Recruitment of the m6A/m6Am demethylase FTO to target RNAs by the telomeric zinc finger protein ZBTB48.

BACKGROUND: N6-methyladenosine (m6A), the most abundant internal modification on eukaryotic mRNA, and N6, 2'-O-dimethyladenosine (m6Am), are epitranscriptomic marks that function in multiple aspects of posttranscriptional regulation. Fat mass and obesity-associated protein (FTO) can remove both m6A and m6Am; however, little is known about how FTO achieves its substrate selectivity. RESULTS: Here, we demonstrate that ZBTB48, a C2H2-zinc finger protein that functions in telomere maintenance, associates with FTO and binds both mRNA and the telomere-associated regulatory RNA TERRA to regulate the functional interactions of FTO with target transcripts. Specifically, depletion of ZBTB48 affects targeting of FTO to sites of m6A/m6Am modification, changes cellular m6A/m6Am levels and, consequently, alters decay rates of target RNAs. ZBTB48 ablation also accelerates growth of HCT-116 colorectal cancer cells and modulates FTO-dependent regulation of Metastasis-associated protein 1 (MTA1) transcripts by controlling the binding to MTA1 mRNA of the m6A reader IGF2BP2. CONCLUSIONS: Our findings thus uncover a previously unknown mechanism of posttranscriptional regulation in which ZBTB48 co-ordinates RNA-binding of the m6A/m6Am demethylase FTO to control expression of its target RNAs.

C2H2-zinc-finger transcription factors bind RNA and function in diverse post-transcriptional regulatory processes.

Cys2-His2 zinc-finger proteins (C2H2-ZNFs) constitute the largest class of DNA-binding transcription factors (TFs) yet remain largely uncharacterized. Although certain family members, e.g., GTF3A, have been shown to bind both DNA and RNA, the extent to which C2H2-ZNFs interact with-and regulate-RNA-associated processes is not known. Using UV crosslinking and immunoprecipitation (CLIP), we observe that 148 of 150 analyzed C2H2-ZNFs bind directly to RNA in human cells. By integrating CLIP sequencing (CLIP-seq) RNA-binding maps for 50 of these C2H2-ZNFs with data from chromatin immunoprecipitation sequencing (ChIP-seq), protein-protein interaction assays, and transcriptome profiling experiments, we observe that the RNA-binding profiles of C2H2-ZNFs are generally distinct from their DNA-binding preferences and that they regulate a variety of post-transcriptional processes, including pre-mRNA splicing, cleavage and polyadenylation, and m6A modification of mRNA. Our results thus define a substantially expanded repertoire of C2H2-ZNFs that bind RNA and provide an important resource for elucidating post-transcriptional regulatory programs.

Establishment of orthotopic osteosarcoma animal models in immunocompetent rats through muti-rounds of in-vivo selection.

Immunodeficient murine models are usually used as the preclinical models of osteosarcoma. Such models do not effectively simulate the process of tumorigenesis and metastasis. Establishing a suitable animal model for understanding the mechanism of osteosarcoma and the clinical translation is indispensable. The UMR-106 cell suspension was injected into the marrow cavity of Balb/C nude mice. Tumor masses were harvested from nude mice and sectioned. The tumor fragments were transplanted into the marrow cavities of SD rats immunosuppressed with cyclosporine A. Through muti-rounds selection in SD rats, we constructed orthotopic osteosarcoma animal models using rats with intact immune systems. The primary tumor cells were cultured in-vitro to obtain the immune-tolerant cell line. VX2 tumor fragments were transplanted into the distal femur and parosteal radius of New Zealand white rabbit to construct orthotopic osteosarcoma animal models in rabbits. The rate of tumor formation in SD rats (P1 generation) was 30%. After four rounds of selection and six rounds of acclimatization in SD rats with intact immune systems, we obtained immune-tolerant cell lines and established the orthotopic osteosarcoma model of the distal femur in SD rats. Micro-CT images confirmed tumor-driven osteolysis and the bone destruction process. Moreover, the orthotopic model was also established in New Zealand white rabbits by implanting VX2 tumor fragments into rabbit radii and femurs. We constructed orthotopic osteosarcoma animal models in rats with intact immune systems through muti-rounds in-vivo selection and the rabbit osteosarcoma model.

