Development and validation of a clinical prediction model for early ventilator weaning in post-cardiac surgery.

Background: Weaning patients from mechanical ventilation is a critical clinical challenge post cardiac surgery. The effective liberation of patients from the ventilator significantly improves their recovery and survival rates. This study aimed to develop and validate a clinical prediction model to evaluate the likelihood of successful extubation in post-cardiac surgery patients. Method: A predictive nomogram was constructed for extubation success in individual patients, and receiver operating characteristic (ROC) and calibration curves were generated to assess its predictive capability. The superior performance of the model was confirmed using Delong's test in the ROC analysis. A decision curve analysis (DCA) was conducted to evaluate the clinical utility of the nomogram. Results: Among 270 adults included in our study, 107 (28.84%) experienced delayed extubation. A predictive nomogram system was derived based on five identified risk factors, including the proportion of male patients, EuroSCORE II, operation time, pump time, bleeding during operation, and brain natriuretic peptide (BNP) level. Based on the predictive system, five independent predictors were used to construct a full nomogram. The area under the curve values of the nomogram were 0.880 and 0.753 for the training and validation cohorts, respectively. The DCA and clinical impact curves showed good clinical utility of this model. Conclusion: Delayed extubation and weaning failure, common and potentially hazardous complications following cardiac surgery, vary in timing based on factors such as sex, EuroSCORE II, pump duration, bleeding, and postoperative BNP reduction. The nomogram developed and validated in this study can accurately predict when extubation should occur in these patients. This tool is vital for assessing risks on an individual basis and making well-informed clinical decisions.

Application of neutrophil-to-lymphocyte-to-monocyte ratio in predicting mortality risk in adult patients with septic shock: A retrospective cohort study conducted at a single center.

Background: Sepsis is a life-threatening condition characterized by an aberrant host response to infection, resulting in multi-organ dysfunction. The application of currently available prognostic indicators for sepsis in primary hospitals is challenging. In this retrospective study, we established a novel index, the neutrophil-to-lymphocyte-to-monocyte ratio (NLMR), based on routine blood examination upon admission, and assessed its prognostic value for early mortality risk in adult patients with septic shock. Methods: This study included clinical data from adult patients with septic shock who were admitted to the hospital between January 1, 2018, and December 31, 2022. Training and validation sets were constructed, and patients were categorized into "survival" and "death" groups based on their survival status within the 28-day hospitalization period. Baseline data, including demographic characteristics and comorbidities, and laboratory results, such as complete blood count parameters, were collected for analysis. The Sequential Organ Failure Assessment (SOFA) and Acute Physiology and Chronic Health Evaluation II (APACHE II) scores were documented.The NLMR was determined through the utilization of multivariate binary logistic regression analysis, leading to the development of a risk model aimed at predicting early mortality in adult patients suffering from septic shock. Results: Overall, 112 adult patients with septic shock were enrolled in this study, with 84 and 28 patients in the training and validation sets, respectively. Multivariate binary logistic analysis revealed that the neutrophil, lymphocyte, and monocyte counts independently contributed to the mortality risk (odds ratios = 1.22, 0.08, and 0.16, respectively). The NLMR demonstrated an area under the receiver operating characteristic curve (ROC-AUC) of 0.83 for internal validation in the training set and 0.97 for external validation in the validation set. Both overall model quality values were significantly high at 0.74 and 0.91, respectively (P < 0.05). NLMR exhibited a higher ROC-AUC value of 0.88 than quick SOFA (ROC-AUC = 0.71), SOFA (ROC-AUC = 0.83), and APACHE II (ROC-AUC = 0.78). Conclusion: NLMR may be a potential marker for predicting the risk of early death in adult patients with septic shock, warranting further exploration and verification.

Inhibition of colon C5a/C5a receptor signalling pathway confers protection against LPS-induced acute kidney injury via gut microbiota-kidney axis.

