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Aims: To investigate the molecular mechanism of osteoclast-derived exosomes in osteoporosis. Main methods: RANKL induced osteoclast model was screened for significantly differentially expressed lncRNAs and mRNAs by whole RNA sequencing. Exosomes were characterized using electron microscopy, western blotting and nanosight. Overexpression or knockdown of AW011738 was performed to explore its function. The degree of osteoporosis in an osteoporosis model was assessed by mirco-CT. The osteoclast model, osteoblast differentiation ability and the molecular mechanism of lncRNA AW011738/miR-24-2-5p/TREM1 axis in osteoporosis were assessed by dual luciferase reporter gene assay, Western blotting (WB), immunofluorescence and ALP staining. Bioinformatics was used to predict interactions of key osteoporosis-related genes with miRNAs, transcription factors, and potential drugs after upregulation of AW011738. To predict the protein-protein interaction (PPI) network associated with key genes, GO and KEGG analyses were performed on the key genes. The ssGSVA was used to predict changes in the immune microenvironment. Key findings: Osteoclast-derived exosomes containing lncRNA AW011738 decreased the osteogenesis-related markers and accelerated bone loss in OVX mice. Osteoclast (si-AW011738)-derived exosomes showed a significant increase in biomarkers of osteoblast differentiation in vitro compared to the si-NC group. As analyzed by mirco-CT, tail vein injected si-AW011738 OVX mice were less osteoporotic than the control group. AW011738 inhibited osteoblast differentiation by regulating TREM1 expression through microRNA. Meanwhile, overexpression of miR-24-2-5p inhibited TREM1 expression to promote osteoblast differentiation. Significance: Osteoclast-derived exosomes containing lncRNA AW011738 inhibit osteogenesis in MC3T3-E1 cells through the lncRNA AW011738/miR-24-2-5p/TREM1 axis and exacerbate osteoporosis in OVX mice.

The inflammatory microenvironment and neurotoxicity can hinder neuronal regeneration and functional recovery after spinal cord injury. Ruxolitinib, a JAK-STAT inhibitor, exhibits effectiveness in autoimmune diseases, arthritis, and managing inflammatory cytokine storms. Although studies have shown the neuroprotective potential of ruxolitinib in neurological trauma, the exact mechanism by which it enhances functional recovery after spinal cord injury, particularly its effect on astrocytes, remains unclear. To address this gap, we established a mouse model of T10 spinal cord contusion and found that ruxolitinib effectively improved hindlimb motor function and reduced the area of spinal cord injury. Transcriptome sequencing analysis showed that ruxolitinib alleviated inflammation and immune response after spinal cord injury, restored EAAT2 expression, reduced glutamate levels, and alleviated excitatory toxicity. Furthermore, ruxolitinib inhibited the phosphorylation of JAK2 and STAT3 in the injured spinal cord and decreased the phosphorylation level of nuclear factor kappa-B and the expression of inflammatory factors interleukin-1β, interleukin-6, and tumor necrosis factor-α. Additionally, in glutamate-induced excitotoxicity astrocytes, ruxolitinib restored EAAT2 expression and increased glutamate uptake by inhibiting the activation of STAT3, thereby reducing glutamate-induced neurotoxicity, calcium influx, oxidative stress, and cell apoptosis, and increasing the complexity of dendritic branching. Collectively, these results indicate that ruxolitinib restores glutamate homeostasis by rescuing the expression of EAAT2 in astrocytes, reduces neurotoxicity, and effectively alleviates inflammatory and immune responses after spinal cord injury, thereby promoting functional recovery after spinal cord injury.

Abstract Background An increasing amount of research has speculated that necroptosis could be a therapeutic strategy for treating cancer. However, understanding the prognostic value of the necroptosis-related long non-coding RNAs (NRLs) in skin cutaneous melanoma (SKCM, hereafter referred to as melanoma) remains poor and needs to be developed. Our research aims to construct a model based on NRLs for the prognosis of patients with melanoma. Methods We obtained the RNA-seq and clinical data from The Cancer Genome Atlas (TCGA) database and retrieved 86 necroptosis-related genes from the GeneCards database. The lncRNAs associated with necroptosis were identified via the Pearson correlation coefficient, and the prognostic model of melanoma was constructed using LASSO regression. Next, we employed multiple approaches to verify the accuracy of the model. Melanoma patients were categorized into two groups (high-risk and low-risk) according to the results of LASSO regression. The relationships between the risk score and survival status, clinicopathological correlation, functional enrichment, immune infiltration, somatic mutation, and drug sensitivity were further investigated. Finally, the functions of AL162457.2 on melanoma proliferation, invasion, and migration were validated by in vitro experiments. Results The prognostic model consists of seven NRLs (EBLN3P, AC093010.2, LINC01871, IRF2-DT, AL162457.2, AC242842.1, HLA-DQB1-AS1) and shows high diagnostic efficiency. Overall survival in the high-risk group was significantly lower than in the low-risk group, and risk scores could be used to predict melanoma survival outcomes independently. Significant differences were evident between risk groups regarding the expression of immune checkpoint genes, immune infiltration, immunotherapeutic response and drug sensitivity analysis. A series of functional cell assays indicated that silencing AL162457.2 significantly inhibited cell proliferation, invasion, and migration in A375 cells. Conclusion Our prognostic model can independently predict the survival of melanoma patients while providing a basis for the subsequent investigation of necroptosis in melanoma and a new perspective on the clinical diagnosis and treatment of melanoma.

Vaccination by inactivated vaccine is an effective strategy to prevent the COVID-19 pandemic. However, the detailed molecular immune response at single-cell level is poorly understood. In this study, we systematically delineated the landscape of the pre- and post-vaccination single-cell transcriptome, TCR (T cell antigen receptor) and BCR (B cell antigen receptor) expression profile of vaccinated candidates. The bulk TCR sequencing analysis of COVID-19 patients was also performed. Enrichment of a clonal CD8+ T cell cluster expressing specific TCR was identified in both vaccination candidates and COVID-19 patients. These clonal CD8+ T cells showed high expression of cytotoxicity, phagosome and antigen presentation related genes. The cell–cell interaction analysis revealed that monocytes and dendritic cells could interact with these cells and initiate phagocytosis via ICAM1-ITGAM and ITGB2 signaling. Together, our study systematically deciphered the detailed immunological response during SARS-CoV-2 vaccination and infection. It may facilitate understanding the immune response and the T-cell therapy against COVID-19.

In the present study, effect of ultrasonic impact treatment (UIT) on the microstructural characterization and mechanical properties of 316L stainless steel (hereinafter referred to as 316L) was investigated experimentally. The fatigue fracture mechanism of 316L before and after UIT was revealed. The experimental results indicated that the martensitic grain size induced at the impact edge was about 2.00 Å. The surface modified 316L formed a gradient nanostructure and induced a martensitic phase transformation. The hardness of the surface layer of the modified 316L was twice the hardness of its matrix. The tensile strengths of 316L before and after UIT were 576 MPa and 703 MPa, respectively. The stretching stripes of 316L were more disordered after UIT. The fatigue strengths of 316L before and after UIT were 267 MPa and 327 MPa, respectively. The fatigue cracking of 316L started from the austenite grain boundaries. The fatigue fracture surface was relatively rough. The fatigue crack sources of the modified 316L came from internal inclusions. The inclusions were oxides dominated by SiO2. As the stress range increased, the crack initiation site migrated to the interior and the fatigue fracture surface became flatter.

