Activation of β-catenin signaling in aggrecan-expressing cells in temporomandibular joint causes osteoarthritis-like defects

β-Catenin plays a critical role in cartilage formation and development. To further understand the role of β-catenin in osteoarthritis（OA） development in temporomandibular joint（TMJ）, we have generated β-catenin conditional activation mice（β-cat（ex3）^Agc1CreER）by breeding Agc1-CreER mice with β-catenin^flox（ex3/＋）mice. Results of histologic analysis showed the progressive TMJ defects in 3-and 6-month-old β-cat（ex3）^Agc1CreERmice（tamoxifen induction was performed at 2 weeks of age）, including decreased chondrocyte numbers in the superficial layer associated with less Alcian blue staining, increased numbers of hypertrophic chondrocytes in deep layers, and rough articular surface. Compared to the TMJ phenotype of β-cat（ex3）^（Col2CreER）mice, β-cat（ex3）^（Agc1CreER）mice showed much severe morphological defects in the superficial layer of TMJ. This may reflect that Agc1-CreER mice could efficiently target cells in the superficial layer of TMJ. Results of immunostaining showed significantly increased expression of MMP13, Col-X, Adamts4,and Adamts5 in TMJ of β-cat（ex3）^（Agc1CreER）mice. Results of proliferating cell nuclear antigen（PCNA）, Ki67, and terminal deoxinucleotidyl transferase-mediated d UTP-fluorescein nick end labeling（TUNEL） staining further demonstrated that cell proliferation was decreased and cell apoptosis was increased in condylar cartilage of β-cat（ex3）^Agc1CreERmice. Our findings indicate that abnormal upregulation of β-catenin in TMJ leads to defects assembling to OA-like phenotype, further demonstrating that β-catenin plays a critical role in TMJ pathogenesis.

Development of a Wireless-Controlled LED Array for the Tunable Optogenetic Control of Cellular Activities

1.Introduction In order to decipher a complex biological process,tools are required to perturb the various players involved to gain information about the important parameters.Optogenetic modules are genetically encoded molecular reagents that,when expressed in cells,allow a specific biological process to be precisely controlled by light in a spatiotemporal manner[1].Optogenetics thus offers cell biologists an unprecedented new way to perturb cellular activities.The application of optogenetic approaches in cellular biology and synthetic biology research has evolved tremendously in the last few years[2–4].

Acute effects of Helicobacter pylori extracts on gastric mucosal blood flow in the mouse

AIM： To investigate the mechanisms underlying the reduction in gastric blood flow induced by a luminal water extract of Hellcobacter pylori （HPE）. METHODS： The stomachs of isoflurane-anesthetized mice were exteriorized, and the mucosal surface exposed. Blood flow was measured with the laserDoppler technique, and systemic arterial blood pressure monitored. C57BL/6 mice were exposed to water extract produced from Hpylori strain 88-23. To investigate the role of a nerveor iNOS-mediated pathway, we used intraluminal lidocaine and iNOS-/- mice. Blood flow response to the endogenous nitric oxide synthase inhibitor asymmetric dimethyl arginine （ADMA） was also assessed. RESULTS： In wild-type mice, HPE decreased mucosal blood flow by approximately 30%. This reduction was abolished in iNOS-deficient mice, and by pre-treatment with lidocaine. Luminally applied ADMA resulted in reduction in blood flow similar to that observed in wildtype mice exposed to HPE. CONCLUSION： A H py/ori water extract reduces gastric mucosal blood flow acutely through iNOS- and nerve-mediated pathways.