Acute kidney injury (AKI) is a critical condition often associated with systemic inflammation and dysregulated gut microbiota. This study aimed to investigate the effects of the C5a receptor antagonist W54011 on lipopolysaccharide (LPS)-induced AKI, focusing on the colon's C5a/C5a receptor pathway, intestinal barrier integrity, and gut microbiota. Our findings demonstrate that W54011 effectively ameliorated kidney injury in the LPS-induced AKI model by selectively inhibiting the colon's C5a/C5a receptor signalling pathway. Additionally, C5a receptor blockade resulted in the inhibition of colonic inflammation and the reconstruction of the intestinal mucosal barrier. Furthermore, W54011 administration significantly impacted the composition and stability of the gut microbiota, restoring the abundance of dominant bacteria to levels observed in the normal state of the intestinal flora and reducing the abundance of potentially harmful bacterial groups. In conclusion, W54011 alleviates LPS-induced AKI by modulating the interplay between the colon, gut microbiota, and kidneys. It preserves the integrity of the intestinal barrier and reinstates gut microbiota, thereby mitigating AKI symptoms. These findings suggest that targeting the colon and gut microbiota could be a promising therapeutic strategy for AKI treatment.

Restoration of constitutional alignment optimizes outcomes of computer navigated total knee arthroplasty: a prospective randomized controlled trial.

PURPOSE: The value of computer navigation in total knee arthroplasty (TKA) for arthritic knees continues to be debated. The purpose of this study was to evaluate the value of navigated TKA associated with updated alignment philosophy. METHODS: This prospective randomized controlled trial enrolled 38 consecutive patients (76 knees) and were randomly assigned to both groups. The demographic data and perioperative data were recorded. The coronal plane alignment of the knee (CPAK) classification was used to classify knee alignment phenotypes. Radiographic outcomes were measured and subgroup analysis was further performed. Clinical outcomes were evaluated using patient-reported outcome measures (PROMs). Surgery-related complications were recorded. RESULTS: The distribution of CPAK phenotypes following constitutional aligned TKA was equivalent to the native cohort, whereas the mechanical aligned TKA dramatically altered the phenotype distribution from type I and type II to type V and type IV. Final implant positioning was different between groups, with constitutional aligned TKA having larger cTCA (P = .004), joint line obliquity (P = .006), joint line distance (P = .033) and smaller sFCA (P = .013). Subgroup analysis showed higher actual accuracy of component positioning was achieved in navigated TKA, especially in knees with deformity of > 10° (P < .05). Patients reported higher HSS score at three months postoperatively in constitutional aligned group (P = .002). One patient in navigated group suffered femoral pin site fracture caused by a minor trauma. CONCLUSION: Computer navigated TKA allows for restoration of constitutional alignment and minimizes soft tissue release, which when compared to mechanical alignment may be associated with superior early outcomes.

[Surgical technique of lateral unicompartmental knee arthroplasty and discussion of the maximum correction value in the treatment of knee valgus deformity].