Targeting nanoplatform synergistic glutathione depletion-enhanced chemodynamic, microwave dynamic, and selective-microwave thermal to treat lung cancer bone metastasis.

Once bone metastasis occurs in lung cancer, the efficiency of treatment can be greatly reduced. Current mainstream treatments are focused on inhibiting cancer cell growth and preventing bone destruction. Microwave ablation (MWA) has been used to treat bone tumors. However, MWA may damage the surrounding normal tissues. Therefore, it could be beneficial to develop a nanocarrier combined with microwave to treat bone metastasis. Herein, a microwave-responsive nanoplatform (MgFe2O4@ZOL) was constructed. MgFe2O4@ZOL NPs release the cargos of Fe3+, Mg2+ and zoledronic acid (ZOL) in the acidic tumor microenvironment (TME). Fe3+ can deplete intracellular glutathione (GSH) and catalyze H2O2 to generate •OH, resulting in chemodynamic therapy (CDT). In addition, the microwave can significantly enhance the production of reactive oxygen species (ROS), thereby enabling the effective implementation of microwave dynamic therapy (MDT). Moreover, Mg2+ and ZOL promote osteoblast differentiation. In addition, MgFe2O4@ZOL NPs could target and selectively heat tumor tissue and enhance the effect of microwave thermal therapy (MTT). Both in vitro and in vivo experiments revealed that synergistic targeting, GSH depletion-enhanced CDT, MDT, and selective MTT exhibited significant antitumor efficacy and bone repair. This multimodal combination therapy provides a promising strategy for the treatment of bone metastasis in lung cancer patients.

Long noncoding RNA X-inactive specific transcript (lncRNA XIST) inhibits hepatic insulin resistance by competitively binding microRNA-182-5p.

BACKGROUND: What is highlighted in this study refers to the role and molecular mechanism of long noncoding RNA (lncRNA) X-inactive specific transcript (XIST) in cells with insulin resistance (IR). METHODS: In this study, LX-2 cells were applied to establish IR model in vitro. The expressions of lncRNA XIST, phosphoenolpyruvate carboxykinase (PEPCK,) and glucose-6-phosphatase (G6Pase) were quantified by quantitative reverse transcription polymerase chain reaction. The 2-deoxy-d-glucose-6-phosphate (2-DG6P) level was detected utilizing 2-deoxy-d-glucose (2-DG) uptake measurement kit. Western blot was adopted to measure the protein expressions of insulin-like growth factor-1 receptor (IGF-1R), G6Pase, PEPCK, and phosphatidylinositol 3-kinase (PI3K)/Akt pathway-related genes. StarBase was used to predict the targeting relationship between lncRNA XIST or IGF-1R with miR-182-5p, the results of which were verified by dual-luciferase reporter, RNA pull-down, and RNA immunoprecipitation assays. Rescue experiments were conducted to investigate the effect of miR-182-5p on IR cells. Next, low-expressed lncRNA XIST and high-expressed miR-182-5p were observed in IR cells. RESULTS: Upregulation of lncRNA XIST increased IGF-1R and 2-DG6P levels, decreased G6Pase and PEPCK expressions, and promoted PI3K/Akt pathway activation in IR cells. LncRNA XIST sponged miR-182-5p which targeted IGF-1R. MiR-182-5p mimic reversed the above effects of lncRNA XIST overexpression on IR cells. CONCLUSIONS: In conclusion, lncRNA XIST/miR-182-5p axis alleviates hepatic IR in vitro via IGF-1R/PI3K/Akt signaling pathway, which could be the promising therapeutic target.

Diagnosis of generalized joint hypermobility with gait patterns using a deep neural network.

