Observation of the nonanalytic behavior of optical phonons in monolayer hexagonal boron nitride.

Phonon splitting of the longitudinal and transverse optical modes (LO-TO splitting), a ubiquitous phenomenon in three-dimensional polar materials, will break down in two-dimensional (2D) polar systems. Theoretical predictions propose that the LO phonon in 2D polar monolayers becomes degenerate with the TO phonon, displaying a distinctive "V-shaped" nonanalytic behavior near the center of the Brillouin zone. However, the full experimental verification of these nonanalytic behaviors has been lacking. Here, using monolayer hexagonal boron nitride (h-BN) as a prototypical example, we report the comprehensive and direct experimental verification of the nonanalytic behavior of LO phonons by inelastic electron scattering spectroscopy. Interestingly, the slope of the LO phonon in our measurements is lower than the theoretically predicted value for a freestanding monolayer due to the screening of the Cu foil substrate. This enables the phonon polaritons in monolayer h-BN/Cu foil to exhibit ultra-slow group velocity (~5 × 10-6 c, c is the speed of light) and ultra-high confinement (~ 4000 times smaller wavelength than that of light). These exotic behaviors of the optical phonons in h-BN presents promising prospects for future optoelectronic applications.

Identifying topological corner states in two-dimensional metal-organic frameworks.

Due to the diversity of molecular building blocks, the two-dimensional (2D) metal-organic frameworks (MOFs) are ideal platforms to realize exotic lattice models in condensed matter theory. In this work, we demonstrate the universal existence of topological corner states in 2D MOFs with a star lattice configuration, and confirm the intriguing higher-order nontrivial topology in the energy window between two Kagome-bands, or between Dirac-band and four-band. Furthermore, combining first-principles calculations and scanning tunneling microscopy measurements, the unique topological corner state is directly identified in monolayer Ni3(HITP)2 (HITP = 2,3,6,7,10,11-hexaiminotriphenylene) grown on the Au(111) substrate. Our results not only illustrate the first organic topological state in the experiments, but also offer an exciting opportunity to study higher-order topology in 2D MOFs with the large insulating band gap.

Synthesizing Cr-Based Two-Dimensional Conjugated Metal-Organic Framework Through On-Surface Substitution Reaction.

A single-layer Cr3 (HITP)2 (HITP = 2,3,6,7,10,11-hexaiminotriphenylene) conjugated metal-organic framework (c-MOF) is synthesized under ultrahigh vacuum conditions by substituting Cr for Ni in Ni3 (HITP)2 template. As revealed by low-temperature scanning tunneling microscopy and scanning tunneling spectroscopy, while codeposition of Cr atoms and 2,3,6,7,10,11-hexaaminotriphenylene precursors produces irregular branches, crystalline Cr3 (HITP)2 frameworks are obtained by depositing Cr atoms to the Ni3 (HITP)2 templates. The density functional theory calculations reveal that the binding energy between Cr and HITP ligands is much higher than that for Ni, which hampers the growth of crystalline Cr3 (HITP)2 frameworks through direct coordination assembly but makes the substitution reaction energetically favorable. This work demonstrates a new strategy to prepare high-quality early-transition-metal-based c-MOFs under ultrahigh vacuum conditions.

CD82 palmitoylation site mutations at Cys5+Cys74 affect EGFR internalization and metabolism through recycling pathway.

Tetraspanin CD82 often participates in regulating the function of epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-Met). Palmitoylation is a post-translational modification that contributes to tetraspanin web formation and affects tetraspanin-dependent cell signaling. However, the molecular mechanisms by which CD82 palmitoylation affects the localization and stability of EGFR and c-Met have not yet been elucidated. This study focuses on the expression and distribution of EGFR and c-Met in breast cancer as well as the related metabolic pathways and molecular mechanisms associated with different CD82 palmitoylation site mutations. The results show that CD82 with a palmitoylation mutation at Cys5+Cys74 can promote the internalization of EGFR. EGFR is internalized and strengthened by direct binding to CD82 with the tubulin assistance and located at the recycling endosome. After studying the recycling pathway marker proteins Rab11a and FIP2, we found that formation of the EGFR/CD82/Rab11a/FIP2 complex promotes the internalization and metabolism of EGFR through the recycling pathway and results in the re-expression of EGFR and CD82 on the cell membrane.

