A study on vertebral refracture and scoliosis after percutaneous kyphoplasty in patients with osteoporotic vertebral compression fractures.

PURPOSE: To analyze the association between scoliosis and vertebral refracture after percutaneous kyphoplasty (PKP) in patients with osteoporotic vertebral compression fractures (OVCFs). METHODS: A retrospective study was conducted on 269 patients meeting the criteria from January 2014 to October 2022. All patients underwent PKP with complete data and were followed-up for > 12 months. First, it was verified that scoliosis was a risk factor in 269 patients. Second, patients with scoliosis were grouped based on the Cobb angle to evaluate the impact of the post-operative angle. The cox proportional hazards regression analysis and survival analysis were used to calculate the hazard ratio and recurrence time. RESULTS: A total of 56 patients had scoliosis, 18 of whom experienced refractures after PKP. The risk factors for vertebral refractures included a T-score < - 3.0 and presence of scoliosis (both p < 0.001). The results indicated that the vertebral fractured arc (T10 - L4) was highly influential in scoliosis and vertebral fractures. When scoliotic and initially fractured vertebrae were situated within T10 - L4, the risk factors for vertebral refracture included a postoperative Cobb angle of ≥ 20° (p = 0.002) and an increased angle (p = 0.001). The mean recurrence times were 17.2 (10.7 - 23.7) months and 17.6 (7.9 - 27.3) months, respectively. CONCLUSION: Osteoporosis combined with scoliosis significantly increases the risk of vertebral refractures after PKP in patients with OVCFs. A postoperative Cobb angle of ≥ 20° and an increased angle are significant risk factors for vertebral refractures when scoliotic and initially fractured vertebrae are situated within T10 - L4.

Brainstem control of vocalization and its coordination with respiration.

Phonation critically depends on precise controls of laryngeal muscles in coordination with ongoing respiration. However, the neural mechanisms governing these processes remain unclear. We identified excitatory vocalization-specific laryngeal premotor neurons located in the retroambiguus nucleus (RAmVOC) in adult mice as being both necessary and sufficient for driving vocal cord closure and eliciting mouse ultrasonic vocalizations (USVs). The duration of RAmVOC activation can determine the lengths of both USV syllables and concurrent expiration periods, with the impact of RAmVOC activation depending on respiration phases. RAmVOC neurons receive inhibition from the preBötzinger complex, and inspiration needs override RAmVOC-mediated vocal cord closure. Ablating inhibitory synapses in RAmVOC neurons compromised this inspiration gating of laryngeal adduction, resulting in discoordination of vocalization with respiration. Our study reveals the circuits for vocal production and vocal-respiratory coordination.

Adenoid Cystic Carcinoma of Esophagus with Lung Metastasis: Case Report.

Adenoid cystic carcinoma (ACC) is a malignant tumor originating in the salivary glands. It most commonly affects the salivary and lacrimal glands, with less frequent occurrences in the esophagus. Esophageal ACC (EACC) typically manifests in the middle or lower parts of the esophagus, with exceedingly rare instances in the upper part. Lung metastasis in EACC is uncommon, and understanding its clinical features and treatment strategies remains challenging. In this study, we present a case of ACC originating in the upper esophagus with lung metastasis. The patient, a middle-aged female, was admitted to the Department of Respiratory and Critical Care Medicine due to an esophageal mass discovered during physical examination that had been present for 4.5 years, along with a newly identified pulmonary nodule for 2 weeks. An X-ray barium meal revealed the presence of a benign esophageal cervical mass. Gastroscopy revealed elevated lesions below the esophageal inlet, and a pathological biopsy confirmed the diagnosis of EACC. The aim of this case report is to enhance understanding of this rare condition and improve clinicians' awareness of the disease. By providing details of the patient's diagnosis, clinical presentation, imaging features and pathological features, we aim to improve diagnostic accuracy and clinical management of similar cases in the future.

A novel stratification framework based on anoikis-related genes for predicting the prognosis in patients with osteosarcoma.