Objective: To investigate the surgical technique and the short-term effectivenss of lateral unicompartmental knee arthroplasty (LUKA) through lateral approach in the treatment of valgus knee and to calculate the maximum value of the theoretical correction of knee valgus deformity. Methods: A retrospective analysis was performed on 16 patients (20 knees) who underwent LUKA and met the selection criteria between April 2021 and July 2022. There were 2 males and 14 females, aged 57-85 years (mean, 71.5 years). The disease duration ranged from 1 to 18 years, with an average of 11.9 years. Knee valgus was staged according to Ranawat classification, there were 6 knees of type Ⅰ, 13 knees of type Ⅱ, and 1 knee of type Ⅲ. All patients were assigned the expected correction value of genu valgus deformity by preoperative planning, including the correction value of lateral approach, intra-articular correction value, and residual knee valgus deformity value. The actual postoperative corrected values of the above indicators were recorded and the theoretical maximum correctable knee valgus deformity values were extrapolated. The operation time, intraoperative blood loss, incision length, hospital stay, hip-knee-ankle angle (HKA), mechanical lateral distal femoral angle (mLDFA), mechanical medial proximal tibia angle (mMPTA), joint line convergence angle (JLCA), posterior tibial slope (PTS), range of motion (ROM), Hospital for Special Surgery (HSS) score, and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score were also recorded for effectiveness evaluation. Results: The patients' incision length averaged 13.83 cm, operation time averaged 85.8 minutes, intraoperative blood loss averaged 74.9 mL, and hospital stay averaged 6.7 days. None of the patients suffered any significant intraoperative neurological or vascular injuries. All patients were followed up 10-27 months, with a mean of 17.9 months. One patient with bilateral knee valgus deformities had intra-articular infection in the left knee at 1 month after operation and the remaining patients had no complication such as prosthesis loosening, dislocation, and infection. The ROM, HSS score, and WOMAC score of knee joint significantly improved at each time point after operation when compared to those before operation, and the indicators further improved with time after operation, the differences were all significant ( P<0.05). Imaging measurement showed that HKA, mLDFA, JLCA, and PTS significantly improved at 3 days after operation ( P<0.05) except for mMPTA ( P>0.05). Postoperative evaluation of the knee valgus deformity correction values showed that the actual intra-articular correction values ranged from 0.54° to 10.97°, with a mean of 3.84°. The postoperative residual knee valgus deformity values ranged from 0.42° to 5.30°, with a mean of 3.59°. The actual correction values of lateral approach ranged from 0.21° to 12.73°, with a mean of 4.26°. Conclusion: LUKA through lateral approach for knee valgus deformity can achieve good early effectiveness. Preoperative planning can help surgeons rationally allocate the correction value of knee valgus deformity, provide corresponding treatment strategies, and the maximum theoretical correction value of knee valgus deformity can reach 25°.

The osteoprotective role of USP26 in coordinating bone formation and resorption.

Bone homeostasis is maintained through a balance of bone formation by osteoblasts and bone resorption by osteoclasts. Ubiquitin-specific proteases (USPs) are involved in regulating bone metabolism by preserving bone formation or antagonizing bone resorption. However, the specific USPs that maintain bone homeostasis by orchestrating bone formation and bone resorption simultaneously are poorly understood. Here, we identified USP26 as a previously unknown regulator of bone homeostasis that coordinates bone formation and resorption. Mechanistically, USP26 stabilizes ß-catenin to promote the osteogenic activity of mesenchymal cells (MSCs) and impairs the osteoclastic differentiation of bone myelomonocytes (BMMs) by stabilizing inhibitors of NF-κBα (IκBα). Gain-of-function experiments revealed that Usp26 supplementation significantly increased bone regeneration in bone defects in aged mice and decreased bone loss resulting from ovariectomy. Taken together, these data show the osteoprotective effect of USP26 via the coordination of bone formation and resorption, suggesting that USP26 represents a potential therapeutic target for osteoporosis.

[Early effectiveness of computer navigation-assisted total knee arthroplasty].