Role of Yes-associated protein (YAP) in regulation of mesenchymal stem cell tenogenic differentiation.

The Yes-associated protein (YAP) transcription co-activator is recognized as a key mediator and has been implicated in the regulation of stem cell fate; however, the role of YAP in the tenogenic differentiation of mesenchymal stem cells (MSCs) is not well understood. In the present study, we characterized tenogenic differentiation of C3H10T1/2 cells induced by BMP-12 and evaluated the function of YAP in the regulation of the BMP-12-induced tenogenesis. We found that the tendon-related proteins Scleraxis, Tenascin C, and Tenomodulin were significantly increased after induction by 10 ng/ml BMP-12 for 48 h in C3H10T1/2 cells. In addition, the expression of YAP was significantly enhanced and transferred from the cytoplasm to the nucleus during tenogenic differentiation. In contrast, downregulation of YAP suppressed the cells' tenogenic differentiation, and the expression of YAP transferred from the nucleus to the cytoplasm. These results indicate that YAP is required for the expression of tenogenic markers in the tenocytic differentiation process of C3H10T1/2 cells. Collectively, we demonstrate that YAP transcription co-activator is a novel regulator in the process of BMP-12-induced MSCs tenogenesis and has a correlation with the other tenogenic regulators and markers. These results shed new light on the function of YAP in tendon healing and regeneration.

Manipulating the Electronic and Magnetic Properties of Coordinated Nickel Atoms in Metal-Organic Frameworks by Hydrogenation.

In the pursuit of manipulating the properties of single atoms, the surface-supported metal-organic frameworks (MOFs) provide us opportunities to individually address the electronic and magnetic properties of coordinated metal atoms by scanning tunneling microscopy. Recently, we have synthesized Ni-TPyP (TPyP = 5,10,15,20-tetra-(4-pyridyl) porphyrin) networks with dinuclear Ni centers on a Au(111) surface, in which the top-Ni atoms are sitting above the molecular plane. Here, we investigate the top-Ni atoms and their hydrogenated derivatives by low-temperature scanning tunneling microscopy and spectroscopy, and show that the electronic and magnetic states of top-Ni atoms can be manipulated by hydrogen adsorption. Specifically, by fitting the spin-flip spectra in vertical magnetic field, we find the spin state of top-Ni atoms is tuned from S = 1/2 to S = 1 by attaching one H atom and S = 3/2 by attaching two H atoms. Our work demonstrates atomic-scale control over the electronic and magnetic properties of coordinated metal atoms in a surface-supported MOF.

[Erratum] Regulation and mechanism of YAP/TAZ in the mechanical microenvironment of stem cells (Review).

Owing to an error that was made during the production stages of the above review article, what was actually Fig. 2 was inadvertently duplicated on p. 7 as Fig. 9. Fig. 9 as it should have appeared in the review is shown below. The Editor apologizes to the authors for this error, and regrets any inconvenience caused to the readership. [the original article was published in Molecular Medicine Reports 24: Article no. 506, 2021; DOI: 10.3892/mmr.2021.12145].

Regulation and mechanism of YAP/TAZ in the mechanical microenvironment of stem cells (Review).