Metabolomics analysis revealed the metabolic heterogeneity of cervical cancer (CC) cell lines C33A and CaSki, and their molecular mechanisms were explored. Using the modified Bligh-Dyer method, the endogenous metabolites of C33A and CaSki cells were divided into polar and nonpolar fractions. The metabolites were analysed by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Then, the differential metabolites were screened by combining multivariate statistical analysis and volcano maps, and functional enrichment and pathway analysis of the differential metabolites were performed. Finally, association analysis was carried out in combination with transcriptomics, and the important differential metabolisms were experimentally verified by real-time PCR (RT−qPCR) and oil red staining. The results showed that between the C33A and CaSki cell lines, there were significant differences in amino acids, nucleotides and lipids, such as in threonine, arachidonic acid and hypoxanthine, in the metabolic pathways. These compounds could be used as markers of differences in cellular metabolism. The heterogeneity of lipid metabolism accounted for 87.8%, among which C33A cells exhibited higher contents of fatty acid polar derivatives, while CaSki cells showed higher contents of free fatty acids and glycerides. Based on correlation analysis of the above metabolic differences in HPV pathways as well as lipid metabolism-related genes, p53 and the genes involved in lipid metabolism pathways, such as Peroxisome Proliferator Activated Receptor Gamma(PPARG) and stearoyl-CoA desaturase (SCD), are relevant to the metabolic heterogeneity of the cells. The differential expression of some genes was validated by RT−qPCR. CaSki cells showed significantly higher glyceride levels than that of C33A cells, as verified by oil red O staining and glyceride assays. The above results showed that the metabolomic differences between C33A and CaSki cells were relatively obvious, especially in lipid metabolism, which might be related to the decreased expression of PPARG and p53 caused by HPV E6. Further studies on the molecular mechanism of lipid metabolism heterogeneity in cervical cancer cell lines with or without HPV could provide a new reference for the development of CC and individualized treatments of tumour patients.

Abstract Mycobacterium tuberculosis (M. tuberculosis) can replicate in the macrophage by interfering with many host protein functions. While it is far from known these host proteins for controlling M. tuberculosis infection. Herein, we infected macrophages including THP-1 and Raw264.7 cells with M. tuberculosis and identified the differentially expressed genes (DEGs) in the interferon signaling pathway. Among them, 2′-5′ oligoadenylate synthetase-like (OASL) underwent the greatest upregulation in M. tuberculosis-infected macrophages. Knockdown of the expression of OASL attenuated M. tuberculosis survival in macrophages. Further, bioinformatics analysis revealed the potential interaction axis of OASL-TAB3- Rv0127, which was further validated by the yeast-two-hybrid (Y2H) assay and Co-IP. This interaction axis might regulate the M. tuberculosis survival and proliferation in macrophages. The study reveals a possible role of OASL during M. tuberculosis infection as a target to control its propagation.

Abstract Background Limited progress in terms of an effective treatment for spinal cord injury (SCI) emphasizes the urgent need for novel therapies. As a vital central nervous system component, the resident astrocytes play crucial roles in regulating recovery after SCI. In this study, recovery after SCI was compared following the transplantation of either A1 or A2 astrocytes. A1 astrocytes are harmful as they upregulate the neurotoxic classical complement cascade genes. Conversely, A2 astrocytes are characterized as neuroprotective as they upregulate the production of many neurotrophic factors. Methods We used different supernatant obtained from microglia stimulated with lipopolysaccharide or interleukin-4 to generate A1 and A2 astrocytes. We detected the influence of astrocytes on neurons by co-culturing A1 and A2 astrocytes with neurons. We transplanted astrocytes into the lesion site of the spinal cord and assessed lesion progression, neural restoration, glia formation and locomotor recovery. Results Astrocytes were polarized into A1 and A2 phenotypes following culture in the supernatant obtained from microglia stimulated with lipopolysaccharide or interleukin-4, respectively. Furthermore, co-culturing A2 astrocytes with neurons significantly suppressed glutamate-induced neuronal apoptosis and promoted the degree of neuron arborization. Transplantation of these A2 astrocytes into the lesion site of the spinal cord of mice significantly improved motor function recovery, preserved spared supraspinal pathways, decreased glia scar deposition, and increased neurofilament formation at the site of injury compared to the transplantation of A1 astrocytes. Additionally, enhanced A2 astrocytes with potentially beneficial A2-like genes were also detected in the A2 group. Moreover, luxol fast blue staining and electron microscopy indicated increased preservation of myelin with organized structure after transplantation of A2 astrocytes than of A1 astrocytes. Conclusions A2 astrocyte transplantation could be a promising potential therapy for SCI. Graphical Abstract Video abstract

Abstract Background Percutaneous kyphoplasty (PKP) has been demonstrated to be effective in the treatment of osteoporotic vertebral compression fractures (OVCF). However, bilateral puncture techniques take more time to accept more X-ray radiation; some spinal surgeons apply unilateral puncture PKP, but the cement cannot be symmetrically distributed in the vertebral body, so we apply a directional bone cement delivery device that undergoes PKP through the unilateral pedicle puncture. This research aims to compare the clinical and radiological results of PKP via unilateral pedicle approach using a traditional bone cement delivery device and a directional bone cement delivery device and determine the value of a directional delivery device for the therapy of thoracolumbar compression fracture in the elderly. Methods We undertook a retrospective analysis of patients with single-level OVCF treated with unilateral pedicle puncture PKP from Jan 2018 to Jan 2020. Operation time, radiation exposure, bone cement injection volume, and the incidence of bone cement leakage were recorded for presentation, and the cement leakage and bone cement distribution were measured by X-ray and computed tomography scan. The patients were followed up postoperatively and were assessed mainly with regard to clinical and radiological outcomes. Results There was no significant difference in the operation time, radiation exposure time, and incidence of bone cement leakage between the two groups. A significant difference was observed in the volume of bone cement injection between the two groups. All patients in both groups had significantly less pain after the procedures, compared with their preoperative period pain. There were no significant differences in Visual Analogue Scale, the relative height of the vertebral body, Cobb angle, and Quality of Life Questionnaire of the European Foundation for Osteoporosis between the two groups at 1 week after PKP, significant difference was observed only 12 months after operation. Conclusion Application of directional bone cement delivery device is safe and feasible, compared with the application of traditional bone cement delivery device, without prolonging the operative time, radiation exposure time, and the incidence of bone cement leakage. It has the advantages of good short- and medium-term effect, excellent bone cement distribution, and low incidence of kyphosis recurrence.

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), which is still the leading cause of mortality from a single infectious disease worldwide. The development of novel anti-TB drugs and vaccines is severely hampered by the complicated and time-consuming genetic manipulation techniques for M. tuberculosis. Here, we harnessed an endogenous type III-A CRISPR/Cas10 system of M. tuberculosis for efficient gene editing and RNA interference (RNAi). This simple and easy method only needs to transform a single mini-CRISPR array plasmid, thus avoiding the introduction of exogenous protein and minimizing proteotoxicity. We demonstrated that M. tuberculosis genes can be efficiently and specifically knocked in/out by this system as confirmed by DNA high-throughput sequencing. This system was further applied to single- and multiple-gene RNAi. Moreover, we successfully performed genome-wide RNAi screening to identify M. tuberculosis genes regulating in vitro and intracellular growth. This system can be extensively used for exploring the functional genomics of M. tuberculosis and facilitate the development of novel anti-TB drugs and vaccines.

