Step-cut osteotomy for cubitus varus deformity with application of patient-specific instrument in children: a preliminary report.

PURPOSE: The step-cut osteotomy has been recognized as a valuable approach for addressing cubitus varus deformity, albeit one that necessitates technical proficiency. This study aims to evaluate the efficacy of the modified step-cut osteotomy technique in conjunction with patient-specific instruments by clinical and radiological assessment. METHODS: We conducted a retrospective review of patients who underwent modified step-cut osteotomy with the use of patient-specific instruments in conjunction with Kirschner wires fixation for the correction of cubitus varus deformity between April 2016 and April 2022. Follow-up was performed for a minimum of two years, during which pre-operative and post-operative clinical and radiological parameters were compared. RESULTS: Fifteen patients were enrolled in this study. The mean pre-operative humeral-elbow-wrist (HEW) of the affected side was -21.7° (ranging from -14° to -34°), while the normal side was 9.4° (ranging from 5° to 15°). The post-operation HEW of affected side was 9° (ranging from 4° to 16°). There was no significant difference between the normal side and affected side after operation (p = 0.74). Pre-operative range of motion in the affected side was 130°, while the post-operative range of motion was 132°. Fourteen patients (93.3%) were pleased with the overall appearance of their elbow. None lazy-S deformity was observed in these cases. There were no major complications. CONCLUSION: The modified step-cut osteotomy technique, utilizing patient-specific instrument in conjunction with Kirschner wires fixation was found to be a safe, reliable, and technically easy procedure for correcting cubitus varus deformity.

METTL3 promotes the osteogenic differentiation of periosteum-derived MSCs via regulation of the HOXD8/ITGA5 axis in congenital pseudarthrosis.

Background: Congenital pseudarthrosis of the tibia (CPT) is a dominant health challenge in pediatric orthopedics. The essential process in the development of CPT is the limited capacity of mesenchymal stem cells (MSCs) derived from CPT to undergo osteogenic differentiation. Our research aimed to elucidate the role and mechanism of methyltransferase-like 3 (METTL3) in the osteogenic differentiation process of CPT MSCs. Methods: The osteogenic differentiation medium was used to culture MSCs, and the detection of osteogenic differentiation was performed using Alizarin Red S and alkaline phosphatase (ALP) assays. Gene or protein expression was assessed by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, or immunofluorescence (IF) staining. The m6A modification of Homeobox D8 (HOXD8) was verified by methylated RNA immunoprecipitation (MeRIP) assay. Interactions between METTL3 and HOXD8 or HOXD8 and integrin alpha 5 (ITGA5) promoter were validated by the luciferase reporter gene, RIP, and chromatin immunoprecipitation (ChIP) assays. Results: METTL3 overexpression enhanced CPT MSCs' osteogenic differentiation. METTL3 stabilized the HOXD8 in an m6A-dependent manner. Moreover, the overexpressed ITGA5 up-regulated the CPT MSCs' osteogenic differentiation. Further, HOXD8 could transcriptionally activate ITGA5. METTL3 increased the transcription of ITGA5 via HOXD8 to enhance the osteogenic differentiation of CPT MSCs. Conclusion: METTL3 promoted osteogenic differentiation via modulating the HOXD8/ITGA5 axis in CPT MSCs.

Folded Alpha Helical Putative New Proteins from Apilactobacillus kunkeei.

The emergence of new proteins is a central question in biology. Most tertiary protein folds known to date appear to have an ancient origin, but it is clear from bioinformatic analyses that new proteins continuously emerge in all organismal groups. However, there is a paucity of experimental data on new proteins regarding their structure and biophysical properties. We performed a detailed phylogenetic analysis and identified 48 putative open reading frames in the honeybee-associated bacterium Apilactobacillus kunkeei for which no or few homologs could be identified in closely-related species, suggesting that they could be relatively new on an evolutionary time scale and represent recently evolved proteins. Using circular dichroism-, fluorescence- and nuclear magnetic resonance (NMR) spectroscopy we investigated six of these proteins and show that they are not intrinsically disordered, but populate alpha-helical dominated folded states with relatively low thermodynamic stability (0-3 kcal/mol). The NMR and biophysical data demonstrate that small new proteins readily adopt simple folded conformations suggesting that more complex tertiary structures can be continuously re-invented during evolution by fusion of such simple secondary structure elements. These findings have implications for the general view on protein evolution, where de novo emergence of folded proteins may be a common event.

