Integrated metabolome and transcriptome analyses reveal the molecular mechanism underlying dynamic metabolic processes during taproot development of Panax notoginseng.

BACKGROUND: Panax notoginseng (Burk) F. H. Chen is one of the most famous Chinese traditional medicinal plants. The taproot is the main organ producing triterpenoid saponins, and its development is directly linked to the quality and yield of the harvested P. notoginseng. However, the mechanisms underlying the dynamic metabolic changes occurring during taproot development of P. notoginseng are unknown. RESULTS: We carried out metabolomic and transcriptomic analyses to investigate metabolites and gene expression during the development of P. notoginseng taproots. The differentially accumulated metabolites included amino acids and derivatives, nucleotides and derivatives, and lipids in 1-year-old taproots, flavonoids and terpenoids in 2- and 3-year-old taproots, and phenolic acids in 3-year-old taproots. The differentially expressed genes (DEGs) are related to phenylpropanoid biosynthesis, metabolic pathway and biosynthesis of secondary metabolites at all three developmental stages. Integrative analysis revealed that the phenylpropanoid biosynthesis pathway was involved in not only the development of but also metabolic changes in P. notoginseng taproots. Moreover, significant accumulation of triterpenoid saponins in 2- and 3-year-old taproots was highly correlated with the up-regulated expression of cytochrome P450s and uridine diphosphate-dependent glycosyltransferases genes. Additionally, a gene encoding RNase-like major storage protein was identified to play a dual role in the development of P. notoginseng taproots and their triterpenoid saponins synthesis. CONCLUSIONS: These results elucidate the molecular mechanism underlying the accumulation of and change relationship between primary and secondary metabolites in P. notoginseng taproots, and provide a basis for the quality control and genetic improvement of P. notoginseng.

Functional characterization of genes related to triterpene and flavonoid biosynthesis in Cyclocarya paliurus.

MAIN CONCLUSION: The oxidosqualene cyclases (OSCs) generating triterpenoid skeletons in Cyclocarya paliurus were identified for the first time, and two uridine diphosphate (UDP)-glycosyltransferases (UGTs) catalyzing the glycosylation of flavonoids were characterized. Cyclocarya paliurus, a native rare dicotyledonous plant in China, contains an abundance of triterpenoid saponins and flavonoid glycosides that exhibit valuable pharmaceutical effects in preventing hypertension, hyperlipidemia, and diabetes. However, the molecular mechanism explaining the biosynthesis of triterpenoid saponin and flavonoid glycoside in C. paliurus remains unclear. In this study, the triterpene content in different tissues and the expression pattern of genes encoding the key enzymes associated with triterpenoid saponin and flavonoid glycoside biosynthesis were studied using transcriptome and metabolome analysis. The eight upstream oxidosqualene cyclases (OSCs) involved in triterpenoid saponin biosynthesis were functionally characterized, among them CpalOSC6 catalyzed 2,3;22,23-dioxidosqualene to form 3-epicabraleadiol; CpalOSC8 cyclized 2,3-oxidosqualene to generate dammarenediol-II; CpalOSC2 and CpalOSC3 produced ß-amyrin and CpalOSC4 produced cycloartenol, while CpalOSC2-CpalOSC5, CpalOSC7, and CpalOSC8 all produced lanosterol. However, no catalytic product was detected for CpalOSC1. Moreover, two downstream flavonoid uridine diphosphate (UDP)-glycosyltransferases (UGTs) (CpalUGT015 and CpalUGT100) that catalyze the last step of flavonoid glycoside biosynthesis were functionally elucidated. These results uncovered the key genes involved in the biosynthesis of triterpenoid saponins and flavonoid glycosides in C. paliurus that could be applied to produce flavonoid glycosides and key triterpenoid saponins in the future via a synthetic strategy.

Dendritic cell type 3 arises from Ly6C+ monocyte-dendritic cell progenitors.