Stem cells receive cues from their physical and mechanical microenvironment via mechanosensing and mechanotransduction. These cues affect proliferation, self­renewal and differentiation into specific cell fates. A growing body of evidence suggests that yes­associated protein (YAP) and transcriptional coactivator with PDZ­binding motif (TAZ) mechanotransduction is key for driving stem cell behavior and regeneration via the Hippo and other signaling pathways. YAP/TAZ receive a range of physical cues, including extracellular matrix stiffness, cell geometry, flow shear stress and mechanical forces in the cytoskeleton, and translate them into cell­specific transcriptional programs. However, the mechanism by which mechanical signals regulate YAP/TAZ activity in stem cells is not fully understand. The present review summarizes the current knowledge of the mechanisms involved in YAP/TAZ regulation on the physical and mechanical microenvironment, as well as its potential effects on stem cell differentiation.

Exosomal protein CD82 as a diagnostic biomarker for precision medicine for breast cancer.

CD82, a member of the tetraspanin superfamily, has been proposed to exert its activity via tetra-transmembrane protein enriched microdomains (TEMs) in exosomes. The present study aimed to explore the potential of the exosome protein CD82 in diagnosing breast cancers of all stages and various histological subtypes in patients. The results strongly suggest that CD82 expression in breast cancer tissue was significantly lower than that in healthy and benign breast disease tissues. There was a significant negative correlation between CD82 expression in tissues and CD82 content in exosomes, which indicated that CD82 expression was redistributed from tissues to the blood with the development and metastasis of breast cancer.

Long non-coding RNA LINC00161 sensitises osteosarcoma cells to cisplatin-induced apoptosis by regulating the miR-645-IFIT2 axis.

Chemotherapeutic insensitivity remains a major obstacle to osteosarcoma treatment. Recently, increasing evidence has suggested that long non-coding RNAs (lncRNAs) play an essential role in tumourigenesis. However, the potential biological roles and regulatory mechanisms of novel lncRNAs in response to cisplatin treatment are poorly understood. Here, we found that lncRNA LINC00161 was induced by cisplatin in osteosarcoma cells. Elevated LINC00161 increased cisplatin-induced apoptosis and reversed the cisplatin-resistant phenotype of osteosarcoma cells by upregulating IFIT2. Further mechanistic studies revealed that LINC00161 could sponge endogenous miR-645 and inhibit its activity leading to IFIT2 increase. In addition, we identified that LINC00161 enhanced cisplatin-induced apoptosis through regulation of the miR-645-IFIT2 pathway. Thus, these findings demonstrate that LINC00161 is an essential regulator in cisplatin-induced apoptosis, and the LINC00161-miR-645-IFIT2 signalling axis plays an important role in reducing osteosarcoma chemoresistance.

Microfluidics­based optimization of neuroleukin­mediated regulation of articular chondrocyte proliferation.

Due to the low proliferative and migratory capacities of chondrocytes, cartilage repair remains a challenging clinical problem. Current therapeutic strategies for cartilage repair result in unsatisfactory outcomes. Autologous chondrocyte implantation (ACI) is a cell based therapy that relies on the in vitro expansion of healthy chondrocytes from the patient, during which proliferation­promoting factors are frequently used. Neuroleukin (NLK) is a multifunctional protein that possesses growth factor functions, and its expression has been associated with cartilage development and bone regeneration, however its direct role in chondrocyte proliferation remains to be fully elucidated. In the current study, the role of NLK in chondrocyte proliferation in vitro in addition to its potential to act as an exogenous factor during ACI was investigated. Furthermore, the concentration of NLK for in vitro chondrocyte culture was optimized using a microfluidic device. An NLK concentration of 12.85 ng/ml was observed to provide optimal conditions for the promotion of chondrocyte proliferation. Additionally, NLK stimulation resulted in an increase in type II collagen synthesis by chondrocytes, which is a cartilaginous secretion marker and associated with the phenotype of chondrocytes. Together these data suggest that NLK is able to promote cell proliferation and type II collagen synthesis during in vitro chondrocyte propagation, and thus may serve as an exogenous factor for ACI.

Neuroleukin/Autocrine Motility Factor Receptor Pathway Promotes Proliferation of Articular Chondrocytes through Activation of AKT and Smad2/3.

