Evaluating stress and displacement in the craniomandibular complex using Twin Block appliances at varied angles: A finite element study.

OBJECTIVES: The objective of this investigation was to assess the stress and displacement pattern of the craniomandibular complex by employing finite element methodology to simulate diverse angulations of inclined planes that are incorporated in the Twin Block appliance. METHODS: A 3D finite element representation was established by use of Cone Beam Computed Tomography (CBCT) scans. This comprehensive structure included craniofacial skeletal components, the articular disc, a posterior disc elastic layer, dental elements, periodontal ligaments, and a Twin Block appliance. This investigation is the first to incorporated inclined planes featuring three distinct angulations (45, 60, and 70°) as the study models. Mechanical impacts were evaluated within the glenoid fossa, tooth, condylar, and articular disc regions. RESULTS: In all simulations, the stress generated by the Twin Block appliance was distributed across teeth and periodontal ligament, facilitating the anterior movement of mandibular teeth and the posterior displacement of maxillary teeth. Within the temporomandibular joint region, compressive forces on the superior and posterior facets of the condyle diminished, coinciding with the stress configuration that fosters condylar and mandibular growth. Stress dispersion homogenized in the condylar anterior facet and articular disc, with considerable tensile stress in the glenoid fossa's posterior aspect conforming to stress distribution that promote fossa reconfiguration. The 70° inclined plane exerts the highest force on the tissues. The condyle's maximum and minimum principal stresses are 0.36 MPa and -0.15 MPa, respectively, while those of the glenoid fossa are 0.54 MPa and -0.23 MPa. CONCLUSION: Three angled appliances serve the purpose of advancing the mandible. A 45° inclined plane relative to the occlusal plane exerts balanced anteroposterior and vertical forces on the mandibular arch. Steeper angles yield greater horizontal forces, which may enhance forward growth and efficient repositioning.

MiR-143-3p regulates chondrogenic differentiation of synovium derived mesenchymal stem cells under mechanical stress through the BMPR2-Smad signalling pathway by targeting BMPR2.

BACKGROUND: Mesenchymal stem cells (MSCs) derived from the synovium, known as synovium mesenchymal stem cells (SMSCs), exhibit significant potential for articular cartilage regeneration owing to their capacity for chondrogenic differentiation. However, the microRNAs (miRNAs) governing this process and the associated mechanisms remain unclear. While mechanical stress positively influences chondrogenesis in MSCs, the miRNA-mediated response of SMSCs to mechanical stimuli is not well understood. OBJECTIVE: This study explores the miRNA-driven mechano-transduction in SMSCs chondrogenesis under mechanical stress. METHODS: The surface phenotype of SMSCs was analysed by flow cytometry. Chondrogenesis capacities of SMSCs were examined by Alcian blue staining. High throughput sequencing was used to screen mechano-sensitive miRNAs of SMSCs. The RNA expression level of COL2A1, ACAN, SOX9, BMPR2 and miR-143-3p of SMSCs were tested by quantitative real-time polymerase chain reaction (qRT-PCR). The interaction between miR-143-3p and TLR4 was confirmed by luciferase reporter assays. The protein expression levels of related genes were assessed by western blot. RESULTS: High-throughput sequencing revealed a notable reduction in miR-143-3p levels in mechanically stressed SMSCs. Gain- or loss-of-function strategies introduced by lentivirus demonstrated that miR-143-3p overexpression hindered chondrogenic differentiation, whereas its knockdown promoted this process. Bioinformatics scrutiny and luciferase reporter assays pinpointed a potential binding site for miR-143-3p within the 3'-UTR of bone morphogenetic protein receptor type 2 (BMPR2). MiR-143-3p overexpression decreased BMPR2 expression and phosphorylated Smad1, 5 and 8 levels, while its inhibition activated BMPR2-Smad pathway. CONCLUSION: This study elucidated that miR-143-3p negatively regulates SMSCs chondrogenic differentiation through the BMPR2-Smad pathway under mechanical tensile stress. The direct targeting of BMPR2 by miR-143-3p established a novel dimension to our understanding of mechano-transduction mechanism during SMSC chondrogenesis. This understanding is crucial for advancing strategies in articular cartilage regeneration.