Physeal bar resection by modified arthroscopically assisted surgery in a closed osteocavity.

Background: Physeal bar resection has been used for partial growth arrest treatment for a decade while removing the bony bar minimally invasively and accurately is challenging. This research aims to illustrate a modified arthroscopically assisted surgery, by which all the procedure was under all-inside visualization, without the constant exchange between burring under fluoroscopy, followed by irrigation, suction, and arthroscopy of the canal. Methods: We retrospectively reviewed the patients who sustained physeal bar resection under direct all-inside visualization of the arthroscope during 2016-2021. Patients who underwent physeal bar resection with the aid of an arthroscope for identifying the physeal cartilage but not resecting and visualizing the physeal bar simultaneously were excluded from this study. Results: In total, nine patients with ten related joints were included in this study. All the patients were followed up for at least two years. The average following time was 28.5 ± 6.7 months. Eight patients with nine related joints had an improvement of angular deformity, averaging 8.3 ± 6.9 degrees, and one had a worsening of the angular deformity. All the patients had a leg length discrepancy improvement, while four patients still had LLD >1 cm. The surgery time was 3.1 ± 0.7 h. There were no postoperative fractures, infections, or intraoperative complications such as neurovascular injury. Conclusions: Using clamps to form a closed osteocavity could make physeal bar resection under all-inside arthroscopic visualization feasible, which is minimally invasive, accurate, and safe.

Intrinsically Disordered Flanking Regions Increase the Affinity of a Transcriptional Coactivator Interaction across Vertebrates.

Interactions between two proteins are often mediated by a disordered region in one protein binding to a groove in a folded interaction domain in the other one. While the main determinants of a certain interaction are typically found within a well-defined binding interface involving the groove, recent studies show that nonspecific contacts by flanking regions may increase the affinity. One example is the coupled binding and folding underlying the interaction between the two transcriptional coactivators NCOA3 (ACTR) and CBP, where the flanking regions of an intrinsically disordered region in human NCOA3 increases the affinity for CBP. However, it is not clear whether this flanking region-mediated effect is a peculiarity of this single protein interaction or if it is of functional relevance in a broader context. To further assess the role of flanking regions in the interaction between NCOA3 and CBP, we analyzed the interaction across orthologs and paralogs (NCOA1, 2, and 3) in human, zebra fish, and ghost shark. We found that flanking regions increased the affinity 2- to 9-fold in the six interactions tested. Conservation of the amino acid sequence is a strong indicator of function. Analogously, the observed conservation of increased affinity provided by flanking regions, accompanied by moderate sequence conservation, suggests that flanking regions may be under selection to promote the affinity between NCOA transcriptional coregulators and CBP.

Rescue of Escherichia coli auxotrophy by de novo small proteins.

Increasing numbers of small proteins with diverse physiological roles are being identified and characterized in both prokaryotic and eukaryotic systems, but the origins and evolution of these proteins remain unclear. Recent genomic sequence analyses in several organisms suggest that new functions encoded by small open reading frames (sORFs) may emerge de novo from noncoding sequences. However, experimental data demonstrating if and how randomly generated sORFs can confer beneficial effects to cells are limited. Here, we show that by upregulating hisB expression, de novo small proteins (≤50 amino acids in length) selected from random sequence libraries can rescue Escherichia coli cells that lack the conditionally essential SerB enzyme. The recovered small proteins are hydrophobic and confer their rescue effect by binding to the 5' end regulatory region of the his operon mRNA, suggesting that protein binding promotes structural rearrangements of the RNA that allow increased hisB expression. This study adds RNA regulatory elements as another interacting partner for de novo proteins isolated from random sequence libraries and provides further experimental evidence that small proteins with selective benefits can originate from the expression of nonfunctional sequences.

The effectiveness of physeal bar resection with or without Hemi-Epiphysiodesis to treat partial growth arrest.