Background: Anoikis resistance is a prerequisite for the successful development of osteosarcoma (OS) metastases, whether the expression of anoikis-related genes (ARGs) correlates with OS prognosis remains unclear. This study aimed to investigate the feasibility of using ARGs as prognostic tools for the risk stratification of OS. Methods: The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases provided transcriptome information relevant to OS. The GeneCards database was used to identify ARGs. Differentially expressed ARGs (DEARGs) were identified by overlapping ARGs with common differentially expressed genes (DEGs) between OS and normal samples from the GSE16088, GSE19276, and GSE99671 datasets. Anoikis-related clusters of patients were obtained by consistent clustering, and gene set variation analysis (GSVA) of the different clusters was completed. Next, a risk model was created using Cox regression analyses. Risk scores and clinical features were assessed for independent prognostic values, and a nomogram model was constructed. Subsequently, a functional enrichment analysis of the high- and low-risk groups was performed. In addition, the immunological characteristics of OS samples were compared between the high- and low-risk groups, and their sensitivity to therapeutic agents was explored. Results: Seven DEARGs between OS and normal samples were obtained by intersecting 501 ARGs with 68 common DEGs. BNIP3 and CXCL12 were significantly differentially expressed between both clusters (P<0.05) and were identified as prognosis-related genes. The risk model showed that the risk score and tumor metastasis were independent prognostic factors of patients with OS. A nomogram combining risk score and tumor metastasis effectively predicted the prognosis. In addition, patients in the high-risk group had low immune scores and high tumor purity. The levels of immune cell infiltration, expression of human leukocyte antigen (HLA) genes, immune response gene sets, and immune checkpoints were lower in the high-risk group than those in the low-risk group. The low-risk group was sensitive to the immune checkpoint PD-1 inhibitor, and the high-risk group exhibited lower inhibitory concentration values by 50% for 24 drugs, including AG.014699, AMG.706, and AZD6482. Conclusion: The prognostic stratification framework of patients with OS based on ARGs, such as BNIP3 and CXCL12, may lead to more efficient clinical management.

Study on the Optimal Surgical Scheme for Very Severe Osteoporotic Vertebral Compression Fractures.

OBJECTIVE: Therapy of very severe osteoporotic compression fractures (VSOVCF) has been a growing challenge for spine surgeons. Opinions vary regarding the optimal surgical procedure for the treatment of VSOVCF and which internal fixation method is more effective is still under debate, and research on this topic is lacking. This retrospective study was conducted to compare the efficacy and safety of various pedicle screw fixation methods for treating VSOVCF. METHODS: This single-center retrospective comparative study was conducted between January 2015 and September 2020. Two hundred and one patients were divided into six groups according to different surgical methods: 45 patients underwent long-segment fixation (Group 1); 39 underwent short-segment fixation (Group 2); 30 received long-segment fixation with cement-reinforced screws (Group 3); 32 received short-segment fixation with cement-reinforced screws (Group 4); 29 had long-segment fixation combined with kyphoplasty (PKP) (Group 5); and 26 cases had short-segment fixation combined with PKP (Group 6). The clinical records were reviewed and the visual analogue scale (VAS) score and the Oswestry Disability Index (ODI) score were used for clinical evaluation. The vertebral height (VH), fractured vertebral body height (FVBH), and Cobb's angle were objectively calculated and analyzed on lateral plain radiographs. Student's t-tests and one-way ANOVA among groups were conducted to analyze the continuous, and the chi-squared test was used to compare the dichotomous or categorical variables. The difference was considered statistically significant when the P-value was less than 0.05. RESULTS: The six groups had similar distributions in age, gender, course of the disease, follow-up period, and injured level. In the postoperative assessment of the VAS score, the surgical intervention most likely to rank first in terms of pain relief was the short-segment fixation with cement-reinforced screws (Group 4). For the functional evaluation, the surgical intervention that is most likely to rank first in terms of ODI score was a short-segment fixation with cement-reinforced screws (Group 4), followed by long-segment fixation (Group 1). The long-segment fixation with cement-reinforced screws was the first-ranked surgical intervention for the maintenance of Cobb's angle and vertebral height, whereas the short-segment fixation performed the worst. The highest overall complication rate was in Group 6 with an incidence of 42.3% (11/26), followed by Group 2 with an incidence of 38.5% (15/39). CONCLUSION: For the treatment of VSOVCF, the short-segment fixation with cement-reinforced screws is the most effective and optimal procedure, and should be used as the preferred surgical method if surgeons are proficient in using cemented screws; otherwise, directly and unquestionably use long-segment fixation to achieve satisfactory clinical results.