Alteration in cartilage matrix stiffness as an indicator and modulator of osteoarthritis.

Osteoarthritis (OA) is characterized by cartilage degeneration and destruction, leading to joint ankylosis and disability. The major challenge in diagnosing OA at early stage is not only lack of clinical symptoms but also the insufficient histological and immunohistochemical signs. Alteration in cartilage stiffness during OA progression, especially at OA initiation, has been confirmed by growing evidences. Moreover, the stiffness of cartilage extracellular matrix (ECM), pericellular matrix (PCM) and chondrocytes during OA development are dynamically changed in unique and distinct fashions, revealing possibly inconsistent conclusions when detecting cartilage matrix stiffness at different locations and scales. In addition, it will be discussed regarding the mechanisms through which OA-related cartilage degenerations exhibit stiffened or softened matrix, highlighting some critical events that generally incurred to cartilage stiffness alteration, as well as some typical molecules that participated in constituting the mechanical properties of cartilage. Finally, in vitro culturing chondrocytes in various stiffness-tunable scaffolds provided a reliable method to explore the matrix stiffness-dependent modulation of chondrocyte metabolism, which offers valuable information on optimizing implant scaffolds to maximally promote cartilage repair and regeneration during OA. Overall, this review systematically and comprehensively elucidated the current progresses in the relationship between cartilage stiffness alteration and OA progression. We hope that deeper attention and understanding in this researching field will not only develop more innovative methods in OA early detection and diagnose but also provide promising ideas in OA therapy and prognosis.

Oral microbiome-driven virulence factors: A novel approach to pancreatic cancer diagnosis.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy, often associated with a poor prognosis for patients. One of the major challenges in managing PDAC is the difficulty in early diagnosis, owing to the limited and invasive nature of current diagnostic methods. Recent studies have identified the oral microbiome as a potential source of non-invasive biomarkers for diseases, including PDAC. In this study, we focused on leveraging the differential expression of virulence factors (VFs) encoded by the oral microbiome to create a diagnostic tool for PDAC. We observed a higher alpha diversity in VF categories among PDAC patients compared to healthy controls. We then identified a panel of VF categories that were significantly upregulated in PDAC patients, these being associated with bacterial adherence, exoenzyme production, and nutritional/metabolic processes. Moreover, Streptococcus-derived VFs were notably enriched in PDAC patients. We developed a diagnostic model using random forest analysis based on the levels of these VFs. The model's diagnostic accuracy was evaluated using receiver operating characteristic (ROC) curve analysis, with an area under the curve (AUC) of 0.88, indicating high accuracy in differentiating PDAC patients from healthy controls. Our findings suggest that VFs encoded by the oral microbiome hold potential as diagnostic tools for PDAC, offering a non-invasive approach that could significantly enhance early detection and prognosis, ultimately leading to improved patient outcomes.

Dual role of endoplasmic reticulum stress-ATF-6 activation in autophagy and apoptosis induced by cyclic stretch in myoblast.