PURPOSE: To evaluate the outcomes of distal femoral, proximal tibial, and distal tibial physeal bar resection combined with or without the Hemi-Epiphysiodesis procedure and provide a better understanding of the application of physeal bar resection combined with Hemi-Epiphysiodesis procedure in the treatment of physeal bar growth arrest. METHODS: We retrospectively reviewed the patients who suffered physeal bar and underwent physeal bar resection with or without the Hemi-Epiphysiodesis technique during 2010-2020. All were followed up for at least 2 years or to maturity. A modified mapping method was used to determine the area of a physeal bar by CT data. The aLDFA, aMPTA, aLDTA, MAD, and LLD were measured to assess the deformity of the lower limb. RESULTS: In total, 19 patients were included in this study. The average age was 8.9 years (range 4.4 to 13.3 years old). During the follow-up, 4 (21.1%) patients had an angular change < 5°; 12 (63.2%) patients had angular deformity improvement > 5° averaging 10.0° (range 5.3° to 23.2°), and 3 (15.8%) patients had improvement of the angular deformity averaging 16.8° (range 7.4° to 27.1°). Eleven patients (57.9%) had significant MAD improvement. After surgery, we found that 7 (36.8%) patients had an LLD change of < 5 mm and were considered unchanged. Only 2 (15%) patients had an LLD improvement > 5 mm averaging 1.0 cm (range 0.7 to 1.3 cm), and 7 (36.8%) patients had increasing of LLD > 5 mm averaging 1.3 cm (range 0.5 to 2.5 cm). There were no postoperative fractures, infections, or intraoperative complications such as neurovascular injury. CONCLUSION: Physeal bar resection combined with Hemi-epiphysiodesis is helpful for partial epiphysis growth arrest. Without statistically verifying, we still believe that patients with limited growth ability could benefit more from physeal bar resection combined with Hemi-epiphysiodesis.

Retrospective Case Analysis of 104 Cases of Talipes Equinus.

A retrospective study of 104 patients (81 male and 23 females, the age range from 2 to 288days) with talipes equinus was conducted to explore the case factors associated with it. We analyzed and discussed the correlation of plaster correction times, age of first visit, gender, and birthplace of patients in the department and understood their correlation and causality. The data were analyzed using frequency analysis, normality test, chi-square goodness-of-fit test, chi-square test, and PLS regression. The findings are set out below. All the distributions of the number of plaster casts in the samples did not have normality. Therefore, we used the nonparametric test and partial least squares regression (PLS regression) and found that the number of plaster casts was more closely related to the age at first visit, gender, and birthplace and had a strong positive correlation. There was a negative correlation between the times of plaster correction and the compliance of braces. The lower the compliance of patients with braces, the more times the plaster correction will be conducted.

miR-30a inhibits the osteogenic differentiation of the tibia-derived MSCs in congenital pseudarthrosis via targeting HOXD8.

Background: Congenital pseudarthrosis of the tibia (CPT) is an uncommon congenital deformity and a special subtype of bone nonunion. The lower ability of osteogenic differentiation in CPT-derived mesenchymal stem cells (MSCs) could result in progression of CPT, and miR-30a could inhibit osteogenic differentiation. However, the role of miR-30a in CPT-derived MSCs remains unclear. Methods: The osteogenic differentiation of CPT-derived MSCs treated with the miR-30a inhibitor was tested by Alizarin Red S staining and alkaline phosphatase (ALP) activity. The expression levels of protein and mRNA were assessed by Western blot or quantitative reverse transcription-polymerase chain reaction (RT-qPCR), respectively. The interplay between miR-30a and HOXD8 was investigated by a dual-luciferase reporter assay. Chromatin immunoprecipitation (ChIP) was conducted to assess the binding relationship between HOXD8 and RUNX2 promoter. Results: CPT-derived MSCs showed a lower ability of osteogenic differentiation than normal MSCs. miR-30a increased in CPT-derived MSCs, and miR-30a downregulation promoted the osteogenic differentiation of CPT-derived MSCs. Meanwhile, HOXD8 is a direct target for miR-30a, and HOXD8 could transcriptionally activate RUNX2. In addition, miR-30a could inhibit the osteogenic differentiation of CPT-derived MSCs by negatively regulating HOXD8. Conclusion: miR-30a inhibits the osteogenic differentiation of CPT-derived MSCs by targeting HOXD8. Thus, this study might supply a novel strategy against CPT.

Diagnostic accuracy of gastric filling ultrasonography in preoperative invasion depth (T stage) of gastric cancer: Meta-analysis.

