Functional enrichment analysis of 10 hub genes showed that these genes are mainly enriched in microtubule-related biological changes, that is sister chromatid segregation, microtubule cytoskeleton organization involved in mitosis, and spindle microtubule. Moreover, immunofluorescence and Western blotting revealed dramatic quantitative and morphological changes in the microtubules during the osteogenic differentiation of human adipose-derived stem cells. In summary, the present results provide novel insights into the microtubule- and cytoskeleton-related biological process changes, identifying candidates for the further study of osteogenic differentiation of the mesenchymal stem cells.

T-cell blast crisis of chronic myelogenous leukemia presented with coexisting p210 and p190 BCR-ABL transcripts and t(10;11)(q11;p15).

BACKGROUND: Blast transformation of chronic myelogenous leukemia (CML) to T lymphoblastic lymphoma/acute lymphoblastic leukemia (T-LBL/ALL) is rare, and the molecular mechanism is still unclear. CASE REPORT: A 28-year-old woman who developed T-ALL with coexpressing both p210 and p190 BCR-ABL transcripts five years after the initial diagnosis of CML in chronic phase. The proliferation of bone marrow was extremely active with blast cells over 20%. Chromosome analysis revealed t(9;22)(q34;q11) and t(10;11)(q25;p15). Flow immunophenotyping showed that blasts expressed CD4, CD7, CD11b, CD38, CD34, CD33, and cCD3. CONCLUSION: It is the first T-cell blast of CML case with coexisting p210 and p190 as well as additional chromosome translocations. Through review this case and previous reports, we will reveal that CML patients with T-lymphocyte transformation depend on potential molecular and pathological mechanism.

Interfacial redox processes in memristive devices based on valence change and electrochemical metallization.

Memristive devices based on electrochemical processes are promising candidates for next-generation memory and neuromorphic applications. The redox processes happening at the interfaces are crucial steps for the ionization as well as generation of counter charges, and are thus indispensable for successful resistive switching, but their detailed mechanism has not been fully clarified. Here, we study the interfacial redox reactions in the forming process of memristive devices based on valence change and electrochemical metallization, using high-resolution electron microscopy and electrostatic force microscopy observations. We show direct evidence for the anodic oxidation of oxygen ions and cathodic reduction of moisture in HfO2- and Ta2O5-based valence change cells, which could take place in different horizontal locations. We further found that the anodic reactions always led to more pronounced structural damage to the electrode, indicating the possibility of additional cathodic reactions without producing gaseous products. When an active electrode is present, oxidation of metal atoms takes place at the anodic interface instead. Further investigations on electrochemical metallization cells have identified Cu ionization and moisture reduction as the anodic and cathodic reactions, respectively, and formation of Cu nuclei at the cathodic interface was directly observed. These findings with microscopic evidence could facilitate future development of memristive devices.

High-speed true random number generation based on paired memristors for security electronics.

True random number generator (TRNG) is a critical component in hardware security that is increasingly important in the era of mobile computing and internet of things. Here we demonstrate a TRNG using intrinsic variation of memristors as a natural source of entropy that is otherwise undesirable in most applications. The random bits were produced by cyclically switching a pair of tantalum oxide based memristors and comparing their resistance values in the off state, taking advantage of the more pronounced resistance variation compared with that in the on state. Using an alternating read scheme in the designed TRNG circuit, the unbiasedness of the random numbers was significantly improved, and the bitstream passed standard randomness tests. The Pt/TaO x /Ta memristors fabricated in this work have fast programming/erasing speeds of ∼30 ns, suggesting a high random number throughput. The approach proposed here thus holds great promise for physically-implemented random number generation.

Encapsulation layer design and scalability in encapsulated vertical 3D RRAM.