OBJECTIVE: Mandibular growth that is induced by functional appliances is closely associated with skeletal and neuromuscular adaptation. Accumulating evidence has proved that apoptosis and autophagy have a vital role in adaptation process. However, little is known about the underlying mechanisms. This study sought to determine whether ATF-6 is involved in stretch-induced apoptosis and autophagy in myoblast. The study also sought to uncover the potential molecular mechanism. MATERIALS AND METHODS: Apoptosis was assessed by TUNEL and Annexin V and PI staining. Autophagy was detected by transmission electron microscopy (TEM) analysis and immunofluorescent staining for autophagy-related protein light chain 3 (LC3). Real time-PCR and western blot were performed to evaluate the expression level of mRNA and proteins that were associated with endoplasmic reticulum stress (ERS), autophagy and apoptosis. RESULTS: Cyclic stretch significantly decreased the cell viability and induced apoptosis and autophagy of myoblasts time-dependently. Stretching stimuli activated ATF-6 pathway and induced ERS-mediated apoptosis. Moreover, using 4-PBA significantly inhibited ERS-related apoptosis, as well as partially decreasing autophagy. In addition, inhibition of autophagy by 3-MA enhanced apoptosis by affecting the expression of CHOP and Bcl-2. However, it had no obvious effects on ERS-related proteins of GRP78 and ATF-6. More importantly, knockdown ATF-6 effectively weakened apoptosis and autophagy. It did so by regulating the expression of Bcl-2, Beclin1 and CHOP, but not cleaved Caspase-12, LC3II and p62 in stretched myoblast. CONCLUSION: ATF-6 pathway was activated in myoblast by mechanical stretch. ATF-6 may regulate the process of stretch-induced myoblast apoptosis and autophagy via CHOP, Bcl-2 and Beclin1 signaling.

Effect of N6-methyladenosine methylation-related gene signature for predicting the prognosis of hepatocellular carcinoma patients.

Background and aims: Hepatocellular carcinoma (HCC) is a common cause of cancer-related death in humans. Increasing evidence indicates that an imbalance in N6-methyladenosine (m6A) methylation is linked to the occurrence and development of cancer. We then developed a prognostic model as an independent risk factor with which predict the prognosis of HCC. Methods: We obtained the gene expression and clinical data of HCC patients from the TCGA databases. The prognostic value of m6A methylation-related genes in patients who had HCC were subjected to comprehensive bioinformatics analysis. We use Risk Score = ∑ i = 1 n Coe f i × X i to construct the risk scoring formula. We collected pathological specimens from 68 patients who had HCC, and conducted immunohistochemical staining experiments on the specimens. Results: There was a significant correlation between candidate m6A methylation-related genes (YTHDF2, METTL14 and ZC3H13) overall survival of HCC patients. Among the 68 HCC patient specimens that underwent immunohistochemical staining, all cancer tissues were positive for METTL14, YTHDF2, and ZC3H13 staining in contrast to the adjacent tissues. We conducted a Kaplan-Meier survival analysis. The results showed that patients who had low METTL14 expression had a longer survival time than those of patients who had high METTL14 expression. Also, patients with low YTHDF2 expression had a longer survival time than patients with high YTHDF2 expression. Finally, patients with high ZC3H13 expression lived longer than those with low ZC3H13 expression. This result is consistent with the bioinformatics analysis conclusion above. Conclusions: Generally, the prognostic model that was based on m6A methylation-related genes in this study can effectively predict the prognosis of HCC patients.

Wnt/ß-catenin signaling pathway is activated in the progress of mandibular condylar cartilage degeneration and subchondral bone loss induced by overloaded functional orthopedic force (OFOF).

Objective: To explore the role of Wnt/ß-catenin signaling pathway in the pathogenesis and progression of temporomandibular joint osteoarthritis (TMJ OA) caused by overloaded force. Materials and methods: We generated a rat model of forward mandibular extension device to induce TMJ OA by overloaded force. Condylar cartilage samples were collected at 2wk, 4wk, and 8wk after appliances were installed. Changes of the condylar cartilage and subchondral bone were evaluated by hematoxylin and eosin (HE), Safranin O and Fast Green staining (SO&FG), micro-CT, tartrate resistant acid phosphatase (TRAP) staining. The expression levels of ß-catenin, COL-2, MMP3 and sclerostin (SOST) were detected by immunohistochemistry (IHC) and PCR. Results: HE, SO&FG, micro-CT, OARSI and Mankin scores showed that the condyle cartilage layer was significantly thinner and proteoglycan loss in the overloded group. TRAP staining exhibited that the number of positive osteoclasts increased and OPG level decreased in the overload group. IHC, PCR showed that the expression of COL2 and SOST decreased, while MMP3 and ß-catenin increased in the overload group. Conclusion: Wnt/ß-catenin signaling pathway is activated in the progress of mandibular condylar cartilage degeneration and subchondral bone loss induced by overloaded functional orthopedic force (OFOF).