OBJECTIVE: This study aimed to evaluate the diagnostic value of gastric filling ultrasonography in the preoperative invasion depth (T staging) of gastric cancer. METHODS: We systematically searched several online electronic databases including CNKI, Wanfang Medical Database, VIP, CBM, Pubmed, Embase, Cochrane Library, and Web of Science from January 2010 to December 2021, identifying the study about gastric filling ultrasonography for diagnostic of invasion depth of gastric cancer. Using bivariate mixed effect model to calculate the sensitivity (Sen), specificity (spe), positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR) with 95% confidence interval (CI). Draw the summary receiver operating characteristic (sROC) curve, likelihood ratio matrix and fagan diagram to evaluate the diagnostic value of gastric filling ultrasonography in the preoperative invasion depth of gastric cancer. Sen analysis and Publication bias tests were performed. RESULTS: This study obtained 21 literatures and the quality were good. The pooled Sen and spe of gastric filling ultrasonography was: T1: 0.63 (95% CI:0.51-0.73), 0.96 (95% CI:0.94-0.98); T2: 0.67 (95% CI:0.62-0.71), 0.90 (95% CI:0.88-0.93); T3: 0.79 (95% CI:0.75-0.82), 0.83 (95% CI:0.80-0.86); T4: 0.80 (95% CI:0.73-0.86), 0.96 (95% CI:0.94-0.97), respectively. In addition, the PLR and NLR of gastric filling ultrasonography was: T1: 16.74 (95% CI:9.98-28.09), 0.39 (95% CI:0.29-0.52); T2: 6.98 (95% CI:5.20-9.38), 0.36 (95% CI:0.31-0.42); T3: 4.65 (95% CI:3.78-5.73), 0.26 (95% CI:0.21-0.31); T4: 18.51 (95% CI:12.77-26.83), 0.20 (95% CI: 0.15-0.29), respectively. The DOR of gastric filling ultrasonography in T1-T4 was: 43.17 (95% CI:20.62-90.41),19.13 (95% CI:12.61-29.03), 18.15 (95% CI:12.86-25.62), 90.63 (95% CI:47.36-173.41), respectively. The sROC curve revealed that the area under the curve (AUC) of T1-T4 was: 0.93, 0.82, 0.87, 0.97, respectively. Sen analysis indicated that the study was steadily. And there is no publication bias in this study. But the study has some heterogeneity. CONCLUSION: Gastric filling ultrasonography is useful for clinical preoperative T staging of gastric cancer, and the result indicate that the accuracy of gastric filling ultrasonography in discriminating T1-T4 is higher than that in discriminating T2 - T3. It can be used as an imaging diagnostic method for preoperative T staging of gastric cancer.

The Establishment of Hypertrophic Cardiomyopathy Diagnosis Model via Artificial Neural Network and Random Decision Forest Method.

Hypertrophic cardiomyopathy is a hereditary disease characterized by asymmetric ventricular hypertrophy as the key anatomical feature. Currently, there exists no effective method for the early diagnosis of hypertrophic cardiomyopathy. In this analysis, we incorporated multiple GEO datasets containing RNA profiles of hypertrophic cardiomyopathic patient tissues, identified 642 differentially expressed genes, and performed GO and KEGG analyses. Furthermore, we narrowed down 46 characteristic genes from these differentially expressed genes using random decision forests and conducted transcription factor regulation analysis on them. Using 40 genes that showed overlap between the training set and the verification set, the artificial neural network was trained, and the final MPS scoring model was constructed, and a receiver-operating characteristic (ROC) curve was drawn. We used the MPS model to predict the verification dataset and drew the ROC curve, which demonstrated the good prediction performance of the model. In conclusion, this study combines a random decision forest and artificial neural network to build a diagnostic model for hypertrophic cardiomyopathy to predict the disease, aiming at early detection and treatment, prolonging the survival time, and improving the quality of life of patients.

CircPVT1 promotes the tumorigenesis and metastasis of osteosarcoma via mediation of miR-26b-5p/CCNB1 axis.

