Vascularization of a bone stump.

Aim To study the character of blood circulation in the bone stump at tight and loose closure of the medullary cavity. Methods Two series of experiments on 39 rabbits with mid-third femoral amputation and muscular plasty were carried out. In the 1st (experimental) series, the bone scapula was closed by thin cortical autograft taken from the epimetaphyseal area, and then the muscles were sutured, and in the 2nd - the scapula was closed by myoplasty only. Follow-up periods: 1st series - 7, 14, 21 days and 1, 3, 6 months, 2nd series - 1, 3, 6 months. Histological methods with infusion of vessels with ink-gelatin mixture and morphometry was used. Results In the 1st series there was a rapid restoration of the disturbed macro- and microcirculation due to the reserve sources of blood circulation and the development of extravascular ways of microcirculation. In the 2nd series, blood circulation recovery was significantly slower and occurred mainly due to the development of extravascular microcirculatory pathways. Conclusion The study established undeniable usefulness of tight closure of the bone marrow cavity during amputation.

A-to-I RNA editing shows dramatic up-regulation in osteosarcoma and broadly regulates tumor-related genes by altering microRNA target regions.

A-to-I RNA editing is a prevalent type of RNA modification in animals. The dysregulation of RNA editing has led to multiple human cancers. However, the role of RNA editing has never been studied in osteosarcoma, a complex bone cancer with unknown molecular basis. We retrieved the RNA-sequencing data from 24 primary osteosarcoma patients and 3 healthy controls. We systematically profiled the RNA editomes in these samples and quantitatively identified reliable differential editing sites (DES) between osteosarcoma and normal samples. RNA editing efficiency is dramatically increased in osteosarcoma, presumably due to the significant up-regulation of editing enzymes ADAR1 and ADAR2. Up-regulated DES in osteosarcoma are enriched in 3'UTRs. Strikingly, such 3'UTR sites are further enriched in microRNA binding regions of gene EMP2 and other oncogenes, abolishing the microRNA suppression on target genes. Accordingly, the expression of these tumor-promoting genes is elevated in osteosarcoma. There might be an RNA editing-dependent pathway leading to osteosarcoma. We expanded our knowledge on the potential roles of RNA editing in oncogenesis. Based on these molecular features, our work is valuable for future prognosis and diagnosis of osteosarcoma.

Bone healing during electrostimulation.

Aim To investigate the effect of direct epineural electrical stimulation of the nerve on the nature of reparative processes in the bone stump. Methods Three series of experiments were carried out with amputation of the thigh in the middle third and muscle plasty. In the 1st and 2nd experimental series a perineural catheter was brought to the stump of the sciatic nerve, through which mechanical irritation of the nerve was performed for 20 days daily for 20 minutes. In the 2nd series, an electrode was added to the nerve and epineural electrical stimulation was performed daily for 20 days. Animals of the 3rd series served as control. The observation periods were 1, 3, 6 months. Histological research method with filling vessels with ink-gelatin mixture was applied. Results In the 1st series, there was a sharp distortion of the reparative process, which consisted of a violation of microcirculation, changes in shape, resorption of the cortical diaphyseal plate, fractures, deformations. In most experiments of the 2nd series, organotypic stumps were formed with normalization of microcirculation. In the 3rd series, results of the formation of the stump were better than in the 1st, but worse than in the 2nd series. Conclusions Painful nerve irritation after amputation leads to a significant disturbance of microcirculation and reparative regeneration at the end of the bone stump with the development of pathological restructuring of bone tissue. Electrostimulation of the nerve improves microcirculation and reparative regeneration of the bone tissue.

An evolutionarily conserved mechanism that amplifies the effect of deleterious mutations in osteosarcoma.

