Wnt/β-catenin signaling components and mechanisms in bone formation,homeostasis,and disease

Wnts are secreted,lipid-modified proteins that bind to different receptors on the cell surface to activate canonical or non-canonical Wnt signaling pathways,which control various biological processes throughout embryonic development and adult life.Aberrant Wnt signaling pathway underlies a wide range of human disease pathogeneses.

Atp6i deficient mouse model uncovers transforming growth factor-β1/Smad2/3 as a key signaling pathway regulating odontoblast differentiation and tooth root formation

The biomolecular mechanisms that regulate tooth root development and odontoblast differentiation are poorly understood.We found that Atp6i deficient mice(Atp6i^(−/−))arrested tooth root formation,indicated by truncated Hertwig’s epithelial root sheath(HERS)progression.Furthermore,Atp6i deficiency significantly reduced the proliferation and differentiation of radicular odontogenic cells responsible for root formation.Atp6i^(−/−)mice had largely decreased expression of odontoblast differentiation marker gene expression profiles(Col1a1,Nfic,Dspp,and Osx)in the alveolar bone.Atp6i^(−/−)mice sample RNA-seq analysis results showed decreased expression levels of odontoblast markers.Additionally,there was a significant reduction in Smad2/3 activation,inhibiting transforming growth factor-β(TGF-β)signaling in Atp6i^(−/−)odontoblasts.Through treating pulp precursor cells with Atp6i^(−/−)or wild-type OC bone resorption-conditioned medium,we found the latter medium to promote odontoblast differentiation,as shown by increased odontoblast differentiation marker genes expression(Nfic,Dspp,Osx,and Runx2).This increased expression was significantly blocked by anti-TGF-β1 antibody neutralization,whereas odontoblast differentiation and Smad2/3 activation were significantly attenuated by Atp6i^(−/−)OC conditioned medium.Importantly,ectopic TGF-β1 partially rescued root development and root dentin deposition of Atp6i^(−/−)mice tooth germs were transplanted under mouse kidney capsules.Collectively,our novel data shows that the prevention of TGF-β1 release from the alveolar bone matrix due to OC dysfunction may lead to osteopetrosis-associated root formation via impaired radicular odontoblast differentiation.As such,this study uncovers TGF-β1/Smad2/3 as a key signaling pathway regulating odontoblast differentiation and tooth root formation and may contribute to future therapeutic approaches to tooth root regeneration.

TGF-β and BMP signaling in osteoblast,skeletal development,and bone formation,homeostasis and disease

INTRODUCTIONThe transforming growth factor-β （TGF-β） superfamily com- prises TGF-βs, Activin, bone morphogenetic proteins （BMPs） and other related proteins. TGF-β superfamily members act through a heteromeric receptor complex,, comprised of type I and type II receptors at the cell surface that transduce intracellular signals via Smad complex or mitogen-activated protein kinase （MAPK） cascade.

Runx1 is a key regulator of articular cartilage homeostasis by orchestrating YAP,TGFβ,and Wnt signaling in articular cartilage formation and osteoarthritis

Runt-related transcription factor 1(Runx1)plays a key role in cartilage formation,but its function in articular cartilage formation is unclear.We generated non-inducible and inducible Runx1-deficient mice(Runx1^(f/f)Col2α1-Cre and Runx1^(f/f)Col2α1-CreER mice)and found that chondrocyte-specific Runx1-deficient mice developed a spontaneous osteoarthritis(OA)-like phenotype and showed exacerbated articular cartilage destruction under OA,characterized by articular cartilage degradation and cartilage ossification,with decreased Col2α1 expression and increased Mmp13 and Adamts5 expression.RNA-sequencing analysis of hip articular cartilage from the Runx1^(f/f)Col2α1-Cre mice compared to that from wild-type mice and subsequent validation analyses demonstrated that Runx1 is a central regulator in multiple signaling pathways,converging signals of the Hippo/Yap,TGFβ/Smad,and Wnt/β-catenin pathways into a complex network to regulate the expression of downstream genes,thereby controlling a series of osteoarthritic pathological processes.RNA-sequencing analysis of mutant knee joints showed that Runx1’s role in signaling pathways in articular cartilage is different from that in whole knee joints,indicating that Runx1 regulation is tissue-specific.Histopathologic analysis confirmed that Runx1 deficiency decreased the levels of YAP and p-Smad2/3 and increased the levels of activeβ-catenin.Overexpression of Runx1 dramatically increased YAP expression in chondrocytes.Adeno-associated virus-mediated Runx1 overexpression in the knee joints of osteoarthritic mice showed the protective effect of Runx1 on articular cartilage damaged in OA.Our results notably showed that Runx1 is a central regulator of articular cartilage homeostasis by orchestrating the YAP,TGFβ,and Wnt signaling pathways in the formation of articular cartilage and OA,and targeting Runx1 and its downstream genes may facilitate the design of novel therapeutic approaches for OA.

