Transcriptome and single-cell analysis reveal disulfidptosis-related modification patterns of tumor microenvironment and prognosis in osteosarcoma.

Osteosarcoma (OS) is the most common malignant bone tumor with high pathological heterogeneity. Our study aimed to investigate disulfidptosis-related modification patterns in OS and their relationship with survival outcomes in patients with OS. We analyzed the single-cell-level expression profiles of disulfidptosis-related genes (DSRGs) in both OS microenvironment and OS subclusters, and HMGB1 was found to be crucial for intercellular regulation of OS disulfidptosis. Next, we explored the molecular clusters of OS based on DSRGs and related immune cell infiltration using transcriptome data. Subsequently, the hub genes of disulfidptosis in OS were screened by applying multiple machine models. In vitro and patient experiments validated our results. Three main disulfidptosis-related molecular clusters were defined in OS, and immune infiltration analysis suggested high immune heterogeneity between distinct clusters. The in vitro experiment confirmed decreased cell viability of OS after ACTB silencing and higher expression of ACTB in patients with lower immune scores. Our study systematically revealed the underlying relationship between disulfidptosis and OS at the single-cell level, identified disulfidptosis-related subtypes, and revealed the potential role of ACTB expression in OS disulfidptosis.

RARRES2 is involved in the "lock-and-key" interactions between osteosarcoma stem cells and tumor-associated macrophages.

Osteosarcoma (OS) is a type of tumor. Osteosarcoma stem cells (OSCs) are responsible for drug resistance, recurrence, and immunosuppression in OS. We aimed to determine the heterogeneity of OSCs and the immunosuppression mechanisms underlying the interactions between OSCs and tumor-associated macrophages (TAMs). The cell components, trajectory changes, and cell communication profiles of OS cells were analyzed by transcriptomics at the single-cell level. The intercellular communication patterns of OSCs were verified, and the role of the cell hub genes was revealed. Hub geneS are genes that play important roles in regulating certain biological processes; they are often defined as the genes with the strongest regulatory effect on differentially expressed gene sets. Moreover, various cellular components of the OS microenvironment were identified. Malignant cells were grouped, and OSCs were identified. Further regrouping and communication analysis revealed that the genes in the stemness maintenance and differentiation subgroups were involved in communication with macrophages. Key receptor-ligand pairs and target gene sets for cell communication were obtained. Transcriptome data analysis revealed the key gene RARRES2, which is involved in intercellular communication between OSCs and TAMs. In vitro studies confirmed that macrophages promote RARRES2-mediated stemness maintenance in OSCs via the TAM-secreted cytokine insulin-like growth factor 1. Patient studies confirmed that RARRES2 could be a biomarker of OS. OSCs are highly heterogeneous, and different subgroups are responsible for proliferation and communication with other cells. The IGF-RARRES2 axis plays a key role in maintaining OSC stemness through communication with TAMs.

Lateral trochlear inclination measured by the transepicondylar axis holds potential for evaluating trochlear dysplasia in patients with lateral patellar dislocation.

PURPOSE: To verify that lateral trochlear inclination (LTI) measured by the transepicondylar axis can reliably be used to evaluate trochlear dysplasia (TD) on MRI and can serve as an objective indication of trochleoplasty for patients with lateral patellar dislocation (LPD). METHODS: Eighty patients with recurrent LPD and eighty healthy subjects were included. TD, posterior condylar angle (PCA), and LTI measured by the posterior condylar reference line (LTIp), surgical transepicondylar axis (LTIs), and anatomical transepicondylar axis (LTIa) were assessed on MRI. The intraclass correlation coefficient (ICC) and Bland-Altman analysis were performed, the correlations and differences amongst the parameters were identified, and a binary logistic regression model was established. RESULTS: Each measurement had excellent inter- and intra-observer agreement. The LTIp, LTIs and LTIa were smaller in the study group than in the control group, with mean differences of 9.0°, 7.2° and 7.0°, respectively (P < 0.001). The PCA was larger in patients with LPD than in the control group (P < 0.001). LTIp was associated with PCA in the study group (r = - 0.41, P < 0.001). The pathological values of LTIp, LTIs and LTIa were 11.7°, 15.3° and 17.4°, respectively. LTIs and LTIa were independent risk factors for LPD, with ORs of 7.33 (95% CI [1.06-52.90], P = 0.048) and 10.29 (95% CI [1.38-76.96], P = 0.023), respectively. CONCLUSION: The LTI can be reliably measured by MRI, but LTIp could potentially decrease the recorded value from the actual inclination angle. LTIs and LTIa are more appropriate to serve as trochleoplasty indications for patients with LPD, which could help orthopedists with surgical decision-making. LEVEL OF EVIDENCE: Level III.