Cartilage defect is an intractable clinical problem. Therapeutic strategies for cartilage repair are far from optimal due to poor proliferation capacity of chondrocytes. Autologous chondrocyte implantation is a cell based therapy that uses in vitro amplified healthy chondrocytes from the patient. However, chondrocyte dedifferentiation during in vitro culture limits its application. Neuroleukin (NLK) is a multifunctional protein that stimulates cell growth and migration, together with its receptor autocrine motility factor receptor (AMFR, also called gp78). We investigated expression of NLK and AMFR/gp78 during cartilage development in vivo and in cultured articular chondrocytes in vitro, and found the pair associates with chondrocyte proliferation and differentiation. While applied to isolated articular chondrocytes, NLK promotes cell proliferation and secretion of type II collagen, a marker of proliferating chondrocytes. Further work demonstrates that NLK up regulates pAKT and pSmad2/3, but down regulates pSmad1/5. In animals, NLK treatment also promotes chondrocyte proliferation while inhibits terminal differentiation, leading to expanded proliferating zone but decreased prehypertrophic and hypertrophic zones in the growth plate region. NLK is therefore a candidate factor that can be applied in the treatment of cartilage defects.

Yes-associated protein regulates the growth of human non-small cell lung cancer in response to matrix stiffness.

The Yes­associated protein (YAP) transcriptional coactivator is recognized as a crucial regulator of human cancer. However, its involvement in human non­small cell lung cancer (NSCLC) in response to physical cues remains unclear. In this study, substrates with different rigidity were generated in order to evaluate the role of YAP, and its upstream regulators in the Hippo pathway, in the regulation of growth of an NSCLC cell line within particular environments. It was shown that the expression of the YAP protein in SPCA-1 NSCLC cells was significantly increased when cultured on a stiff substrate compared to a soft substrate. However, the expression of phospho­YAP protein and large tumor suppressor kinase 1 (LATS1) were markedly decreased after culturing on the stiff substrate. Phosphorylation of YAP by LATS1 leads to cytoplasmic retention of YAP, which inhibits its function as a nuclear transcription coactivator. The study also found that the stiff substrate promoted the growth of NSCLC cells in vitro, and an increase in the transcription levels of Survivin, connective tissue growth factor, amphiregulin and Ki67, as well as a decrease in the expression level of YAP in the cytoplasm, and adecrease in p-YAP. In conclusion, the findings showed that the stiffness of the subcellular matrix altered the behavior of NSCLC cells, and that YAP regulated the growth of NSCLC cells in response to matrix stiffness, thereby suggesting a role for the Hippo­YAP pathway in the response of NSCLC cell growth to specific microenvironments.

[Chondrocyte microenvironment and application of microfluidic chips in constructing chondrocyte microenvironment].

OBJECTIVE: To review the chondrocyte survival microenvironment and the research progress of the application of microfluidic chips in constructing the chondrocyte microenvironment. METHODS: Recent literature about the role of microenvironment in the regulation of chondrocytes and the application of microfluidic chips in constructing the chondrocyte microenvironment was reviewed and analyzed. RESULTS: Regulating the microenvironment of chondrocyte mainly involves extracellular matrix microenvironment, mechanical microenvironment, electric microenvironment, and hypoxic microenvironment. Currently, the related research of chondrocyte microenvironment based on microfluidic system mainly involves biochemical stimuli, mechanical stimuli, production of biomimetic scaffold materials, and so on. CONCLUSION: It will be helpful for constructing cartilage tissue being closer to the physiological function in the future to deeply understand chondrocyte survival environment and to mimic the microenvironment in vivo required by chondrocyte development as possible by using microfluidic chips.

Ganglioside GM3 promotes HGF-stimulated motility of murine hepatoma cell through enhanced phosphorylation of cMet at specific tyrosine sites and PI3K/Akt-mediated migration signaling.