Conventional dendritic cells (cDCs) are professional antigen-presenting cells that control the adaptive immune response. Their subsets and developmental origins have been intensively investigated but are still not fully understood as their phenotypes, especially in the DC2 lineage and the recently described human DC3s, overlap with monocytes. Here, using LEGENDScreen to profile DC vs. monocyte lineages, we found sustained expression of FLT3 and CD45RB through the whole DC lineage, allowing DCs and their precursors to be distinguished from monocytes. Using fate mapping models, single-cell RNA sequencing and adoptive transfer, we identified a lineage of murine CD16/32+CD172a+ DC3, distinct from DC2, arising from Ly6C+ monocyte-DC progenitors (MDPs) through Lyz2+Ly6C+CD11c- pro-DC3s, whereas DC2s develop from common DC progenitors (CDPs) through CD7+Ly6C+CD11c+ pre-DC2s. Corresponding DC subsets, developmental stages, and lineages exist in humans. These findings reveal DC3 as a DC lineage phenotypically related to but developmentally different from monocytes and DC2s.

Effects of a High-Fat Diet on Intestinal and Gonadal Metabolism in Female and Male Sea Cucumber Apostichopus japonicus.

Parental nutrient reserves are directly related to reproductive performance in sea cucumbers. This study focused on the lipid requirements of male and female sea cucumbers Apostichopus japonicus during the reproductive stage and analyzed their physiological responses to a high-fat diet (HFD). The intestinal lipid metabolites and microbiome profile changed significantly in animals fed with the HFD, as given by an upregulation of metabolites related to lipid metabolism and an increase in the predominance of Proteobacteria in the microbiome, respectively. The metabolic responses of male and female sea cucumbers to the HFD differed, which in turn could have triggered sex-related differences in the intestinal microbiome. These results suggest that the lipid content in diets can be differentially adjusted for male and female sea cucumbers to improve nutrition and promote reproduction. This data contributes to a better understanding of the reproductive biology and sex differences of sea cucumbers.

Biosynthetic pathway of prescription bergenin from Bergenia purpurascens and Ardisia japonica.

Bergenin is a typical carbon glycoside and the primary active ingredient in antitussive drugs widely prescribed for central cough inhibition in China. The bergenin extraction industry relies on the medicinal plant species Bergenia purpurascens and Ardisia japonica as their resources. However, the bergenin biosynthetic pathway in plants remains elusive. In this study, we functionally characterized a shikimate dehydrogenase (SDH), two O-methyltransferases (OMTs), and a C-glycosyltransferase (CGT) involved in bergenin synthesis through bioinformatics analysis, heterologous expression, and enzymatic characterization. We found that BpSDH2 catalyzes the two-step dehydrogenation process of shikimic acid to form gallic acid (GA). BpOMT1 and AjOMT1 facilitate the methylation reaction at the 4-OH position of GA, resulting in the formation of 4-O-methyl gallic acid (4-O-Me-GA). AjCGT1 transfers a glucose moiety to C-2 to generate 2-Glucosyl-4-O-methyl gallic acid (2-Glucosyl-4-O-Me-GA). Bergenin production ultimately occurs in acidic conditions or via dehydration catalyzed by plant dehydratases following a ring-closure reaction. This study for the first time uncovered the biosynthetic pathway of bergenin, paving the way to rational production of bergenin in cell factories via synthetic biology strategies.

Spata2L Suppresses TLR4 Signaling by Promoting CYLD-Mediated Deubiquitination of TRAF6 and TAK1.

Toll-like receptor 4 (TLR4) is a key pattern recognition receptor that can be activated by bacterial lipopolysaccharide to elicit inflammatory response. Proper activation of TLR4 is critical for the host defense against microbial infections. Since overactivation of TLR4 causes deleterious effects and inflammatory diseases, its activation needs to be tightly controlled by negative regulatory mechanisms, among which the most pivotal could be deubiquitination of key signaling molecules mediated by deubiquitinating enzymes (DUBs). CYLD is a member of the USP family of DUBs that acts as a critical negative regulator of TLR4-depedent inflammatory responses by deconjugating polyubiquitin chains from signaling molecules, such as TRAF6 and TAK1. Dysregulation of CYLD is implicated in inflammatory diseases. However, how the function of CYLD is regulated during inflammatory response remains largely unclear. Recently, we and other authors have shown that Spata2 functions as an important CYLD partner to regulate enzymatic activity of CYLD and substrate binding by this protein. Here, we show that a Spata2-like protein, Spata2L, can also form a complex with CYLD to inhibit the TLR4-dependent inflammatory response. We found that Spata2L constitutively interacts with CYLD and that the deficiency of Spata2L enhances the LPS-induced NF-κB activation and proinflammatory cytokine gene expression. Mechanistically, Spata2L potentiated CYLD-mediated deubiquitination of TRAF6 and TAK1 likely by promoting CYLD enzymatic activity. These findings identify Spata2L as a novel CYLD regulator, provide new insights into regulatory mechanisms underlying CYLD role in TLR4 signaling, and suggest potential targets for modulating TLR4-induced inflammation.