Generalized joint hypermobility (GJH) describes the situation that the range of joint motion exceeds the normal range. GJH is found to increase the risk of knee-related injury and osteoarthritis, challenging the athletic ability of the population. Gait signals are directly related to hip and knee athletic conditions, and have been shown to have significant changes with GJH by our previous research. But gait data are noisy, and vary with age, gender, weight, and ethnicity, which makes them hard to analyze with traditional statistical methods. In this study, we proposed an end-to-end deep learning model to recognize the patterns of the gait signals. The model consists of convolutional network blocks, residual network blocks, and attention blocks. Our dataset is composed of 452 samples of gait data obtained by a three-dimension motion capture system, with the six-degree-of-freedom kinematic data of hip, knee, and ankle joints during level walking, downhill, and uphill walking. The model achieves 95.77% accuracy and 98.68% specificity with a recall of 76.84% while is more efficient than traditional machine learning methods. The trained model can be run on economical friendly devices, and provide help for immediate and precise diagnosis of GJH. It is also meaningful to consider its application in large-scale GJH screening, which can contribute to sports medicine.

Premetastatic Niche Mimicking Bone-On-A-Chip: A Microfluidic Platform to Study Bone Metastasis in Cancer Patients.

Primary cancer modulates the bone microenvironment to sow the seeds of dormancy and metastasis in tumor cells, leading to multiple organ metastasis and death. In this study, 3D printing and bone-on-a-chip (BOC) are combined to develop a BOC platform that mimics the pre-metastatic niches (PMNs) and facilitates elucidation of the interactions between bone-resident cells and metastatic tumor cells under the influence of primary cancer. Photocrosslinkable gelatin methacrylate (GelMA) is used as a 3D culturing hydrogel to encapsulate cells, and circulate tumor culture medium (CM) adjacent to the hydrogel to verify the critical role of mesenchymal stem cells (MSCs) and osteoclasts (RAW264.7s). Three niches: the dormancy niche, the perivascular niche, and the "vicious cycle" niche, are devised to recapitulate bone metastasis in one chip with high cell viability and excellent nutrient exchange. With respect to tumor dormancy and reactivation, the invadopodia formation of A549 lung cancer cells in communication with MSCs and RAW264.7 via the cortactin pathway is researched. As a proof of concept, the functionality and practicality of the platform are demonstrated by analyzing the invadopodia formation and the influence of various cells, and the establishment of the dynamic niches paves the way to understanding PMN formation and related drug discovery.

Multifunctional 3D-printed scaffolds eradiate orthotopic osteosarcoma and promote osteogenesis via microwave thermo-chemotherapy combined with immunotherapy.

Tumor recurrence and a lack of bone-tissue integration are two critical concerns in the surgical treatment of osteosarcoma. Thus, an advanced multifunctional therapeutic platform capable of simultaneously eliminating residual tumor cells and promoting bone regeneration is urgently needed for efficient osteosarcoma treatment. Herein, to thoroughly eliminate tumors and simultaneously promote bone regeneration, an intelligent multifunctional therapeutic scaffold has been engineered by integrating microwave-responsive zeolitic imidazolate framework 8 (ZIF-8) nanomaterials loaded with a chemotherapeutic drug and an immune checkpoint inhibitor onto 3D-printed titanium scaffolds. The constructed scaffold features distinct microwave-thermal sensitization and tumor microenvironment-responsive characteristics, which can induce tumor immunogenic death by microwave hyperthermia and chemotherapy. Orthotopic implantation of the nanocomposite scaffold results in an enhanced immune response against osteosarcoma that may effectively inhibit tumor recurrence through synergistic immunotherapy. During long-term implantation, the zinc ions released from the degradation of ZIF-8 can induce the osteogenic differentiation of stem cells. The porous structure and mechanical properties of the 3D-printed titanium scaffolds provide a structural microenvironment for bone regeneration. This study provides a paradigm for the design of multifunctional microwave-responsive composite scaffolds for use as a therapy for osteosarcoma, which could lead to improved strategies for the treatment of the disease.

Comparing the Accuracy of Seven Scoring Systems in Predicting Survival of Lung Cancer Patients With Spinal Metastases: An External Validation From Two Centers.