Capsaicin Reduces Cancer Stemness and Inhibits Metastasis by Downregulating SOX2 and EZH2 in Osteosarcoma.

Metastasis of osteosarcoma is an important adverse factor affecting patients' survival, and cancer stemness is the crucial cause of distant metastasis. Capsaicin, the main component of pepper, has been proven in our previous work to inhibit osteosarcoma proliferation and enhance its drug sensitivity to cisplatin at low concentrations. This study aims to further explore the anti-osteosarcoma effect of capsaicin at low concentrations (100[Formula: see text][Formula: see text]M, 24[Formula: see text]h) on stemness and metastasis. The stemness of human osteosarcoma (HOS) cells was decreased significantly by capsaicin treatment. Additionally, the capsaicin treatment's inhibition of cancer stem cells (CSCs) was dose-dependent on both sphere formation and sphere size. Meanwhile, capsaicin inhibited invasion and migration, which might be associated with 25 metastasis-related genes. SOX2 and EZH2 were the most two relevant stemness factors for capsaicin's dose-dependent inhibition of osteosarcoma. The mRNAsi score of HOS stemness inhibited by capsaicin was strongly correlated with most metastasis-related genes of osteosarcoma. Capsaicin downregulated six metastasis-promoting genes and up-regulated three metastasis-inhibiting genes, which significantly affected the overall survival and/or disease-free survival of patients. In addition, the CSC re-adhesion scratch assay demonstrated that capsaicin inhibited the migration ability of osteosarcoma by inhibiting its stemness. Overall, capsaicin exerts a significant inhibitory effect on the stemness expression and metastatic ability of osteosarcoma. Moreover, it can inhibit the migratory ability of osteosarcoma by suppressing its stemness via downregulating SOX2 and EZH2. Therefore, capsaicin is expected to be a potential drug against osteosarcoma metastasis due to its ability to inhibit cancer stemness.

Role of amino acid metabolism in mitochondrial homeostasis.

Mitochondria are central hubs for energy production, metabolism and cellular signal transduction in eukaryotic cells. Maintenance of mitochondrial homeostasis is important for cellular function and survival. In particular, cellular metabolic state is in constant communication with mitochondrial homeostasis. One of the most important metabolic processes that provide energy in the cell is amino acid metabolism. Almost all of the 20 amino acids that serve as the building blocks of proteins are produced or degraded in the mitochondria. The synthesis of the amino acids aspartate and arginine depends on the activity of the respiratory chain, which is essential for cell proliferation. The degradation of branched-chain amino acids mainly occurs in the mitochondrial matrix, contributing to energy metabolism, mitochondrial biogenesis, as well as protein quality control in both mitochondria and cytosol. Dietary supplementation or restriction of amino acids in worms, flies and mice modulates lifespan and health, which has been associated with changes in mitochondrial biogenesis, antioxidant response, as well as the activity of tricarboxylic acid cycle and respiratory chain. Consequently, impaired amino acid metabolism has been associated with both primary mitochondrial diseases and diseases with mitochondrial dysfunction such as cancer. Here, we present recent observations on the crosstalk between amino acid metabolism and mitochondrial homeostasis, summarise the underlying molecular mechanisms to date, and discuss their role in cellular functions and organismal physiology.