Ganglioside GM3 plays a well-documented and important role in the regulation of tumor cell proliferation, invasion, and metastasis by modulating tyrosine kinase growth factor receptors. However, the effect of GM3 on the hepatocyte growth factor receptor (HGFR, cMet) has not been fully delineated. In the current study, we investigated how GM3 affects cMet signaling and HGF-stimulated cell motility and migration using three hepatic cancer cell lines of mouse (Hca/A2, Hca/16A3, and Hepa1-6). Decreasing GM3 expression with the use of P4, a specific inhibitor for ganglioside synthesis inhibited the HGF-stimulated phosphorylation of cMet and activity of PI3K/Akt signaling pathway. In contrast, the increased expression of GM3 as a result of adding exogenous GM3 enhanced the HGF-stimulated phosphorylation of cMet and activity of PI3K/Akt signaling pathway. Furthermore, HGF-stimulated cell motility and migration in vitro were inhibited by reduced expression of GM3 and enhanced by increased expression of GM3. All the observations indicate that ganglioside GM3 promotes HGF-stimulated motility of murine hepatoma cell through enhanced phosphorylation of cMet at specific tyrosine sites and PI3K/Akt-mediated migration signaling.

Regulation of fibrochondrogenesis of mesenchymal stem cells in an integrated microfluidic platform embedded with biomimetic nanofibrous scaffolds.

In native fibrocartilage, mechanotransduction allows the cells to perceive the physical microenvironment not only through topographical cues from the extracellular matrix, but also through mechanical cues, such as interstitial flow. To create a microenvironment that simultaneously integrates nanotopography and flow stimulus, we developed a biomimetic microfluidic device embedded with aligned nanofibers to contain microchambers of different angles, which enabled the flow direction to form different angles with the fibers. Using this device, we investigated the effects of microfluidic and nanotopographical environment on the morphology and fibrochondrogenesis of mesenchymal stem cells (MSCs) and the involvement of RhoA/ROCK pathway and Yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ). The results showed that the flow direction perpendicular to aligned nanofibers was conducive to fibrochondrogenesis of MSCs. In addition, ROCK inhibitor and knockdown of YAP/TAZ disrupted fibrochondrogenic differentiation of MSCs. In conclusion, our data suggest the crucial role of mechanotransduction in regulating fibrochondrogenic differentiation of MSCs, which may be mediated by RhoA/ROCK pathway and YAP/TAZ.

An integrated microfluidic device in marine microalgae culture for toxicity screening application.

Algal assay using marine microalgae has emerged as an important method to evaluate the toxicity of chemicals, which is currently undertaken using conventional culture and additional detection of physiological cellular endpoints. While effective, this approach can be labor-intensive and thus could benefit from a more streamlined, integrated approach. Microfluidics offers a way to accomplish this goal. Here, we demonstrate a microfluidic device which consists of a concentration gradient generator (CGG), diffusible culturing module and power-free valve system. It allows the processes of chemical liquid dilution and diffusion, micro-scale microalgal culture (in batch or chemostatic conditions), cell stimulation and on-lined screening to be integrated into a single device. Using the device, marine microalgae were successfully cultured and stressed on-chip. The simple assay provides multi-biological response measurements of cell division rate, autofluorescence and esterase activity. This work showed promising in developing a microfluidic platform for toxicity screening based on marine microalgal culture.

YAP-mediated regulation of the chondrogenic phenotype in response to matrix elasticity.