STUDY DESIGN: Retrospective case series. OBJECTIVE: To investigate the accuracy of seven scoring systems for the prediction of survival in lung cancer patients with spinal metastases (SPM). SUMMARY OF BACKGROUND DATA: Although survival scoring systems have been developed for surgical decision-making, the reliability and validity of these models are unclear for specific cancer types. As the prevalence of patients with lung cancer increases, it is imperative to determine the accuracy of these models for lung cancer patients with SPM. MATERIALS AND METHODS: This is a retrospective study of a cohort of lung cancer patients with SPM who underwent spine surgery between 2019 and 2021 at two centers. The optimal area under the curve (AUC) was calculated to evaluate the accuracy of seven candidate scoring systems at 3, 6, and 12 months. Calibration and decision curve analysis was used for further validation. RESULTS: A total of 166 patients (mean age: 58.98±10.94; 105 males and 61 females) with SPM were included. The median postoperative survival was 12.87±0.93 months. The modified Bauer score, revised Tokuhashi score, Linden score, Tomita score, the Skeletal Oncology Research Group nomogram, and the New England Spinal Metastasis Score in prediction survival at 3, 6, and 12 months showed a slightly weaker AUC (range 0.464-0.659). The AUC of the Katagiri-New score in predicting 1-year survival for lung cancer patients was the highest (0.708; range 0.619-0.798). The decision curve analysis showed that the Katagiri-New score led to a greater net benefit than the strategies of changing management for all patients or none of the patients. CONCLUSIONS: This study suggests that the most commonly used models have limitations in predicting survival in patients undergoing spinal surgery for metastatic lung cancer and underestimate survival. In this sample of lung cancer patients, the Katagiri-New Scoring system score had the best performance in predicting 1-year survival. LEVEL OF EVIDENCE: 4.

Morphological feature recognition of different differentiation stages of induced ADSCs based on deep learning.

In order to accurately identify the morphological features of different differentiation stages of induced Adipose Derived Stem Cells (ADSCs) and judge the differentiation types of induced ADSCs, a morphological feature recognition method of different differentiation stages of induced ADSCs based on deep learning is proposed. Using the super-resolution image acquisition method of ADSCs differentiation based on stimulated emission depletion imaging, after obtaining the super-resolution images at different stages of inducing ADSCs differentiation, the noise of the obtained image is removed and the image quality is optimized through the ADSCs differentiation image denoising model based on low rank nonlocal sparse representation; The denoised image is taken as the recognition target of the morphological feature recognition method for ADSCs differentiation image based on the improved Visual Geometry Group (VGG-19) convolutional neural network. Through the improved VGG-19 convolutional neural network and class activation mapping method, the morphological feature recognition and visual display of the recognition results at different stages of inducing ADSCs differentiation are realized. After testing, this method can accurately identify the morphological features of different differentiation stages of induced ADSCs, and is available.

Generalized joint hypermobility subjects without knee hyperextension have greater walking anterior tibial translation and flexion angle than those with knee hyperextension.

BACKGROUND: The walking knee kinematic results of generalized joint hypermobility (GJH) subjects were controversial in previous studies. We proposed that this could be related to the knee statuses of GJH subjects with/without knee hyperextension (KH) and assumed that there are significant sagittal knee kinematic differences between GJH subjects with/without KH during gait. RESEARCH QUESTION: Do GJH subjects with KH exhibit significantly different kinematic characteristics than those without KH during walking? METHODS: 35 GJH subjects without KH, 34 GJH subjects with KH, and 30 healthy controls were recruited in this study. A three-dimensional gait analysis system was used to record and compare the knee kinematics of the participants. RESULTS: Significant walking knee kinematics differences were found between GJH subjects with/without KH during walking. GJH subjects without KH had greater flexion angles (4.7-6.0°, 24-53 % gait cycle (GC), p < 0.001; 5.1-6.1°, 65-77 % GC, p = 0.008) and anterior tibial translation (ATT) (3.3-4.1 mm, 0-4 % GC, p = 0.015; 3.8-4.3 mm, 91-100 % GC, p = 0.01) than those with KH. Compared to controls, GJH without KH exhibited increased ATT (4.0-5.7 mm, 0-26 % GC, p < 0.001; 5.1-6.7 mm, 78-100 % GC, p < 0.001), and range of motion of ATT (3.3 mm, p = 0.028) whereas GJH with KH only exhibited increased extension angle (6.9-7.3°, 62-66 % GC, p = 0.015) during walking. SIGNIFICANCE: The findings confirmed the hypothesis and suggested that GJH subjects without KH had more walking ATT and flexion angle asymmetries than those with KH. This may raise concerns about the differences in knee health and risk of knee diseases between GJH subjects with/without KH. However, further investigations should be done to explore the exact influence of walking ATT and flexion angle asymmetries in GJH subjects without KH.