Femoral Anteversion Measured by the Surgical Transepicondylar Axis Is Correlated with the Tibial Tubercle-Roman Arch Distance in Patients with Lateral Patellar Dislocation.

Background and Objectives: Various predisposing factors for lateral patellar dislocation (LPD) have been identified, but the relation between femoral rotational deformity and the tibial tubercle-Roman arch (TT-RA) distance remains elusive. Materials and Methods: We conducted this study including 72 consecutive patients with unilateral LPD. Femoral anteversion was measured by the surgical transepicondylar axis (S-tAV), and the posterior condylar reference line (P-tAV), TT-RA distance, trochlear dysplasia, knee joint rotation, patellar height, and hip-knee-ankle angle were measured by CT images or by radiographs. The correlations among these parameters were analyzed, and the parameters were compared between patients with and without a pathological TT-RA distance. Binary regression analysis was performed, and receiver operating characteristic curves were obtained. Results: The TT-RA distance was correlated with S-tAV (r = 0.360, p = 0.002), but the correlation between P-tAV and the TT-RA distance was not significant. S-tAV had an AUC of 0.711 for predicting a pathological TT-RA, with a value of >18.6° indicating 54.8% sensitivity and 82.9% specificity. S-tAV revealed an OR of 1.13 (95% CI [1.04, 1.22], p = 0.003) with regard to the pathological TT-RA distance by an adjusted regression model. Conclusions: S-tAV was significantly correlated with the TT-RA distance, with a correlation coefficient of 0.360, and was identified as an independent risk factor for a pathological TT-RA distance. However, the TT-RA distance was found to be independent of P-tAV.

Cholesterol in the cargo membrane amplifies tau inhibition of kinesin-1-based transport.

Intracellular cargos are often membrane-enclosed and transported by microtubule-based motors in the presence of microtubule-associated proteins (MAPs). Whereas increasing evidence reveals how MAPs impact the interactions between motors and microtubules, critical questions remain about the impact of the cargo membrane on transport. Here we combined in vitro optical trapping with theoretical approaches to determine the effect of a lipid cargo membrane on kinesin-based transport in the presence of MAP tau. Our results demonstrate that attaching kinesin to a fluid lipid membrane reduces the inhibitory effect of tau on kinesin. Moreover, adding cholesterol, which reduces kinesin diffusion in the cargo membrane, amplifies the inhibitory effect of tau on kinesin binding in a dosage-dependent manner. We propose that reduction of kinesin diffusion in the cargo membrane underlies the effect of cholesterol on kinesin binding in the presence of tau, and we provide a simple model for this proposed mechanism. Our study establishes a direct link between cargo membrane cholesterol and MAP-based regulation of kinesin-1. The cholesterol effects uncovered here may more broadly extend to other lipid alterations that impact motor diffusion in the cargo membrane, including those associated with aging and neurological diseases.

Targeting Ferroptosis Holds Potential for Intervertebral Disc Degeneration Therapy.

Intervertebral disc degeneration (IVDD) is a common pathological condition responsible for lower back pain, which can significantly increase economic and social burdens. Although considerable efforts have been made to identify potential mechanisms of disc degeneration, the treatment of IVDD is not satisfactory. Ferroptosis, a recently reported form of regulated cell death (RCD), is characterized by iron-dependent lipid peroxidation and has been demonstrated to be responsible for a variety of degenerative diseases. Accumulating evidence suggests that ferroptosis is implicated in IVDD by decreasing viability and increasing extracellular matrix degradation of nucleus pulposus cells, annulus fibrosus cells, or endplate chondrocytes. In this review, we summarize the literature regarding ferroptosis of intervertebral disc cells and discuss its molecular pathways and biomarkers for treating IVDD. Importantly, ferroptosis is verified as a promising therapeutic target for IVDD.

Role of ferroptosis-associated genes in ankylosing spondylitis and immune cell infiltration.