Study on Risk Factors of Primary Non-traumatic OVCF in Chinese Elderly and a Novel Prediction Model.

OBJECTIVE: Prevention of fragility fractures is one of the public health priorities worldwide, whilst the incidence of osteoporotic vertebral compression fractures (OVCF) continues to rise and lacks the corresponding accurate prediction model. This study aimed to screen potential causes and risk factors for primary non-traumatic osteoporotic vertebral compression fractures (NTOVCF) in the elderly by characterizing a patient population with NTOVCF and comparing it with a population of osteoporotic patients. METHODS: Between January 2013 and January 2022, 208 elderly patients with unequivocal evidence of bone fragility manifested as painful NTOVCF were enrolled, and compared with 220 patients with osteoporosis and no fractures. The demographic data, bone turnover markers, blood routine, serum biochemical values, and radiological findings were investigated. Differences between the fracture and non-fracture groups were analyzed, and variables significant in univariate analysis and correlation analysis were included in the logistic analysis to build the risk prediction model for osteoporotic vertebral fractures. Univariate analysis using student's t-tests for continuous variables or a chi-squared test for categorical variables was conducted to identify risk factors. RESULTS: No significant differences were revealed regarding age, gender, BMI, smoking, alcohol consumption, blood glucose, propeptide of type I procollagen (P1NP), and N-terminal middle segment osteocalcin (N-MID) (P > 0.05). Parathyroid Hormone (PTH), 25(OH)D, serum albumin (ALB), hemoglobin (HB), bone mineral density (BMD), and cross-sectional area (CSA) of the paraspinal muscle in the fracture group were significantly lower than those in the control group; however, b-C-terminal telopeptide of type I collagen (ß-CTX), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), non-prostatic acid phosphatase (NACP), and fatty degeneration ratio (FDR) were significantly higher than those in the control group (P < 0.05). Logistic regression analysis showed that ALB, HB, CSA, and BMD were negatively correlated with NTOVCF, while ß-CTX, HDL-C, NACP, and FDR were positively correlated with NTOVCF. CONCLUSION: Decreased physical activity, anemia, hypoproteinemia, imbalances in bone metabolism, abnormal lipid metabolism, and degenerative and decreased muscle mass, were all risk factors for OVCF in the elderly, spontaneous fractures may be the consequence of cumulative declines in multiple physiological systems over the lifespan. Based on this risk model, timely detection of patients with high OVCF risk and implementation of targeted preventive measures is expected to improve the effect of fracture prevention.

Comprehensive bioinformatics analysis reveals the hub genes and pathways associated with multiple myeloma.

PURPOSE: While the prognosis of multiple myeloma (MM) has significantly improved over the last decade because of new treatment options, it remains incurable. Aetiological explanations and biological targets based on genomics may provide additional help for rational disease intervention. MATERIALS AND METHODS: Three microarray datasets associated with MM were downloaded from the Gene Expression Omnibus (GEO) database. GSE125364 and GSE39754 were used as the training set, and GSE13591 was used as the verification set. The differentially expressed genes (DEGs) were obtained from the training set, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to annotate their functions. The hub genes were derived from the combined results of a protein-protein interaction (PPI) network and weighted gene coexpression network analysis (WGCNA). The receiver operating characteristic (ROC) curves of hub genes were plotted to evaluate their clinical diagnostic value. Biological processes and signaling pathways associated with hub genes were explained by gene set enrichment analysis (GSEA). RESULTS: A total of 1759 DEGs were identified. GO and KEGG pathway analyses suggested that the DEGs were related to the process of protein metabolism. RPN1, SEC61A1, SPCS1, SRPR, SRPRB, SSR1 and TRAM1 were proven to have clinical diagnostic value for MM. The GSEA results suggested that the hub genes were widely involved in the N-glycan biosynthesis pathway. CONCLUSION: The hub genes identified in this study can partially explain the potential molecular mechanisms of MM and serve as candidate biomarkers for disease diagnosis.

Phactr1 negatively regulates bone mass by inhibiting osteogenesis and promoting adipogenesis of BMSCs via RhoA/ROCK2.