Intensive stretch-activated CRT-PMCA1 feedback loop promoted apoptosis of myoblasts through Ca2+ overloading.

Mechanical stretch exerted pro-apoptotic effect on myoblasts, the mechanism of which is currently unknown. Intracellular Ca2+ accumulation has been implicated in stretch-induced apoptosis. calreticulin (CRT) and plasma membrane Ca2+ transporting ATPase 1 (PMCA1) are two critical components of Ca2+ signaling system participating in intracellular Ca2+ homeostasis. In this study, we explored the contribution of CRT and PMCA1 in mediating stretch-induced Ca2+ accumulation and apoptosis of myoblasts. Stretching stimuli elevated level of CRT while inhibited activity of PMCA1. Moreover, there were bidirectional regulations between CRT and PMCA1, which formed the positive feedback loop leading to continuous increment of CRT level and repression of PMCA1 activity, in stretched myoblasts. Specifically, increased CRT level inhibited PMCA1 activity via suppressing Calmodulin (CaM), while reduced PMCA1 activity promoted CRT expression through activating p38MAPK pathway. Thus, the CRT-CaM-PMCA1 and PMCA1-p38MAPK-CRT pathways constituted a close cycle comprising CRT, PMCA1, CaM and p38MAPK. Inhibition of both CaM and p38MAPK affected the other three factors in stretched myoblasts. Circulation of the vicious cycle resulted in escalated Ca2+ overloading in myoblasts under continuous stretching stimuli. CRT knock-down, PMCA1 overexpression, and p38MAPK inhibition all attenuated the raised intracellular Ca2+ level and ameliorated myoblast apoptosis in the stretching environment. Conversely, CRT overexpression, PMCA1 knock-down, and CaM inhibition all aggravated stretch-induced Ca2+ overloading and myoblast apoptosis. A positive feedback loop between CRT and PMCA1 was activated in stretched myoblasts, which contributed to intracellular Ca2+ accumulation and resultant myoblast apoptosis.

The effects of specialized emergency and intensive nursing team on arterial blood gas and pulmonary function in pulmonary infection with respiratory failure.

OBJECTIVE: To investigate effects of specialized emergency and intensive nursing team on arterial blood gas and pulmonary function in pulmonary infected patients with respiratory failure. METHODS: 126 patients with pulmonary infection and respiratory failure admitted to our hospital were chosen and randomly divided into observation group and control group, with 63 cases in each group. The control-group received specialized routine nursing care, and the observation-group was treated with the emergency and intensive nursing care. Subsequently, the arterial blood gas, pulmonary function, inflammatory biomarkers, complication rate, recovery course and nursing satisfaction between the two groups were compared accordingly. RESULTS: After nursing care, the arterial blood gas and pulmonary function indexes of the two groups were remarkably improved than before, and the improvement in observation-group was superior to that in control-group (P<0.05); The inflammatory indicators of hs-CRP and PCT in two groups decreased substantially than before, and observation-group had remarkably lower indicators than that of the control-group (P<0.05); The incidence of complications in observation-group was 4.76%, significantly lower than 19.05% in control-group (P<0.05); The objects in observation-group spent exactly shorter time on ventilator than whom in control-group, and the difference was statistically significant (P<0.05); The observation-group had critically shorter length of hospital stay than those in control-group (P<0.05). The satisfaction of the observation-group with nursing care was 93.65%, which was dramatically higher than 73.02% in control-group (P<0.05). CONCLUSIONS: For pulmonary infection and respiratory failure, the nursing intervention carried by the specialized emergency and intensive nursing team can remarkably improve the arterial blood gas and pulmonary function, reduce the patients' inflammatory indicators and incidence of complications. The application of the nursing team can reduce the time on ventilator and length of hospital stay, and improve patients' satisfaction with nursing care.

Effect of standardized enteral nutrition on AECOPD patients with respiratory failure.