Elicitation of strong immune responses by a DNA vaccine expressing a secreted form of hepatitis C virus envelope protein E2 in murine and porcine animal models

AIM： To characterize the immunogenicity of a hepatitis C virus （HCV） E2 DNA vaccine alone or with a protein vaccine boost in murine and porcine animal models. METHODS： A DNA vaccine expressing a secreted form of HCV E2 protein was constructed and used to vaccinate mice and piglets with or without boosting with a recombinant E2 protein vaccine formulated with CpG ODN and 10% Emulsigen. The immunogenicity of HCV E2 vaccines was analyzed by ELISA for antibody responses, MTT assay for lymphocyte proliferation, ELISPOT for the number of interferon-γ secreting cells, and cytotoxic T lymphocyte assays. RESULTS： Intradermal injection of E2 DNA vaccine induced strong Th1-1ike immune responses in mice. In piglets, E2 DNA vaccine elicited moderate and more balanced immune responses. A DNA vaccine prime and protein boost vaccination strategy induced significantly higher E2-specific antibody levels and shifted the immune response towards Th2-1ike ones in piglets. CONCLUSION： A DNA vaccine expressing a secreted form of HCV E2 protein elicited E2-specific immune responses in mice and piglets. Recombinant E2 protein vaccination following DNA immunization significantly increased the antibody response in piglets. These HCV E2 vaccines may represent promising hepatitis C vaccine candidates for further investigations.

A hiddenly high hepatitis C virus related liver disease burden among Chinese patients with non-liver disease complaints: A hospital based study from 2013 to 2017

Objective: To determine the frequency of anti-HCV antibody positivity in patients with nonliver disease complaints, to explore whether anti-HCV positive patients had been properly advised and visited hepatologists for further assessments, and to investigate their clinical characteristics as well as the HCV treatment status.Methods: A hospital based survey of nonliver disease patients with anti-HCV positive and their attending physicians was conducted to determine: 1.were the patients adequately advised of the implication of anti-HCV positive finding; 2.to what extent the patients were aware of potential chronic liver disease associated with HCV infection and whether they sought for further assessments and care of hepatologists.Results: A total of 295 294 non-liver disease patients were tested for anti-HCV antibody, and 2 778 of them were found to be positive(0.94%).However, only 45.10%(1 253/2 778) of the anti-HCV antibody(+) patients were referred to hepatologists and received HCV RNA test.In addition, 34.10%(312/915) and 1.42%(13/915) of them had already advanced to cirrhosis and hepatocellular carcinoma(HCC), respectively.Further analysis showed that the patients who declined antiviral therapy were older, with lower education and lower income, possessed poorer knowledge on the risk of chronic hepatitis C, and had more severe liver diseases.Surprisingly, 65% of the surveyed physicians did not know the genotype-guided treatment duration suggested by the guidelines.Alarmingly, 22% of the surveyed physicians did not know the standard assays for the diagnosis of HCV infection.Conclusions: Our findings highlight the challenge and hidden enormous burden of chronic HCV infection among patients with non-liver disease complaints in China.