The imbalance between osteogenic and adipogenic differentiation of Bone marrow-derived mesenchymal stem cells (BMSCs) is involved in the occurrence and development of osteoporosis (OP). Previous studies have indicated the potential of phosphatase and actin regulator 1 (Phactr1) in regulating osteogenic and adipogenic differentiation of BMSCs. The present study aims to investigate the function and mechanism of Phactr1 in regulating osteogenic and adipogenic differentiation of BMSCs. Herein, the expression of Phactr1 in bone and adipose tissue of OP rats was determined by immunohistochemical. BMSCs were subjected to osteogenic and adipogenic differentiation, and transfected with Phactr1 overexpression lentivirus, small interference RNA (siRNA) and KD025 (selective ROCK2 inhibitor). The relationship between Phactr1 and ROCK2 was detected by Co-IP experiment. The expression of Phactr1, Runx2, C/EBPα, RhoA and ROCK2 was detected by Western blot. Calcium nodule and lipid droplets were determined by alizarin red and Oil red O staining. Interestingly, Phactr1 increased in both bone and adipose tissue of OP rats. During osteogenic differentiation, Phactr1 decreased and active RhoA, ROCK2 increased, while overexpression Phactr1 inhibits the increase of Runx2. Phactr1 increased and active RhoA decreased, ROCK2 did not changed during adipogenic differentiation. While, Knockdown Phactr1 inhibits the increase of C/EBPα. Phactr1 and ROCK2 were combined in osteogenic differentiation, but not in adipogenic differentiation. By using KD025, the decrease of Phactr1 and increase of Runx2 were inhibited respectively in osteogenic differentiation. Meanwhile, when ROCK2 was inhibited, Phactr1, C/EBPα were significantly increased in adipogenic differentiation. These findings indicated that Phactr1 negatively regulates bone mass by inhibiting osteogenesis and promoting adipogenesis of BMSCs by activating RhoA/ROCK2.

Comparison of Percutaneous Kyphoplasty and Pedicle Screw Fixation for Treatment of Thoracolumbar Severe Osteoporotic Vertebral Compression Fracture with Kyphosis.

BACKGROUND: Pedicle screw fixation (PSF) has been considered the preferred surgery for the treatment of severe osteoporotic vertebral compression fracture (sOVCF), and sOVCF was traditionally regarded as a relative contraindication to minimally invasive percutaneous kyphoplasty (PKP). Debate has continued regarding the selection of the best surgical method for sOVCF. In the present study, we compared the efficacy and safety between PKP and PSF. METHODS: PKP was performed in 376 patients in group 1 and PSF in 121 patients in group 2. The visual analog scale (VAS), Oswestry disability index (ODI), local kyphotic angle, fractured vertebral body height, and complications were evaluated. RESULTS: In the immediate postoperative analysis, the mean VAS score for group 1 was 2.4, significantly lower than the VAS score of 4.7 for group 2. The mean ODI score was 44.4% for group 1, lower than the ODI score of 57.1% for group 2. In addition, group 1 had had a significantly better ODI score at 1 year of follow-up. The local kyphotic angle and fractured vertebral body height had recovered better in group 2. In group 1, 113 patients had experienced cement leakage, and 29 patients had undergone PKP for adjacent new vertebral fractures. In group 2, 2 patients had developed wound infections, 4 had developed pneumonia, 2 had developed urinary tract infection, 3 had experienced asymptomatic screw loosening, and 7 had undergone PKP to treat new vertebral fractures and 1 had undergone removal of internal fixation because of back pain. CONCLUSIONS: The results of the clinical and radiological evaluations showed that PKP is comparable to PSF for the treatment of sOVCF with kyphosis, with PKP having the advantages of minimal invasion, quick postoperative pain relief, and functional recovery.

High prevalence of osteoporosis in patients undergoing spine surgery in China.