Here the authors delineate the dynamic changes in 3D genome and epigenome of differentiating macrophages and during infection with Mycobacterium tuberculosis. They reveal a role for NF-κB upon infection and identify SNPs in disease-susceptible loci, including rs1873613 that is located in the anchor of a dynamic chromatin loop.

The human stress hormones catecholamines play a critical role in communication between human microbiota and their hosts and influence the outcomes of bacterial infections. However, it is unclear how M. tuberculosis senses and responds to certain types of human stress hormones. In this study, we screened several human catecholamine stress hormones (epinephrine, norepinephrine, and dopamine) for their effects on Mycobacterium growth. Our results showed that epinephrine significantly stimulated the growth of M. tuberculosis in the serum-based medium as well as macrophages. In silico analysis and molecular docking suggested that the extra-cytoplasmic domain of the MprB might be the putative adrenergic sensor. Furthermore, we showed that epinephrine significantly enhances M. tuberculosis biofilm formation, which has distinct texture composition, antibiotic resistance, and stress tolerance. Together, our data revealed the effect and mechanism of epinephrine on the growth and biofilm formation of M. tuberculosis, which contributes to the understanding of the environmental perception and antibiotic resistance of M. tuberculosis and provides important clues for the understanding of bacterial pathogenesis and the development of novel antibacterial therapeutics.

This paper presents a theoretical and numerical study of angle steel joints for conductors in transmission tower structures in the medium and heavy icing area. The results show that the deformation of the angle steel joint in suspension towers is controlled by the vertical load, and the deformation of the angle steel joint in strain towers is controlled by the tension load. The bolt of the angle steel joint for conductors is tension-controlled. A theoretical method considering the bending stiffness ratio of the angle steel plate and the bolt is derived. The proposed theoretical method quantifies the bending stiffness difference in the angle steel plate and bolt group, and the effects of bolt layout and load cases are considered. The numerical results are in good agreement with theoretical results by validating the internal forces and neutral axis depth. The accuracy of the proposed theoretical method is higher than that of theoretical methods based on steel structure principles. The proposed theoretical method is of great significance to the development of transmission tower structure design.

Abstract Foams are substances widely used the foam flooding technology, which aim to greatly improve the residual oil recovery. In the present study, we perform a comprehensive investigation on the oil removal process driven by the foam embedded with magnetic particles, under the action of the magnetic force. The experiment shows that the addition of magnetic particles has little effect on the stability of the foam. During the motion of the foam, its maximum displacement and maximum acceleration are fully explored. Such factors as the volume of the foam, the volume of the oil droplet, the mass concentration of magnetic particles, and the Young’s contact angle of surfactant on solid are surveyed in detail. The function curves of the maximum displacement and the maximum acceleration with respect to these variables are obtained in the experiment, and the selection of some optimal parameters is advised. Moreover, the dimensional analysis has been conducted and several scaling laws are given, which are in agreement with the experimental results. These findings are beneficial to understand the oil displacement with the aid of magnetic field, which also provide some inspirations on drug delivery, robots and micro-fluidics.

Labyrinth seals are widely used in turbomachinery devices to prevent the leakage of fluid medium in seal structures. Under high parametrical environments, the heat generated by frictional resistance of the turbulence between the stator and rotor walls not only increases the temperature of the fluid and the solid, but also reduces the operation efficiency of the device. Therefore, it is necessary to implement the fluid–solid-thermal multi-field analysis of labyrinth seals and propose a comprehensive optimization design of the load-bearing and heat insulation. In the present study, inspired by a bionic snail shell, a sandwich structure with load-bearing material on both upper and lower surfaces and the heat insulation material in the middle is obtained through the genetic algorithm optimization. Then, the luid-solid-thermal multi-field analysis of the labyrinth seal with the sandwich structure is carried out. It is verified that the layer arrangement and layer thickness ratio of the bionic structure ensured the optimal performance of load-bearing and heat insulation. After changing the layer arrangement or layer thickness ratio, the comprehensive performance of load-bearing and heat insulation is obviously weakened. The result shows that the bionic multilayer sandwich structure can be applied to the labyrinth seal to achieve optimal comprehensive performance of load-bearing and heat insulation.

Treatment of spinal cord injury (SCI) remains challenging. Considering the rapid developments in neurorestorative therapies for SCI, we have revised and updated the Clinical Therapeutic Guidelines for Neurorestoration in Spinal Cord Injury (2016 Chinese version) of the Chinese Association of Neurorestoratology (Preparatory) and China Committee of International Association of Neurorestoratology. Treatment of SCI is a systematic multimodal process that aims to improve survival and restore neurological function. These guidelines cover real-world comprehensive neurorestorative management of acute, subacute, and chronic SCI and include assessment and diagnosis, pre-hospital first aid, treatment, rehabilitation, and complication management.

Abstract Background Bilateral decompression via unilateral approach (BDUA) is an effective surgical approach for treating lumbar degenerative diseases. However, no studies of prognosis, especially the recovery of the soft tissue, have reported using BDUA in an elderly population. The aims of these research were to investigate the early efficacy of the bilateral decompression via unilateral approach versus conventional approach transforaminal lumbar interbody fusion (TLIF) for the treatment of lumbar degenerative disc disease in the patients over 65 years of age, especially in the perioperative factors and the recovery of the soft tissue. Methods The clinical data from 61 aging patients with lumbar degenerative disease who received surgical treatment were retrospectively analyzed. 31 cases who received the lumbar interbody fusion surgery with bilateral decompression via unilateral approach (BDUA) were compared with 30 cases who received conventional approach transforaminal lumbar interbody fusion. The radiographic parameters were measured using X-ray including lumbar lordosis angle and fusion rate. Japanese Orthopedic Association (JOA), Visual Analogue Scale (VAS) and Oswestry Disability Index (ODI) scores were used to evaluate the clinical outcomes at different time points. Fatty degeneration ratio and area of muscle/vertebral body were used to detect recovery of soft tissue. Results The BDUA approach group was found to have significantly less intraoperative blood loss(p 0.05) in two sides of conventional transforaminal lumbar interbody fusion approach group six months after surgery. Conclusions Bilateral decompression via unilateral approach (BDUA) is able to reduce the intraoperative and postoperative body fluid loss in the elderly. The opposite side of decompression in BDUA shows less fatty degeneration in 6 months, which indicates better recovery of the soft tissue of the aging patients.

Abstract Background Percutaneous kyphoplasty is the main method in the treatment of thoracolumbar osteoporotic compression fractures. However, much radiation exposure during the operation harms the health of surgeons and patients. In addition, the accuracy of this surgery still needs to be improved. This study aimed to assess the radiation exposure and clinical efficacy of Tirobot-assisted vertebroplasty in treating thoracolumbar osteoporotic compression fracture. Methods Included in this retrospective cohort study were 60 patients (60–90 years) who had undergone unilateral vertebroplasty for thoracolumbar osteoporotic compression fracture at our hospital between June 2019 and June 2020. All showed no systemic diseases and were assigned to Tirobot group (treated with Tirobot-assisted approach) and control group (treated with traditional approach). Fluoroscopic frequency, operative duration, length of stay (LOS), post-operative complications (cement leakage, infection, and thrombosis), and pre-operative and pre-discharge indexes (VAS score, JOA score, and Cobb’s angle) were compared. Results The fluoroscopic frequency (P < 0.001) and post-operative complications (P = 0.035) in Tirobot group were significantly lower than those in control group. The operative duration and LOS in the Tirobot group were shorter than those in the control group, but the differences were not statistically significant (P = 0.183). Pre-discharge VAS score and Cobb’s angle decreased, and JOA increased after surgeries in both groups. These three indexes showed a significant difference after surgery in each group (P < 0.001), but not between groups (P VAS = 0.175, P Cobb’s = 0.585, P JOA = 0.448). Conclusion The Tirobot-assisted vertebroplasty can reduce surgery-related trauma, post-operative complications, and patients’ and operators’ exposure to radiation. As a safe and effective strategy, this surgery can realize the quick recovery from thoracolumbar osteoporotic compression fracture.