OBJECTIVE: To investigate the effects of standardized enteral nutrition (EN) on nutritional indicators and immunological functioning of acute exacerbations of chronic obstructive pulmonary disease (AECOPD) patients with respiratory failure. METHODS: We selected 92 cases of AECOPD patients with respiratory failure as the research objects, and classified them into two groups (control/observation group, n=46 respectively) according to random number table. Both groups were given conventional anti-infection and symptomatic treatment. In addition, the control group received diet support therapy, and the observation group was given standard EN treatment. Afterwards, the changes of nutritional indicators, immunological functioning, inflammatory indicators and cardiopulmonary function of the two groups before and after therapy were compared. RESULTS: Hemoglobin (HB), serum albumin (ALB), and total protein (TP) of the two groups after treatment were critically higher than those before treatment, and the indicators in observation group in post-treatment were remarkably higher than those in control group (P<0.05); lymphocyte count (TLC), immunoglobulin A (IgA) and immunoglobulin G (IgG) of the two groups tremendously increased in post-treatment than before receiving treatment, and the post-treatment indicators of observation group were obviously higher than those of control group (P<0.05); high sensitivity C-reactive protein (hs-CRP) and procalcitonin (PCT) in two groups sharply decreased after treatment comparing to which before treatment, and the observation group had notably lower post-treatment indexes than that of the control group (P<0.05); Ejection minutes (LVEF), NT proBNP, partial pressure of carbon dioxide (PaCO2), partial pressure of blood oxygen (PaO2) and pH of the two groups had remarkably improved after treatment, and the improvement effect in observation group was superior to that in control group (P<0.05). CONCLUSION: The standard EN can substantially improve the nutritional status and immunological functioning of AECOPD patients with respiratory failure, reduce the inflammatory indicators, and promote their cardiopulmonary function.

Molecular and Biomechanical Adaptations to Mechanical Stretch in Cultured Myotubes.

Myotubes are mature muscle cells that form the basic structural element of skeletal muscle. When stretching skeletal muscles, myotubes are subjected to passive tension as well. This lead to alterations in myotube cytophysiology, which could be related with muscular biomechanics. During the past decades, much progresses have been made in exploring biomechanical properties of myotubes in vitro. In this review, we integrated the studies focusing on cultured myotubes being mechanically stretched, and classified these studies into several categories: amino acid and glucose uptake, protein turnover, myotube hypertrophy and atrophy, maturation, alignment, secretion of cytokines, cytoskeleton adaption, myotube damage, ion channel activation, and oxidative stress in myotubes. These biomechanical adaptions do not occur independently, but interconnect with each other as part of the systematic mechanoresponse of myotubes. The purpose of this review is to broaden our comprehensions of stretch-induced muscular alterations in cellular and molecular scales, and to point out future challenges and directions in investigating myotube biomechanical manifestations.

Risk factors for emergence agitation in adults after general anesthesia: A systematic review and meta-analysis.

BACKGROUND: Emergence agitation (EA) is an adverse post-operative complication that increases the risk for injury, self-extubation, hemorrhages, and prolonged hospitalization. This meta-analysis aims to define the risk factors for adult EA after general anesthesia and provide recommendations for clinical practice. METHODS: Embase, PubMed, Medline, and the Cochrane Library databases were comprehensive retrieved. Observational studies that reported the risk factors for adult EA were enrolled. Review Manager 5.4 was used to analyze the extracted data. RESULTS: Eighteen observational studies involving 16, 678 adult patients were enrolled in this study. Eighteen pre-operative and nineteen intraoperative factors with unadjusted data, and five pre-operative and five intraoperative factors with adjusted data were meta-analyzed separately. Among them, seven factors (age, male, smoking, history of substance misuse, inhalational anesthesia, urinary catheter, complain of pain, or need analgesic drug use in post-anesthetic care unit) were the risk factors no matter meta-analyzed by unadjusted data or adjusted data. Intraoperative use of benzodiazepines was the risk factor when meta-analyzed by adjusted data, but not unadjusted data. Moreover, age and inhalational anesthesia were not the risk factors when omitted one study for sensitivity analysis, and history of substance misuse could not do sensitivity analysis. CONCLUSION: Based on this meta-analysis, male, smoking, urinary catheter, and post-operative pain are the risk factors, while age, inhalational anesthesia, history of substance misuse, and intraoperative use of benzodiazepines are the possible risk factors for adult EA. EDITORIAL COMMENT: This systematic review and meta-analysis identify risk factors associated with the occurrence of agitation during emergence from general anesthesia. As might be expected, the strongest factors are generally things which are irritating or painful for patients, but cannot necessarily be avoided.

