Inhibition of TOPORS ubiquitin ligase augments the efficacy of DNA hypomethylating agents through DNMT1 stabilization.

DNA hypomethylating agents (HMAs) are used for the treatment of myeloid malignancies, although their therapeutic effects have been unsatisfactory. Here we show that CRISPR-Cas9 screening reveals that knockout of topoisomerase 1-binding arginine/serine-rich protein (TOPORS), which encodes a ubiquitin/SUMO E3 ligase, augments the efficacy of HMAs on myeloid leukemic cells with little effect on normal hematopoiesis, suggesting that TOPORS is involved in resistance to HMAs. HMAs are incorporated into the DNA and trap DNA methyltransferase-1 (DNMT1) to form DNA-DNMT1 crosslinks, which undergo SUMOylation, followed by proteasomal degradation. Persistent crosslinking is cytotoxic. The TOPORS RING finger domain, which mediates ubiquitination, is responsible for HMA resistance. In TOPORS knockout cells, DNMT1 is stabilized by HMA treatment due to inefficient ubiquitination, resulting in the accumulation of unresolved SUMOylated DNMT1. This indicates that TOPORS ubiquitinates SUMOylated DNMT1, thereby promoting the resolution of DNA-DNMT1 crosslinks. Consistently, the ubiquitination inhibitor, TAK-243, and the SUMOylation inhibitor, TAK-981, show synergistic effects with HMAs through DNMT1 stabilization. Our study provides a novel HMA-based therapeutic strategy that interferes with the resolution of DNA-DNMT1 crosslinks.

Capsular and fascial closure with barbed sutures reduces blood loss compared to traditional interrupted sutures in total hip arthroplasty.

BACKGROUND: STRATAFIX, a recently introduced barbed suture device, incorporates self-anchoring, knotless sutures with higher tensile strength and enhanced tissue-holding capacity compared to traditional braided absorbable sutures. This study aimed to compare the efficacy of barbed sutures and interrupted sutures in capsular and fascial closure during total hip arthroplasty. METHODS: We retrospectively reviewed the records of patients who underwent total hip arthroplasty between April 2017 and March 2021. Overall, 547 patients were evaluated, comprising 77 men and 470 women (mean age: 64.5 years). Among them, 330 patients were in the interrupted suture (control) group, while 217 were in the barbed suture (BS) group. Data on surgical time, perioperative hemoglobin, length of hospital stay, complications such as transfusions and delayed wound healing, and dislocation rates were collected during the latest outpatient follow-up and compared between the two groups. RESULTS: No differences were observed in intraoperative blood loss between the groups. However, the BS group exhibited significantly longer operative time, as well as significantly lower postoperative blood loss, total blood loss, and postoperative hemoglobin drop compared to the control group. Dislocation was reported in two cases within the control group, whereas no instances were recorded in the BS group. CONCLUSION: The use of barbed sutures demonstrated effectiveness in reducing perioperative blood loss for capsular and fascial closure during total hip arthroplasty.

A Supramolecular Biosensor for Rapid and High-Throughput Quantification of a Disease-Associated Niacin Metabolite.

Metabolic abnormalities play a pivotal role in various pathological conditions, necessitating the quantification of specific metabolites for diagnosis. While mass spectrometry remains the primary method for metabolite measurement, its limited throughput underscores the need for biosensors capable of rapid detection. Previously, we reported that pillar[6]arene with 12 carboxylate groups (P6AC) forms host-guest complexes with 1-methylnicotinamide (1-MNA), which is produced in vivo by nicotinamide N-methyltransferase (NNMT). P6AC acts as a biosensor by measuring the fluorescence quenching caused by photoinduced electron transfer upon 1-MNA binding. However, the low sensitivity of P6AC makes it impractical for detecting 1-MNA in unpurified biological samples. In this study, we found that P6A with 12 sulfonate groups (P6AS) is a specific and potent supramolecular host for 1-MNA interactions even in biological samples. The 1-MNA binding affinity of P6AS in water was found to be (5.68 ± 1.02) × 106 M-1, which is approximately 700-fold higher than that of P6AC. Moreover, the 1-MNA detection limit of P6AS was determined to be 2.84 × 10-7 M, which is substantially lower than that of P6AC. Direct addition of P6AS to culture medium was sufficient to quantify 1-MNA produced by cancer cells. Furthermore, this sensor was able to specifically detect 1-MNA even in unpurified human urine. P6AS therefore enables rapid and high-throughput quantification of 1-MNA, and further improvement of our strategy will contribute to the establishment of high-throughput screening of NNMT inhibitors, diagnosis of liver diseases, and imaging of human cancer cells in vivo.