Japanese encephalitis virus (JEV) is one of the most important members of the flavivirus family. It is a typical zoonotic pathogen that has caused substantial social and economic losses worldwide. The relation between JEV-induced immunosuppression and inflammatory responses has not been thoroughly investigated. In this study, cells infiltrating the brain tissue of JEV-infected mice were mainly identified as monocytic myeloid-derived suppressor cells (M-MDSCs), which subsequently differentiated into CD3+ macrophages. Co-culture with T cells showed that both splenic M-MDSCs and brain infiltrated M-MDSCs isolated from JEV-infected mice inhibited T cell proliferation through ARG1 and iNOS. The splenectomy model revealed that JEV-induced M-MDSCs were mainly derived from bone marrow and migrated to the spleen and central nervous system (CNS). The results of the transcriptome analysis and IRF7-deficient mice indicated that the ZBP1-IRF7 signaling pathway stimulated by JEV RNA played a central role in the induction of M-MDSCs. M-MDSCs migrated into the CNS through the chemokine CCL2/N-CCL2 derived from astrocytes and brain infiltrated M-MDSCs differentiated into CD3+ macrophages through a mechanism mediated by M-CSF, IL-6 and IFN-γ in the brain microenvironment. These findings provide evidence for the mechanism that JEV regulates the differentiation of M-MDSCs and thereby exacerbates pathogenicity, which represents a potential therapeutic target for Japanese encephalitis (JE).

The technology of pile wall combination in a deep foundation pit is a kind of technology to resist soil and water pressure by using the great stiffness of the retaining pile and basement wall. As a part of basement wall, the retaining pile can reduce the thickness of the basement wall. However, the influence of excavation deformation and the interaction mechanism between the retaining pile and basement wall are ignored in the current design theory of the pile wall combination in a deep foundation pit. Therefore, we proposed a composite structure model of an elastic subgrade beam associated with a continuous beam, and introduced an elastic spring to simulate the interaction between the pile and basement wall. The finite difference equation and calculation method were established to figure out the internal force and displacement of the pile wall combination structure, which was able to accurately calculate the internal force and deformation of the retaining pile during the excavation stage. Based on the above results, further investigation was carried on the interaction between the pile and basement wall during the construction and normal service stage of the basement structure. It was concluded that the basement wall could reduce the lateral displacement on the top of the retaining pile caused by removal of the strut and affect the bending moment of the upper part of the retaining pile among the depth of the basement wall. The bending moment of the basement wall was closely related to the deformation of the retaining pile and changes with the working condition. In addition, the results of factor sensitivity analysis showed that the pile-wall relative stiffness ratio, stiffness of the horizontal beam, and slab of the basement had a significant influence on the interaction between the pile and basement wall.

Coronaviruses are a great source of threat to public health which could infect various species and cause diverse diseases. However, the epidemic’s spreading among different species remains elusive. This study proposed an in silico infection analysis (iSFA) system that includes pathogen genome or transcript mining in transcriptome data of the potential host and performed a comprehensive analysis about the infection of 38 coronaviruses in wild animals, based on 2,257 transcriptome datasets from 89 mammals’ lung and intestine, and revealed multiple potential coronavirus infections including porcine epidemic diarrhea virus (PEDV) infection in Equus burchellii. Then, through our transmission network analysis, potential intermediate hosts of five coronaviruses were identified. Notably, iSFA results suggested that the expression of coronavirus receptor genes tended to be downregulated after infection by another virus. Finally, binding affinity and interactive interface analysis of S1 protein and ACE2 from different species demonstrated the potential inter-species transmission barrier and cross-species transmission of SARS-CoV-2. Meanwhile, the iSFA system developed in this study could be further applied to conduct the source tracing and host prediction of other pathogen-induced diseases, thus contributing to the epidemic prevention and control.

Under starvation conditions, bacteria tend to slow down their translation rate by reducing rRNA synthesis, but the way they accomplish that may vary in different bacteria. In Mycobacterium species, transcription of rRNA is activated by the RNA polymerase (RNAP) accessory transcription factor CarD, which interacts directly with RNAP to stabilize the RNAP-promoter open complex formed on rRNA genes. The functions of CarD have been extensively studied, but the mechanisms that control its expression remain obscure. Here, we report that the level of CarD was tightly regulated when mycobacterial cells switched from nutrient-rich to nutrient-deprived conditions. At the translational level, an antisense RNA of carD (AscarD) was induced in a SigF-dependent manner to bind with carD mRNA and inhibit CarD translation, while at the post-translational level, the residual intracellular CarD was quickly degraded by the Clp protease. AscarD thus worked synergistically with Clp protease to decrease the CarD level to help mycobacterial cells cope with the nutritional stress. Altogether, our work elucidates the regulation mode of CarD and delineates a new mechanism for the mycobacterial starvation response, which is important for the adaptation and persistence of mycobacterial pathogens in the host environment.

Determination of the mechanical properties of rock containing pre-existing cracks under tension condition is of great significance to understand the failure process of rock in engineering. This paper presents the experimental results of sandstone containing pre-existing cracks under Brazilian compression. The characteristics of the microcracks were analyzed by a scanning electron microscope. The results show that the rock containing pre-existing cracks has an obvious anisotropic characteristic. When the crack inclination is 45°, the rock has the minimum tensile strength and the weakest axial deformation resistance.

The computational fluid dynamics (CFD) combined with radial basis function (RBF) method were adopted to obtain the response surface of the Muszynska nonlinear seal force model coefficient with two variables: eccentricity and rotation speed. During the implementation of the simulation, three coefficients of the seal force model were calculated in each sub-step according to the current state of the rotor-bearing seal system; following which the rotor dynamics analysis with varying parameters was realized. As with the traditional constant coefficient method, the first-order critical speed of the system was obtained, and the bifurcation point and oil film whirl of the system response were identified. The difference is that the coefficients of the traditional method ordinarily do not change with the state of the system. Comparing the results of the varying parameter method with those of the traditional method, it can be seen that the speeds of the system corresponding to the bifurcation and oil film whirl are different. The varying parameter rotor dynamics simulation method proposed in this paper provides a new way of analyzing the nonlinear characteristics of rotor-bearing-seal systems.