External validation of the SORG machine learning algorithms for predicting 90-day and 1-year survival of patients with lung cancer-derived spine metastases: a recent bi-center cohort from China.

BACKGROUND CONTEXT: The survival prediction of lung cancer-derived spinal metastases is often underestimated by several scores. The SORG machine learning (ML) algorithm is considered a promising tool to predict the risk of 90-day and 1-year mortality in patients with spinal metastases, but not been externally validated for lung cancer. PURPOSE: This study aimed to externally validate the SORG ML algorithms on lung cancer-derived spinal metastases patients from two large-volume, tertiary medical centers between 2018 and 2021. STUDY DESIGN/SETTING: Retrospective, cohort study. PATIENT SAMPLE: Patients aged 18 years or older at two tertiary medical centers in China are treated surgically for spinal metastasis. OUTCOME MEASURES: Mortality within 90 days of surgery, mortality within 1 year of surgery. METHODS: The baseline characteristics were compared between the development cohort and our validation cohort. Discrimination (receiver operating curve), calibration (calibration plot, intercept, and slope), the overall performance (Brier score), and decision curve analysis was used to assess the overall performance of the SORG ML algorithms. RESULTS: This study included 150 patients with lung cancer-derived spinal metastases from two medical centers in China. Ninety-day and 1-year mortality rates were 12.9% (19/147) and 51.3% (60/117), respectively. Lung Cancer with targeted therapies had the lowest Hazard Ratio (HR=0.490), showing an optimal protecting factor. The AUC of the SORG ML algorithm for 90-day mortality prediction in lung cancer-derived spinal metastases is 0.714. While the AUC for 1-year mortality prediction is 0.832 (95CI%, 0.758-0.906). The algorithm for 1-year mortality was well-calibrated with an intercept of 0.13 and a calibration slope of 1.00. However, the 90-day mortality prediction was underestimated with an intercept of 0.60 and a slope of 0.37. The SORG ML algorithms for 1-year mortality showed a greater net benefit than the "treats all or no patients" strategies. CONCLUSIONS: In the latest cohort of lung cancer-derived spinal metastases in China, the SORG algorithms for predicting 1-year mortality performed well on external validation. However, 90-day mortality was underestimated. The algorithm should be further validated by single primary tumor-derived metastasis treated with the latest comprehensive treatment in diverse populations.

6DOF knee kinematic alterations due to increased load levels.

Whether load carriage leads to six-degrees-of-freedom (6DOF) knee kinematic alterations remains unclear. Exploring this mechanism may reveal meaningful knee kinematic information that can be used to improve load carriage conditions, the design of protective devices, and the knowledge of the effects of load carriage on knees. We recruited 44 subjects to explore kinematic alterations from an unloaded state to 60% bodyweight (BW) load carriage. A three-dimensional gait analysis system was used to collect the knee kinematic data. One-way repeated analysis of variance (ANOVA) was used to explore the effects of load levels on knee kinematics. The effects of increasing load levels on knee kinematics were smooth with decreased or increased trends. We found that knees significantly exhibited increased lateral tibial translation (up to 1.2 mm), knee flexion angle (up to 1.4°), internal tibial rotation (up to 1.3°), and tibial proximal translation (up to 1.0 mm) when they went from an unloaded state to 60%BW load carriage during the stance phase (p < 0.05). Significant small knee adduction/abduction angle and posterior tibial translation alterations (<1°/mm) were also identified (p < 0.05). Load carriage can cause significant 6DOF knee kinematic alterations. The results showed that knee kinematic environments are challenging during increased load. Our results contain kinematic information that could be helpful for knee-protection-related activities, such as target muscle training to reduce abnormal knee kinematics and knee brace design.

Knee Kinematic Patterns and Early Cartilage Lesion Characteristics in Patients with Anterior Cruciate Ligament Reconstruction.