BACKGROUND: With the increase in life expectancy, a large number of patients with osteoporosis (OP) are undergoing spine surgery, which may adversely affect the surgical success rate. The prevalence of OP varies in different regions, and no data are available that represent the prevalence of OP among Chinese patients over 50 years of age who are undergoing spine surgery. It was the first multicenter study to assess OP in these patients. Aiming to obtain comprehensive data, this study combined bone mineral density (BMD) measurements and visual radiography assessment (VRA) to analyze the prevalence of OP in patients aged > 50 years who underwent spine surgery. METHODS: Data from 1,856 patients aged over 50 years undergoing spine surgery who resided in northern, central, and southern China were reviewed between 2018 and 2019. Based on the perioperative BMD and X-ray data, we calculated the prevalence of OP in this special population according to sex, age, and spine degenerative disease. RESULTS: A total of 1,245 patients (678 females and 567 males) were included in the study. The prevalence of OP diagnosed by BMD was 52.8 % in females and 18.7 % in males. When we combined with BMD and VRA, the prevalence of OP increased from 52.8 to 65.9 % in females and from 18.7 to 40.6 % in males. Although OP was more severe in females than in males, a significant difference in the rate of vertebral fracture (VF) was not observed between females and males with a normal BMD and osteopenia (females vs. males: aged 50-59 years, P = 0.977; 60-69 years, P = 0.302; >70 years, P = 0.172). Similarly, no significant difference in the vertebral fracture rate was observed within different age groups of patients with a normal BMD and osteopenia (females: P = 0.210; males, P = 0.895). The incidence of OP in patients with degenerative scoliosis was higher than that in the remaining patients (females: 63.6 % vs. 42.4 %, P = 0.018; males: 38.9 % vs. 13.8 %, P = 0.004). CONCLUSIONS: A high prevalence of OP was identified in patients aged > 50 years undergoing spine surgery, especially in patients whose primary diagnosis was degenerative scoliosis. BMD and VRA evaluations should be included in the clinical routine for these patients prior to surgery.

Ultrasound-Targeted Microbubble Destruction Enhances the Inhibitive Efficacy of miR-21 Silencing in HeLa Cells.

BACKGROUND Previous studies have shown that miR-21 upregulation is related to the aggressive development of cervical cancer. Ultrasound-targeted microbubble destruction (UTMD) is a method that increases the absorption of targeted genes or drugs by cells. We focus on the role of UTMD-mediated miR-21 transfection in HeLa cells, a cervical cancer cell line. MATERIAL AND METHODS The effects of different ultrasound intensities on the transfection efficiency of miR-21-enhanced green fluorescent protein (EGFP) and miR-21 inhibitor-EGFP plasmids were determined by flow cytometry. The effects of UTMD-mediated miR-21 transfection on HeLa cell proliferation, apoptosis, migration, and invasion were measured by CCK-8, flow cytometry, wound healing experiments, and transwell migration assay, respectively. Western blot and real-time quantitative PCR were used to detect the expression of tumor-related genes. RESULTS When the ultrasound intensity was 1.5 W/cm², the miR-21 plasmid had the highest transfection efficiency. Exogenous miR-21 promoted cell proliferation, migration, and invasion, and inhibited cell apoptosis in HeLa cells. Treatment of cells with UTMD further enhanced the effects of miR-21-EGFP and miR-21 inhibitor-EGFP. In addition, miR-21 overexpression significantly increased the expression of p-Akt, Akt, Bcl-2, Wnt, ß-catenin, matrix metalloprotein-9 (MMP-9), and epidermal growth factor (EGFR) levels, and decreased Bax expression. The regulatory role of miR-21 inhibitor-EGFP was opposite to that of miR-21-EGFP. After UTMD, miR-21-EGFP and miR-21 inhibitor-EGFP had more significant regulatory effects on these genes. CONCLUSIONS Our research revealed that an ultrasound intensity of 1.5 W/cm² is the best parameter for miR-21 transfection. UTMD can enhance the biological function of miR-21 in HeLa cells, and alter the effect of miR-21 on apoptosis, metastasis, and phosphorylation genes.

Gas Embolism After Hydrogen Peroxide Use During Spine Surgery: Case Report and Literature Review.