Ankylosing spondylitis (AS) is a chronic progressive autoimmune disease with insidious onset, high rates of disability among patients, unknown pathogenesis, and no effective treatment. Ferroptosis is a novel type of regulated cell death that is associated with various cancers and diseases. However, its relation to AS is not clear. In the present study, we identified two potential therapeutic targets for AS based on genes associated with ferroptosis and explored their association with immune cells and immune cell infiltration (ICI). We studied gene expression profiles of two cohorts of patients with AS (GSE25101 and GSE41038) derived from the gene expression omnibus database, and ferroptosis-associated genes (FRGs) were obtained from the FerrDb database. LASSO regression analysis was performed to build predictive models for AS based on FRGs, and the ferroptosis level in each sample was assessed via single-sample gene set enrichment analysis. Weighted gene co-expression network and protein-protein interaction network analyses were performed for screening; two key genes, DDIT3 and HSPB1, were identified in patients with AS. The relationship between key genes and ICI levels was assessed using the CIBERSORT algorithm, followed by gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. Finally, DDIT3 and HSPB1 were identified as diagnostic markers and potential therapeutic targets for AS. DDIT3 was highly positively correlated with the infiltration levels of various immune cells, while HSPB1 was negatively correlated with the infiltration levels of several different types of immune cells. In conclusion, DDIT3 and HSPB1 may induce ferroptosis in the cells of patients with AS via changes in the inflammatory response in the immune microenvironment, and these genes could serve as molecular targets for AS therapy.

Theaflavin-3,3'-Digallate Plays a ROS-Mediated Dual Role in Ferroptosis and Apoptosis via the MAPK Pathway in Human Osteosarcoma Cell Lines and Xenografts.

Globally, osteosarcoma (OS) is the most prevalent form of primary bone cancer in children and adolescents. Traditional neoadjuvant chemotherapy regimens have reached a bottleneck; thus, OS survivors have unsatisfactory outcomes. Theaflavin-3,3'-digallate (TF3) exhibits potent anticancer properties against many human cancers. Nevertheless, the biological effects and the underlying molecular mechanism of TF3 in human OS remain unclear. The objective of this study was to investigate the effects of TF3 on human OS cell lines and mouse xenograft models. The results showed that TF3 reduced cell viability, suppressed cell proliferation, and caused G0/G1 cell cycle arrest in both MG63 and HOS cell lines in a concentration-dependent manner. TF3 also altered the homeostatic mechanisms for iron storage in the examined cell lines, resulting in an excess of labile iron. Unsurprisingly, TF3 caused oxidative stress through reduced glutathione (GSH) exhaustion, reactive oxygen species (ROS) accumulation, and the Fenton reaction, which triggered ferroptosis and apoptosis in the cells. TF3 also induced MAPK signalling pathways, including the ERK, JNK, and p38 MAPK pathways. Furthermore, oxidative stress was shown to be the primary reason for TF3-induced proliferation inhibition, programmed cell death, and MAPK pathway activation in vitro. Moreover, TF3 exhibited markedly strong antitumour efficacy in vivo in mouse models. In summary, this study demonstrates that TF3 concomitantly plays dual roles in apoptotic and ferroptotic cell death by triggering the ROS and MAPK signalling pathways in both in vitro and in vivo models.

Thermosensation in Caenorhabditis elegans is linked to ubiquitin-dependent protein turnover via insulin and calcineurin signalling.

Organismal physiology and survival are influenced by environmental conditions and linked to protein quality control. Proteome integrity is achieved by maintaining an intricate balance between protein folding and degradation. In Caenorhabditis elegans, acute heat stress determines cell non-autonomous regulation of chaperone levels. However, how the perception of environmental changes, including physiological temperature, affects protein degradation remains largely unexplored. Here, we show that loss-of-function of dyf-1 in Caenorhabditis elegans associated with dysfunctional sensory neurons leads to defects in both temperature perception and thermal adaptation of the ubiquitin/proteasome system centered on thermosensory AFD neurons. Impaired perception of moderate temperature changes worsens ubiquitin-dependent proteolysis in intestinal cells. Brain-gut communication regulating protein turnover is mediated by upregulation of the insulin-like peptide INS-5 and inhibition of the calcineurin-regulated forkhead-box transcription factor DAF-16/FOXO. Our data indicate that perception of ambient temperature and its neuronal integration is important for the control of proteome integrity in complex organisms.