Novel Insights Into the Role of N6-Methyladenosine RNA Modification in Bone Pathophysiology.

Thus far, there are more than known 150 modifications to RNA, in which common internal modifications of mRNA include N6-methyladenosine (m6A), N1-methyladenosine, and 5-methylcytosine. Among them, m6A RNA modification is one of the highest abundance modifications in eukaryotes, regulating mechanisms controlling gene expression at the post-transcription level. As an invertible and dynamic epigenetic marker, m6A base modification influences almost all vital biological processes, cellular components, and molecular functions. Once the m6A modification process is abnormal, a series of diseases-including cancer, neurological diseases, and growth disorders-will be caused. Besides, several base modification activities also have been created by noncoding RNAs (ncRNAs), for instance, microRNAs, and circular RNAs, long ncRNAs, which were dynamically regulated during bone and cartilage pathophysiology processes. Therefore, it has now been clear that dynamic modification on coding RNAs and ncRNAs represents a completely new way to modulate genetic information. In this review, we highlight up-to-date progress and applications of m6A RNA modification in bone and cartilage pathophysiology, and we discuss the pathological roles and underlying molecular mechanism of m6A modifications in osteoarthritis and osteoporosis and osteosarcoma pathogenesis.

Overloaded Orthopedic Force Induces Condylar Subchondral Bone Absorption by Stimulating Rat Mesenchymal Stem Cells Differentiating into Osteoclasts via mTOR-Regulated RANKL/OPG Secretion in Osteoblasts.

Appropriate orthopedic force led to bone remodeling of mandibular condyle, while overloaded orthopedic force (OOF) induced condylar bone absorption. Bone absorption is ascribed to the imbalanced activities between osteoclasts (OCs) and osteoblasts (OBs), mechanism of which remains unclear. This study aimed to observe the condylar changes induced by OOF by mandible advancement appliance and to further investigate the role of mammalian target of Rapamycin (mTOR) and RANKL/OPG in osteoclastic differentiation of stem cells in vivo and in vitro. In vivo, the results of micro-CT analysis indicated that condylar bone resorption was induced by OOF through mandibular advancement appliance for 2 weeks and worsened time dependently. Morphologically, cartilage thickness was reduced, subchondral cortical bone line appeared not continuous, and subchondral bone exhibited irregular-shaped and owned uneven surface. The bone mineral density (BMD), bone volume/tissue volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) were decreased accomplished with the increased trabecular separation (Tb.Sp) determined by micro-CT. In addition, based on immunofluorescent labeling, OOF activated both OCs and OBs, but osteoclastogenesis prevailed over osteogenesis. The mTOR activation and ratio of RANKL/OPG in OBs were elevated by OOF. In vitro, the results of western blot and polymerase chain reaction (PCR) consistently suggested that the mTOR and RANKL/OPG ratio were upregulated by overloaded mechanical stretch. Pretreatment with mTOR inhibitor, rapamycin, could attenuate the activation of mTOR and the secretion of RANKL in OBs. Interestingly, based on the Trap staining, the supernatant of OBs exposed to OOF could promote osteoclastic differentiation of mesenchymal stem cells (MSCs), while its role was weakened by inhibition of mTOR in OBs. Collectively, OOF induced condylar bone absorption; in the process, osteoclastogenesis was prominent than osteogenesis. The activation of mTOR and secretion of RANKL/OPG were enhanced by OOF and were involved in promoting MSCs differentiating into OCs.