ABSTRACT Mycobacterium tuberculosis can invade different cells with distinct persistence fates because cells are equipped with different host restriction factors. However, the underlying mechanisms remain elusive. Here, we infected THP1 and Raw264.7 macrophages cell lines, A549 epithelial cell line, and hBMEC and bEnd.3 endothelial cell lines with M. tuberculosis and demonstrated that M. tuberculosis significantly inhibited lysosome acidification in THP1, hBMEC, A549, and Raw264.7 cells, while, in bEnd.3 cells, M. tuberculosis was mainly delivered into acidified phagolysosomes and auto-lysosomes. The systematic gene profile analysis of different cells and intracellular M. tuberculosis showed that the phagosome autophagy-pathway-related genes itgb3 and atg3 were highly expressed in bEnd.3 cells. Knockdown of these genes significantly increased the number of viable intracellular M. tuberculosis bacilli by altering phagosomal trafficking in bEnd.3 cells. Treatment with itgb3 agonist significantly decreased M. tuberculosis survival in vivo. These findings could facilitate the identification of anti-M. tuberculosis host genes and guide M. tuberculosis-resistant livestock breeding. IMPORTANCE As an intracellular pathogen, Mycobacterium tuberculosis could avoid host cell immune clearance using multiple strategies for its long-term survival. Understanding these processes could facilitate the development of new approaches to restrict intracellular M. tuberculosis survival. Here, we characterized the detailed molecular events occurring during intracellular trafficking of M. tuberculosis in macrophage, epithelial, and endothelial cell lines and found that ITGB3 facilitates M. tuberculosis clearance in endothelial cells through altering phagosomal trafficking. Meanwhile, the treatment with ITGB3 agonist could reduce bacterial load in vivo. Our results identified new anti-M. tuberculosis restriction factors and illuminated a new anti-M. tuberculosis defense mechanism.

Tuberculosis (TB) is a debilitating infectious disease responsible for more than one million deaths per year. The emergence of drug-resistant TB poses an urgent need for the development of new anti-TB drugs. In this study, we screened a library of over 4,000 small molecules and found that orbifloxacin and the peptide AK15 possess significant bactericidal activity against Mycobacterium tuberculosis (Mtb) in vitro. Orbifloxacin also showed an effective ability on the clearance of intracellular Mtb and protect mice from a strong inflammatory response but not AK15. Moreover, we identified 17 nucleotide mutations responsible for orbifloxacin resistance by whole-genome sequencing. A critical point mutation (D94G) of the DNA gyrase (gyrA) gene was found to be the key role of resistance to orbifloxacin. The computational docking revealed that GyrA D94G point mutation can disrupt the orbifloxacin–protein gyrase interactions mediated by magnesium ion bridge. Overall, this study indicated the potential ability of orbifloxacin as an anti-tuberculosis drug, which can be used either alone or in combination with first-line antibiotics to achieve more effective therapy on TB.

Mycobacterium tuberculosis (M.tb) secretes numerous proteins to interfere with host immune response for its long-term survival. As one of the top abundant M.tb secreted proteins, Rv3722c was found to be essential for bacilli growth. However, it remains elusive how this protein interferes with the host immune response and regulates M.tb survival. Here, we confirmed that Rv3722c interacted with host TRAF3 to promote M.tb replication in macrophages. Knock-down of TRAF3 attenuated the effect of Rv3722c on the intracellular M.tb survival. The interaction between Rv3722c and TRAF3 hampered MAPK and NF-κB pathways, resulting in a significant increase of IFN-β expression and decrease of IL-1β, IL-6, IL-12p40, and TNF-α expression. Our study revealed that Rv3722c interacted with TRAF3 and interrupted its downstream pathways to promote M.tb survival in macrophages. These findings facilitate further understanding of the mechanism of M.tb secreted proteins in regulating the host cell immune response and promoting its intracellular survival.

Skeletal muscle atrophy with high prevalence can induce weakness and fatigability and place huge burden on both health and quality of life. During skeletal muscle degeneration, excessive fibroblasts and extracellular matrix (ECM) accumulated to replace and impair the resident muscle fiber and led to loss of muscle mass. Cyclooxygenase-2 (COX-2), the rate-limiting enzyme in synthesis of prostaglandin, has been identified as a positive regulator in pathophysiological process like inflammation and oxidative stress. In our study, we found injured muscles of human subjects and mouse model overexpressed COX-2 compared to the non-damaged region and COX-2 was also upregulated in fibroblasts following TGF-β stimulation. Then we detected the effect of selective COX-2 inhibitor celecoxib on fibrogenesis. Celecoxib mediated anti-fibrotic effect by inhibiting fibroblast differentiation, proliferation and migration as well as inactivating TGF-β-dependent signaling pathway, non-canonical TGF-β pathways and suppressing generation of reactive oxygen species (ROS) and oxidative stress. In vivo pharmacological inhibition of COX-2 by celecoxib decreased tissue fibrosis and increased skeletal muscle fiber preservation reflected by less ECM formation and myofibroblast accumulation with decreased p-ERK1/2, p-Smad2/3, TGF-βR1, VEGF, NOX2 and NOX4 expression. Expression profiling further found that celecoxib could suppress PDK1 expression. The interaction between COX-2 and PDK1/AKT signaling remained unclear, here we found that COX-2 could bind to PDK1/AKT to form compound. Knockdown of COX-2 in fibroblasts by pharmacological inactivation or by siRNA restrained PDK1 expression and AKT phosphorylation induced by TGF-β treatment. Besides, si-COX-2 prevented TGF-β-induced K63-ubiquitination of AKT by blocking the interaction between AKT and E3 ubiquitin ligase TRAF4. In summary, we found blocking COX-2 inhibited fibrogenesis after muscle atrophy induced by injury and suppressed AKT signaling pathway by inhibiting upstream PDK1 expression and preventing the recruitment of TRAF4 to AKT, indicating that COX-2/PDK1/AKT signaling pathway promised to be target for treating muscle atrophy in the future.

The recovery of lower-urinary-tract activity is a top priority for patients with spinal-cord injury. Historically, locomotor training improved micturition function in both patients with spinal cord injury and animal models. We explore whether training augments such as the supraspinal control of the external urethral sphincter results in enhanced coordination in detrusor-sphincter activity. We implemented a clinically relevant contusive spinal-cord injury at the 12th thoracic level in rats and administered forced wheel running exercise for 11 weeks. Awake rats then underwent bladder cystometrogram and sphincter electromyography recordings to examine the micturition reflex. Subsequently, pseudorabies-virus-encoding red fluorescent protein was injected into the sphincter to trans-synaptically trace the supraspinal innervation of Onuf’s motoneurons. Training in the injury group reduced the occurrence of bladder nonvoiding contractions, decreased the voiding threshold and peak intravesical pressure, and shortened the latency of sphincter bursting during voiding, leading to enhanced voiding efficiency. Histological analysis demonstrated that the training increased the extent of spared spinal-cord tissue around the epicenter of lesions. Compared to the group of injury without exercise, training elicited denser 5-hydroxytryptamine-positive axon terminals in the vicinity of Onuf’s motoneurons in the cord; more pseudorabies virus-labeled or c-fos expressing neurons were detected in the brainstem, suggesting the enhanced supraspinal control of sphincter activity. Thus, locomotor training promotes tissue sparing and axon innervation of spinal motoneurons to improve voiding function following contusive spinal-cord injury.

Accumulating evidence has indicated that abnormal microRNAs (miRNAs) serve critical roles in carcinogenesis and development of osteosarcoma (OS). The purpose of the present study was to elucidate the relationship between miR-766-3p and development of osteosarcoma and explore the potential mechanism. In this study, we found that miR-766-3p was the most downregulated miRNA by analyzing GSE65071 from the GEO database. miR-766-3p was lowly expressed in OS tissue samples and cells, and high miR-766-3p expression repressed the malignant level of OS, including cell proliferation, EMT, migration, and invasion in vitro and in vivo. B-Cell Lymphoma 9-Like Protein (BCL9L) was negatively associated with miR-766-3p expression in OS cells and tissue samples and was validated as the downstream target by luciferase reporter assay and western blotting. Rescue experiment indicated that BCL9L could restore the influence of miR-766-3p on OS cells. In addition, the β-Catenin/TCF-4 signal pathway was demonstrated to be related to the miR-766-3p/BCL9L axis. In summary, miR-766-3p, a negative regulator of BCL9L, plays the role of tumor metastasis suppressor via the β-catenin signaling pathway in the progression of OS.