Illustrating the molecular consequence of deleterious mutations is essential for bridging the gap between genotype and phenotype. In the cancer field, differential expression of the two alleles on heterozygous sites could directly reflect the effect of a mutation under certain trans environment. We retrieved transcriptomes of osteosarcoma and normal tissues in human and mouse. We defined tumor-specific heterozygous mutations with stringent criteria by considering sequencing depth and ancestral state. We calculated the relative expression of mutated alleles and normal alleles on the missense mutation sites in osteosarcoma. There is a conserved pattern that the mutated alleles have globally higher expression levels than the normal alleles in tumors. In the shared genes with missense mutations in both human and mouse, the relative expression of mutated alleles is highly correlated. Moreover, shared genes are functionally more important than unshared genes, and are enriched in oncogenes. The oncogenic role of mutations in oncogene KMT2A is experimentally verified. We systematically illustrate the deleterious effects of missense mutations by showing the over-expression of mutated alleles. We partially bridge the gap between genotype and phenotype by surmising that the over-expression of the mutated alleles might break the cellular equilibrium and lead to tumorigenesis.

Cost-Efficiency Optimization Serves as a Conserved Mechanism that Promotes Osteosarcoma in Mammals.

Mutations that reduce the biosynthetic cost of ATP production or increase the gene translation efficiency (tAI) are favorable for rapid cell growth and proliferation and therefore likely to be observed in tumors. Whether the mutations in tumors optimize the trade-off between the ATP biosynthesis cost and gene translation efficiency by increasing the tAI/ATP ratio is currently unknown. We retrieved transcriptome data of normal and osteosarcoma tissue samples from humans and mice and identified tumor-specific mutations in each species by using stringent cutoffs and outgroup information. We compared the tAI/ATP values of genes before and after mutation. The tAI/ATP profile was found to be highly conserved in humans and mice, and also correlated with the essentiality of genes. Tumor-specific rather than shared mutations were found to lead to increased tAI/ATP values in both species. Thus, tumor-specific mutations were found to optimize the cost-efficiency trade-off by increasing the tAI/ATP ratio of genes in osteosarcoma. This may indicate an evolutionarily conserved mechanism that promotes tumorigenesis by facilitating rapid cell growth and proliferation.

Comparative genomic analysis of a naturally born serpentized pig reveals putative mutations related to limb and bone development.

BACKGROUND: It is believed that natural selection acts on the phenotypical changes caused by mutations. Phenotypically, from fishes to amphibians to reptiles, the emergence of limbs greatly facilitates the landing of ancient vertebrates, but the causal mutations and evolutionary trajectory of this process remain unclear. RESULTS: We serendipitously obtained a pig of limbless phenotype. Mutations specific to this handicapped pig were identified using genome re-sequencing and comparative genomic analysis. We narrowed down the causal mutations to particular chromosomes and even several candidate genes and sites, such like a mutation-containing codon in gene BMP7 (bone morphogenetic protein) which was conserved in mammals but variable in lower vertebrates. CONCLUSIONS: We parsed the limbless-related mutations in the light of evolution. The limbless pig shows phenocopy of the clades before legs were evolved. Our findings might help deduce the emergence of limbs during vertebrate evolution and should be appealing to the broad community of human genetics and evolutionary biology.

BIOMECHANICAL FACTORS IN RESIDUAL LIMB FORMATION AFTER AMPUTATION.

OBJECTIVE: The aim: To study the influence of biomechanical factors on the character of morphological disorders in the process of reparative regeneration at the end of the residual limb after amputation. PATIENTS AND METHODS: Materials and methods: 10 series of experiments on 144 rabbits were conducted. We used myodesis with normal, insufficient and excessive muscle tension, their electrical stimulation, tight and leaky closure of the bone marrow canal. Terms of observation 1, 3, 6 months. The method of research - histological with the filling of vessels with inkgelatin mixture. RESULTS: Results: Dense closure of the meduallary cavity and uniform muscle tension during plasty in the first three series of experiments allow to obtain a cylindrical residual limb with preservation of the cortical diaphyseal plate, formation of the bone closing plate, normalization of intraosseous microcirculation, completion of the reparative process. In the majority of observations of the IV-X series there was a reparative regeneration disorder connected with the incorrect tension of muscles and the absence of normalization of intraosseous circulation, the reparative process was not observed to be complete, which led to the pathological reorganization of bone tissue with the formation of stumps of various shapes. CONCLUSION: Conclusions: Uneven muscle tension and lack of closure of the intramedually canal except for microcirculation disorders leads to increased periosteum bone formation, formation of periosteum cartilage exostases, clavate stumps, resorption and fractures of the cortical diaphyseal plate with curvature and stump axis disorders, formation of a conical stump.