Specific knee kinematic alterations have been theorized to correlate with the progression of cartilage degeneration, and therefore, post-traumatic osteoarthritis in patients with anterior cruciate ligament reconstruction (ACLR). However, how specific knee kinematic alterations contribute to knee joint cartilage degenerations remains to be unclear. To solve this problem, we hypothesized that there are specific cartilage-degenerating kinematic gait patterns that could be supported by the specific areas of cartilage lesions in ACLR knees. Thirty patients with unilateral ACLR knees and 30 healthy controls were recruited for the study. The kinematic differences between the ACLR knees and the healthy control knees during the stance phase were calculated to identify the kinematic patterns. Cartilage lesion distribution characteristics were acquired for patients with ACLR knees to validate the kinematic patterns using magnetic resonance images. Two kinematic patterns were modeled, i.e., sagittal (increased flexion angle and posterior tibial translation) and coronal (increased lateral tibial translation and abduction angle) kinematic patterns. For the sagittal pattern, the cartilage lesion distributions showed that there were more cartilage lesions (CLs) in the superoposterior regions than the posterior regions in the femoral condyles (p = 0.001), and more CLs in the posterior regions than the middle regions in the tibial plateau (p < 0.001). For the coronal pattern, the cartilage lesion distributions showed that there were more CLs in the lateral compartments near the tibial spine than the medial compartments near the tibial spine (tibial sides, p = 0.005 and femoral sides, p = 0.290). To conclude, the cartilage degeneration distribution evidence largely supports that the two kinematic patterns may contribute to cartilage degeneration in ACLR knees. These findings may provide a potential strategy of delaying early cartilage degeneration in ACLR knees by using motion (kinematic) pattern modification or training. However, investigations should be conducted on the actual effects of this potential strategy.

Kinematic alterations of the ankle in subjects with generalized joint hypermobility compared with the controls: A cross-sectional study.

INTRODUCTION: Generalized joint hypermobility (GJH) is a hereditary connective tissue disease in which the range of motion (ROM) of multiple joints exceeds the normal range, and the ROM varies with age, gender, and ethnicity. At present, the six-degree-of-freedom (6-DOF) of ankle kinematics among people with GJH have not been studied. To investigate the kinematic characteristics in the ankle during treadmill gait of university students with generalized joint hypermobility compared to normal participants. We hypothesized that compared to the participants in the control group, those with GJH would exhibit kinematic characteristics of poorer active motion stability in the ankle during treadmill gait. METHODS: Healthy university student volunteers aged 18-24 (excluding those with a history of ankle trauma, etc.) were recruited and divided into a control group (50 volunteers) and a GJH group (Beighton score ≥4, 50 volunteers). Data of the 6-DOF kinematics of ankle was collected using a 3D gait analysis system. Variables were evaluated using independent t-tests and Wilcoxon signed-rank tests. RESULTS: In the proximal/distal parameter, proximal displacement was significantly increased in the GJH group compared with the control group during 4-9% and 96-97% of the gait phase (loading response and terminal swing phase), with an increase of (0.1-0.2 cm, p < .05). Regarding the proximal/distal, internal/external, plantarflexion/dorsiflexion, and anterior/posterior parameters, the participants with GJH exhibited greater ROM than those in the control group throughout the gait cycle (0.24 ± 0.22 cm vs. 0.19 ± 0.15 cm, p = 0.047, 5.56 ± 2.90° vs. 4.48 ± 3.30°, p = .020, 23.05 ± 5.75° vs. 20.36 ± 4.91°, p < .001, 0.65 ± 0.30 cm vs. 0.55 ± 0.27 cm, p = .018). However, ROM of inversion/eversion translation was found to be decreased in the GJH group compared to the control group (8.92 ± 1.59° vs. 9.47 ± 1.37°, p = .009). In addition, there was no statistical difference between the GJH group and the control group in ROM of medial/lateral translation (0.05 ± 0.06 cm vs. 0.04 ± 0.05 cm, p = .131). CONCLUSION: Our results confirm that our hypothesis is not valid. Although there were a few differences in each gait parameter of the ankle between the GJH group and the control group, the difference was not significant. These results indicate that the presence of GJH has less effect on ankle kinematics and enhance our knowledge of the relationship between GJH and 6-DOF of ankle kinematics.