Heterologous Expression of Rat Testis GABA_A Receptor β3t Splicing Variant in CHO Cells

Objective To characterize a possible retention function of unique sequence in the 5＇end of rat testis GABAA receptor β3t splicing variant Methods Rat testis GABAA receptor β3t splicing variant cDNA was cloned and two eukaryotic expression recombinant plasmids of pEGFP-N1 and pEGFP-C1 were constructed respectively by fusing green fluorescent protein to the N or C-terminus of β3t isoform. The recombinant plasmids were transfected into CHO cells by calcium phosphate co-precipitation method Fluorescence microscope and laser confocal microscope were used to analyze localization of β3t in the transfected cells. ConA-Texas-Red was used to label cell ER and the localization of rat testis β3t splicing variant in CHO cells was determined. Results When rat testis β3t splicing variant was expressed in CHO cells, two expression patterns were delineated, the distributions of uniform and mainly discrete intracellular compartments respectively, The chimera product failed to be translocated into the cell surface when expressed in ClIO cells; whereas the β3 subunit of rat brain was incorporated into the plasma membrane. Conclusion The inability of β3t to target into the ER may be a consequence of the unique 25 specific amino acid segments in the N terminus.

Simulation of internal nitrogen release from bottom sediments in an urban lake using a nitrogen flux model

Nutrient release from sediment is considered a significant source for overlying water. Given that nutrient release mechanisms in sediment are complex and difficult to simulate, traditional approaches commonly use assigned parameter values to simulate these processes. In this study, a nitrogen flux model was developed and coupled with the water quality model of an urban lake. After parameter sensitivity analyses and model calibration and validation, this model was used to simulate nitrogen exchange at the sediment–water interface in eight scenarios. The results showed that sediment acted as a buffer in the sediment–water system. It could store or release nitrogen at any time, regulate the distribution of nitrogen between sediment and the water column, and provide algae with nitrogen. The most effective way to reduce nitrogen levels in urban lakes within a short time is to reduce external nitrogen loadings. However, sediment release might continue to contribute to the water column until a new balance is achieved. Therefore, effective measures for reducing sediment nitrogen should be developed as supplementary measures. Furthermore, model parameter sensitivity should be individually examined for different research subjects.

三种不同手术对屈光不正患者术后视觉质量及高阶像差的比较

目的:探讨飞秒激光制瓣的准分子激光原位角膜磨镶术(FS-LASIK)、角膜地形图引导LASIK(TG-LASIK)和飞秒激光小切口角膜基质透镜取出术(SMILE)对屈光不正患者术后视觉质量及高阶像差的效果。方法:纳入2019-12/2020-02本院屈光中心收治的296例586眼单纯近视或近视散光患者。在明确手术适应证的前提下,患者自行选择接受FS-LASIK、TG-LASIK、SMILE手术,其中FS-LASIK组95例189眼,TG-LASIK组104例205眼,SMILE组97例192眼。收集患者术前及术后6、12mo时视力、屈光度、角膜地形图、高阶像差及对比敏感度。结果:术后6、12mo各组患者裸眼视力、最佳矫正视力及球镜度数、柱镜度数、等效球镜度数均优于术前(P<0.05),组间比较均无差异(P>0.05)。术后6、12mo TG-LASIK组角膜表面规则指数、表面非对称指数均显著低于SMILE组、FS-LASIK组(P<0.05);SMILE组、FS-LASIK组组间比较无差异(P>0.05)。术后6、12mo时FS-LASIK组总高阶像差、彗差、球差及术后6mo时三叶草像差高于TG-LASIK组、SMILE组(P<0.05);术后6、12mo时SMILE组球差显著低于其他两组(P<0.05)。术后6、12mo时TG-LASIK组在无眩光及有眩光时12.0、18.0c/d频率下对比敏感度显著高于SMILE组、FS-LASIK组(P<0.05)。结论:FS-LASIK、TG-LASIK、SMILE三种手术均可获得较为满意的治疗效果,但TG-LASIK术后像差增加较小,角膜形态更规则,视觉质量更高。

Statistical description of depth-dependent turbulent velocity measured in Taihu Lake, China

Quantitative description of turbulence using simple physical/mathematical models remains a challenge in classical physics and hydrologic dynamics. This study monitored the turbulence velocity field at the surface and bottom of Taihu Lake, in China, a large shallow lake with a heterogeneous complex system, and conducted a statistical analysis of the data for the local turbulent structure. Results show that the measured turbulent flows with finite Reynolds numbers exhibit properties of non-Gaussian distribution. Compared with the normal distribution, the Levy distribution with meaningful parameters can better characterize the tailing behavior of the measured turbulence. Exit-distance statistics and multiscaling extended self-similarity(ESS) were used to interpret turbulence dynamics with different scale structures. Results show that the probability density function of the reverse structure distance and the multiscaling ESS can effectively capture the turbulent flow dynamics varying with water depth. These results provide an approach for quantitatively analyzing multiscale turbulence in large natural lakes.