ALA-Net: Adaptive Lesion-Aware Attention Network for 3D Colorectal Tumor Segmentation.

Accurate and reliable segmentation of colorectal tumors and surrounding colorectal tissues on 3D magnetic resonance images has critical importance in preoperative prediction, staging, and radiotherapy. Previous works simply combine multilevel features without aggregating representative semantic information and without compensating for the loss of spatial information caused by down-sampling. Therefore, they are vulnerable to noise from complex backgrounds and suffer from misclassification and target incompleteness-related failures. In this paper, we address these limitations with a novel adaptive lesion-aware attention network (ALA-Net) which explicitly integrates useful contextual information with spatial details and captures richer feature dependencies based on 3D attention mechanisms. The model comprises two parallel encoding paths. One of these is designed to explore global contextual features and enlarge the receptive field using a recurrent strategy. The other captures sharper object boundaries and the details of small objects that are lost in repeated down-sampling layers. Our lesion-aware attention module adaptively captures long-range semantic dependencies and highlights the most discriminative features, improving semantic consistency and completeness. Furthermore, we introduce a prediction aggregation module to combine multiscale feature maps and to further filter out irrelevant information for precise voxel-wise prediction. Experimental results show that ALA-Net outperforms state-of-the-art methods and inherently generalizes well to other 3D medical images segmentation tasks, providing multiple benefits in terms of target completeness, reduction of false positives, and accurate detection of ambiguous lesion regions.

Mutation profiling of a limbless pig reveals genome-wide regulation of RNA processing related to bone development.

Mutation is the basis of phenotypic changes and serves as the source of natural selection. The development of limbs has been the milestone in vertebrate evolution. Several limb and bone-related genes were verified experimentally, but other indirect and regulatory factors of limb development remained untested, especially very few cases were observed in natural environment. We report a naturally born serpentized pig without hindlimbs. Whole genome sequencing followed by comparative genomic analysis revealed multiple interesting patterns on the handicapped pig-specific mutations. Although the bone-related genes are not directly subjected to mutations, other regulatory factors such as the RNA deaminase genes Adar are damaged in the handicapped pig, leading to the abolished A-to-I deamination in many functional, conserved genes as well as the bone-related genes. This is a precious case that the limbless phenotype is observed in naturally born non-model organisms. Our study broadened the generality of the limbless phenotype across mammals and extended the regulation of hindlimb development to other non-bone-related genes. Our knowledge of limb and bone-related mutations and regulation would also contribute to human genetics.

Reamputation stumps below knee.

Aim To investigate rehabilitation outcomes of patients with malformed tibial stumps. Methods Observations included 421 patients with residual limb diseases and malformations (extensive inactive scars adhered to the bone, excessively long or short stumps, bone filing, osteomyelitis of the stump, muscle attachment to the skin scar, excessive mobility and deviations of the fibula, improper filing). Four hundred and thirty-six (436) reconstructive surgeries were performed. A follow-up period was from 6 months to 15 years. Radiological, ultrasonic, tensometrical, and histological methods were used. Results Due to the frequent combination of several malformations and diseases in the same patient, non-free skin grafting with displaced dermal-subcutaneous flaps, which cover rather large defects, was widely used. The surgeries were performed simultaneously and allowed for reconstruction without shortening the bone lever stump. Complications in the form of marginal necrosis were obtained in three (0.71%) patients. The improved technique of muscle grafting with the fixation of muscles to the bone provided an elastic stump covering the bone filing. The authors have developed original methods of surgery to create a bone block of the tibia, which make it possible to obtain painless, highly functional stumps using partial support and ensure long-term use of modern prosthesis designs in 100% of cases. Conclusion Non-free dermal plasty with cutaneous-subcutaneous flap is the method of choice for closing skin defects on the residual limb. Muscle-bone fixation permits to eliminate some residual limb defects and to form an elastic muscular residual limb with closed bone filaments. Synostosis formation in different ways considerably improves functional quality of the residual limb.