Osteosarcoma accounts for about 0.2% in human malignant solid tumors. The mortality and metastatic rates of osteosarcoma remain relatively high. MicroRNA (miRNA) is a kind of non-coding small-molecular RNA discovered in recent years. Various studies have identified the involvement of miRNA in the occurrence and development of tumor as an oncogene or tumor-suppressor gene. This study aims to investigate the effect of hsa-miR-889-3p on the progression of osteosarcoma and its underlying mechanism. Through the bioinformatics methods, we first found that hsa-miR-889-3p was upregulated in osteosarcoma, and it was a prognostic risk factor for osteosarcoma. Additionally, the gene set enrichment analysis (GSEA) revealed that hsa-miR-889-3p mainly affected cell cycle progression and proliferation of osteosarcoma. To verify the bioinformatics results, regulatory effects of hsa-miR-889-3p on osteosarcoma both in vitro and in vivo experiments were investigated. It is found that hsa-miR-889-3p could promote the proliferation of osteosarcoma cells though regulating cell cycle progression. Tumor size and growth rate of osteosarcoma were influenced by hsa-miR-889-3p in xenograft models. To further explore its potential mechanism, the target gene of hsa-miR-889-3p was predicted. Furthermore, hsa-miR-889-3p was confirmed to inhibit the expression of myeloid cell nuclear differentiation antigen (MNDA) in a targeted manner. In conclusion, hsa-miR-889-3p could promote the proliferation of osteosarcoma through inhibiting MNDA expression, which provides a potential therapeutic strategy in treatment for osteosarcoma.

By means of the ultrasonic surface impact (amplitude of 30 μm, strike number of 48,000 times/mm2), nanograins have been achieved in the surfaces of both Ti6Al4V(TC4) and Ti3Zr2Sn3Mo25Nb(TLM) titanium alloys, mainly because of the dislocation motion. Many mechanical properties are improved, such as hardness, residual stress, and roughness. The rotating–bending fatigue limits of TC4 and TLM subjected to ultrasonic impact are improved by 13.1% and 23.7%, separately. Because of the bending fatigue behavior, which is sensitive to the surface condition, cracks usually initiate from the surface defects under high stress amplitude. By means of an ultrasonic impact tip with the size of 8 mm, most of the inner cracks present at the zone with a depth range of 100~250 μm in the high life region. The inner crack core to TC4 usually appears as a deformed long and narrow α-phase, while the cracks in TLM specimens prefer to initiate at the triple grain boundary junctions. This zone crosses the grain refined layer and the deformed coarse grain layer. With the gradient change of elastic parameters, the model shows an increase of normal stress at this zone. Combined with the loss of plasticity and toughness, it is easy to understand these fatigue behaviors.

Background/Aims: Hypertrophic scars (HS) formation results from reduced apoptosis and increased proliferation of fibroblasts. Therefore, apoptosis of fibroblasts is a key target for the development of novel therapeutic strategies for HS. Previous reports demonstrated that FK506 could attenuate scar formation in vivo and FK506 could also induce endoplasmic reticulum stress (ER stress). However, the effects of FK506 on ER stress-mediated apoptosis in fibroblasts remain unclear. Methods: Rat skin fibroblasts were used in the study. Cell viability was examined using cell counting Kit-8. Apoptosis was detected by Annexin V/Propidium Iodide Double Staining. Gene silencing was performed using Small Interfering RNAs (siRNAs) or via lentiviral infection. The expression of apoptosis-related proteins was determined via Western blot. Interaction between proteins was explored by co-immunoprecipitation. Results: FK506 significantly reduced cell viability and induced apoptosis in fibroblasts. Interestingly, ER stress was also activated after FK506 treatment. We further demonstrated that FK506-induced apoptosis was mediated by ER stress via activating CHOP, evidenced by decreased apoptosis after inhibition of ER stress using TUDCA or silencing expression of CHOP. Furthermore, Co-immunoprecipitation results indicated that treatment of FK506 induced disassociation of FKBP12.6 from RyR2 and its translocation from ER membrane to cytosol, consequently promoting ER stress-mediated apoptosis. Conclusion: FK506-induced fibroblasts apoptosis was mediated by ER stress via CHOP signaling pathway.

Background/Aims: Epidural fibrosis, a common complication after laminectomy, has been demonstrated to be closely associated with poor surgical outcomes. Previous studies showed that taurine had remarkable anti-fibrotic effects on lung and liver fibrosis. We performed this study to investigate the effects of taurine in rat models of epidural fibrosis after laminectomy and to explore the potential molecular mechanism. Methods: Laminectomy was performed on each rat to establish epidural fibrosis model. After taurine treatment, Masson's trichrome and immunohistochemistry staining were used to examine epidural fibrosis. Cell viability was determined using the Cell Counting Kit-8 assay. Annexin V/Propidium Iodide double staining was performed to detect fibroblasts apoptosis. Microarray was adopted to identify significantly changed mRNAs. mRNA expression was measured by qRT-PCR. Lentivirus infection was performed to establish stable knockdown and overexpression cell lines. The expression of fibrosis-related proteins was determined via Western blot. Results: Taurine treatment markedly reduced laminectomy-induced epidural fibrosis in rat models. However, this effect of taurine was independent on TGF-β/Smad pathway, evidenced by no change in the expression of TGF-β and its receptors. Besides, taurine had almost no effect on cell apoptosis. Interestingly, taurine treatment significantly decreased expression of EGR1 (Early growth response protein 1), an enhancer of fibrosis, both in vivo and in vitro. Furthermore, overexpression of EGR1 increased activation of fibroblasts, while EGR1 knockdown achieved an opposite effect, indicating that EGR1 plays a key role in the inhibitory effect of taurine on TGF-β-induced fibrosis. Conclusions: Reduced epidural fibrosis in vivo and decreased activation of fibroblasts in vitro after taurine treatment was mediated by EGR1. Taurine promises to be a potential prevention for epidural fibrosis after laminectomy.

Background/Aims: Postmenopausal osteoporosis is closely associated with reduction in the differentiation of mesenchymal stem cells (MSCs) into osteoblasts. Previous studies have demonstrated that miR-214 plays an important role in the genesis and development of postmenopausal osteoporosis. Here, we performed this study to investigate the potential mechanism by which miR-214 regulates osteoblast differentiation of MSCs. Methods: First, we explored the expression of miR-214 in MSCs of osteoporotic mice. Next, we examined the change of miR-214 during osteoblast differentiation of MSCs. Then, MSCs were infected with lentiviral vectors expressing miR-214 or miR-214 sponge to investigate the effect of miR-214 on osteoblast differentiation of MSCs. Further, bioinformatics analysis and luciferase reporter assay were performed to identify and validate the target gene of miR-214. Results: MiR-214 was up-regulated in MSCs of osteoporotic mice and down-regulated during osteoblast differentiation of MSCs. Furthermore, overexpression of miR-214 inhibited osteoblast differentiation of MSCs in vitro, whereas inhibition of miR-214 function promoted this process, evidenced by increased expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Bioinformatics, Western blot analysis and luciferase reporter assay demonstrated that FGFR1 is a direct target of miR-214. Conclusions: MiR-214 attenuates osteogenesis by inhibiting the FGFR1/FGF signaling pathway. Our findings suggest that targeting miR-214 promises to be a potential therapy in treatment of postmenopausal osteoporosis.