Inhibition of Rgs10 Expression Prevents Immune Cell Infiltration in Bacteria-induced Inflammatory Lesions and Osteoclast-mediated Bone Destruction

Regulator of G-protein Signaling 10 （Rgsl0） plays an important function in osteoclast differentiation. However, the role of Rgsl0 in immune cells and inflammatory responses, which activate osteoclasts in inflam- matory lesions, such as bacteria-induced periodontal disease lesions, remains largely unknown. In this study, we used an adeno-associated virus （AAV-） mediated RNAi （AAV-shRNA-Rgs10） knockdown approach to study Rgsl0＇s function in immune cells and osteoclasts in bacteria-induced inflammatory lesions in a mouse model of periodontal disease. We found that AAV-shRNA-Rgs10 mediated Rgs10 knockdown impaired osteoclastogenesis and osteoclast-mediated bone resorption, in vitro and in vivo. Interestingly, local injection of AAV-shRNA-Rgs10 into the periodontal tissues in the bacteria-induced inflammatory lesion greatly decreased the number of dendritic cells, T-cells and osteoclasts, and protected the periodontal tissues from local inflammatory damage and bone destruction. Importantly, AAV-mediated Rgs10 knockdown also reduced local expression of osteoclast markers and pro-inflammatory cytokines. Our results demonstrate that AAV- shRNA-Rgs10 knockdown in periodontal disease tissues can prevent bone resorption and inflammation simultaneously. Our data indicate that Rgsl0 may regulate dendritic cell proliferation and maturation, as well as the subsequent stimulation of T-cell proliferation and maturation, and osteoclast differentiation and acti- vation. Our study suggests that AAV-shRNA-Rgs10 can be useful as a therapeutic treatment of periodontal disease.

Identification of broad neutralizing antibodies against Omicron subvariants from COVID-19 convalescents and vaccine recipients

Dear Editor,Omicron(B.1.1.529)was designated a variant of concern(VOC)on November 26,2021(Callaway,2021),and its subvariants BA.1,BA.2,and BA.3 emerged and circulated almost simultaneously(Desingu et al.,2022).BA.2 was more efficient in transmission and quickly overtook BA.1 to become the variant most frequently detected worldwide(Yamasoba et al.,2022a).Compared to the prototype SARS-CoV-2 spike protein(S),the BA.1 and BA.2 spike proteins harbor more than 30 mutations,of which 21 are identical between the two subvariants,while the BA.3 spike differs from BA.1 and BA.2 by 3 mutations in the receptor binding domain(RBD)(Fig.1A).More recently,BA.4 and BA.5(hereafter BA.4/5)emerged,sharing the same spike sequence and containing four additional mutations,Del69–70,L452R,F486V,and R493Q,compared with BA.2.BA.4/5 were detected first in South Africa and evolved independently of BA.2;they have spread widely and replaced BA.2 as the predominant VOC(Gruell et al.,2022b;Tegally et al.,2022).In addition,BA.2.75,derived from the BA.2 subvariant,harbors nine additional mutations in the spike protein compared with BA.2(Fig.1A).BA.4/5 and BA.2.75 have led to the continuous emergence of novel Omicron subvariants,including BF.7 and BQ.1.These new subvariants may be driving waves of pandemics.

Isolation and characterization of spike S2-specific monoclonal antibodies with reactivity to pan-coronaviruses

Dear Editor,Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),theseventh coronavirus known to jump from intermediate hosts to humans,has resulted in a worldwide pandemic and caused immense economicdamage(Tan et al.,2020;Zhu et al.,2020).It is believed that coronavirus spillover may occur again in the future.Human exposure to a bat coro-navirus has been serologically confimed in a rural area of China,indi-cating that the coronavirus spillover occurred,although the origin andfrequency of spillovers are unclear(Wang et al.,2018).