CHIP regulates bone mass by targeting multiple TRAF family members in bone marrow stromal cells

Carboxyl terminus of Hsp70-interacting protein（CHIP or STUB1） is an E3 ligase and regulates the stability of several proteins which are involved in different cellular functions. Our previous studies demonstrated that Chip deficient mice display bone loss phenotype due to increased osteoclast formation through enhancing TRAF6 activity in osteoclasts. In this study we provide novel evidence about the function of CHIP. We found that osteoblast differentiation and bone formation were also decreased in Chip KO mice. In bone marrow stromal（BMS） cells derived from Chip^-/- mice, expression of a panel of osteoblast marker genes was significantly decreased. ALP activity and mineralized bone matrix formation were also reduced in Chip-deficient BMS cells. We also found that in addition to the regulation of TRAF6, CHIP also inhibits TNFα-induced NF-κB signaling through promoting TRAF2 and TRAF5 degradation. Specific deletion of Chip in BMS cells downregulated expression of osteoblast marker genes which could be reversed by the addition of NF-κB inhibitor. These results demonstrate that the osteopenic phenotype observed in Chip^-/- mice was due to the combination of increased osteoclast formation and decreased osteoblast differentiation. Taken together, our findings indicate a significant role of CHIP in bone remodeling.

Association of gene and protein expression and genetic polymorphism of CC chemokine ligand 4 in colorectal cancer

BACKGROUND Leukocytes,such as T cells and macrophages,play an important role in tumorigenesis.CC chemokine ligand(CCL)4,which is produced by lymphocytes and macrophages,has been found to be expressed in the mucosa of the gastrointestinal tract and is a potent chemoattractant for various leukocytes.AIM To examine CCL4 expression and its genetic polymorphism rs10491121 in patients with colorectal cancer(CRC)and evaluate their prognostic significance.METHODS Luminex technology was used to determine CCL4 Levels in CRC tissue(n=98),compared with paired normal tissue,and in plasma from patients with CRC(n=103),compared with healthy controls(n=97).Included patients had undergone surgical resection for primary colorectal adenocarcinomas between 1996 and 2019 at the Department of Surgery,Ryhov County Hospital,Jönköping,Sweden.Reverse transcription quantitative PCR was used to investigate the CCL4 gene expression in CRC tissue(n=101).Paired normal tissue and TaqMan single nucleotide polymorphism assays were used for the CCL4 rs10491121 polymorphism in 610 CRC patients and 409 healthy controls.RESULTS The CCL4 protein and messenger RNA expression levels were higher in CRC tissue than in normal paired tissue(90%,P<0.001 and 45%,P<0.05,respectively).CRC tissue from patients with localized disease had 2.8-fold higher protein expression levels than that from patients with disseminated disease.Low CCL4 protein expression levels in CRC tissue were associated with a 30%lower cancer-specific survival rate in patients(P<0.01).The level of plasma CCL4 was 11%higher in CRC patients than in healthy controls(P<0.05)and was positively correlated(r=0.56,P<0.01)with the CCL4 protein level in CRC tissue.The analysis of CCL4 gene polymorphism rs10491121 showed a difference(P<0.05)between localized disease and disseminated disease in the right colon,with a dominance of allele A in localized disease.Moreover,the rate of the A allele was higher among CRC patients with mucinous cancer than among those with nonmucinous cancer.CONCLUSION The present study indicates that the CRC tissue levels of CCL4 and CCL4 gene polymorphism rs10491121,particularly in the right colon,are associated with clinical outcome in CRC patients.

Protective Effects of α-Lipoic Acid on Vascular Oxidative Stress in Rats with Hyperuricemia