Whole-genome resequencing reveals the origin of tea in Lincang.

Phylogeographic, population genetics and diversity analysis are crucial for local tea resource conservation and breeding programs. Lincang in Yunnan has been known as the possible place of domestication for tea worldwide, yet, its genetic makeup and unique Lincang origin are little understood. Here, we reported a large-scale whole-genome resequencing based population genomic analysis in eight main tea-producing areas of Lincang in Yunnan (1,350 accessions), and the first comprehensive map of tea genome variation in Lincang was constructed. Based on the population structure, tea sample in Lincang was divided into three subgroups, and inferred Xigui and Nahan Tea Mountain in Linxiang, Baiying Mountain Ancient Tea Garden in Yun, and Jinxiu Village of Xiaowan Town in Fengqing, which belong to the birthplace of the three subgroups, were all likely to be the origin center of Lincang tea. Meanwhile, the history population sizes analysis show that similar evolutionary patterns were observed for the three subgroups of Lincang. It also was observed that the hybrid among eight areas of Lincang was noticeable, resulting in insignificant genetic differentiation between geographical populations and low genetic diversity. The findings of this study clarified the genetic make-up and evolutionary traits of the local population of tea, which gave some insight into the development of Lincang tea.

Integrated Metabolomic and Transcriptomic Analysis and Identification of Dammarenediol-II Synthase Involved in Saponin Biosynthesis in Gynostemma longipes.

Gynostemma longipes contains an abundance of dammarane-type ginsenosides and gypenosides that exhibit extensive pharmacological activities. Increasing attention has been paid to the elucidation of cytochrome P450 monooxygenases (CYPs) and UDP-dependent glycosyltransferases (UGTs) that participate downstream of ginsenoside biosynthesis in the Panax genus. However, information on oxidosqualene cyclases (OSCs), the upstream genes responsible for the biosynthesis of different skeletons of ginsenoside and gypenosides, is rarely reported. Here, an integrative study of the metabolome and the transcriptome in the leaf, stolon, and rattan was conducted and the function of GlOSC1 was demonstrated. In total, 46 triterpenes were detected and found to be highly abundant in the stolon, whereas gene expression analysis indicated that the upstream OSC genes responsible for saponin skeleton biosynthesis were highly expressed in the leaf. These findings indicated that the saponin skeletons were mainly biosynthesized in the leaf by OSCs, and subsequently transferred to the stolon via CYPs and UGTs biosynthesis to form various ginsenoside and gypenosides. Additionally, a new dammarane-II synthase (DDS), GlOSC1, was identified by bioinformatics analysis, yeast expression assay, and enzyme assays. The results of the liquid chromatography-mass spectrometry (LC-MS) analysis proved that GlOSC1 could catalyze 2,3-oxidosqualene to form dammarenediol-II via cyclization. This work uncovered the biosynthetic mechanism of dammarenediol-II, an important starting substrate for ginsenoside and gypenosides biosynthesis, and may achieve the increased yield of valuable ginsenosides and gypenosides produced under excess substrate in a yeast cell factory through synthetic biology strategy.

Inflammasome Meets Centrosome: Understanding the Emerging Role of Centrosome in Controlling Inflammasome Activation.

Inflammasomes are multi-protein platforms that are assembled in response to microbial and danger signals to activate proinflammatory caspase-1 for production of active form of IL-1ß and induction of pyroptotic cell death. Where and how an inflammasome is assembled in cells has remained controversial. While the endoplasmic reticulum, mitochondria and Golgi apparatus have been reported to be associated with inflammasome assembly, none of these sites seems to match the morphology, number and size of activated inflammasomes that are microscopically observable as one single perinuclear micrometer-sized punctum in each cell. Recently, emerging evidence shows that NLRP3 and pyrin inflammasomes are assembled, activated and locally regulated at the centrosome, the major microtubule organizing center in mammalian cells, elegantly accounting for the singularity, size and perinuclear location of activated inflammasomes. These new exciting findings reveal the previously unappreciated importance of the centrosome in controlling inflammasome assembly and activation as well as inflammasome-related diseases.