OBJECTIVE: To estimate the early effectivenss of computer navigation-assisted total knee arthroplasty (TKA) by comparing with traditional TKA. METHODS: The clinical data of 89 patients (100 knees) underwent primary TKA between October 2017 and July 2018 were analyzed retrospectively, including 44 patients (50 knees) who completed the TKA under the computer-assisted navigation system as the navigation group and 45 patients (50 knees) treated with traditional TKA as the control group. There was no significant difference between the two groups ( P>0.05) in gender, age, body mass index, diagnosis, side, disease duration, Kellgren-Lawrence classification of osteoarthritis, and preoperative American Hospital for Special Surgery (HSS) score, range of motion (ROM), hip-knee-ankle angle (HKA) deviation. The operation time, incision length, difference in hemoglobin before and after operation, postoperative hospital stay, and the complications were recorded and compared between the two groups. The HSS score, ROM, and joint forgetting score (FJS-12) were used to evaluate knee joint function in all patients. Unilateral patients also underwent postoperative time of up and go test and short physical performance battery (SPPB) test. At 1 day after operation, the HKA, mechanical lateral distal femoral angle (mLDFA), mechanical medial proximal tibial angle (mMPTA), sagittal femoral component angle (sFCA), and sagittal tibial component angle (sTCA) were measured and calculated the difference between the above index and the target value (deviation); and the joint line convergence angle (JLCA) was also measured. RESULTS: The operations of the two groups were successfully completed, and the incisions healed by first intention. The operation time and incision length of the navigation group were longer than those of the control group ( P<0.05); the difference in difference of hemoglobin before and after the operation and the postoperative hospital stay between groups was not significant ( P>0.05). Patients in the two groups were followed up 27-40 months, with an average of 33.6 months. Posterior tibial vein thrombosis occurred in 1 case in each of the two groups, and 1 case in the control group experienced repeated knee joint swelling. The HSS scores of the two groups gradually increased after operation ( P<0.05); HSS scores in the navigation group at 1 and 2 years after operation, and knee ROM and FJS-12 scores at 2 years were significantly higher than those in the control group ( P<0.05). There was no significant difference in the postoperative time of up and go test and SPPB results between the two groups at 7 days after operation ( P>0.05); the postoperative time of up and go test of the navigation group was shorter than that of the control group at 2 years ( t=-2.226, P=0.029), but there was no significant difference in SPPB ( t=0.429, P=0.669). X-ray film measurement at 1 day after operation showed that the deviation of HKA after TKA in the navigation group was smaller than that of the control group ( t=-7.392, P=0.000); among them, the HKA deviations of 50 knees (100%) in the navigation group and 36 knees (72%) in the control group were less than 3°, showing significant difference between the two groups ( χ 2=16.279, P=0.000). The JLCA and the deviations of mLDFA, mMPTA, sFCA, and sTCA in the navigation group were smaller than those in the control group ( P<0.05). CONCLUSION: Compared with traditional TKA, computer navigation-assisted TKA can obtain more accurate prosthesis implantation position and lower limb force line and better early effectiveness. But there is a certain learning curve, and the operation time and incision length would be extended in the early stage of technology application.

Osteocytic HIF-1α Pathway Manipulates Bone Micro-structure and Remodeling via Regulating Osteocyte Terminal Differentiation.

The activation of hypoxia-inducible factor 1α (HIF-1α) signaling has promising implications for the treatment of bone diseases such as osteoporosis and skeletal fractures. However, the effects of manipulating HIF-1α pathway on bone micro-structure and remodeling should be fully studied before the clinical application of therapeutics that interfere with the HIF-1α pathway. In this study, we found that osteocyte-specific HIF-1α pathway had critical role in manipulating bone mass accrual, bone material properties and micro-structures, including bone mineralization, bone collagen fiber formation, osteocyte/canalicular network, and bone remodeling. In addition, our results suggest that osteocyte-specific HIF-1α pathway regulates bone micro-structure and remodeling via impairing osteocyte differentiation and maturation.

Inhibition of Long Non-coding RNA CTD-2574D22.4 Alleviates LPS-induced Apoptosis and Inflammatory Injury of Chondrocytes.

BACKGROUND: Osteoarthritis (OA) is a common joint disease characterized by cartilage degeneration. Long non-coding RNAs (lncRNAs) have been associated with inflammatory diseases, including OA. Here, we investigated the potential molecular role of lncRNAs in OA pathogenesis. METHODS: ATDC5 cells were treated with lipopolysaccharides (LPS), and qPCR was used to identify and determine expression of potential lncRNAs involved in LPS-induced chondrocyte injury. Cell viability, apoptosis, and expression of cartilage-related genes and inflammatory cytokines were assessed after CTD-2574D22.4 knockdown. RESULTS: After LPS stimulation, CTD-2574D22.4 was found to be the second highest up-regulated gene, and the enhanced expression was validated in OA chondrocytes. Moreover, CTD-2574D22.4 inhibition significantly rescued cell viability, suppressed by LPS stress, and markedly attenuated LPS-induced apoptosis. The expression of cartilage-degrading enzymes MMP-13 and ADAMTS-5 were increased, while type II collagen was reduced after LPS treatment. This trend was largely reversed by CTD-2574D22.4 knockdown. Additionally, mRNA and protein levels of key inflammatory cytokines (TNF-a, IL-6, and IL-1ß) were significantly elevated in the LPS group and partially relieved upon CTD-2574D22.4 knockdown. CONCLUSION: CTD2574D22.4 knockdown ameliorates LPS-induced cartilage injury by protecting chondrocytes from apoptosis via anti-inflammation and anti- cartilage-degrading pathways. Thus, CTD2574D22.4 might be a potential diagnostic and therapeutic target for OA.