Multiple Effects of Mechanical Stretch on Myogenic Progenitor Cells.

Mechanically stretched skeletal muscle undergoes dramatic shifts in structure, mass, and function. In vitro tensile strain models have demonstrated that myogenic progenitor cells, including satellite cells and myoblasts, are highly mechanosensitive cells, and respond to mechanical strain in a wide variety of aspects. However, the experimental results from different researchers and laboratories are not always in support of each other. Moreover, some specific molecules or signaling pathways were reported to play distinct roles in stretched myogenic cells, according to the statements of different studies. The purpose of this review is to integrate the researches conducting in vitro culture of satellite cells or myoblasts and exploring their mechanoresponses using in vitro stretching apparatus. These responses will be categorized into several groups, such as activation, proliferation, myogenic differentiation, cellular damage or apoptosis, properties of plasma membrane, transdifferentiation, reorientation, etc. In addition, detailed experimental designs like culturing conditions and straining regimens will be displayed and compared, to interpret some contradictory statements in different studies. Furthermore, the currently known interconnections among some mechanosensitive pathways will be pictured to give a better understanding about the complex regulations of myogenic cell responses to mechanical stretch. Hopefully, by summarizing the published studies about mechanoresponses of myogenic progenitor cells, future directions, and perspectives would be made clearer to researchers in this field.

Rational design to improve activity of the Est3563 esterase from Acinetobacter sp. LMB-5.

Acinetobacter sp. strain LMB-5 can produce a kind of esterase degrading phthalate esters. However, low activity of Est3563 esterase limited its large-scale application. In this study, computer-aided simulation mutagenesis was used to improve the esterase activity with a tightened screening library and enlarged success rate. Two positive mutants, P218R and A242R, were obtained with 2.5 and 2.1 folds higher than the WT Est3563 esterase, with 11.96 ± 0.45 U·mg-1 and 9.90 ± 0.52 U·mg-1, respectively. With the help of bioinformatics analysis and three-dimensional printing technology, it was found that the mutations could increase the 240-280 residues swing distance and make them deviate from the catalytic pocket. The instability and deviation of these residues on the lid-like structure of the esterase could deteriorate the seal of the binding pocket and expose the active site. Thus, the catalytic efficiency of the mutants became higher. This result demonstrates that the instability and deviation of the lid-like structure could expand the binding pocket of the esterase and enhance the esterase activity.

Stretching magnitude-dependent inactivation of AKT by ROS led to enhanced p53 mitochondrial translocation and myoblast apoptosis.

Previously, we had shown that high magnitude stretch (HMS), rather than low magnitude stretch (LMS), induced significant apoptosis of skeletal muscle C2C12 myoblasts. However, the molecular mechanism remains obscure. In this study, we found that p53 protein accumulated in the nucleus of LMS-loaded cells, whereas it translocated into mitochondria of HMS-loaded cells. Knocking down endogenous p53 by shRNA abrogated HMS-induced apoptosis. Furthermore, we demonstrated that overaccumulation of reactive oxygen species (ROS) during HMS-inactivated AKT that was activated in LMS-treated cells, which accounted for the distinct p53 subcellular localizations under HMS and LMS. Blocking ROS generation by N-acetylcysteine (NAC) or overexpressing constitutively active AKT vector (CA-AKT) inhibited HMS-incurred p53 mitochondrial translocation and promoted its nuclear targeting. Moreover, both NAC and CA-AKT significantly attenuated HMS-induced C2C12 apoptosis. Finally, we found that Ser389 phosphorylation of p53 was a downstream event of ROS-inactivated AKT pathway, which was critical to p53 mitochondrial trafficking during HMS stimuli. Transfecting p53-shRNA C2C12s with the mutant p53 (S389A) that was unable to target p53 to mitochondria underwent significantly lower apoptosis than transfection with wild-type p53. Altogether, our study uncovered that mitochondrial localization of p53, resulting from p53 Ser389 phosphorylation through ROS-inactivated AKT pathway, prompted C2C12 myoblast apoptosis during HMS stimulation.