Cell-Free Fat Extract Prevents Tail Suspension-Induced Bone Loss by Inhibiting Osteocyte Apoptosis.

Introduction: As the space field has developed and our population ages, people engaged in space travel and those on prolonged bed rest are at increasing risk for bone loss and fractures. Disuse osteoporosis occurs frequently in these instances, for which the currently available anti-osteoporosis agents are far from satisfactory and have undesirable side effects. CEFFE is a cell-free fraction isolated from nanofat that is enriched with a variety of growth factors, and we aim to investigate its potential therapeutic effects on disuse osteoporosis. Methods: A tail suspension-induced osteoporosis model was applied in this study. Three weeks after tail suspension, CEFFE was intraperitoneally injected, and PBS was used as a control. The trabecular and cortical bone microstructures of the tibia in each group were assessed by µCT after 4 weeks of administration. Osteocyte lacunar-canalicularity was observed by HE and silver staining. In vitro, MLO-Y4 cell apoptosis was induced by reactive oxygen species (ROSUP). TUNEL staining and flow cytometry were used to detect apoptosis. CCK-8 was used to detect cell proliferation, and Western blotting was used to detect MAPK signaling pathway changes. Results: CEFFE increased the bone volume (BV/TV) and trabecular number (Tb.N) of the trabecular bone and increased the thickness of the cortical bone. HE and silver staining results showed that CEFFE reduced the number of empty lacunae and improved the lacuna-canalicular structure. CEFFE promoted osteocyte proliferative capacity in a dose-dependent manner. CEFFE protected MLO-Y4 from apoptosis by activating the serine/threonine-selective protein kinase (ERK) signaling pathways. Conclusion: CEFFE attenuated immobilization-induced bone loss by decreasing osteocyte apoptosis. CEFFE increased the survival of osteocytes and inhibited osteocyte apoptosis by activating the ERK signaling pathway in vitro.

Osteocyte Apoptosis Contributes to Cold Exposure-induced Bone Loss.

Emerging evidence indicates that bone mass is regulated by systemic energy balance. Temperature variations have profound effects on energy metabolism in animals, which will affect bone remodeling. But the mechanism remains unclear. 2-month-old C57BL/6J male mice were exposed to cold (4°C) and normal (23°C) temperatures for 28 days and the effects of cold exposure on bone mass was investigated. Micro-computed tomography results showed that bone volume fraction was significantly reduced after 14 days of exposure to cold temperature, and it was recovered after 28 days. Ploton silver staining and immunohistochemical results further revealed that exposure to cold decreased canalicular length, number of E11-and MMP13-positive osteocytes after 14 days, but they returned to the baseline levels after 28 days, different from the normal temperature control group. In addition, change of Caspase-3 indicated that exposure to cold temperature augmented apoptosis of osteocytes. In vitro results confirmed the positive effect of brown adipocytes on osteocyte's dendrites and E11 expression. In conclusion, our findings indicate that cold exposure can influence bone mass in a time-dependent manner, with bone mass decreasing and recovering at 2 and 4 weeks respectively. The change of bone mass may be caused by the apoptosis osteocytes. Brown adipocyte tissue could influence bone remodeling through affecting osteocyte.

Large-Scale Characterization of the Soil Microbiome in Ancient Tea Plantations Using High-Throughput 16S rRNA and Internal Transcribed Spacer Amplicon Sequencing.