Regulation of alternative polyadenylation by the C2H2-zinc-finger protein Sp1.

Alternative polyadenylation (APA) enhances gene regulatory potential by increasing the diversity of mRNA transcripts. 3' UTR shortening through APA correlates with enhanced cellular proliferation and is a widespread phenomenon in tumor cells. Here, we show that the ubiquitously expressed transcription factor Sp1 binds RNA in vivo and is a common repressor of distal poly(A) site usage. RNA sequencing identified 2,344 genes (36% of the total mapped mRNA transcripts) with lengthened 3' UTRs upon Sp1 depletion. Sp1 preferentially binds the 3' UTRs of such lengthened transcripts and inhibits cleavage at distal sites by interacting with the subunits of the core cleavage and polyadenylation (CPA) machinery. The 3' UTR lengths of Sp1 target genes in breast cancer patient RNA-seq data correlate with Sp1 expression levels, implicating Sp1-mediated APA regulation in modulating tumorigenic properties. Taken together, our findings provide insights into the mechanism for dynamic APA regulation by unraveling a previously unknown function of the DNA-binding transcription factor Sp1.

Frequently expressed glypican-3 as a promising novel therapeutic target for osteosarcomas.

Osteosarcoma (OS) is the most common bone malignancy without a reliable therapeutic target. Glypican-3 (GPC3) mutation and upregulation have been detected in multidrug resistant OS, and anti-GPC3 immunotherapy can effectively suppress the growth of organoids. Further profiling of GPC3 mutations and expression patterns in OS is of clinical significance. To address these issues, fresh OS specimens were collected from 24 patients for cancer-targeted next-generation sequencing (NGS) and three-dimensional patient-derived organoid (PDO) culture. A tumor microarray was prepared using 37 archived OS specimens. Immunohistochemical (IHC) staining was performed on OS specimens and microarrays to profile GPC3 and CD133 expression as well as intratumoral distribution patterns. RT-PCR was conducted to semiquantify GPC3 and CD133 expression levels in the OS tissues. Anti-GPC3 immunotherapy was performed on OS organoids with or without GPC3 expression and its efficacy was analyzed using multiple experimental approaches. No OS cases with GPC3 mutations were found, except for the positive control (OS-08). IHC staining revealed GPC3 expression in 73.77% (45/61) of OSs in weak (+; 29/45), moderate (++; 8/45), and strong (+++; 8/45) immunolabeling densities. The intratumoral distribution of GPC3-positive cells was variable in the focal (+; 10%-30%; 8/45), partial (++; 31%-70%; 22/45), and the most positive patterns (+++; >71%; 15/45), which coincided with CD133 immunolabeling (P = 9.89 × 10-10 ). The anti-GPC3 antibody efficiently inhibits Wnt/ß-catenin signaling and induces apoptosis in GPC3-positive PDOs and PDXs, as opposed to GPC3-negative PDOs and PDXs. The high frequency of GPC3 and CD133 co-expression and the effectiveness of anti-wild-type GPC3-Ab therapy in GPC3-positive OS models suggest that GPC3 is a novel prognostic parameter and a promising therapeutic target for osteosarcoma.

Upslope walking increases anterior tibial translation deficiency in patients with generalized joint hypermobility.