INTRODUCTION: Osteosarcoma (OS) is the most aggressive malignancy among the bone tumors in the world. Circular RNAs (circRNAs) have been reported to be participated in multiple cancers, including OS. Meanwhile, circPVT1 has been proved to be upregulated in OS. However, the mechanism by which circPVT1 mediates the tumorigenesis of OS remains to be further explored. MATERIALS AND METHODS: Protein and gene expressions in OS cells were measured by western blot and RT-qPCR, respectively. Cell growth was assessed by flow cytometry and colony formation, respectively. In addition, cell migration was assessed by wound healing, and invasion was evaluated by Transwell assay. Meanwhile, the correlation among circPVT1, miR-26b-5p and CCNB1 was explored by RNA pull-down and dual luciferase assay. Finally, in vivo model was established to explore the role of circPVT1 in OS in vivo. RESULTS: CircPVT1 and CCNB1 were significantly upregulated in OS cells, while miR-26b-5p was downregulated. Knockdown of circPVT1 notably inhibited proliferation and induced apoptosis of OS cells. CircPVT1 shRNA significantly suppressed the OS cell invasion and migration. Meanwhile, circPVT1 sponged miR-26b-5p and CCNB1 was found to be the direct target of miR-26b-5p. Furthermore, silencing of circPVT1 inhibited the growth and metastasis of OS in vivo. CONCLUSION: Silencing of circPVT1 notably suppressed the tumorigenesis and metastasis of OS via miR-26b-5p/CCNB1 axis. Therefore, circPVT1 might be used as a target for OS treatment.

Value of Ultrasound-Guided Biopsy in Evaluating Internal Mammary Lymph Node Metastases in Breast Cancer.

OBJECTIVES: This retrospective study aimed to assess the value of a real-time, ultrasound-guided biopsy in evaluating internal mammary lymph nodes (IMLNs) in breast cancer. METHODS: Patients who were diagnosed with breast cancer and underwent real-time, ultrasound-guided core-needle biopsy (CNB) or fine-needle aspiration (FNA) in suspected IMLN metastasis were retrospectively analyzed. Patient information and ultrasonographic images were reviewed and correlated with pathology results. RESULTS: Of the 164 IMLNs that were subjected to CNB, 131 were positive for metastasis by histopathologic confirmation, 8 were negative, and 25 were insufficient. By FNA, 84 IMLNs were regarded as positive for metastasis, 4 were negative, and 4 were insufficient. In total, there were 215 (83.98%) metastatic IMLNs, 12 benign IMLNs, and 29 unconfirmed by histopathology. There were statistically significant differences in the success of puncture sampling and detection of IMLN metastasis between the CNB and FNA groups (P < .05). There were no significant complications reported after FNA or CNB, including bleeding, nerve injury, infection, pneumothorax, or hemothorax. CONCLUSIONS: Our study showed that ultrasonography accurately detected nodes that were likely to be malignant IMLNs, and that real-time, ultrasound-guided CNB and FNA are accurate and valuable techniques for the determination of status in breast cancer patients. Moreover, performing ultrasound-guided CNB and FNA on suspicious IMLN metastasis does not have additional severe complications.

Surgical treatment of congenital left ventricular diverticulum.

BACKGROUND: Congenital left ventricular diverticulum (LVD) is a rare cardiac malformation. Its prevalence rate is less than 0.1% of the congenital heart diseases requiring surgery. Some scholars suggest that all LVD should be actively removed to prevent possible risks, including diverticulum rupture, arterial embolism, and malignant arrhythmia. However, others believe that asymptomatic LVD can be followed up without immediate surgery. We reviewed and reported the diagnosis, clinical features, and surgical treatment of four cases of congenital LVD to provide clinical experience and a reference for the treatment of such patients. METHODS: Four patients (aged 3-32 years old) were diagnosed with congenital LVD and received surgical treatment at the Department of Cardiovascular Surgery of PLA General Hospital, Beijing, China from September 2009 to July 2019. All four patients had complete long-term postoperative follow-up data, including echocardiogram, enhanced cardiac computed tomography (CT), and electrocardiogram to monitor changes in left ventricular structure, heart function, and heart rhythm. RESULTS: In the first case, the fibrodiverticulum under the aortic valve squeezed the right ventricular outflow tract and the right main coronary artery; the morphology of the right ventricle and coronary artery returned to normal after surgery. The second patient was complicated with a huge lipoma in the apex of the left ventricle and underwent lipoma resection during LVD resection surgery. The third and fourth cases had muscular diverticula in the left ventricular apexes and received LVD removal surgery. All four patients recovered well after surgery and their left ventricular morphology and cardiac function were normal without adverse complications, such as atrial fibrillation, ventricular arrhythmia, and cerebrovascular accident. CONCLUSIONS: Although the morphology and character of congenital LVD were different in each case, the use of effective diagnostic and follow-up tools, including echocardiogram, enhanced CT, and magnetic resonance imaging (MRI), allowed for successful surgical treatment of the left ventricular diverticula and symptoms or other malformations. We propose that congenital LVD should be actively treated with surgery, especially considering effectiveness and low risk associated with this therapeutic option.