Background/Aims: Schwann cells (SCs) which were demonstrated to be responsible for axonal myelination and ensheathing are widely studied and commonly used for cell transplantation to treat spinal cord injury (SCI). We performed this meta-analysis to summarize the effects of SCs versus controls for locomotor recovery in rat models of traumatic SCI. Methods: Studies of the BBB scores after transplantation of SCs were searched out from Pubmed, Cochrane Library Medline databases and analyzed by Review Manager 5.2.5. Results: Thirteen randomized controlled animal trials were selected with 283 rats enrolled. The studies were divided to different subgroups by different models of SCI, different cell doses for transplantation, different sources of SCs and different transplantation ways. The pooled results of this meta-analysis suggested that SCs transplantation cannot significantly improve the locomotor recovery at a short time after intervention (1 week after transplantation) in both impacted and hemi-sected SCI models. However, at a longer time after intervention (3, 5-7 and over 8 weeks after transplantation), significant improvement of BBB score emerged in SCs groups compared with control groups. Subgroup analyses revealed that SCs transplantation can significantly promote locomotor recovery regardless of in high or low doses of cells, from different sources (isolated from sciatic nerves or differentiated from bone marrow stromal cells(BMSCs)) and with or without scaffolding. Conclusion: SCs seem to demonstrate substantial beneficial effects on locomotor recovery in a widely-used animal models of SCI.

The effect of nano grain surface layer generated by ultrasonic impact on the fatigue behaviors of a titanium alloy Ti3Zr2Sn3Mo25Nb (TLM) was investigated. Three vibration strike-numbers of 24,000 times, 36,000 times and 48,000 times per unit are chosen to treat the surface of TLM specimens. Nanocrystals with an average size of 30 nm are generated. The dislocation motion plays an important role in the transformation of nanograins. Ultrasonic surface impact improves the mechanical properties of TLM, such as hardness, surface residual stress, tensile strength and fatigue strength. More vibration strike numbers will cause a higher enhancement. With a vibration strike number of 48,000 times per square millimeter the rotating-bending fatigue strength of TLM at 107 cycles is improved by 23.7%. All the fatigue cracks initiate from the surface of untreated specimens, while inner cracks appear after the fatigue life of 106 cycles with the ultrasonic surface impact. The crystal slip in the crack initiation zone is the main way of growth for microcracks. Crack cores are usually formed at the junction of crystals. The stress intensity factor of TLM titanium alloy is approximately 7.0 MPa·m1/2.

The non-pathogenic bacterium Mycobacterium smegmatis mc2155 has been widely used as a model organism in mycobacterial research, yet a detailed study about its transcription landscape remains to be established. Here we report the transcriptome, expression profiles and transcriptional structures through growth-phase-dependent RNA sequencing (RNA-seq) as well as other related experiments. We found: (1) 2,139 transcriptional start sites (TSSs) in the genome-wide scale, of which eight samples were randomly selected and further verified by 5′-RACE; (2) 2,233 independent monocistronic or polycistronic mRNAs in the transcriptome within the operon/sub-operon structures which are classified into five groups; (3) 47.50% (1016/2139) genes were transcribed into leaderless mRNAs, with the TSSs of 41.3% (883/2139) mRNAs overlapping with the first base of the annotated start codon. Initial amino acids of MSMEG_4921 and MSMEG_6422 proteins were identified by Edman degradation, indicating the presence of distinctive widespread leaderless features in M. smegmatis mc2155. (4) 150 genes with potentially wrong structural annotation, of which 124 proposed genes have been corrected; (5) eight highly active promoters, with their activities further determined by β-galactosidase assays. These data integrated the transcriptional landscape to genome information of model organism mc2155 and lay a solid foundation for further works in Mycobacterium.

Background/Aims: Rheumatoid arthritis (RA) is a systemic chronic inflammatory disease characterised by prominent synoviocyte hyperplasia and a potential imbalance between the growth and death of fibroblast-like synoviocytes (FLS). Mitomycin C (MMC) has previously been demonstrated to inhibit fibroblast proliferation and to induce fibroblast apoptosis. However, the effects of MMC on the proliferation and apoptosis of human RA FLS and the potential mechanisms underlying its effects remain unknown. Methods: Cell viability was determined using the Cell Counting Kit-8 assay. Apoptotic cell death was analysed via Annexin V-FITC/PI double staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling. The production of intracellular reactive oxygen species (ROS) was assessed via flow cytometry, and the changes in mitochondrial membrane potential (ΔΨm) were visualized based on JC-1 staining via fluorescence microscopy. The expression of apoptosis-related proteins was determined via Western blot. Results: Treatment with MMC significantly reduced cell viability and induced apoptosis in RA FLS. Furthermore, MMC exposure was found to stimulate the production of ROS and to disrupt the ΔΨm compared to the control treatment. Moreover, MMC increased the release of mitochondrial cytochrome c, the ratio of Bax/Bcl-2, the activation of caspase-9 and caspase-3, and the subsequent cleavage of poly(ADP-ribose) polymerase. Conclusion: Our findings suggest that MMC inhibits cell proliferation and induces apoptosis in RA FLS, and the mechanism underlying this MMC-induced apoptosis may involve a mitochondrial signalling pathway.

Identification of motor and sensory nerves is important in applications such as nerve injury repair. Conventional practice relies on time consuming staining methods for this purpose. Here, we use laser scanning infrared differential interference contrast (IR-DIC) microscopy for label-free observation of the two types of nerve. Ventral and dorsal nerve roots of adult beagle dogs were collected and sections of different thicknesses were imaged with an IR-DIC microscope. Different texture patterns of the IR-DIC images of the motor and sensory nerve can be distinguished when the section thickness increases to 40μm. This suggests that nerve fibers in motor and sensory nerves have different distribution patterns. The result hints a potential new way for more rapid identification of nerve type in peripheral nerve repair surgery.

The influence of ultrasonic nanocrystal surface modification (UNSM) on the fatigue fracture characteristics of Ti6Al4V was investigated. Two groups of specimens were separated due to different heat treatment conditions. Group one was stress-relief annealed at 650 °C, and group two was then treated with solid solution-aging. UNSM with the conditions of a static load of 25 N, vibration amplitude of 30 μm, and 36,000 strikes per unit produced about 40 μm surface severe plastic deformation (SPD) layers on both groups of specimens. UNSM improved the microhardness and the compressive residual stress. UNSM also helped achieve a neat surface, almost without changing the surface roughness. The fatigue strengths of these two groups were improved by 7% and 11.7%, respectively. After UNSM, fatigue cracks mainly initiated from the surface of the specimen before the fatigue life of 106 cycles, while they appeared at the internal compress deformed α-phase at the zone between the SPD layer and the core after the fatigue life of 106 cycles. The cracks usually extended along the deformation overflow bands and the process traces on the surface. Through the change of micro-dimples in the fatigue final rupture region, nanocrystals were achieved in the SPD layer. The crystal slip and the surface remodeling together influenced the energy field of crack evolution.