Aerotaxis Assay in Caenorhabditis elegans to Study Behavioral Plasticity.

C. elegans shows robust and reproducible behavioral responses to oxygen. Specifically, worms prefer O 2 levels of 5-10% and avoid too high or too low O 2 . Their O 2 preference is not fixed but shows plasticity depending on experience, context, or genetic background. We recently showed that this experience-dependent plasticity declines with age, providing a useful behavioral readout for studying the mechanisms of age-related decline of neural plasticity. Here, we describe a technique to visualize behavioral O 2 preference and its plasticity in C. elegans , by creating spatial gradients of [O 2 ] in a microfluidic polydimethylsiloxane (PDMS) chamber and recording the resulting spatial distribution of the animals.

Reprograming of proteasomal degradation by branched chain amino acid metabolism.

Branched-chain amino acid (BCAA) metabolism is a central hub for energy production and regulation of numerous physiological processes. Controversially, both increased and decreased levels of BCAAs are associated with longevity. Using genetics and multi-omics analyses in Caenorhabditis elegans, we identified adaptive regulation of the ubiquitin-proteasome system (UPS) in response to defective BCAA catabolic reactions after the initial transamination step. Worms with impaired BCAA metabolism show a slower turnover of a GFP-based proteasome substrate, which is suppressed by loss-of-function of the first BCAA catabolic enzyme, the branched-chain aminotransferase BCAT-1. The exogenous supply of BCAA-derived carboxylic acids, which are known to accumulate in the body fluid of patients with BCAA metabolic disorders, is sufficient to regulate the UPS. The link between BCAA intermediates and UPS function presented here sheds light on the unexplained role of BCAAs in the aging process and opens future possibilities for therapeutic interventions.

Effects of exercise training in hypoxia versus normoxia on fat-reducing in overweight and/or obese adults: A systematic review and meta-analysis of randomized clinical trials.

Objective: Fat loss theory under various oxygen conditions has been disputed, and relevant systematic review studies are limited. This study is a systematic review and meta-analysis to assess whether hypoxic exercise training (HET) leads to superior fat-reducing compared with normoxic exercise training (NET). Methods: We searched PubMed, Web of Science, CNKI, ProQuest, Google Scholar, Cochrane Library, and EBSCOhost from inception to June 2022 for articles comparing the effects of hypoxic and normoxic exercise on body composition indicators, glycometabolism, and lipometabolism indicators in obese and overweight adults. Only randomized controlled trials (RCTs) were included. The effect sizes were expressed as standardized mean difference (SMD) and 95% confidence intervals (CI). Between-study heterogeneity was examined using the I 2 test and evaluated publication bias via Egger's regression test. The risk of bias assessment was performed for each included trial using Cochrane Evaluation Tool second generation. The meta-analysis was performed by using R 4.1.3 and RevMan 5.3 analytic tools. Results: A total of 19 RCTs with 444 subjects were analyzed according to the inclusion and exclusion criteria. Among them, there were 14 English literature and five Chinese literature. No significant difference in body composition (SMD -0.10, 95% CI -0.20 to -0.01), glycometabolism and lipid metabolism (SMD -0.01, 95% CI -0.13 to -0.10) has been observed when comparing the HET and NET groups. We only found low heterogeneity among trials assessing glycometabolism and lipometabolism (I 2 = 20%, p = 0.09), and no publication bias was detected. Conclusion: The effects of HET and NET on fat loss in overweight or obese people are the same. The application and promotion of HET for fat reduction need further exploration.

Elevated intraspinal pressure drives edema progression after acute compression spinal cord injury in rabbits.