Real-time ultrasound-guided internal jugular vein cannulation by oblique-axis in-plane: Practice at the Fourth Hospital of Hebei Medical University.

OBJECTIVE: We aimed to evaluate oblique-axis in-plane (OA-IP) techniques for real-time ultrasound-guided internal jugular vein (IJV) cannulation. METHODS: We retrospectively analysed 1065 patients who underwent ultrasound (US)-guided IJV cannulation. We recorded demographic characteristics of patients, success rate, access time, cannulation time, number of attempts and the incidence of acute complications. RESULTS: The overall success rate of the procedure was 100% (n = 1605). In total, 1594 cases (99.3%) were successful at the first attempt, and 11 (0.7%) were successful at the second attempt; no patient required three or more attempts. The mean access time was 18.7 ± 19.3 seconds. The mean cannulation time was 349.0 ± 103.8 seconds. There were 54 (3.4%) acute complications out of the total 1605 cannulations: 23 cases of puncture site bleeding (1.4%), 20 cases allergic to dressing (1.3%), 10 cases of local cervical hematomas (0.6%), and one catheter misplacement (0.1%). There were no major complications 12 hours following the procedure. CONCLUSIONS: The results of our study suggest that OA-IP techniques can improve ultrasound-guided IJV cannulation with a high success rate and safety in clinical practice. Clinicians should consider adopting these methods.

Serum-derived exosomes from neurofibromatosis type 1 congenital tibial pseudarthrosis impaired bone by promoting osteoclastogenesis and inhibiting osteogenesis.

Treatment of congenital pseudarthrosis of the tibia (CPT) still is full of challenges in pediatric orthopedist. Serum-derived exosomes (SDEs) have been proven to be participated in bone remodeling. However, the molecular changes in SDEs of CPT children and their pathologies have not been elucidated. In this study, SDEs were isolated and purified from CPT patients (CPT-SDEs) associated with neurofibromatosis type 1 (NF1) and normal children (Norm-SDEs). Then we obtained the proteomics profile of SDEs by combining liquid chromatography-tandem mass spectrometry (LC-MS/MS) and tandem mass tag label-based quantitation. In vitro, the efficacy of SDEs on osteoblastic differentiation of MC3T3-E1 cells and osteoclastogenesis ability of RAW264.7 cells were evaluated by quantitative real-time PCR (qRT-PCR) and cytochemical staining. In vivo, we used micro-CT to assess cortical bone mass and trabecular microstructures to reflect the influence of SDEs on bone remodeling after injection into the tail vein of rats. Based on proteomics analysis, 410 differentially expressed proteins, including 289 downregulated proteins and 121 upregulated proteins, were identified in the CPT-SDEs. These proteins have multiple biological functions associated with cellular metabolic processes, catalytic activity, and protein binding, which are important for cell differentiation and proliferation. In vitro, CPT-SDEs decreased the osteogenic differentiation of MC3T3-E1 cells and promoted the osteoclastogenesis of RAW264.7 cells. Injection of CPT-SDEs into the tail vein for two months resulted in bone loss in rats, as indicated by the decrease in trabecular and cortical bone mass. Our findings demonstrated the differences in proteins in SDEs between normal and CPT children with NF1. These differentially expressed proteins in CPT-SDEs contributed to deteriorating trabecular bone microstructures by inhibiting bone formation and stimulating bone resorption.

Structure and Characterization of Phosphoglucomutase 5 from Atlantic and Baltic Herring-An Inactive Enzyme with Intact Substrate Binding.