There is a special interaction between the environment, soil microorganisms, and tea plants, which constitute the ecosystem of tea plantations. Influenced by environmental factors and human management, the changes in soil microbial community affected the growth, quality, and yield of tea plants. However, little is known about the composition and structure of soil bacterial and fungal communities in 100-year-old tea plantations and the mechanisms by which they are affected. In this regard, we characterized the microbiome of tea plantation soils by considering the bacterial and fungal communities in 448 soil samples from 101 ancient tea plantations in eight counties of Lincang city, which is one of the tea domestication centers in the world. 16S and Internal Transcribed Spacer (ITS) rRNA high-throughput amplicon sequencing techniques were applied in this study. The results showed that the abundance, diversity, and composition of the bacterial and fungal communities have different sensitivity with varying pH, altitude, and latitude. pH and altitude affect soil microbial communities, and bacterial communities are more sensitive than fungi in terms of abundance and diversity to pH. The highest α-diversity of bacterial communities is shown in the pH 4.50-5.00 and 2,200-m group, and fungi peaked in the pH 5.00-5.50 and 900-m group. Because of environmental and geographical factors, all microbes are similarly changing, and further correlations showed that the composition and structure of bacterial communities are more sensitive than fungal communities, which were affected by latitude and altitude. In conclusion, the interference of anthropogenic activities plays a more important role in governing fungal community selection than environmental or geographical factors, whereas for the bacterial community, it is more selective to environment adaptation than to adaptation to human activities.

FTO-stabilized lncRNA HOXC13-AS epigenetically upregulated FZD6 and activated Wnt/ß-catenin signaling to drive cervical cancer proliferation, invasion, and EMT.

PURPOSE: Cervical cancer (CC) is the third most prevalent malignancy in women. Frizzled class receptor 6 (FZD6) is demonstrated to either activate or repress the activity of Wnt/ß-catenin pathway, a crucial signaling involved in cancer development. However, the role of FZD6 in CC is unknown. The present study explored the function of FZD6 and its mechanism in CC. METHODS: The levels of FZD6, HOXC13-AS were detected in CC specimens and CC cell lines via qRT-PCR. Cell proliferation and invasion was explored via CCK-8 assay, colony formation assay and transwell assay. Luciferase reporter analysis, FISH, subcellular fractionation, chromatin immunoprecipitation and RNA immunoprecipitation were performed for investigating the molecular mechanism. RESULTS: FZD6 was up-regulated in CC. FZD6 silence retarded proliferation, invasion, and epithelial-to-mesenchymal transition (EMT), and inactivated Wnt/ß-catenin. HOXC13 antisense RNA (HOXC13-AS) was up-regulated in CC and positively correlated with FZD6. Mechanistically, HOCX13-AS1 augmented FZD through cAMP-response element binding protein-binding protein (CBP)-modulated histone H3 on lysine 27 acetylation (H3K27ac). Additionally, fat mass and obesity-associated protein (FTO) reduced N6-methyladenosine (m6A) and stabilized HOXC13-AS in CC. CONCLUSIONS: In conclusion, this study firstly showed that FTO-stabilized HOXC13-AS epigenetically up-regulated FZD6 and activated Wnt/ß-catenin signaling to drive CC proliferation, invasion, and EMT, suggesting HOXC13-AS as a potential target for CC treatment.

Influence of an L-type SALMFamide neuropeptide on locomotory performance and muscle physiology in the sea cucumber Apostichopus japonicus.

Neuropeptides in the SALMFamide family serve as muscle relaxants in echinoderms and may affect locomotion, as the motor behavior in sea cucumbers involves alternating contraction and extension of the body wall, which is under the control of longitudinal muscle. We evaluated the effect of an L-type SALMFamide neuropeptide (LSA) on locomotory performance of Apostichopus japonicus. We also investigated the metabolites of longitudinal muscle tissue using ultra performance liquid chromatography and quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) to assess the potential physiological mechanisms underlying the effect of LSA. The hourly distance, cumulative duration and number of steps moved significantly increased in sea cucumbers in the fourth hour after injection with LSA. Also, the treatment enhanced the mean and maximum velocity by 9.8% and 17.8%, respectively, and increased the average stride by 12.4%. Levels of 27 metabolites in longitudinal muscle changed after LSA administration, and the increased concentration of pantothenic acid, arachidonic acid and lysophosphatidylethanolamine, and the altered phosphatidylethanolamine/phosphatidylcholine ratio are potential physiological mechanisms that could explain the observed effect of LSA on locomotor behavior in A. japonicus.

Transcriptome analysis of gender-biased CYP genes in gonads of the sea cucumber Apostichopus japonicus.