Elevated intraspinal pressure (ISP) following traumatic spinal cord injury (tSCI) can be an important factor for secondary SCI that may result in greater tissue damage and functional deficits. Our present study aimed to investigate the dynamic changes in ISP after different degrees of acute compression SCI in rabbits with closed canals and explore its influence on spinal cord pathophysiology. Closed balloon compression injuries were induced with different inflated volumes (40 µl, 50 µl or no inflation) at the T7/8 level in rabbits. ISP was monitored by a SOPHYSA probe at the epicenter within 7 days post-SCI. Edema progression, spinal cord perfusion and damage severity were evaluated by serial multisequence MRI scans, somatosensory evoked potentials (SEPs) and behavioral scores. Histological and blood spinal cord barrier (BSCB) permeability results were subsequently analyzed. The results showed that the ISP waveforms comprised three peaks, significantly increased after tSCI, peaked at 72 h (21.86 ± 3.13 mmHg) in the moderate group or 48 h (31.71 ± 6.02 mmHg) in the severe group and exhibited "slow elevated and fast decreased" or "fast elevated and slow decreased" dynamic changes in both injured groups. Elevated ISP after injury was correlated with spinal cord perfusion and edema progression, leading to secondary lesion enlargement. The secondary damage aggravation can be visualized by diffusion tensor tractography (DTT). Moreover, the BSCB permeability was significantly increased at the epicenter and rostrocaudal segments at 72 h after SCI; by 14 days, notable permeability was still observed at the caudal segment in the severely injured rabbits. Our results suggest that the ISP of rabbits with closed canals increased after acute compression SCI and exhibited different dynamic change patterns in moderately and severely injured rabbits. Elevated ISP exacerbated spinal cord perfusion, drove edema progression and led to secondary lesion enlargement that was strongly associated with BSCB disruption. For severe tSCI, early intervention targeting elevated ISP may be an indispensable choice to rescue spinal cord function.

YAP knockdown in combination with ferroptosis induction increases the sensitivity of HOS human osteosarcoma cells to pyropheophorbide-α methyl ester-mediated photodynamic therapy.

BACKGROUND AND AIMS: This study was designed to explore the effects of Yes-associated protein (YAP) knockdown on human osteosarcoma (HOS) cell sensitivity to Pyropheophorbide-α methyl ester-mediated photodynamic therapy (MPPa-PDT), and to assess how YAP silencing in combination with treatment with the ferroptosis inducer Erastin improves HOS cell sensitivity to MPPa-PDT in an effort to better clarify the molecular mechanisms underlying these phenotypes. METHODS: At 12 h post-MPPa-PDT, Hoechst staining and flow cytometry were conducted to evaluate the apoptotic death of HOS cells. The expression of YAP in these cells at 12 h post-MPPa-PDT treatment was assessed via Western blotting and immunofluorescent staining. BODIPY581/591-C11 was used to evaluate lipid peroxidation. Following shYAP lentiviral transduction, Western blotting was conducted to assess the expression of proteins associated with proliferation, apoptosis, and ferroptosis. EdU assays and clonogenic assays were performed to analyze cellular proliferation. Erastin-treated HOS cells were used to establish a ferroptosis model. Western blotting was used to measure ferroptosis-associated protein levels following shYAP and erastin treatment, while changes in proliferation and MDA levels in each group were examined using an MDA kit. RESULTS: At 12 h post-MPPa-PDT, HOS cells exhibited apoptotic characteristics including nuclear fragmentation and pyknosis, with concomitant increases in apoptosis-associated proteins as detected via Western blotting and apoptotic induction as measured via flow cytometry. Phosphorylated YAP levels fell and non-phosphorylated YAP levels rose following such treatment. Transfection with shYAP was successful as a means of generating stable HOS cell lines, and Western blotting analyses of these cells revealed reductions in proteins associated with cellular proliferation together with the upregulation of apoptosis-related proteins.  MDA assays indicated that erastin combined with YAP knockdown enhanced the sensitivity of HOS cells to MPPa-PDT treatment. CONCLUSIONS: These data indicate that ferroptosis and YAP knockdown can enhance osteosarcoma cell sensitivity to MPPa-PDT therapy.