Effects of silencing the HIF-1α gene on the expression of BSP and osterix in rat BMMSCs under tension / 口腔疾病防治

Objective@#To explore the effect of hypoxia inducible factor 1α (HIF-1α) gene silencing in rat bone marrow mesenchymal stem cells (BMMSCs) under mechanical distraction on the expression of bone sialoprotein (BSP) and osterix and to provide a new idea for repairing bone defects with BMMSCs.@*Methods @#The shRNA sequence was designed according to the rat HIF-1α gene, and the pGMLV-SC1RNAi lentiviral vector was cloned after PCR amplification. After screening positive clones and identifying competent transformed cells by sequencing, 293T cells were packaged and titered, rat BMMSCs were transfected and cultured in vitro. Clones with stably silenced HIF-1α expression were screened by inverted fluorescence microscopy. The RNAi response experiment was divided into four groups: the blank control group, the HIF-1α shRNA group, the negative control group, and the response group. Western blot was used to detect the expression of HIF-1α protein in the four groups to verify the response of the target genes and exclude off-target effects. A Flexcell FX-5000T cell stress loading system was used to intervene in the mechanical stretch of the cells. qRT-PCR and Western blot were used to detect the expression of BSP and osterix in the blank control group, HIF-1α shRNA group, and negative control group.@*Results@#The HIF-1α shRNA lentiviral vector was successfully constructed. The results of the RNAi response showed no significant difference in the expression of HIF-1α between the response and the blank control group (P > 0.05). The recombinant lentivirus could effectively silence HIF-1α in BMMSCs. After mechanical distraction of the BMMSCs, compared with the blank and negative control groups, the HIF-1α shRNA group showed significantly increased mRNA and protein expression of the bone-related factors BSP and osterix (P < 0.05); there was no significant difference in the mRNA and protein expression of BSP or osterix between the blank and negative control groups (P > 0.05).@*Conclusion @#Silencing HIF-1α in BMMSCs under mechanical distraction can promote the expression of BSP and osterix.

Cleavage of caspase-12 at Asp94, mediated by endoplasmic reticulum stress (ERS), contributes to stretch-induced apoptosis of myoblasts.

Mechanical overloading can lead to skeletal muscle damage instead of remodeling. This is attributed to the excessive apoptosis of myoblasts, mechanism of which remains to be elucidated. The present study aimed to investigate the involvement of endoplasmic reticulum stress (ERS) and caspase-12 in mediating the stretch-induced apoptosis of myoblasts. Myoblast apoptosis was evaluated by Hoechst staining, DNA fragmentation assay, Annexin V binding, and propidium iodide staining, as well as caspase-3 and poly-ADP-ribose polymerase 1 cleavage. First, our results showed that apoptosis was elevated in a time-dependent manner when myoblasts were subjected to cyclic mechanical stretch (CMS) for 12, 24, and 36 hr. Concomitantly, CMS triggered the ERS and caspase-12 cleavage; ERS inhibitor GSK 2606414 suppressed the CMS-induced cleavage of caspase-12 and myoblast apoptosis. Silencing caspase-12 attenuated the apoptosis of myoblasts under CMS. Furthermore, CMS-induced myoblast apoptosis was partially recovered by overexpressing wild-type caspase-12 in caspase-12-silenced myoblasts. In contrast, overexpressing mutant caspase-12 (D94N), which cannot be cleaved into the active caspase-12 fragments, failed to accomplish the same effect. Finally, C2C12 overexpressing truncated caspase-12 segment (TC-casp12-D94), which starts from Asp94 and ends at Asn419, underwent apoptosis under both static and stretched conditions. Interestingly, C2C12 myoblasts seemed to be resistant to stretch-induced apoptosis upon low-serum-induced differentiation. In conclusion, our study provided evidence that caspase-12 cleavage at Asp94, induced by ERS under mechanical stimuli, is the key molecule in initiating the stretch-triggered apoptosis of myoblasts.