Analyzing the Influence of Modic Changes on Patients with Lower Back Pain Undergoing Conservative Treatment.

Objective: This study aimed to investigate if the presence of Modic changes (MCs) was correlated with lower back pain (LBP) and LBP-related disability in patients who underwent nonsurgical treatment. Methods: In this study, 129 patients who experienced consecutive LBP and underwent lumbar spine magnetic resonance imaging in our institute were divided into three groups according to the presence or type of MCs. The Oswestry Disability Index (ODI) and visual analog scale (VAS) were used to assess the outcomes of the treatment. Results: Based on the achieved results, there was no significant difference between three groups before treatment (P > 0.05). Three months after undergoing nonsurgical treatment, the rates of improved ODI and VAS scores were statistically significantly different (P=0.014, 0.023). After an additional 3 months of treatment, in patients with Modic type I changes, the symptoms significantly improved in comparison with those 3 months prior (P=0.037, 0.026), while that improvement did not occur in patients with Modic type II changes (P > 0.05). Conclusions: The existence of MCs affects the outcomes of nonsurgical treatment in patients with LBP. However, symptoms can be improved after an additional round of treatment for Modic type I changes, while this is not confirmed for Modic type II changes.

TRAF6 neddylation drives inflammatory arthritis by increasing NF-κB activation.

Neddylation is a process similar to ubiquitination, and is critical in various inflammatory diseases; however, its importance in the pathogenesis of inflammatory arthritis is not well understood. Here, we investigated the role of neddylation in collagen-induced arthritis (CIA) and its clinical relevance. We showed that neddylation-related genes, including NEDD8 and CULLIN-1, were significantly upregulated in inflamed arthritic synovia. Functionally, neddylation activation was crucial for synovitis of CIA, as the inhibition of neddylation by MLN4924 significantly suppressed synovial cell proliferation and inflammatory responses. Mechanistically, neddylation mediated inflammatory arthritis by regulating NF-κB activation in fibroblast-like synovial cells (FLSs). Furthermore, TNF receptor-associated factor 6 (TRAF6) neddylation at Lys124 was essential for IL-17A-induced NF-κB activation. Replacing the Lys-124 residue with Arg (K124R) resulted in significantly impaired conjugation of NEDD8 to TRAF6, as well as markedly attenuated IL-17A-induced NF-κB activity. Therefore, the pathogenic role of neddylation in CIA as well as its mechanism of action demonstrated here provides a new insight into understanding the role of post-transcriptional modifications in the arthritis inflammatory response.

Cytisine induces apoptosis of HepG2 cells.

Cytisine is a quinolizidine alkaloid, which has been reported to be among the major bioactive components of Sophora alopecuraides L. Quinolizidine alkaloids have previously been demonstrated to inhibit the proliferation of several types of tumor cells. However, few studies have investigated the effects of cytisine on cancer cells. The present study was performed to further investigate the molecular mechanisms underlying cytisine­induced apoptosis of HepG2 human hepatocellular carcinoma cells. The results of an MTT assay demonstrated that cytisine inhibited the growth of HepG2 cells in a dose­dependent manner. In addition, the induction of apoptosis was detected, as determined by morphological observation and flow cytometry. As determined by fluorescence microscopy, apoptotic morphological alterations were detected following cytisine administration. Flow cytometric analyses demonstrated that cytisine induced cytotoxicity through apoptosis­like mechanisms in HepG2 cells. Furthermore, western blot analysis was performed to investigate the release of cytochrome c (Cyt­c) and activation of the caspase cascade, and the results indicated that treatment of HepG2 cells with cytisine induced caspase­dependent apoptosis via the release of Cyt­c from the mitochondria, upregulation of caspase­3 and downregulation of pro­caspase­3. These results indicated that cytisine may induce apoptosis of HepG2 cells through the mitochondrial pathway.