Phosphoglucomutase 5 (PGM5) in humans is known as a structural muscle protein without enzymatic activity, but detailed understanding of its function is lacking. PGM5 belongs to the alpha-D-phosphohexomutase family and is closely related to the enzymatically active metabolic enzyme PGM1. In the Atlantic herring, Clupea harengus, PGM5 is one of the genes strongly associated with ecological adaptation to the brackish Baltic Sea. We here present the first crystal structures of PGM5, from the Atlantic and Baltic herring, differing by a single substitution Ala330Val. The structure of PGM5 is overall highly similar to structures of PGM1. The structure of the Baltic herring PGM5 in complex with the substrate glucose-1-phosphate shows conserved substrate binding and active site compared to human PGM1, but both PGM5 variants lack phosphoglucomutase activity under the tested conditions. Structure comparison and sequence analysis of PGM5 and PGM1 from fish and mammals suggest that the lacking enzymatic activity of PGM5 is related to differences in active-site loops that are important for flipping of the reaction intermediate. The Ala330Val substitution does not alter structure or biophysical properties of PGM5 but, due to its surface-exposed location, could affect interactions with protein-binding partners.

The neurofibromatosis type I gene promotes autophagy via mTORC1 signalling pathway to enhance new bone formation after fracture.

Bone fracture is one of the most common injuries. Despite the high regenerative capacity of bones, failure of healing still occurs to near 10% of the patients. Herein, we aim to investigate the modulatory role of neurofibromatosis type I gene (NF1) to osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) and new bone formation after fracture in a rat model. We studied the NF1 gene expression in normal and non-union bone fracture models. Then, we evaluated how NF1 overexpression modulated osteogenic differentiation of BMSCs, autophagy activity, mTORC1 signalling and osteoclastic bone resorption by qRT-PCR, Western blot and immunostaining assays. Finally, we injected lentivirus-NF1 (Lv-NF1) to rat non-union bone fracture model and analysed the bone formation process. The NF1 gene expression was significantly down-regulated in non-union bone fracture group, indicating NF1 is critical in bone healing process. In the NF1 overexpressing BMSCs, autophagy activity and osteogenic differentiation were significantly enhanced. Meanwhile, the NF1 overexpression inhibited mTORC1 signalling and osteoclastic bone resorption. In rat non-union bone fracture model, the NF1 overexpression significantly promoted bone formation during fracture healing. In summary, we proved the NF1 gene is critical in non-union bone healing, and NF1 overexpression promoted new bone formation after fracture by enhancing autophagy and inhibiting mTORC1 signalling. Our results may provide a novel therapeutic clue of promoting bone fracture healing.

The AMPK pathway triggers autophagy during CSF1-induced microglial activation and may be implicated in inducing neuropathic pain.

The development and maintenance of neuropathic pain is now given far more attention in the clinic work. Increasing evidence has shown that colony-stimulating factor 1 (CSF1) is involved in microglial activation and may further induce pain. Here, we observed the signaling events that link the CSF1-induced microglial activated and consequences for pain processing. For the in vitro study, flow cytometry showed the microglial activity was markedly increased after CSF1 stimulation. Western blot showed the increased expression of p-PRKAA1/PRKAA1, p-AMPK/AMPK, p-ULK1/ULK1, p-S6k/S6k and LC3-II/LC3-I. QRT-PCR showed the IL-1, TNF-α and BDNF were simultaneously upregulated in the activated microglia cells, whereas the specific AMPK inhibitor compound C exhibited reverse effects in microglia. Using immunofluorescence staining and electron microscopy, we found CSF1 decreased microglial p62 expression and induced the number of autophagosomes, whereas compound C significantly exhibited the reverse effects. For the in vivo study, compared with the control and AMPK-siRNA transfection, the mice under CSF1 intrathecal injection increased CSF1 receptor and LC3 expressed in the activated spinal microglia. More importantly, qRT-PCR showed CSF1 intrathecal injection substantially upregulated BDNF and c-Fos mRNA expression as well as the ensuing neuropathic pain. Our findings demonstrated that CSF1 induced a significant upregulation of microglial activation via the AMPK signaling pathway and resulted in an increasing microglial autophagic level. An increasing CSF1 level in the central nervous system can mimic and cause pain syndromes by up-regulation of AMPK-depended autophagy, thus offering a new target for the therapy of neuropathic pain.