The aim of the present study was to observe the protective effects of α-lipoic acid(ALA)on vascular injury in rats with hyperuricemia(HUA).The ALA treatment groups(10,30 and 90 mg/kg,respectively)were administered with ALA via gavage for 2 weeks.Subsequently,the levels of blood urea nitrogen(BUN),creatinine(CREA),uric acid(UA),total cholesterol(TC),high density lipoprotein-C(HDL-C)and low density lipoprotein-C(LDL-C)were measured;the activities of glutathione peroxidase(GSH-Px),catalase(CAT),malonaldehyde(MDA),superoxide dismutase(SOD)and xanthine oxidase(XOD)were also determined.The thoracic aorta of rats in each experimental group was observed under a light microscope;ultrastructural analysis was performed.SOD and CAT protein contents were investigated by Western blotting.The results revealed that:i)Compared with the model group,the levels of UA were decreased in the ALA groups and the levels of BUN,CREA,TC,and LDL-C decreased in the 30 and 90 mg/kg ALA groups(P<0.05);ii)compared with the model group,the activities of GSH-Px,SOD and XOD were increased and the levels of MDA were reduced in the 90 mg/kg ALA group (P<0.05);and iii)in the model and 10 mg/kg ALA groups,edema and shedding were observed in endothelial cells.Compared with the model and 10 mg/kg ALA groups,the 30 and 90 mg/kg ALA groups exhibited fewer swollen endothelial cells.In summary,the results of the present study indicated that HUA resulted in vascular oxidative stress injury and decreased the activity of antioxidative enzymes,which leads to endothelial cell damage and vascular lesions.ALA may serve as a therapeutic agent for the treatment of HUA-induced endothelial dysfunction.

Protective role of FoxO transcription factors against oxidative stress-induced chondrocyte dysfunction:a new therapeutic target for osteoarthritis

Chondrocyte dysfunction has been demonstrated to be a major inducer of osteoarthritis(OA).The pathological mechanism of chondrocyte dysfunction is definitely multifactoral,but oxidative stressis regarded as one of the leading causes of apoptosis,autophagy,senescence,and mitochondrial dysfunctionin chondrocytes.Strategies for arresting oxidative stress-induced chondrocyte dysfunction have been considered as potential therapeutic targets for OA.Recently,fork head box O(Fox O)transcription factors have been determined to play a protective role in chondrocytes through the regulation of autophagy and defense against oxidative stress;they also regulate growth,maturation,and matrix synthesis.To explore Fox O′s potential role in the treatment of OA,we first discussed the recent advances in the field of oxidative stress-induced chondrocyte dysfunction and then emphasized the protective role of fox otranscription factors as a potential molecular target for the treatment of OA.Understanding the function of fox otranscription factors will be important in designing next-generation therapies to prevent or reverse the development of OA.

Sex differences and effects of oestrogen in rat gastric mucosal defence

AIM To evaluate sex differences and the effects of oestrogen administration in rat gastric mucosal defence.METHODS Sex differences in gastric mucus thickness and accumulation rate, absolute gastric mucosal blood flow using microspheres, the integrity of the gastric mucosal epithelium in response to a chemical irritant and the effects of oestrogen administration on relative gastric mucosal blood flow in an acute setting was assessed in an in vivo rat experimental model. Subsequently, sex differences in the distribution of oestrogen receptors and calcitonin gene related peptide in the gastric mucosa of animals exposed to oestrogen in the above experiments was evaluated using immunohistochemistry.RESULTS The absolute blood flow in the GI-tract was generally higher in males, but only significantly different in the corpus part of the stomach (1.12 ± 0.12 m L/min·g in males and 0.51 ± 0.03 m L/min·g in females) (P = 0.002). After removal of the loosely adherent mucus layer the thickness of the firmly adherent mucus layer in males and females was 79 ± 1 μm and 80 ± 3 μm respectively. After 60 min the mucus thickness increased to 113 ± 3 μm in males and 121 ± 3 μm in females with no statistically significant difference seen between the sexes. Following oestrogen administration(0.1 followed by 1 μg/kg·min), mean blood flow in the gastric mucosa decreased by 31% [68 ± 13 perfusion units (PFU)] in males which was significantly different compared to baseline(P = 0.02). In females however, mean blood flow remained largely unchanged with a 4% (5 ± 33 PFU) reduction. The permeability of the gastric mucosa increased to a higher level in females than in males (P = 0.01) after taurocholate challenge. However, the calculated mean clearance increase did not significantly differ between the sexes [0.1 ± 0.04 to 1.1 ± 0.1 m L/min·100 g in males and 0.4 ± 0.3 to 2.1 ± 0.3 m L/min·100 g in females(P = 0.065)]. There were no significant differences between 17β-Estradiol treated males (mean ratio of positive staining ± SEM)(0.06 ± 0.07) and females(0.11 ± 0.11) in the staining of ERα (P = 0.24). Also, there were no significant differences between 17β-Estradiol treated males (0.18 ± 0.21) and females (0.06 ± 0.12) in the staining of ERβ (P = 0.11). Finally, there were no significant differences between 17β-Estradiol treated males (0.04 ± 0.05) and females (0.11 ± 0.10) in the staining of CGRP(P = 0.14).CONCLUSION Gastric mucosal blood flow is higher in male than in female rats and is reduced in male rats by oestrogen administration.