Over-Expression of POU Class 1 Homeobox 1 Transcription Factor (Pit-1) Predicts Poor Prognosis for Breast Cancer Patients.

BACKGROUND: The POU class 1 homeobox 1 transcription factor (POU1F1, also known as Pit-1) was reported to be associated with tumor progression and metastasis. The purpose of this study was to evaluate the prognostic value of Pit-1 in breast cancer patients. MATERIAL AND METHODS: The relative expression levels of Pit-1 in breast cancer patients were detected by quantitative real-time PCR (qRT-PCR). Chi-square analysis was used to analyze the association between Pit-1 expression and clinical features. The Kaplan-Meier method was used to estimate the overall survival of the patients and Cox regression analysis was used to analyze the prognostic value of Pit-1. RESULTS: Increased expression of Pit-1 was detected in the tumor tissues compared with the normal tissues (1.086 vs. 0.541) and the abnormal expression was associated with tumor size, clinical stage, tumor grade, and lymph node metastasis (P<0.05). High expression level of Pit-1 was significantly associated with poor overall survival of the patients (P=0.001) and Cox regression analysis indicated that Pit-1 might be a prognostic factor for breast cancer prognosis (HR=1.955, 95% CI=1.295-3.035, P=0.003). CONCLUSIONS: Pit-1 may be a potential prognostic biomarker for breast cancer patients and it is associated with tumor progression.

Biphasic photoelectrochemical sensing strategy based on in situ formation of CdS quantum dots for highly sensitive detection of acetylcholinesterase activity and inhibition.

Herein, we reported a facile and highly sensitive biphasic photoelectrochemical (PEC) sensing strategy based on enzymatic product-mediated in situ formation of CdS quantum dots (QDs), and assayed the activity and inhibition of acetylcholinesterase (AChE) in its optimal state. Upon the hydrolysis of acetylthiocholine catalyzed by AChE, the product thiocholine stabilizes the in situ formation of CdS QDs in homogenous solution. Due to the electrostatic attraction, the resulting tertiary amino group-functionalized CdS QDs are attached to the surface of the negatively charged indium tin oxide (ITO) electrode, generating significant PEC response upon illumination in the presence of electron donors. By taking full advantage of the in situ formation of CdS QDs in homogenous solution, this strategy is capable of detecting AChE activity and inhibition in its optimal state. A directly measured detection limit of 0.01mU/mL for AChE activity is obtained, which is superior to those obtained by some fluorescence methods. The inhibition of AChE activity by aldicarb is successfully detected, and the corresponding IC50 is determined to be 13µg/L. In addition to high sensitivity and good selectivity, this strategy also exhibits additional advantages of simplicity, low cost and easy operation. To the best of our knowledge, the as-proposed strategy is the first example demonstrating the application of CdS QDs formed in situ for biphasic PEC detection of enzyme activity and inhibition. More significantly, it opens up a new horizon for the development of homogenous PEC sensing platforms, and has great potential in probing many other analytes.

A versatile and highly sensitive homogeneous electrochemical strategy based on the split aptamer binding-induced DNA three-way junction and exonuclease III-assisted target recycling.