Terrain matching localization for hybrid underwater vehicle in the Challenger Deep of the Mariana Trench

The maximum ocean depth so far reported is about 11000 m,and is located in the Mariana Trench in the Western Pacific Ocean.The hybrid unmanned underwater vehicle,Haidou,is developed to perform scientific survey at the deepest parts of the Earth oceans.For vehicles working at the full-ocean depth,acoustic positioning is the most effective and popular method.The 11000 m class acoustic positioning system is relatively massive and complex,and it requires specialized research vessels equipped with compatible acoustic instruments.As a compact testbed platform,it is impractical for Haidou to carry an LBL/USBL beacon with its large volume and weight.During the descent to about 11000 m,horizontal drift could not be eliminated because of the hydrodynamics and uncertain ocean currents in the sea trials.The maximum depth recorded by Haidou is 10905 m,and determining the precise location of the deepest point is challenging.With the bathymetric map produced by a multibeam sonar,the terrain contour matching(TERCOM)method is adopted for terrain matching localization.TERCOM is stable in providing an accurate position because of its insensitivity to the initial position errors.The final matching results show the best estimate of location in the reference terrain map.

Mechanistic target of rapamycin kinase(Mtor)is required for spermatogonial proliferation and differentiation in mice

Spermatogonial development is a vital prerequisite for spermatogenesis and male fertility.However,the exact mechanisms underlying the behavior of spermatogonia,including spermatogonial stem cell(SSC)self-renewal and spermatogonial proliferation and differentiation,are not fully understood.Recent studies demonstrated that the mTOR complex 1(mTORC1)signaling pathway plays a crucial role in spermatogonial development,but whether MTOR itself was also involved in any specific process of spermatogonial development remained undetermined.In this study,we specifically deleted Mtor in male germ cells of mice using Stra8-Cre and assessed its effect on the function of spermatogonia.The Mtor knockout(KO)mice exhibited an age-dependent perturbation of testicular development and progressively lost germ cells and fertility with age.These age-related phenotypes were likely caused by a delayed initiation of Mtor deletion driven by Stra8-Cre.Further examination revealed a reduction of differentiating spermatogonia in Mtor KO mice,suggesting that spermatogonial differentiation was inhibited.Spermatogonial proliferation was also impaired in Mtor KO mice,leading to a diminished spermatogonial pool and total germ cell population.Our results show that MTOR plays a pivotal role in male fertility and is required for spermatogonial proliferation and differentiation.

Prmt7 regulates epiboly by facilitating 2-OST and modulating actin cytoskeleton

Dear Editor,Epiboly,the thinning and spreading of blastoderm over the yolk cells,is the first morphogenetic movement in zebrafish.Studies of zebrafish epiboly have provided insights into basic cellular properties and mechanisms of morphogenesis that are widely used in animal development(Lepage and Bruce,2010).

SARS-CoV-2 spike-specific TFH cells exhibit unique responses in infected and vaccinated individuals

Long-term humoral immunity to SARS-CoV-2 is essential for preventing reinfection. The production of neutralizing antibody (nAb)and B cell differentiation are tightly regulated by T follicular help (T_(FH)) cells. However, the longevity and functional role of T_(FH) cellsubsets in COVID-19 convalescents and vaccine recipients remain poorly defined. Here, we show that SARS-CoV-2 infection andinactivated vaccine elicited both spike-specific CXCR3^(+) T_(FH) cell and CXCR3^(-) T_(FH) cell responses, which showed distinct responsepatterns. Spike- specific CXCR3^(+) T_(FH) cells exhibit a dominant and more durable response than CXCR3^(-) T_(FH) cells that positivelycorrelated with antibody responses. A third booster dose preferentially expands the spike-specific CXCR3^(+) T_(FH) cell subset inducedby two doses of inactivated vaccine, contributing to antibody maturation and potency. Functionally, spike-specific CXCR3^(+) T_(FH) cellshave a greater ability to induce spike-specific antibody secreting cells (ASCs) differentiation compared to spike-specific CXCR3^(-) T_(FH)cells. In conclusion, the persistent and functional role of spike-specific CXCR3^(+) T_(FH) cells following SARS-CoV-2 infection andvaccination may play an important role in antibody maintenance and recall response, thereby conferring long-term protection. Thefindings from this study will inform the development of SARS-CoV-2 vaccines aiming to induce long-term protective immunememory.