Effects of diabetes type 2 and metformin treatment in Swedish patients with colorectal cancer

The association between type 2 diabetes mellitus(DM)and colorectal cancer(CRC)has been thoroughly investigated and reports have demonstrated that the risk of CRC is increased in DM patients.The association between DM and the survival of patients with CRC is controversial.Evidence suggests that metformin with its anti-inflammatory effects is a protective factor against the development of CRC among DM patients and that metformin therapy is associated with a better prognosis in patients with DM.In our cohort,we did not find any associations between the presence of DM or metformin and cancer specific survival or any relation to plasma levels of a panel of 40 inflammatory factors and irisin.On the other hand,we identified that the insulin-like growth factor binding protein 7 single nucleotide polymorphism rs2041437 was associated with DM in CRC patients.The dominance of the T bearing genotypes in patients with DM was statistically significant(P=0.038),with an odds ratio of 1.66(95%confidence interval:1.03-2.69).

A single-cell transcriptome of mesenchymal stromal cells to fabricate bioactive hydroxyapatite materials for bone regeneration

The osteogenic microenvironment of bone-repairing materials plays a key role in accelerating bone regeneration but remains incompletely defined,which significantly limits the application of such bioactive materials.Here,the transcriptional landscapes of different osteogenic microenvironments,including three-dimensional(3D)hydroxyapatite(HA)scaffolds and osteogenic medium(OM),for mesenchymal stromal cells(MSCs)in vitro were mapped at single-cell resolution.Our findings suggested that an osteogenic process reminiscent of endochondral ossification occurred in HA scaffolds through sequential activation of osteogenic-related signaling pathways,along with inflammation and angiogenesis,but inhibition of adipogenesis and fibrosis.Moreover,we revealed the mechanism during OM-mediated osteogenesis involves the ZBTB16 and WNT signaling pathways.Heterogeneity of MSCs was also demonstrated.In vitro ossification of LRRC75A+MSCs was shown to have better utilization of WNT-related ossification process,and PCDH10+MSCs with superiority in hydroxyapatite-related osteogenic process.These findings provided further understanding of the cellular activity modulated by OM conditions and HA scaffolds,providing new insights for the improvement of osteogenic biomaterials.This atlas provides a blueprint for research on MSC heterogeneity and the osteogenic microenvironment of HA scaffolds and a database reference for the application of bioactive materials for bone regeneration.

Function-oriented design:A novel strategy for advanced biomedical materials

It has always been a dream to construct tissues and even organs for transplantation to replace those with defects caused by diseases or injuries.Tissue engineering is another milestone in the developmental history of life science after cellular and molecular bioscience.Nevertheless,despite decades of rapid de-velopment,tissue-engineered biomaterials have not been widely used clinically.Biomaterials constructed by physical and chemical methods have lots of difficulty in precisely mimicking the macroscopic and mi-croscopic structures of human tissues.The ultimate way to build organoid tissue for regeneration is to enable the cells to take the initiative and build suitable functions.Based on the thoughts of tissue engi-neering,organoid technology holds great potential as a research tool for a wide range of fields,including developmental biology,disease pathology,cell biology,precision medicine,and drug toxicity and efficacy testing.This technology also holds tremendous potential for regenerative medicine,as organoids present the possibility for autologous and allogeneic cell therapy through the replacement of damaged or dis-eased tissues with organoid-propagated tissue or stem cell populations.In this review work,we briefly outlook the development history of organoid technology,summarize the current bottlenecks and the un-derlying reasons,and propose the unified term“function-oriented design in tissue engineering”,a new topic that may provide a solution to overcome these bottlenecks.