Herein, a highly sensitive and versatile homogeneous electrochemical biosensing strategy is proposed, based on the split aptamer-incorporated DNA three-way junction and the exonuclease (Exo) III-assisted target recycling. The aptamer of adenosine triphosphate (ATP, chosen as the model analyte) is split into two fragments and embedded in single-stranded DNA1 and DNA2, respectively. ATP specifically binds with the split aptamers, bringing DNA1 and DNA2 close to each other, thus inducing the DNA three-way junction formation through the partial hybridization among DNA1, DNA2 and the methylene blue-labelled MB-DNA. Subsequently, MB-DNA is specifically digested by Exo III, releasing a MB-labelled mononucleotide, as well as a DNA1-ATP-DNA2 complex, which acts as the recycled target and hybridizes with another intact MB-DNA to initiate the subsequent cycling cleavage process. As a result, large amounts of MB-labelled mononucleotides are released, generating a significantly amplified electrochemical signal toward the ATP assay. To the best of our knowledge, it is the first example to successfully incorporate split aptamers into DNA three-way junctions and to be adopted in a homogeneous electrochemical assay. In addition to high sensitivity, this strategy also exhibits the advantages of simplicity and convenience, because it is carried out in a homogeneous solution, and sophisticated electrode modification processes are avoided. By simply changing the sequences of the split aptamer fragments, this versatile strategy can be easily adopted to assay a large spectrum of targets. Due to its advantages of high sensitivity, excellent selectivity, versatility and simple operation, the as-proposed approach has great potential to be applied in biochemical research and clinical practices.

A highly sensitive homogeneous electrochemical assay for alkaline phosphatase activity based on single molecular beacon-initiated T7 exonuclease-mediated signal amplification.

Alkaline phosphatase (ALP), a class of enzymes that catalyzes the dephosphorylation of a variety of substrates, is one of the most commonly assayed enzymes in routine clinical practice, and an important biomarker related to many human diseases. Herein, a facile and highly sensitive homogeneous electrochemical biosensing strategy was proposed for the ALP activity detection based on single molecular beacon-initiated T7 exonuclease-assisted signal amplification. One 3'-phosphorylated and 5'-methylene blue (MB) labeled hairpin probe (HP) is ingeniously designed. In the presence of ALP, the dephosphorylation of HP, the subsequent Klenow fragment (KF) polymerase-catalyzed elongation and T7 exonuclease-catalyzed digestion of the duplex stem of HP take place, releasing MB-labeled mononucleotides and the trigger DNA (tDNA). tDNA then hybridizes with another HP and initiates the subsequent cycling cleavage process. As a result, a large amount of MB-labeled mononucleotides are released, generating a significantly amplified electrochemical signal toward the ALP activity assay. A directly measured detection limit as low as 0.1 U L(-1) is obtained, which is comparable to that of the fluorescence method and up to three orders of magnitude lower than that of the immobilization-based electrochemical strategy previously reported. In addition to high sensitivity and good selectivity, the as-proposed strategy also exhibits the advantages of simplicity and convenience, because the assay is carried out in the homogeneous solution phase and sophisticated electrode modification processes are avoided. Therefore, the homogeneous electrochemical method we proposed here is an ideal candidate for ALP activity detection in biochemical research and clinical practices.

HIF-1α disturbs osteoblasts and osteoclasts coupling in bone remodeling by up-regulating OPG expression.

Hypoxia-inducible factor 1α (HIF-1α) is one of the master regulators of hypoxia reactions, playing an important role in bone modeling, remodeling, and homeostasis. And overexpression of HIF-1α in mature osteoblasts through conditional deletion of the von Hippel-Lindau (VHL) gene profoundly increases angiogenesis and osteogenesis. Studies showed that mice with osteoblasts lacking Vhl had a high level of Hif-1α and increased bone mass and density. On the contrary, Hif-1α conditional knockout mice had decreased bone mass and density. Our in vitro study showed that osteoprotegerin (OPG), an essential regulator of osteoclastic activity, can be upregulated by HIF-1α and in turn downregulate the resorption activity of osteoclasts. We showed that HIF-1α may directly bind to the upstream site of OPG and enhance its expression. Our study suggested that a novel mechanism, which works via OPG signaling, may mediate the function of HIF-1α in bone remodeling.

Activation of HIFa pathway in mature osteoblasts disrupts the integrity of the osteocyte/canalicular network.