Elevated intraspinal pressure in traumatic spinal cord injury is a promising therapeutic target.

The currently recommended management for acute traumatic spinal cord injury aims to reduce the incidence of secondary injury and promote functional recovery. Elevated intraspinal pressure (ISP) likely plays an important role in the processes involved in secondary spinal cord injury, and should not be overlooked. However, the factors and detailed time course contributing to elevated ISP and its impact on pathophysiology after traumatic spinal cord injury have not been reviewed in the literature. Here, we review the etiology and progression of elevated ISP, as well as potential therapeutic measures that target elevated ISP. Elevated ISP is a time-dependent process that is mainly caused by hemorrhage, edema, and blood-spinal cord barrier destruction and peaks at 3 days after traumatic spinal cord injury. Duraplasty and hypertonic saline may be promising treatments for reducing ISP within this time window. Other potential treatments such as decompression, spinal cord incision, hemostasis, and methylprednisolone treatment require further validation.

Prediction of Subsequent Contralateral Patellar Dislocation after First-Time Dislocation Based on Patellofemoral Morphologies.

The subsequent dislocation of a contralateral patellofemoral joint sometimes occurs after a first-time lateral patellar dislocation (LPD). However, the anatomic risk factors for subsequent contralateral LPD remain elusive. This study included 17 patients with contralateral LPD and 34 unilateral patellar dislocators. The anatomic parameters of the contralateral patellofemoral joints were measured using CT images and radiographs that were obtained at the time of the first dislocation. The Wilcoxon rank-sum test was performed, and a binary regression model was established to identify the risk factors. The receiver operating characteristic curves and the area under the curve (AUC) were analyzed. The tibial tubercle-Roman arch (TT-RA) distance was significantly different between patients with and without contralateral LPD (24.1 vs. 19.5 mm, p < 0.001). The hip−knee−ankle (HKA) angle, patellar tilt, congruence angle, and patellar displacement were greater in the study group than in the control group (p < 0.05). The TT-RA distance revealed an OR of 1.35 (95% CI (1.26−1.44]), p < 0.001) and an AUC of 0.727 for predicting contralateral LPD. The HKA angle revealed an OR of 1.74 (95% CI (1.51−2.00), p < 0.001) and an AUC of 0.797. The Patellar tilt, congruence angle, and patellar displacement had AUC values of 0.703, 0.725, and 0.817 for predicting contralateral LPD, respectively. In conclusion, the contralateral patellofemoral anatomic parameters were significantly different between patients with and without subsequent contralateral LPD. Increased TT-RA distance and excessive valgus deformity were risk factors and could serve as predictors for contralateral LPD. At first-time dislocation, the abnormal position of the patella relative to the trochlea may also be an important cause of subsequent LPD.

RhoA enhances osteosarcoma resistance to MPPa-PDT via the Hippo/YAP signaling pathway.

