Dynamics of N6-methyladenosine modification during aging and their potential roles in the degeneration of intervertebral disc.

Background: The N6-methyladenosine (m6A) dynamics in the progression of intervertebral disc (IVD) aging remain largely unknown. This study aimed to explore the distribution and pattern of m6A modification in nucleus pulpous (NP) tissues of rats at different ages. Methods: Histological staining and MRI were performed to evaluate the degeneration of IVD. The expression of m6A modifiers was analyzed using qRT-PCR and western blot. Subsequently, methylated RNA immunoprecipitation next generation sequencing and RNA-seq were conducted to identify differences in m6A methylome and transcriptome of NP tissues. Results: Compared to 2-month-old rats, we found significant changes in the global m6A level and the expression of Mettl3 and FTO in NP tissues from 20-month-old rats. During the progression of NP aging, there were 1126 persistently differentially m6A peaks within 931 genes, and 51 persistently differentially expressed genes. GO and KEGG analyses showed that these m6A peaks and m6A modified genes were mainly engaged in the biological processes and pathways of intervertebral disc degermation (IDD), such as extracellular matrix metabolism, angiogenesis, inflammatory response, mTOR and AMPK signaling pathways. Meanwhile, conjoint analyses and Venn diagram revealed a total of 405 aging related genes contained significant methylation and expression levels in 20-month-old rats in contrast to 2-month-old and 10-month-old rats. Moreover, it was found that four aging related genes with hypermethylated modification including BUB1, CA12, Adamts1, and Adamts4 depicted differentially expressed at protein level, of which BUB1 and CA12 were decreased, while Adamts1 and Adamts4 were increased during the progression of NP aging. Conclusion: Collectively, this study elucidated the distribution and pattern of m6A modification during the aging of IVD. Furthermore, the m6A modified genes were involved in the IDD related biological processes and pathways. These findings may provide novel insights into the mechanisms and therapies of IDD from the perspective of aging.

The sonic hedgehog pathway suppresses oxidative stress and senescence in nucleus pulposus cells to alleviate intervertebral disc degeneration via GPX4.

Disruption of intervertebral disc (IVD) homeostasis caused by oxidative stress and nucleus pulposus cell (NPC) senescence is a main cause of intervertebral disc degeneration (IDD). The sonic hedgehog (Shh) pathway plays an important role in IVD development, but its roles in IDD are unknown. This study aimed to investigate the effects of the Shh pathway on the alleviation of IDD and the related mechanisms. In vivo, the effect of the Shh pathway on IVD homeostasis was studied by intraperitoneal injection of recombinant Shh (rShh) and GANT61 based on puncture-induced IDD. GANT61, lentivirus-coated sh-Gli1 and rShh were used to investigate the role and mechanism of the Shh pathway in NPCs based on senescence induced by Braco19 and oxidative stress induced by TBHP. Shh pathway expression decreased, and senescence and oxidative stress increased with age. Intraperitoneal injection of rShh activated the Shh pathway to suppress oxidative stress and NPC senescence and consequently alleviated needle puncture-induced IDD. In vitro, the Shh pathway upregulated glutathione peroxidase 4 (GPX4) expression to suppress oxidative stress and senescence in NPCs. Moreover, GPX4 suppression in NPCs by si-GPX4 significantly reduced the protective effect of the Shh pathway on oxidative stress and senescence in NPCs. Our results demonstrate for the first time that the Shh pathway plays a key role in the alleviation of IDD by suppressing oxidative stress and cell senescence in NP tissues. This study provides a new potential target for the prevention and reversal of IDD.

Demonstration of Germanium Vertical Gate-All-Around Field-Effect Transistors Featured by Self-Aligned High-κ Metal Gates with Record High Performance.

A special Ge nanowire/nanosheet (NW/NS) p-type vertical sandwich gate-all-around (GAA) field-effect transistor (FET) (Ge NW/NS pVSAFET) with self-aligned high-κ metal gates (HKMGs) is proposed. The Ge pVSAFETs were fabricated by high-quality GeSi/Ge epitaxy, an exclusively developed self-limiting isotropic quasi atomic layer etching (qALE) of Ge selective to both GeSi and the (111) plane, top-drain implantation, and ozone postoxidation (OPO) channel passivation. The Ge pVSAFETs, which have hourglass-shaped (111) channels with the smallest size range from 5 to 20 nm formed by qALE, have reached a record high Ion of ∼291 µA/µm and exhibited good short channel effects (SCEs) control. The integration flow is compatible with mainstream CMOS processes, and Ge pVSAFETs with precise control of gate lengths/channel sizes were obtained.