BACKGROUND: As an irrigant, an antiseptic, and a hemostatic agent, hydrogen peroxide (H2O2) is widely used in surgical treatment, but it has been surrounded by persistent controversy. Fatal or near-fatal embolic events caused by H2O2 have been reported sporadically in spine surgery. CASE DESCRIPTION: In this report, we present an 87-year-old man who underwent lumbar instrumentation removal and debridement consequent to surgical site infection in a prone position. H2O2 was used to irrigate the infected screw tracks and surrounding tissues during the procedures. Soon after irrigation, the patient suddenly developed tachycardia, hypotension, and rapid oxygen desaturation, followed by bradycardia. Transesophageal echocardiography indicated gas embolism. After prompt first aid treatment, the patient's condition improved and the gas embolus disappeared within a few minutes without any evidence of organ embolism. CONCLUSIONS: Spine surgeons should reconsider the pending results of using H2O2 during surgery. Prolonged prone positioning and semiclosed cavities may increase the risk of gas embolism. An early diagnosis and timely intervention may be the key measures to prevent the occurrence of fatal consequences caused by gas embolism.

A Common Neuroendocrine Substrate for Diverse General Anesthetics and Sleep.

How general anesthesia (GA) induces loss of consciousness remains unclear, and whether diverse anesthetic drugs and sleep share a common neural pathway is unknown. Previous studies have revealed that many GA drugs inhibit neural activity through targeting GABA receptors. Here, using Fos staining, ex vivo brain slice recording, and in vivo multi-channel electrophysiology, we discovered a core ensemble of hypothalamic neurons in and near the supraoptic nucleus, consisting primarily of neuroendocrine cells, which are persistently and commonly activated by multiple classes of GA drugs. Remarkably, chemogenetic or brief optogenetic activations of these anesthesia-activated neurons (AANs) strongly promote slow-wave sleep and potentiates GA, whereas conditional ablation or inhibition of AANs led to diminished slow-wave oscillation, significant loss of sleep, and shortened durations of GA. These findings identify a common neural substrate underlying diverse GA drugs and natural sleep and reveal a crucial role of the neuroendocrine system in regulating global brain states. VIDEO ABSTRACT.

[Vascular endothelial growth factor/polylactide-polyethyleneglycol-polylactic acid copolymer/basic fibroblast growth factor mixed microcapsules in promoting angiogenic differentiation of rat bone marrow mesenchymal stem cells in vitro].

Objective: To observe the effect of vascular endothelial growth factor/polylactide-polyethyleneglycol-polylactic acid copolymer/basic fibroblast growth factor (VEGF/PELA/bFGF) mixed microcapsules in promoting the angiogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. Methods: The BMSCs were isolated by the method of whole bone marrow adherent, and sub-cultured. The passage 3 BMSCs were identified by Wright-Giemsa staining and flow cytometry, and used for subsequent experiments. VEGF/PELA/bFGF (group A), PELA/bFGF (group B), VEGF/PELA (group C), and PELA (group D) microcapsules were prepared. The biodegradable ability and cytotoxicity of PELA microcapsule were determined，and the slow-released ability of VEGF/PELA/bFGF mixed microcapsules was measured. The passage 3 BMSCs were co-cultured with the extracts of groups A, B, C, and D, separately. At 1, 3, 7, 14, and 20 days after being cultured, the morphological changes of induced BMSCs were recorded. At 21 days, the induced BMSCs were tested for DiI-labeled acetylated low density lipoprotein (Dil-ac-LDL) and FITC-labeled ulex europaeus agglutinin I (FITC-UEA-I) uptake ability. The tube-forming ability of the induced cells on Matrigel was also verified. The differences of the vascularize indexes in nodes, master junctions, master segments, and tot.master segments length in 4 groups were summarized and analyzed. Results: The isolated and cultured cells were identified as BMSCs. The degradation time of PELA was more than 20 days. There was no significant effect on cell viability under co-culture conditions. At 20 days, the cumulative release of VEGF in the mixed microcapsules exceeded 95%, and the quantity of bFGF exceeded 80%. The morphology of cells in groups A, B, and C were changed. The cells in groups A and B showed the typical change of cobble-stone morphology. The numbers of double fluorescent labeled cells observed by fluorescence microscope were the most in group A, and decreases from group B and group C, with the lowest in group D. The cells in groups A and B formed a grid-like structure on Matrigel. Quantitative analysis showed that the differences in the number of nodes, master junctions, master segments, and tot.master segments length between groups A, B and groups C, D were significant ( P<0.05). The number of nodes and the tot.master segments length of group A were more than those of group B ( P<0.05). There was no significant differences in the number of master junctions and master segments between group A and group B ( P>0.05). Conclusion: VEGF/PELA/bFGF mixed microcapsules have significantly ability to promote the angiogenic differentiation of rat BMSCs in vitro.