REPARATIVE REGENERATION AT THE END OF BONE FILING AFTER OSTOPLASTIC AMPUTATION.

OBJECTIVE: The aim: To study the role and place of bone grafting in the formation of bone stump after amputation. PATIENTS AND METHODS: Materials and methods: 3 series of experiments were carried out on 44 rabbits with amputation of the thigh in the middle third and stump grafting using osteoplastic hermetic closure of the canal with a thin cortical plate (series I), closure of the canal with a spongy bone (series II), and loose closure of the canal with a cortical graft located at the entrance to the canal at an angle of 30° (ІІІ series). Observation period: 1, 3, 6 months. Histological examination method with vascular filling with 10% mascara-gelatin mixture. RESULTS: Results: In series I, in the majority of observations, a stump of a cylindrical shape with a bone locking plate of an osteon-beam structure and normalization of intraosseous microcirculation was formed. A slight displacement of the graft caused a violation of microcirculation. In series II, organotypic stumps were formed in all observations. In series III, incomplete closure of the bone marrow cavity led to sharp microcirculatory disorders and the course of the reparative process with pathological bone remodeling. CONCLUSION: Conclusions: The parameters of the favorable course of the reparative process and the formation of the organotypic bone stump are the safety of its cylindrical shape, the presence of a compact bone structure, normalization of intraosseous microcirculation.

Long non-coding RNA GAS5 suppresses rheumatoid arthritis progression via miR-128-3p/HDAC4 axis.

Rheumatoid arthritis (RA) is a highly relevant public health problem. RA fibroblast-like synoviocytes (RAFLSs) play an important role in RA progression. Long non-coding RNA growth arrest-specific transcript 5 (GAS5) could improve RA by inducing RAFLSs apoptosis. However, the mechanism of GAS5 in RA remains unclear. RT-qPCR detected the expressions of GAS5, microRNA-128-3p (miR-128-3p), and histone deacetylase 4 (HDAC4) in RA synovial tissues and RAFLSs. Proliferation, apoptosis, migration, and invasion were measured by Cell Counting Kit-8 assay (CCK-8), flow cytometry, and transwell assays, severally. The protein levels of B-cell lymphoma-2 (Bcl-2), C-caspase 3, Bcl-2 related X protein (Bax), Tumor Necrosis factor-α (TNF-α), Interleukin 6 (IL-6), Interleukin 17 (IL-17), HDAC4, phosphorylation-protein kinase B (p-AKT), AKT, a phosphorylation-mechanistic target of rapamycin (p-mTOR), and mTOR were assessed by western blot assay. The interaction between miR-128-3p and GAS5 or HDAC4 was predicted by ENCORI or TargetScan Human and verified by the dual-luciferase reporter, RNA Immunoprecipitation (RIP), and RNA pull-down assays. GAS5 and HDAC4 were downregulated, and miR-128-3p was upregulated in RA synovial tissues and RAFLSs. Function analysis indicated that GAS5 curbed proliferation, migration, invasion, inflammation, and facilitated apoptosis of RAFLSs. Rescue assay confirmed that miR-128-3p overexpression or HDAC4 knockdown weakened the inhibitory effect of GAS5 or anti-miR-128-3p on RA development. GAS5 acted as a miR-128-3p sponge to upregulate HDAC4 expression. Besides, GAS5/miR-128-3p/HDAC4 axis regulated RA progression partially through the AKT/mTOR pathway. Our studies disclosed that GAS5 restrained inflammation in synovial tissue partly through regulating HDAC4 via miR-128-3p, suggesting a potential lncRNA-targeted therapy for RA treatment.

LRVRG: a local region-based variational region growing algorithm for fast mandible segmentation from CBCT images.