A Biomimetic Peptide Functions as Specific Extracellular Matrix for Quiescence of Stem Cells against Intervertebral Disc Degeneration.

Maintaining quiescence of stem cells is a potential way to decrease cell nutrition demand for restoring the organization. Herein, a biomimetic peptide to maintain quiescence of stem cells through C-X-C motif chemokine ligand 8 (CXCL8)-C-X-C motif chemokine receptor 1 (CXCR1) pathway against intervertebral disc degeneration (IVDD) is developed. First, it is confirmed that quiescence can be induced via inhibiting phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway in nucleus pulposus stem cells (NPSCs). Meanwhile, it is well known that CXCR1, a chemokine receptor, can be targeted by CXCL8, resulting in cell proliferation via activating PI3K/Akt/mTOR pathway. Second, a biomimetic peptide (OAFF) that can bind to CXCR1 and form fibrous networks on NPSCs, mimicking extracellular matrix formation is developed. The multivalent effect and long-term binding to CXCR1 on NPSCs of OAFF fibers offer forcefully competitive inhibition with natural CXCL8, which induces NPSCs quiescence and ultimately overcomes obstacle in intradiscal injection therapy. In rat caudal disc puncture model, OAFF nanofibers still maintain at 5 weeks after operation and inhibit degeneration process of intervertebral disc in terms of histopathology and imageology. In situ fibrillogenesis of biomimetic peptide on NPSCs provides promising stem cells for intradiscal injection therapy against IVDD.

Gli1 depletion induces oxidative stress and apoptosis of nucleus pulposus cells via Fos in intervertebral disc degeneration.

Background: Intervertebral disc degeneration (IDD) is the most common chronic disease. Oxidative stress and apoptosis of nucleus pulposus (NP) cells disrupt intervertebral disc (IVD) homeostasis, which is the main cause of IDD. Glioma-associated oncogene 1 (Gli1) is an important transcription factor in the Hedgehog (Hh) pathway. Depletion of Gli1 accelerates the occurrence and development of degenerative diseases. This study aimed to explore the role of aging related Gli1 depletion in the progression of IDD. Methods: The relationship between aging related Gli1 depletion and IDD was studied in the NP tissues of human and rats of different ages, and the levels of oxidative stress and NP cell apoptosis during IDD were explored. Gli1 depletion of NP cells were established by targeting inhibitor GANT61 or lentivirus-coated Gli1 sh-RNA (sh-Gli1) to explore the role of Gli1 in NP cells and underlying mechanism. Exogenous Gli1 depletion induced IDD of rats was established by intraperitoneal injection of GANT61. Also, the roles of Fos in the Gli1 depletion induced NP cell oxidative stress, apoptosis and IDD were investigated. Results: Gli1 was down-regulated in the tissues of degenerative NP, and the level of Gli1 was negatively correlated with the severity of aging related IDD in human and rats. Furthermore, we found enhanced oxidative stress and apoptosis in degenerative NP tissues. Gli1 depletion promoted oxidative stress and apoptosis of NP cells and resulted in the degradation of extracellular matrix (ECM) and decreased ECM synthesis. Transcriptome sequencing showed that Gli1 depletion caused Fos activation in NP cells. the effect of Gli1 depletion on the oxidative stress and apoptosis of NP cells were retarded by Fos inhibitor. In vivo, Fos inhibition alleviated the IDD induced by exogenous Gli1 depletion. Conclusions: This study revealed for the first time that Gli1 is gradually depleted in NP with IDD progression. Exogenous Gli1 depletion causes oxidative stress and apoptosis of NP cells both in vivo and in vitro. Fos suppression effectively retards the destructive effects of Gli1 depletion on IVD homoeostasis.The translational potential of this article: This study may provide new potential targets for preventing and reversing IDD. Maintaining Gli1 expression in NP and suppressing Fos activation may be an effective treatment strategy for IDD.

Investigation on Ge0.8Si0.2-Selective Atomic Layer Wet-Etching of Ge for Vertical Gate-All-Around Nanodevice.