BACKGROUND: Generalized joint hypermobility (GJH) is a highly prevalent disease that frequently affects the knee joint. The current literature has conflicting results about whether patients with GJH had knee kinematics deficiency during gait. This could be because most of the testing environment (level walking) was gentle and low-demanding for patients when studying their knee kinematics. With a high-demanding knee function and sagittal firm structure requirement, upslope walking was thought to stimulate sagittal knee kinematics deficiency in patients with GJH. RESEARCH QUESTIONS: However, only little investigation reported whether upslope walking could stimulate knee kinematic deficiency or not. We hypothesize that upslope walking can increase sagittal knee kinematic deficiency between GJH subjects and healthy controls. METHODS: A three-dimensional motion analysis was conducted to explore whether upslope walking could stimulate sagittal knee kinematic deficiency in patients with GJH. A total of 44 patients with GJH and 44 healthy controls were recruited. Subjects walked on both level and upslope (15%) conditions when the kinematic data were collected. SPM1D analysis was taken to explore the differences between groups. RESULTS: Our results showed that upslope walking could significantly increase knee flexion angle and anterior tibial translation in both GJH patients and healthy controls (p < 0.05). The increments of anterior tibial translation (values in upslope walking minus values in level walking) of GJH patients were greater than those of healthy controls (magnitude varying from 2.5 to 2.9 mm during 0-3% gait cycles (GC), p = 0.034; 1.4-2.9 mm during 93-100%GC, p = 0.012). SIGNIFICANCES: The findings partially confirmed our hypothesis and suggested that upslope walking could increase anterior tibial translation deficiency in patients with GJH. Upslope walking may be a practical motion task in studying the weakness of knee kinematics of GJH subjects for researchers and scholars. Patients with GJH may face a more challenging knee kinematic environment than healthy controls in up-sloped activities.

Two Cannulated Screws Provide Sufficient Biomechanical Strength for Prophylactic Fixation in Adult Patients With an Aggressive Benign Femoral Neck Lesion.

Background: Two cannulated screws were proposed for prophylactic fixation in adult patients with an aggressive benign femoral neck lesion in recent literature. However, the biomechanical properties of this intervention have not yet been investigated. Methods: After the evaluation of the heterogeneity of bone mineral density and geometry via quantitative computed tomography, 24 embalmed adult human cadaver femurs were randomized into the control, inferior half of the anterior cortical (25%) bone defect, entire anterior cortical (50%) bone defect, and the 50% bone defect and two cannulated screw group. Biomechanical analysis was conducted to compare the stiffness and failure load among the four groups when mimicking a one-legged stance. A CT-based finite element analysis (FEA) was performed to mimic the cortical and cancellous bone defect and the implantation of two cannulated screws of the four groups. Measurements of the maximal displacement and von Mises stress were conducted with the longitudinal load force and boundary conditions being established for a one-leg-standing status. Results: We noted a significant improvement in the failure load after the insertion of two 6.5 mm cannulated screws in femurs with 50% bone defect (+95%, p = 0.048), and no significant difference was found between the screw group and the intact femur. Similar trends were also found in the measurements of stiffness (+23%, p > 0.05) via biomechanical testing and the von Mises stresses (-71%, p = 0.043) by FEA when comparing the screw group and the 50% bone defect group. Conclusion: Our findings suggest that two cannulated screws provided sufficient biomechanical strength for prophylactic fixation in adult patients with an aggressive benign femoral neck lesion even when the entire anterior cortical bone is involved.

Oxyhydroxide-Coated PEO-Treated Mg Alloy for Enhanced Corrosion Resistance and Bone Regeneration.

Plasma electrolytic oxidation (PEO) is widely used as a surface modification method to enhance the corrosion resistance of Mg alloy, the most likely applied biodegradable material used in orthopedic implants. However, the pores and cracks easily formed on the PEO surface are unfavorable for long-term corrosion resistance. In this study, to solve this problem, we used simple immersion processes to construct Mn and Fe oxyhydroxide duplex layers on the PEO-treated AZ31 (PEO-Mn/Fe). As control groups, single Mn and Fe oxyhydroxide layers were also fabricated on PEO (denoted as PEO-Mn and PEO-Fe, respectively). PEO-Mn showed a similar porous morphology to the PEO sample. However, the PEO-Fe and PEO-Mn/Fe films completely sealed the pores on the PEO surfaces, and no cracks were observed even after the samples were immersed in water for 7 days. Compared with PEO, PEO-Mn, and PEO-Fe, PEO-Mn/Fe exhibited a significantly lower self-corrosion current, suggesting better corrosion resistance. In vitro C3H10T1/2 cell culture showed that PEO-Fe/Mn promoted the best cell growth, alkaline phosphatase activity, and bone-related gene expression. Furthermore, the rat femur implantation experiment showed that PEO-Fe/Mn-coated Mg showed the best bone regeneration and osteointegration abilities. Owing to enhanced corrosion resistance and osteogenesis, the PEO-Fe/Mn film on Mg alloy is promising for orthopedic applications.