OBJECTIVES: To segment the mandible from cone-beam computed tomography (CBCT) images efficiently and accurately for the 3D mandible model is essential for subsequent research and diagnosis. METHODS: This paper proposes a local region-based variational region growing algorithm, which integrates local region and shape prior to segment the mandible accurately. Firstly, we select initial seeds in the CBCT image and then calculate candidate point sets and the local region energy function of each point. If a point reduces the energy, it is selected to be a pixel of the foreground region. By multiple iterations, the mandible segmentation of the slice can be obtained. Secondly, the segmented result of the previous slice is adopted as the shape prior to the next slice until all of the slices in CBCT are segmented. At last, the final mandible model is reconstructed by the Marching Cubes algorithm. RESULTS: The experimental results on CBCT datasets illustrate the LRVRG algorithm can obtain satisfied 3D mandible models from CBCT images and it can solve the fuzzy problem effectively. Furthermore, quantitative comparisons with other methods demonstrate the proposed method achieves the state-of-the-art performance in mandible segmentation. CONCLUSIONS: Experiments demonstrate that our method is efficient and accurate for the mandible model segmentation.

Deep Learning for Accurate Diagnosis of Liver Tumor Based on Magnetic Resonance Imaging and Clinical Data.

Background: Early-stage diagnosis and treatment can improve survival rates of liver cancer patients. Dynamic contrast-enhanced MRI provides the most comprehensive information for differential diagnosis of liver tumors. However, MRI diagnosis is affected by subjective experience, so deep learning may supply a new diagnostic strategy. We used convolutional neural networks (CNNs) to develop a deep learning system (DLS) to classify liver tumors based on enhanced MR images, unenhanced MR images, and clinical data including text and laboratory test results. Methods: Using data from 1,210 patients with liver tumors (N = 31,608 images), we trained CNNs to get seven-way classifiers, binary classifiers, and three-way malignancy-classifiers (Model A-Model G). Models were validated in an external independent extended cohort of 201 patients (N = 6,816 images). The area under receiver operating characteristic (ROC) curve (AUC) were compared across different models. We also compared the sensitivity and specificity of models with the performance of three experienced radiologists. Results: Deep learning achieves a performance on par with three experienced radiologists on classifying liver tumors in seven categories. Using only unenhanced images, CNN performs well in distinguishing malignant from benign liver tumors (AUC, 0.946; 95% CI 0.914-0.979 vs. 0.951; 0.919-0.982, P = 0.664). New CNN combining unenhanced images with clinical data greatly improved the performance of classifying malignancies as hepatocellular carcinoma (AUC, 0.985; 95% CI 0.960-1.000), metastatic tumors (0.998; 0.989-1.000), and other primary malignancies (0.963; 0.896-1.000), and the agreement with pathology was 91.9%.These models mined diagnostic information in unenhanced images and clinical data by deep-neural-network, which were different to previous methods that utilized enhanced images. The sensitivity and specificity of almost every category in these models reached the same high level compared to three experienced radiologists. Conclusion: Trained with data in various acquisition conditions, DLS that integrated these models could be used as an accurate and time-saving assisted-diagnostic strategy for liver tumors in clinical settings, even in the absence of contrast agents. DLS therefore has the potential to avoid contrast-related side effects and reduce economic costs associated with current standard MRI inspection practices for liver tumor patients.

Quantitative Assessment of Hippocampal Tau Pathology in AD and PART.

To quantitatively assess the distribution pattern of hippocampal tau pathology in Alzheimer's disease (AD) and primary age-related tauopathy (PART), we investigated the distribution of phosphorylated tau protein (AT8) in 6 anatomically defined subregions of the hippocampal formation and developed a mathematical algorithm to compare the patterns of tau deposition in PART and AD. We demonstrated regional patterns of selective vulnerability as distinguishing features of PART and AD in functionally relevant structures of the hippocampus. In AD cases, tau pathology was high in both CA1 and subiculum, followed by CA2/3, entorhinal cortex (EC), CA4, and dentate gyrus (DG). In PART, the severity of tau pathology in CA1 and subiculum was high, followed by EC, CA2/3, CA4, and DG. There are significant differences between sector DG and CA1, DG and subiculum in both AD and PART.