Gender differences in physiological characteristics are widespread in animals. Herein, differentially expressed genes (DEGs) in gonads of the sea cucumber Apostichopus japonicus were analysed by transcriptomics, and the results showed that 19,973 genes were commonly expressed in the males and females, 4186 were female-biased, and 2540 were male-biased, 4695 genes were up-regulated in the females and 3436 genes were up-regulated in the males. These DEGs were mainly associated with metabolism, including lipid metabolism, amino acid metabolism, nucleotide metabolism, energy metabolism, and cofactor and vitamin metabolism. 29 Cytochrome P450 (CYP) superfamily genes with gender differential expression were selected, and performed gene identification, phylogenetic, and functional analyses. The results indicated significant roles in multiple metabolic pathways, such as steroid hormone biosynthesis, ovarian steroidogenesis, cortisol synthesis and secretion, arachidonic acid metabolism, linoleic acid metabolism, and retinol metabolism. The findings provide insight into the molecular characteristics of physiological gender differences in sea cucumbers, and will help lay the foundation for the establishment of effective sea cucumber breeding technologies.

Brown Adipose Tissue Rescues Bone Loss Induced by Cold Exposure.

Cold temperature activates the sympathetic nervous system (SNS) to induce bone loss by altering bone remodeling. Brown adipose tissue (BAT) is influenced by the SNS in cold environments. Many studies have confirmed a positive relationship between BAT volume and bone mass, but the influence and mechanism of BAT on bone in vivo and in vitro is still unknown. Two-month-old C57/BL6j male mice were exposed to cold temperature (4°C) to induce BAT generation. BAT volume, bone remodeling and microstructure were assessed after 1 day, 14 days and 28 days of cold exposure. CTX-1, P1NP and IL-6 levels were detected in the serum by ELISA. To determine the effect of BAT on osteoclasts and osteoblasts in vitro, brown adipocyte conditional medium (BAT CM) was collected and added to the differentiation medium of bone marrow-derived macrophages (BMMs) and bone marrow mesenchymal stem cells (BMSCs). Micro-CT results showed that the bone volume fraction (BV/TV, %) significantly decreased after 14 days of exposure to cold temperature but recovered after 28 days. Double labeling and TRAP staining in vivo showed that bone remodeling was altered during cold exposure. BAT volume enlarged after 14 days of cold stimulation, and IL-6 increased. BAT CM promoted BMSC mineralization by increasing osteocalcin (Ocn), RUNX family transcription factor 2 (Runx2) and alkaline phosphatase (Alp) expression, while bone absorption was inhibited by BAT CM. In conclusion, restoration of bone volume after cold exposure may be attributed to enlarged BAT. BAT has a beneficial effect on bone mass by facilitating osteogenesis and suppressing osteoclastogenesis.

Analysis of Myeloid Cells in Mouse Tissues with Flow Cytometry.

Myeloid cells, including dendritic cells (DCs), granulocytes, monocytes, monocyte-derived cells and macrophages, are important players in the immune response, but their identification is not as clear as lymphocytes, especially in tissues. This protocol details the step-by-step procedure for the analysis of myeloid populations in various mouse tissues by flow cytometry. For complete details on the use and execution of this protocol, please refer to Liu et al. (2019).

Irisin inhibits osteocyte apoptosis by activating the Erk signaling pathway in vitro and attenuates ALCT-induced osteoarthritis in mice.