SPECT imaging of distribution and retention of a brain-penetrating bispecific amyloid-β antibody in a mouse model of Alzheimer's disease

Background:Alzheimer's disease(AD)immunotherapy with antibodies targeting amyloid-B(AB)has been extensively explored in clinical trials.The aim of this study was to study the long-term brain distribution of two radiolabeled monoclonal Aβ antibody variants-RmAb158,the recombinant murine version of BAN2401,which has recently demonstrated amyloid removal and reduced cognitive decline in AD patients,and the bispecific RmAb158-scFv8D3,which has been engineered for enhanced brain uptake via transferrin receptor-mediated transcytosis.Methods:A single intravenous injection of iodine-125(251)-labeled RmAb158-scFv8D3 or RmAb158 was administered to AD transgenic mice(tg-ArcSwe).In vivo single photon emission computed tomography was used to investigate brain retention and intrabrain distribution of the antibodies over a period of 4 weeks.Activity in blood and brain tissue was measured ex vivo and autoradiography was performed in combination with Aβand CD31 immunostaining to investigate the intrabrain distribution of the antibodies and their interactions with AB.Results:Despite faster blood clearance,[125]RmAb158-scFv8D3 displayed higher brain exposure than[25]RmAb158 throughout the study.The brain distribution of[l25]RmAb158-scFv8D3 was more uniform and coincided with parenchymal Aβ pathology,while[2 I]RmAb158 displayed a more scattered distribution pattern and accumulated in central parts of the brain at later times.Ex vivo autoradiography indicated greater vascular escape and parenchymal Aβ interactions for[25]RmAb158-scFv8D3,whereas[25]RmAb158 displayed retention and Aβ interactions in lateral ventricles.Conclusions:The high brain uptake and uniform intrabrain distribution of RmAb158-scFv8D3 highlight the benefits of receptor-mediated transcytosis for antibody-based brain imaging.Moreover,it suggests that the alternative transport route of the bispecific antibody contributes to improved efficacy of brain-directed immunotherapy.

Expression of leukotriene B4 receptor 1 defines functionally distinct DCs that control allergic skin inflammation

Leukotriene B4(LTB4)receptor 1(BLT1)is a chemotactic G protein-coupled receptor expressed by leukocytes,such as granulocytes,macrophages,and activated T cells.Although there is growing evidence that BLT1 plays crucial roles in immune responses,its role in dendritic cells remains largely unknown.Here,we identified novel DC subsets defined by the expression of BLT1,namely,BLT1hi and BLT1lo DCs.We also found that BLT1hi and BLT1lo DCs differentially migrated toward LTB4 and CCL21,a lymph node-homing chemoattractant,respectively.By generating LTB4-producing enzyme LTA4H knockout mice and CD11c promoter-driven Cre recombinase-expressing BLT1 conditional knockout(BLT1 cKO)mice,we showed that the migration of BLT1hi DCs exacerbated allergic contact dermatitis.Comprehensive transcriptome analysis revealed that BLT1hi DCs preferentially induced Th1 differentiation by upregulating IL-12p35 expression,whereas BLT1lo DCs accelerated T cell proliferation by producing IL-2.Collectively,the data reveal an unexpected role for BLT1 as a novel DC subset marker and provide novel insights into the role of the LTB4-BLT1 axis in the spatiotemporal regulation of distinct DC subsets.