Matrix elasticity exerts considerable influence on the phenotype of terminally differentiated chondrocytes via physical cues. The Yes-associated protein (YAP) transcription co-activator is recognized as a key mediator that may be involved in the nuclear transduction of physical cues controlling cellular behavior and function. However, whether substrate elasticity in the regulation of the chondrocyte phenotype is associated with YAP remains unclear. In this work, we developed micropatterned substrates with varying stiffnesses to investigate the function of YAP and its related Hippo pathway kinases in the regulation of chondrocyte phenotype on soft and stiff substrates. We found that the phenotypic variation of chondrocytes in response to substrate stiffness is concomitant with the changes in YAP localization. The downregulation of YAP expression helps to maintain the chondrogenic phenotype while inhibiting chondrocyte proliferation. Furthermore, the change in the chondrocyte phenotype response to LATS1 kinase inactivation in the Hippo pathway varies significantly between soft and stiff substrates. We also found that LATS1 kinase inactivation promotes chondrocyte dedifferentiation only on stiff substrate. Collectively, these findings reveal that YAP may be involved in the changes that occur in chondrocytes cultured on substrates with different stiffnesses and that these changes do not entirely depend on the Hippo pathway kinase LATS1. Importantly, our findings indicate that YAP inactivation is conducive to the maintenance of the chondrogenic phenotype, thereby providing new insight into articular cartilage repair and regeneration mechanisms.

Mesenchymal stem cell and chondrocyte fates in a multishear microdevice are regulated by Yes-associated protein.

Mechanical cues exert considerable influence on the fates of stem cells and terminally differentiated chondrocytes. The elucidation of the interactions between cell fate and mechanical cues in nuclear mechanotransduction will provide new clues to modulate tissue homeostasis and regeneration. In this study, we used an integrated microfluidic perfusion device to simultaneously generate multiple-parameter fluid shear stresses to investigate the role of fluid flow stimuli in the regulation of Yes-associated protein (YAP) expression and the fates of mesenchymal stem cells (MSCs) and primary chondrocytes. YAP expression was regulated by the level of fluid flow stimulus in both MSCs and chondrocytes. An increase in the magnitude of stimulation enhanced the expression of YAP, ultimately resulting in an increase in osteogenesis and a decrease in adipogenesis for MSCs, and initiating dedifferentiation for chondrocytes. Cytochalasin D not only repressed nuclear YAP accumulation in the flow state, but also abrogated flow-induced effects on MSC differentiation and the chondrocyte phenotype, resulting in MSC adipogenesis and the maintenance of the chondrocyte phenotype. Our findings reveal the connection between YAP and MSC/chondrocyte fates in a fluid flow-induced mechanical microenvironment and provide new insights into the mechanisms by which mechanical cues regulate the fates of MSCs and chondrocytes.

Platelet-derived growth factors-BB and fibroblast growth factors-base induced proliferation of Schwann cells in a 3D environment.

The proliferation of neonatal Schwann cells (SCs) in response to mitogenic agents has been well analyzed in vitro (mono-layer-culture method, 2D environment), but not in vivo (3D environment). To assess the mitogenic effect of platelet-derived growth factors-BB (PDGF-BB), Fibroblast Growth Factors-base (bFGF), and their combinations for SCs in collagen gel (three-dimensional, 3D environment), we have developed an integrated microfluidic device on which can reproducibly measure the proliferation from small number of cells (1-100). The rat SCs were cultured for 4 week at the different concentrations of growth factors generated by concentration gradient generator. In the collagen gel culture, almost all of the cells in colonies presented a round cell morphology and maintained their round morphology by the 4th week. The results showed that PDGF-BB and bFGF are all capable of moderately stimulating SCs growth and every group reached the peak in the growth curve at 3 weeks. Moreover, the proliferation test using the conventional method was performed simultaneously and revealed similar results. The biggest difference between 2D and 3D was that cells decrease more remarkable in 3D than that in 2D at 4 weeks. And at 2 and 3 weeks, the growth rate in the collagen gel with 7.14/2.86 and 8.57/1.43 ng/mL groups was higher than that in the mono-layer culture. Our results showed that PDGF-BB and bFGF are capable of moderately stimulating neonatal SCs growth, respectively and synergistically, and the microfluidic technique is highly controllable, contamination free, fully automatic, and inexpensive.