Moderate exercise can alleviate symptoms of osteoarthritis (OA) such as pain, stiffness, and joint deformities that are associated with progressive cartilaginous degeneration, osteophyte formation, subchondral bone changes, and synovial inflammation. Irisin is an exercise-related myokine that reportedly plays a crucial role in bone remodeling. However, its role in OA remains unknown. This study aimed to determine whether irisin can attenuate OA progression and the mechanism of its therapeutic effect. Three-month-old male C57BL/6J mice were randomized to groups that underwent sham operation, and anterior cruciate ligament transection (ACLT) intraperitoneally injected with vehicle or irisin in vivo. Apoptosis was induced by stretching murine osteocyte-like MLO-Y4 cells in vitro. Irisin reduced wear, maintained the proportion of hyaline cartilage, a more complete cartilage structure, and lower Osteoarthritis Research Society International (OARSI) scores at 4 weeks after ACLT. Irisin reduced the expression of matrix metalloproteinase (MMP)-13 in cartilage and caspase 3 in the subchondral bone. Irisin exerted rescue effects in microstructural parameters of subchondral trabecular bone including bone volume fraction (BV/TV), trabecular number (Tb.N), connection density (Conn. D), and the structure model index (SMI) compared with ACLT-vehicle group. Bone histomorphometry showed that irisin increased subchondral bone remodeling. The decreasing ratio (%) of the eroded surface (ES/BS) was reversed by irisin in the ACLT+vehicle group. Staining with tartrate-resistant acid phosphatase showed a decreased number of osteoclasts. Irisin significantly increased the proliferation of osteocytes, protected them from apoptosis, and maintained cellular activity by regulating the expression of Bax, Bcl-2, and osteoprotegerin/receptor activator of nuclear factor (NF)-kB-ligand (OPG/Rankl). Irisin activated serine/threonine-selective protein kinases (Erk) and p38 signaling, and its anti-apoptosis function depended on the Erk signaling pathway. Irisin attenuated OA progression by decreasing osteocyte apoptosis and improving the microarchitecture of subchondral bone. Activation of the Erk pathway by irisin plays an important role in reducing osteocyte apoptosis in vitro.

Chiral Ru(ii) complexes act as a potential non-viral gene carrier for directional transportation to the nucleus and cytoplasm.

Guanine-rich DNA sequences can spontaneously fold into four-stranded structures called G-quadruplexes (G4s). G4s have been identified extensively in the promoter regions of several proto-oncogenes, including c-myc, as well as telomeres. G4s have attracted an increasing amount of attention in the field of nanotechnology because of their use as versatile building blocks of DNA-based nanostructures. In this study, we report the self-assembly of c-myc G-quadruplex DNA controlled by a pair of chiral ruthenium(ii) complexes coordinated by 2-(4-phenyacetylenephenyl)-1H-imidazo[4,5f][1,10]phenanthroline (PBEPIP), Λ-[Ru(bpy)2(PBEPIP)](ClO4)2 (Λ-RM0627, bpy = bipyridine) and Δ-[Ru(bpy)2(PBEPIP)](ClO4)2 (Δ-RM0627). Λ-RM0627 could promote the high-order self-assembly of c-myc G-quadruplex DNA into a nanowire structure, whereas Δ-RM0627 could induce DNA condensation into G-quadruplex aggregates. Moreover, in vitro studies on human liver carcinoma HepG2 cells showed that the nanowire of c-myc G-quadruplex DNA promoted by Λ-RM0627 could be localized in the nuclei of cells, whereas the nanoparticle of c-myc G-quadruplex DNA generated by Δ-RM0627 was taken up and localized in the cytoplasm. This study provides examples of the enantioselective self-assembly of G4 DNA molecules controlled by chiral ruthenium(ii) complexes and suggests the potential applications of assembled nanostructures as non-viral DNA vectors for gene therapy.

PLK4 deubiquitination by Spata2-CYLD suppresses NEK7-mediated NLRP3 inflammasome activation at the centrosome.

The innate immune sensor NLRP3 assembles an inflammasome complex with NEK7 and ASC to activate caspase-1 and drive the maturation of proinflammatory cytokines IL-1ß and IL-18. NLRP3 inflammasome activity must be tightly controlled, as its over-activation is involved in the pathogenesis of inflammatory diseases. Here, we show that NLRP3 inflammasome activation is suppressed by a centrosomal protein Spata2. Spata2 deficiency enhances NLRP3 inflammasome activity both in the macrophages and in an animal model of peritonitis. Mechanistically, Spata2 recruits the deubiquitinase CYLD to the centrosome for deubiquitination of polo-like kinase 4 (PLK4), the master regulator of centrosome duplication. Deubiquitination of PLK4 facilitates its binding to and phosphorylation of NEK7 at Ser204. NEK7 phosphorylation in turn attenuates NEK7 and NLRP3 interaction, which is required for NLRP3 inflammasome activation. Pharmacological or shRNA-mediated inhibition of PLK4, or mutation of the NEK7 Ser204 phosphorylation site, augments NEK7 interaction with NLRP3 and causes increased NLRP3 inflammasome activation. Our study unravels a novel centrosomal regulatory pathway of inflammasome activation and may provide new therapeutic targets for the treatment of NLRP3-associated inflammatory diseases.