For the formation of nano-scale Ge channels in vertical Gate-all-around field-effect transistors (vGAAFETs), the selective isotropic etching of Ge selective to Ge0.8Si0.2 was considered. In this work, a dual-selective atomic layer etching (ALE), including Ge0.8Si0.2-selective etching of Ge and crystal-orientation selectivity of Ge oxidation, has been developed to control the etch rate and the size of the Ge nanowires. The ALE of Ge in p+-Ge0.8Si0.2/Ge stacks with 70% HNO3 as oxidizer and deionized (DI) water as oxide-removal was investigated in detail. The saturated relative etched amount per cycle (REPC) and selectivity at different HNO3 temperatures between Ge and p+-Ge0.8Si0.2 were obtained. In p+-Ge0.8Si0.2/Ge stacks with (110) sidewalls, the REPC of Ge was 3.1 nm and the saturated etching selectivity was 6.5 at HNO3 temperature of 20 °C. The etch rate and the selectivity were affected by HNO3 temperatures. As the HNO3 temperature decreased to 10 °C, the REPC of Ge was decreased to 2 nm and the selectivity remained at about 7.4. Finally, the application of ALE in the formation of Ge nanowires in vGAAFETs was demonstrated where the preliminary Id-Vds output characteristic curves of Ge vGAAFET were provided.

The Effect of Doping on the Digital Etching of Silicon-Selective Silicon-Germanium Using Nitric Acids.

Gate-all-around (GAA) field-effect transistors have been proposed as one of the most important developments for CMOS logic devices at the 3 nm technology node and beyond. Isotropic etching of silicon-germanium (SiGe) for the definition of nano-scale channels in vertical GAA CMOS and tunneling FETs has attracted more and more attention. In this work, the effect of doping on the digital etching of Si-selective SiGe with alternative nitric acids (HNO3) and buffered oxide etching (BOE) was investigated in detail. It was found that the HNO3 digital etching of SiGe was selective to n+-Si, p+-Si, and intrinsic Si. Extensive studies were performed. It turned out that the selectivity of SiGe/Si was dependent on the doped types of silicon and the HNO3 concentration. As a result, at 31.5% HNO3 concentration, the relative etched amount per cycle (REPC) and the etching selectivity of Si0.72Ge0.28 for n+-Si was identical to that for p+-Si. This is particularly important for applications of vertical GAA CMOS and tunneling FETs, which have to expose both the n+ and p+ sources/drains at the same time. In addition, the values of the REPC and selectivity were obtained. A controllable etching rate and atomically smooth surface could be achieved, which enhanced carrier mobility.

First Demonstration of Novel Vertical Gate-All-Around Field-Effect-Transistors Featured by Self-Aligned and Replaced High-κ Metal Gates.

A novel n-type nanowire/nanosheet (NW/NS) vertical sandwich gate-all-around field-effect-transistor (nVSAFET) with self-aligned and replaced high-κ metal gates (HKMGs) is presented for the first time, aiming at a 3 nm technology node and beyond. The nVSAFETs were fabricated by an integration flow of Si/SiGe epitaxy, quasi-atomic layer etching (qALE) of SiGe selective to Si, formation of SiGe/Si core/shell NS/NW structure, building of nitride dummy gate, and replacement of the dummy gate. This fabrication method is complementary metal oxide semiconductor (CMOS)-compatible, simple, and reproducible, and NWs with a diameter of 17 nm and NSs with a thickness of 20 nm were obtained. Excellent control of short-channel-effects was presented. The device performance was also investigated and discussed. The proposed integration scheme has great potential for applications in chip manufacturing, especially with vertical channel devices.

Cartilage intermediate layer protein affects the progression of intervertebral disc degeneration by regulating the extracellular microenvironment (Review).

Intervertebral disc degeneration (IDD), which is caused by multiple factors, affects the health of individuals and contributes to low back pain. The pathology of IDD is complicated, and changes in the extracellular microenvironment play an important role in promoting the process of degeneration. Cartilage intermediate layer protein (CILP) is a matrix protein that resides in the middle of human articular cartilage and is involved in numerous diseases that affect cartilage. However, there is no detailed review of the relationship between CILP and degenerative disc disease. Growing evidence has revealed the presence of CILP in the extracellular microenvironment of intervertebral discs (IVDs) and has suggested that there is a gradual increase in CILP in degenerative discs. Specifically, CILP plays an important role in regulating the metabolism of the extracellular matrix (ECM), an important component of the extracellular microenvironment. CILP can combine with transforming growth factor­ß or insulin­like growth factor­1 to regulate the ECM synthesis of IVDs and influence the balance of ECM metabolism, which leads to changes in the extracellular microenvironment to promote the process of IDD. It may be possible to show the correlation of CILP with IDD and to target CILP to interfere with IDD. For this purpose, in the present study, the current knowledge on CILP was summarized and a detailed description of CILP in discs was provided.