Niclosamide-induced Wnt signaling inhibition in colorectal cancer is mediated by autophagy.

The Wnt signaling pathway, known for regulating genes critical to normal embryonic development and tissue homeostasis, is dysregulated in many types of cancer. Previously, we identified that the anthelmintic drug niclosamide inhibited Wnt signaling by promoting internalization of Wnt receptor Frizzled 1 and degradation of Wnt signaling pathway proteins, Dishevelled 2 and ß-catenin, contributing to suppression of colorectal cancer growth in vitro and in vivo Here, we provide evidence that niclosamide-mediated inhibition of Wnt signaling is mediated through autophagosomes induced by niclosamide. Specifically, niclosamide promotes the co-localization of Frizzled 1 or ß-catenin with LC3, an autophagosome marker. Niclosamide inhibition of Wnt signaling is attenuated in autophagosome-deficient ATG5-/- MEF cells or cells expressing shRNA targeting Beclin1, a critical constituent of autophagosome. Treatment with the autophagosome inhibitor 3MA blocks niclosamide-mediated Frizzled 1 degradation. The sensitivity of colorectal cancer cells to growth inhibition by niclosamide is correlated with autophagosome formation induced by niclosamide. Niclosamide inhibits mTORC1 and ULK1 activities and induces LC3B expression in niclosamide-sensitive cell lines, but not in the niclosamide-resistant cell lines tested. Interestingly, niclosamide is a less effective inhibitor of Wnt-responsive genes (ß-catenin, c-Myc, and Survivin) in the niclosamide-resistant cells than in the niclosamide-sensitive cells, suggesting that deficient autophagy induction by niclosamide compromises the effect of niclosamide on Wnt signaling. Our findings provide a mechanistic understanding of the role of autophagosomes in the inhibition of Wnt signaling by niclosamide and may provide biomarkers to assist selection of patients whose tumors are likely to respond to niclosamide.

A craniofacial-specific monosynaptic circuit enables heightened affective pain.

Humans often rank craniofacial pain as more severe than body pain. Evidence suggests that a stimulus of the same intensity induces stronger pain in the face than in the body. However, the underlying neural circuitry for the differential processing of facial versus bodily pain remains unknown. Interestingly, the lateral parabrachial nucleus (PBL), a critical node in the affective pain circuit, is activated more strongly by noxious stimulation of the face than of the hindpaw. Using a novel activity-dependent technology called CANE developed in our laboratory, we identified and selectively labeled noxious-stimulus-activated PBL neurons and performed comprehensive anatomical input-output mapping. Surprisingly, we uncovered a hitherto uncharacterized monosynaptic connection between cranial sensory neurons and the PBL-nociceptive neurons. Optogenetic activation of this monosynaptic craniofacial-to-PBL projection induced robust escape and avoidance behaviors and stress calls, whereas optogenetic silencing specifically reduced facial nociception. The monosynaptic circuit revealed here provides a neural substrate for heightened craniofacial affective pain.

[FGF-2/PELA/BMP-2 microcapsule scaffold promotes osteogenic differentiation of rat periosteum-derived stem cells in vitro].

OBJECTIVE: To observe the effect of a microencapsule scaffold capable of sustained release of fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2) in promoting the osteogenic differentiation of rat periosteum-derived stem cells (PDSCs) in vitro. METHODS: PDSCs from 4-week-old SD rats, after identification of the surface markers using flow cytometry, were induced to differentiate into osteoblast, chondroblast, and adipocyte lineages. The differentiated cells were verified by staining with Alizarin red, toluidine blue, alcian blue, oil red O and by immunofluorescence assay. FGF-2/PELA/BMP-2, FGF-2/PELA, PELA/BMP-2 and PELA microcapsules were prepared, examined for surface morphologies using scanning electron microscopy (SEM), and tested for controlled release of FGF-2 and BMP-2 using ELISA. The third passage of PDSCs were cultured in the presence of the aqueous extracts of one of the 4 materials, and alkaline phosphatase (AKP) activity in the culture media was detected at 7 and 14 days of culture; the expression levels of osteogenesis-related genes were quantified with quantitative real-time PCR (qRT-PCR). The osteogenic differentiation ability of the PDSCs cultured with the extracts was compared. RESULTS: The PDSCs, which expressed mesenchymal stem cell surface markers, were shown to have osteogenic, chondrogenic and adipogenic differentiation potentials. The cells cultured with the extract of FGF-2/PELA/BMP-2 microcapsules showed the highest AKP activity at 7 and 14 days of culture, and their expression levels of OCN and RunX-2 mRNA were the highest among the 4 groups; RunX-2 expression reached its peak level on day 14, and OCN mRNA expression level increased progressively as the culture time extended. CONCLUSION: FGF-2/PELA/BMP-2 biomimetic controlled release microcapsules preserve the cytokine activities and are capable of promoting the osteogenic differentiation of rat PDSCs.