The effect of ultrasonic nanocrystal surface modification (UNSM) on the fatigue behavior of Ti6Al4V (TC4) in simulated body fluid (SBF) was investigated. UNSM with the condition of a static load of 25 N, vibration amplitude of 30 μm and 36,000 strikes per unit produced about 35 μm surface severe plastic deformation (SPD) layers on the TC4 specimens. One group was treated with a hybrid surface treatment (UNSM + TiN film). UNSM technique improves the micro hardness and the compressive residual stress. The surface roughness is increased slightly, but it can be remarkably improved by the TiN film. The fatigue strength of TC4 is improved by about 7.9% after UNSM. Though the current density of corrosion is increased and the pitting corrosion is accelerated, UNSM still improved the fatigue strength of TC4 after pre-soaking in SBF by 10.8%. Interior cracks initiate at the deformed carbide and oxide inclusions due to the ultrasonic impacts of UNSM. Corrosion products are always observed at the edge of fracture surface to both interior cracks and surface cracks. Coating a TiN film on the UNSMed surface helps to improve the whole properties of TC4 further.

Endoplasmic reticulum (ER) stress-mediated cell apoptosis has been implicated in various cell types, including fibroblasts. Previous studies have shown that mitomycin C (MMC)-induced apoptosis occurs in fibroblasts, but the effects of MMC on ER stress-mediated apoptosis in fibroblasts have not been examined. Here, MMC-induced apoptosis in human primary fibroblasts was investigated by exposing cells to a single dose of MMC for 5 minutes. Significant inhibition of cell proliferation and increased apoptosis were observed using a cell viability assay, Annexin V/propidium iodide double staining, cell cycle analysis, and TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining. Upregulation of proapoptotic factors, including cleaved caspase-3 and poly ADP-ribose polymerase (PARP), was detected by Western blotting. MMC-induced apoptosis was correlated with elevation of 78-kDa glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP), which are hallmarks of ER stress. Three unfolded protein response (UPR) sensors (inositol-requiring enzyme 1, IRE1; activating transcription factor 6, ATF6; and PKR-like ER kinase, PERK) and their downstream signaling pathways were also activated. Knockdown of CHOP attenuated MMC-induced apoptosis by increasing the ratio of BCL-2/BAX and decreasing BIM expression, suggesting that ER stress is involved in MMC-induced fibroblast apoptosis. Interestingly, knockdown of PERK significantly decreased ER stress-mediated apoptosis by reducing the expression of CHOP, BIM and cleaved caspase-3. Reactive oxygen species (ROS) scavenging also decreased the expression of GRP78, phospho-PERK, CHOP, and BIM. These results demonstrate that MMC-induced apoptosis is triggered by ROS generation and PERK activation.

Mechanical stress has detrimental effects on cartilaginous endplate chondrocytes due to apoptosis in vivo and in vitro. In this study, we investigated the possible apoptosis signaling pathways induced by mechanical stress in cultured rat cervical endplate chondrocytes. Static mechanical load significantly reduced cell viability in a time- and load-dependent manner, as demonstrated by the Cell Counting Kit-8 (CCK-8) assay. Chondrocyte apoptosis induced by mechanical stress was confirmed by annexin V/propidium iodide (PI) staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Western blot analysis revealed that static load-induced chondrocyte apoptosis was accompanied by increased phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 mitogen-activated protein kinase (MAPK). The loss of mitochondrial membrane potential (ΔΨm), increased Cytochrome c release, and activated Caspase-9 and Caspase-3, indicating that the mitochondrial pathway is involved in mechanical stress-induced chondrocyte apoptosis. Treatment with inhibitors of JNK (SP600125), p38 MAPK (SB203580), and ERK (PD98059) prior to mechanical stimulation reversed both the static load-induced chondrocyte apoptosis and the activation of JNK, p38 MAPK, and ERK. Taken together, the data presented in this study demonstrate that mechanical stress induces apoptosis in rat cervical endplate chondrocytes through the MAPK-mediated mitochondrial apoptotic pathway.

Differences in the levels of acetylcholinesterase (AChE) in ventral and dorsal spinal roots can be used to differentiate the spinal nerves. Although many methods are available to assay AChE, a rapid and sensitive method has not been previously developed. Here, we describe an antibody-based quartz crystal microbalance (QCM) assay and its application for the quantification of AChE in the solutions of ventral and dorsal spinal roots. The frequency variation of the QCM device corresponds to the level of AChE over a wide dynamic range (0.5-10 µg/ml), which is comparable to the response range of the ELISA method. The frequency shift caused by the ventral roots is 3-fold greater than that caused by the dorsal roots. The antibody-based QCM sensor was stable across many successive replicate samples, and the method required less than 10 min, including the AChE extraction and analysis steps. This method is a rapid and convenient means for the quantification of AChE in biological samples and may be applicable for distinguishing the ventral and dorsal roots during surgical operations.

Although the capacity of exogenous PTH1-34 to enhance the rate of bone repair is well established in animal models, our understanding of the mechanism(s) whereby PTH induces an anabolic response during skeletal repair remains limited. Furthermore it is unknown whether endogenous PTH is required for fracture healing and how the absence of endogenous PTH would influence the fracture-healing capacity of exogenous PTH.Closed mid-diaphyseal femur fractures were created and stabilized with an intramedullary pin in 8-week-old wild-type and Pth null (Pth(-/-)) mice. Mice received daily injections of vehicle or of PTH1-34 (80 µg/kg) for 1-4 weeks post-fracture, and callus tissue properties were analyzed at 1, 2 and 4 weeks post-fracture. Cartilaginous callus areas were reduced at 1 week post-fracture, but were increased at 2 weeks post-fracture in vehicle-treated and PTH-treated Pth(-/-) mice compared to vehicle-treated and PTH-treated wild-type mice respectively. The mineralized callus areas, bony callus areas, osteoblast number and activity, osteoclast number and surface in callus tissues were all reduced in vehicle-treated and PTH-treated Pth(-/-) mice compared to vehicle-treated and PTH-treated wild-type mice, but were increased in PTH-treated wild-type and Pth(-/-) mice compared to vehicle-treated wild-type and Pth(-/-) mice.Absence of endogenous PTH1-84 impedes bone fracture healing. Exogenous PTH1-34 can act in the absence of endogenous PTH but callus formation, including accelerated endochondral bone formation and callus remodeling as well as mechanical strength of the bone are greater when endogenous PTH is present. Results of this study suggest a complementary role for endogenous PTH1-84 and exogenous PTH1-34 in accelerating fracture healing.

Leukocyte telomere length (LTL) is a complex genetic trait. It shortens with age and is associated with a host of aging-related disorders. Recent studies have observed that offspring of older fathers have longer LTLs. We explored the relation between paternal age and offspring's LTLs in 4 different cohorts. Moreover, we examined the potential cause of the paternal age on offspring's LTL by delineating telomere parameters in sperm donors. We measured LTL by Southern blots in Caucasian men and women (n=3365), aged 18-94 years, from the Offspring of the Framingham Heart Study (Framingham Offspring), the NHLBI Family Heart Study (NHLBI-Heart), the Longitudinal Study of Aging Danish Twins (Danish Twins), and the UK Adult Twin Registry (UK Twins). Using Southern blots, Q-FISH, and flow-FISH, we also measured telomere parameters in sperm from 46 young (50 years) donors. Paternal age had an independent effect, expressed by a longer LTL in males of the Framingham Offspring and Danish Twins, males and females of the NHLBI-Heart, and females of UK Twins. For every additional year of paternal age, LTL in offspring increased at a magnitude ranging from half to more than twice of the annual attrition in LTL with age. Moreover, sperm telomere length analyses were compatible with the emergence in older men of a subset of sperm with elongated telomeres. Paternal age exerts a considerable effect on the offspring's LTL, a phenomenon which might relate to telomere elongation in sperm from older men. The implications of this effect deserve detailed study.