Here we propose a novel encapsulated vertical 3D RRAM structure with each resistive switching cell encapsulated by dielectric layers, contributing to both the reliability improvement of individual cells and thermal disturbance reduction of adjacent cells due to the effective suppression of unwanted oxygen vacancy diffusion. In contrast to the traditional vertical 3D RRAM, encapsulated bar-electrodes are adopted in the proposed structure substituting the previous plane-electrodes, thus encapsulated resistive switching cells can be naturally formed by simply oxidizing the tip of the metal bar-electrodes. In this work, TaO x -based 3D RRAM devices with SiO2 and Si3N4 as encapsulation layers are demonstrated, both showing significant advantages over traditional unencapsulated vertical 3D RRAM. Furthermore, it was found thermal conductivity and oxygen blocking ability are two key parameters of the encapsulation layer design influencing the scalability of vertical 3D RRAM. Experimental and simulation data show that oxygen blocking ability is more critical for encapsulation layers in the relatively large scale, while thermal conductivity becomes dominant as the stacking layers scale to the sub-10 nm regime. Finally, based on the notable impacts of the encapsulation layer on 3D RRAM scaling, an encapsulation material with both excellent oxygen blocking ability and high thermal conductivity such as AlN is suggested to be highly desirable to maximize the advantages of the proposed encapsulated structure. The findings in this work could pave the way for reliable ultrahigh-density storage applications in the big data era.

Identification of a Highly Conserved Allosteric Binding Site on Mnk1 and Mnk2.

Elevated levels of phosphorylated eukaryotic initiation factor 4E (eIF4E) have been implicated in many tumor types, and mitogen activated protein kinase-interacting kinases (Mnks) are the only known kinases that phosphorylate eIF4E at Ser209. The phosphorylation of eIF4E is essential for oncogenic transformation but is of no significance to normal growth and development. Pharmacological inhibition of Mnks therefore provides a nontoxic and effective strategy for cancer therapy. However, a lack of specific Mnk inhibitors has confounded pharmacological target validation and clinical development. Herein, we report the identification of a novel series of Mnk inhibitors and their binding modes. A systematic workflow has been established to distinguish between type III and type I/II inhibitors. A selection of 66 compounds was tested for Mnk1 and Mnk2 inhibition, and 9 out of 20 active compounds showed type III interaction with an allosteric site of the proteins. Most of the type III inhibitors exhibited dual Mnk1 and Mnk2 activities and demonstrated potent antiproliferative properties against the MV4-11 acute myeloid leukemia cell line. Interestingly, ATP-/substrate-competitive inhibitors were found to be highly selective for Mnk2, with little or no activity for Mnk1. Our study suggests that Mnk1 and Mnk2 share a common structure of the allosteric inhibitory binding site but possess different structural features of the ATP catalytic domain. The findings will assist in the future design and development of Mnk targeted anticancer therapeutics.

Pharmacologic Inhibition of MNKs in Acute Myeloid Leukemia.

The Ras/Raf/MAPK and PI3K/Akt/mTOR pathways are key signaling cascades involved in the regulation of cell proliferation and survival, and have been implicated in the pathogenesis of several types of cancers, including acute myeloid leukemia (AML). The oncogenic activity of eIF4E driven by the Mnk kinases is a convergent determinant of the two cascades, suggesting that targeting the Mnk/eIF4E axis may provide therapeutic opportunity for the treatment of cancer. Herein, a potent and selective Mnk2 inhibitor (MNKI-85) and a dual-specific Mnk1 and Mnk2 inhibitor (MNKI-19), both derived from a thienopyrimidinyl chemotype, were selected to explore their antileukemic properties. MNKI-19 and MNKI-85 are effective in inhibiting the growth of AML cells that possess an M5 subtype with FLT3-internal tandem duplication mutation. Further mechanistic studies show that the downstream effects with respect to the selective Mnk1/2 kinase inhibition in AML cells causes G1 cell cycle arrest followed by induction of apoptosis. MNKI-19 and MNKI-85 demonstrate similar Mnk2 kinase activity and cellular antiproliferative activity but exhibit different time-dependent effects on cell cycle progression and apoptosis. Collectively, this study shows that pharmacologic inhibition of both Mnk1 and Mnk2 can result in a more pronounced cellular response than targeting Mnk2 alone. However, MNKI-85, a first-in-class inhibitor of Mnk2, can be used as a powerful pharmacologic tool in studying the Mnk2/eIF4E-mediated tumorigenic mechanism. In conclusion, this study provides a better understanding of the mechanism underlying the inhibition of AML cell growth by Mnk inhibitors and suggests their potential utility as a therapeutic agent for AML.