Strained Si0.2Ge0.8/Ge multilayer Stacks Epitaxially Grown on a Low-/high-Temperature Ge Buffer Layer and Selective Wet-Etching of Germanium.

With the development of new designs and materials for nano-scale transistors, vertical Gate-All-Around Field Effect Transistors (vGAAFETs) with germanium as channel materials have emerged as excellent choices. The driving forces for this choice are the full control of the short channel effect and the high carrier mobility in the channel region. In this work, a novel process to form the structure for a VGAA transistor with a Ge channel is presented. The structure consists of multilayers of Si0.2Ge0.8/Ge grown on a Ge buffer layer grown by the reduced pressure chemical vapor deposition technique. The Ge buffer layer growth consists of low-temperature growth at 400 °C and high-temperature growth at 650 °C. The impact of the epitaxial quality of the Ge buffer on the defect density in the Si0.2Ge0.8/Ge stack has been studied. In this part, different thicknesses (0.6, 1.2 and 2.0 µm) of the Ge buffer on the quality of the Si0.2Ge0.8/Ge stack structure have been investigated. The thicker Ge buffer layer can improve surface roughness. A high-quality and atomically smooth surface with RMS 0.73 nm of the Si0.2Ge0.8/Ge stack structure can be successfully realized on the 1.2 µm Ge buffer layer. After the epitaxy step, the multilayer is vertically dry-etched to form a fin where the Ge channel is selectively released to SiGe by using wet-etching in HNO3 and H2O2 solution at room temperature. It has been found that the solution concentration has a great effect on the etch rate. The relative etching depth of Ge is linearly dependent on the etching time in H2O2 solution. The results of this study emphasize the selective etching of germanium and provide the experimental basis for the release of germanium channels in the future.

Selective Digital Etching of Silicon-Germanium Using Nitric and Hydrofluoric Acids.

A digital etching method was proposed to achieve excellent control of etching depth. The digital etching characteristics of p+-Si and Si0.7Ge0.3 using a combination of HNO3 oxidation and buffered oxide etching oxide removal processes were investigated. Experimental results showed that oxidation saturates as time goes on because of low activation energy and its diffusion-limited characteristic. An oxidation model was developed to describe the wet oxidation process with nitric acid. The model was calibrated with experimental data, and the oxidation saturation time, final oxide thickness, and selectivity between Si0.7Ge0.3 and p+-Si were obtained. In Si0.7Ge0.3/p+-Si stacks, the saturated relative etched depth per cycle was 0.5 nm (four monolayers), and variation between experiments was about 4% after saturation. A corrected selectivity calculation formula was also proposed, and the calculated selectivity was 3.7-7.7 for different oxidation times, which was the same as the selectivity obtained from our oxidation model. The proposed model can be used to analyze process variations and repeatability, and it can provide credible guidance for the design of other wet digital etching experiments.

A positive feedback loop between EZH2 and NOX4 regulates nucleus pulposus cell senescence in age-related intervertebral disc degeneration.

BACKGROUND: The senescence of nucleus pulposus (NP) cells plays a vital role in the pathogenesis of intervertebral disc (IVD) degeneration (IDD). NADPH oxidase 4 (NOX4)-associated oxidative stress has been shown to induce premature NP cell senescence. Enhancer of zeste homolog 2 (EZH2) is a crucial gene regulating cell senescence. The aim of this study was to investigate the roles of EZH2 in NOX4-induced NP cell senescence and a feedback loop between EZH2 and NOX4. RESULTS: The down-regulation of EZH2 and the up-regulation of NOX4 and p16 were observed in the degenerative discs of aging rats. EZH2 regulated NP cell senescence via the H3K27me3-p16 pathway. Also, EZH2 regulated the expression of NOX4 in NP cells through the histone H3 lysine 27 trimethylation (H3K27me3) in the promoter of NOX4 gene. Furthermore, NOX4 down-regulated EZH2 expression in NP cells via the canonical Wnt/ß-catenin pathway. CONCLUSIONS: A positive feedback loop between EZH2 and NOX4 is involved in regulating NP cell senescence, which provides a novel insight into the mechanism of IDD and a potential therapeutic target for IDD.