The hypoxia-inducible factors (HIFs), HIF-1α and HIF-2α, are the central mediators of the homeostatic response that enables cells to survive and differentiate in low-oxygen conditions. Previous studies indicated that disruption of the von Hippel-Lindau gene (Vhl) coincides with the activation of HIFα signaling. Here we show that inactivation of Vhl in mature osteoblasts/osteocytes induces their apoptosis and disrupts the cell/canalicular network. VHL-deficient (ΔVHL) mice exhibited a significantly increased cortical bone area resulting from enhanced proliferation and osteogenic differentiation of the bone marrow stromal cells (BMSCs) by inducing the expression of ß-catenin in the BMSC. Our data suggest that the VHL/HIFα pathway in mature osteoblasts/osteocytes plays a critical role in the bone cell/canalicular network and that the changes of osteocyte morphology/function and cell/canalicular network may unleash the bone formation, The underlying mechanism of which was the accumulation of ß-catenin in the osteoblasts/osteoprogenitors of the bone marrow.

Osteoblast-secreted factors promote proliferation and osteogenic differentiation of bone marrow stromal cells via VEGF/heme-oxygenase-1 pathway.

The hypoxia-inducible factors (HIFα) are the critical factors that couple angiogenesis and osteogenesis by activating transcription of VEGF in osteoblasts. Mice lacking von Hippel-Lindau gene (Vhl), thus overexpressing HIFα in osteoblasts develop extremely dense and highly vascularized long bones. Here we provide evidence that osteoblasts lacking Vhl overexpress and secrete high levels of VEGF, which subsequently promotes the proliferation and osteogenic differentiation of bone marrow stromal cells (BMSC) by promoting expression of Heme oxygenase-1 (HO-1) in BMSC. Conditioned medium from osteoblasts Vhl (CM-CRE) promoted the proliferation and osteogenic differentiation of BMSC, in comparison with conditioned medium derived from normal osteoblasts (CM-GFP). Recombinant VEGF stimulated the proliferation and osteogenic differentiation of BMSC culturing in CM-GFP. By contrast, VEGF-neutralizing antibody inhibited the proliferation and osteogenic differentiation of BMSC culturing in CM-CRE. Treatment with a HO-1 inhibitor, SnPP, significantly inhibited VEGF-induced BMSC proliferation and osteogenic differentiation. On the contrary, activation of HO-1 with CoPP reversed the suppressing of VEGF-antibody on the proliferation and osteogesis of BMSC culturing in CM-CRE. These studies suggest that osteoblasts promote the proliferation and osteogenic differentiation of BMCS by VEGF/HO-1 pathway.

Prolyl hydroxylase inhibitors protect from the bone loss in ovariectomy rats by increasing bone vascularity.

The hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway is involved in skeletal development, bone repair, and postmenopausal osteoporosis. Inhibitors of prolyl hydroxylases (PHD) enhance vascularity, increase callus formation in a stabilized fracture model, and activate the HIF-1α/VEGF pathway. This study examined the effects of estrogen on the HIF-1α/VEGF pathway in osteoblasts and whether PHD inhibitors can protect from bone loss in postmenopausal osteoporosis. Osteoblasts were treated with estrogen, and expressions of HIF-1α and VEGF were measured at mRNA (qPCR) and protein (Western blot) levels. Further, osteoblasts were treated with inhibitors of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway, and levels of VEGF mRNA and protein expression were detected. In addition, ovariectomized rats were treated with PHD inhibitors, and bone microarchitecture and bone mechanical strength were assessed using micro-CT and biomechanical analyses (lower ultimate stress, modulus, and stiffness). Blood vessel formation was measured with India Ink Perfusion and immunohistochemistry. Estrogen, in a dose- and time-dependent manner, induced VEGF expression at both mRNA and protein levels and enhanced HIF-1α protein stability. Further, the estrogen-induced VEGF expression in osteoblasts involved the PI3K/Akt pathway. PHD inhibitors increased bone mineral density, bone microarchitecture and bone mechanical strength, and promoted blood vessel formation in ovariectomized rats. In conclusion, estrogen and PHD inhibitors activate the HIF-1α/VEGF pathway in osteoblasts. PHD inhibitors can be utilized to protect bone loss in postmenopausal osteoporosis by improving bone vascularity and angiogenesis in bone marrow.