Capturing and Manipulating Activated Neuronal Ensembles with CANE Delineates a Hypothalamic Social-Fear Circuit.

We developed a technology (capturing activated neuronal ensembles [CANE]) to label, manipulate, and transsynaptically trace neural circuits that are transiently activated in behavioral contexts with high efficiency and temporal precision. CANE consists of a knockin mouse and engineered viruses designed to specifically infect activated neurons. Using CANE, we selectively labeled neurons that were activated by either fearful or aggressive social encounters in a hypothalamic subnucleus previously known as a locus for aggression, and discovered that social-fear and aggression neurons are intermixed but largely distinct. Optogenetic stimulation of CANE-captured social-fear neurons (SFNs) is sufficient to evoke fear-like behaviors in normal social contexts, whereas silencing SFNs resulted in reduced social avoidance. CANE-based mapping of axonal projections and presynaptic inputs to SFNs further revealed a highly distributed and recurrent neural network. CANE is a broadly applicable technology for dissecting causality and connectivity of spatially intermingled but functionally distinct ensembles.

Fluorescent labeling of newborn dentate granule cells in GAD67-GFP transgenic mice: a genetic tool for the study of adult neurogenesis.

Neurogenesis in the adult hippocampus is an important form of structural plasticity in the brain. Here we report a line of BAC transgenic mice (GAD67-GFP mice) that selectively and transitorily express GFP in newborn dentate granule cells of the adult hippocampus. These GFP(+) cells show a high degree of colocalization with BrdU-labeled nuclei one week after BrdU injection and express the newborn neuron marker doublecortin and PSA-NCAM. Compared to mature dentate granule cells, these newborn neurons show immature morphological features: dendritic beading, fewer dendritic branches and spines. These GFP(+) newborn neurons also show immature electrophysiological properties: higher input resistance, more depolarized resting membrane potentials, small and non-typical action potentials. The bright labeling of newborn neurons with GFP makes it possible to visualize the details of dendrites, which reach the outer edge of the molecular layer, and their axon (mossy fiber) terminals, which project to the CA3 region where they form synaptic boutons. GFP expression covers the whole developmental stage of newborn neurons, beginning within the first week of cell division and disappearing as newborn neurons mature, about 4 weeks postmitotic. Thus, the GAD67-GFP transgenic mice provide a useful genetic tool for studying the development and regulation of newborn dentate granule cells.

Cytoplasmic transport signal is involved in phogrin targeting and localization to secretory granules.

Phogrin is an integral glycoprotein primarily expressed in neuroendocrine cells. The predominant localization of phogrin is on dense-core secretory granules, and the lumenal domain has been shown to be involved in its efficient sorting to the regulated secretory pathway. Here, we present data showing that a leucine-based sorting signal [EExxxIL] within the cytoplasmic tail contributes its steady-state localization to secretory granules. Deletion mutants in the tail region failed to represent granular distribution in pancreatic beta-cell line, MIN6, and anterior pituitary cell line, AtT-20. A sorting signal mutant with two glutamic acids substituted into alanines (EE/AA) is primarily accumulated in the Golgi area instead of secretory granules, and another mutant (IL/AA) is trapped at the plasma membrane due to a defect in endocytosis. We further demonstrate that the leucine-based sorting signal of phogrin specifically interacts with both adaptor protein (AP)-1 and AP-2 clathrin adaptor complexes in vitro. These observations, along with previous studies, suggest that distinct domains of phogrin mediate proper localization of this transmembrane protein on secretory granules.