BACKGROUND: Osteosarcoma (OS) is the most prevalent primary bone malignancy affecting adolescents, yet the emergence of chemoradiotherapeutic resistance has limited efforts to cure affected patients to date. Pyropheophorbide-α methyl ester-mediated photodynamic therapy (MPPa-PDT) is a recently developed, minimally invasive treatment for OS that is similarly constrained by such therapeutic resistance. This study sought to explore the mechanistic basis for RhoA-activated YAP1 (YAP)-mediated resistance in OS. METHODS: The relationship between YAP expression levels and patient prognosis was analyzed, and YAP levels in OS cell lines were quantified. Immunofluorescent staining was used to assess YAP nuclear translocation. OS cell lines (HOS and MG63) in which RhoA and YAP were knocked down or overexpressed were generated using lentiviral vectors. CCK-8 assays were used to examine OS cell viability, while the apoptotic death of these cells was monitored via Hoechst staining, Western blotting, and flow cytometry. Tumor-bearing nude mice were additionally used to assess the relationship between lentivirus-mediated alterations in RhoA expression and MPPa-PDT treatment outcomes. TUNEL and immunohistochemical staining approaches were leveraged to assess apoptotic cell death in tissue samples. RESULTS: OS patients exhibited higher levels of YAP expression, and these were correlated with a poor prognosis. MPPa-PDT induced apoptosis in OS cells, and such MPPa-PDT-induced apoptosis was enhanced following YAP knockdown whereas it was suppressed by YAP overexpression. RhoA and YAP expression levels were positively correlated in OS patients, and both active and total RhoA protein levels rose in OS cells following MPPa-PDT treatment. When RhoA was knocked down, levels of unphosphorylated YAP and downstream target genes were significantly reduced, while RhoA/ROCK2/LIMK2 pathway phosphorylation was suppressed, whereas RhoA overexpression resulted in the opposite phenotype. MPPa-PDT treatment was linked to an increase in HMGCR protein levels, and the inhibition of RhoA or HMGCR was sufficient to suppress RhoA activity and to decrease the protein levels of YAP and its downstream targets. Mevalonate administration partially reversed these reductions in the expression of YAP and YAP target genes. RhoA knockdown significantly enhanced the apoptotic death of OS cells in vitro and in vivo following MPPa-PDT treatment, whereas RhoA overexpression had the opposite effect. CONCLUSIONS: These results suggest that the mevalonate pathway activates RhoA, which in turn activates YAP and promotes OS cell resistance to MPPa-PDT therapy. Targeting the RhoA/ROCK2/LIMK2/YAP pathway can significantly improve the efficacy of MPPa-PDT treatment for OS.

Penetration of Carbon Nanotubes into the Retinoblastoma Tumor after Intravitreal Injection in LH BETA T AG Transgenic Mice Reti-noblastoma Model.

PURPOSE: To evaluate the penetration of carbon nanotubes (CNTs) throughout retinoblastoma in a transgenic mice model. METHODS: CNTs functionalized with fluorescein isothiocyanate and targeting ligands biotin (CTN-FITC-Bio, 0.5mg/ml), or folic acid (CNT-FITC-FA, 0.5mg/ml) were injected into the vitreous of one eye of LH BETA T AG transgenic mice. Other eye did not receive any injection and was used as control. Three mice were sacrificed at days 1, 2, and 3. Eyes were enucleated and stained with 4,6-diamidino-2-phenylindole. The sections were imaged by fluorescent microscope. The images were transformed into grey-scale in MATLAB for intensity analysis. Background intensity was normalized by marking squares outside the eyeball and using the mean intensity of these squares. Fluorescent intensity (FI) for each image was measured by calculating the intensity of a same-sized square within retinoblastoma. RESULTS: Nine eyes of nine mice were included in each CNT-FITC-Bio and CNT-FITC-FA groups. The mean FI in CNT-FITC-Bio was 52.08 ± 6.33, 53.62 ± 9.00, and 65.54 ± 5.14 in days 1, 2, and 3, respectively. The mean FI in CNT-FITC-FA was 50.28 ± 7.37, 59.21 ± 6.43, and 58.38 ± 2.32 on days 1, 2, and 3, respectively. FI was significantly higher in eyes injected with CNT-FITC-Bio and CNT-FITC-FA compared to the control eyes (P = 0.02). There was no difference in FI between eyes with CNT-FITC-Bio and CNT-FITC-FA, and FI remained stable on days 1-3 in CNT-FITC-Bio, CNT-FITC-FA, and control eyes (P > 0.05). CONCLUSION: We observed higher FI in eyes with CNT-FITC-Bio and CNT-FITC-FA compared to control eyes, showing penetration of CNTs throughout retinoblastoma. CNTs can be a carrier candidate for imaging or therapeutic purposes in retinoblastoma.