Functional Improvement and Patient Satisfaction Following Conversion of Fused Hip to Total Hip Arthroplasty.

BACKGROUND: This study aimed to determine the clinical outcomes, predictors of suboptimal functional improvement, and factors influencing patient satisfaction following conversion of a fused hip to total hip arthroplasty (THA). METHODS: A retrospective analysis of clinical and radiographic data was performed on 83 patients (83 hips) who underwent fused hip conversion to THA. Implant survival and predictors of poor functional outcome (Harris hip score < 70) were analyzed. In addition, factors associated with patient dissatisfaction (visual analog scale < 25th percentile) were analyzed in 63 patients (63 hips) who completed a patient-reported outcome measures questionnaire. RESULTS: The median Harris hip score improved from 55 (range, 18 to 77) to 78 (range, 36 to 100) at a mean follow-up of 10.2 ± 4.8 years (P < .001). Implant survival was 97.4% at 10 years and 91.3% at 20 years, with any revision as the endpoint. Multivariate analysis identified preoperative reliance on mobility aids as an independent predictor of poor functional outcome (P = .021). There were 48 of 63 patients (76%) satisfied (satisfaction visual analog scale ≥80) with the operated hip. Demographics and preoperative/postoperative clinical data did not differ between satisfied and unsatisfied patients. Among the patient-reported outcome measures, the Forgotten Joint Score-12 emerged as an independent discriminator of patient satisfaction. CONCLUSIONS: Conversion of a fused hip to THA provides functional improvement, favorable implant survival, and high patient satisfaction. However, patients dependent on mobility aids may experience suboptimal functional recovery, underscoring the need for careful preoperative counseling and patient selection.

Factors associated with joint survival after transposition osteotomy of the acetabulum in patients with Tönnis grade 2 osteoarthritis secondary to hip dysplasia.

Aims: The aim of this study was to determine the clinical outcomes and factors contributing to failure of transposition osteotomy of the acetabulum (TOA), a type of spherical periacetabular osteotomy, for advanced osteoarthritis secondary to hip dysplasia. Methods: We reviewed patients with Tönnis grade 2 osteoarthritis secondary to hip dysplasia who underwent TOA between November 1998 and December 2019. Patient demographic details, osteotomy-related complications, and the modified Harris Hip Score (mHHS) were obtained via medical notes review. Radiological indicators of hip dysplasia were assessed using preoperative and postoperative radiographs. The cumulative probability of TOA failure (progression to Tönnis grade 3 or conversion to total hip arthroplasty) was estimated using the Kaplan-Meier product-limited method. A multivariate Cox proportional hazards model was used to identify predictors of failure. Results: This study included 127 patients (137 hips). Median follow-up period was ten years (IQR 6 to 15). The median mHHS improved from 59 (IQR 52 to 70) preoperatively to 90 (IQR 73 to 96) at the latest follow-up (p < 0.001). The survival rate was 90% (95% CI 82 to 95) at ten years, decreasing to 21% (95% CI 7 to 48) at 20 years. Fair joint congruity on preoperative hip abduction radiographs and a decreased postoperative anterior wall index (AWI) were identified as independent risk factors for failure. The survival rate for the 42 hips with good preoperative joint congruity and a postoperative AWI ≥ 0.30 was 100% at ten years, and remained at 83% (95% CI 38 to 98) at 20 years. Conclusion: Although the overall clinical outcomes of TOA in patients with advanced osteoarthritis are suboptimal, favourable results can be achieved in selected cases with good preoperative joint congruity and adequate postoperative anterior acetabular coverage.

Amino acid influx via LAT1 regulates iron demand and sensitivity to PPMX-T003 of aggressive natural killer cell leukemia.

Aggressive natural killer cell leukemia (ANKL) is a rare hematological malignancy with a fulminant clinical course. Our previous study revealed that ANKL cells proliferate predominantly in the liver sinusoids and strongly depend on transferrin supplementation. In addition, we demonstrated that liver-resident ANKL cells are sensitive to PPMX-T003, an anti-human transferrin receptor 1 inhibitory antibody, whereas spleen-resident ANKL cells are resistant to transferrin receptor 1 inhibition. However, the microenvironmental factors that regulate the iron dependency of ANKL cells remain unclear. In this study, we first revealed that the anti-neoplastic effect of PPMX-T003 was characterized by DNA double-strand breaks in a DNA replication-dependent manner, similar to conventional cytotoxic agents. We also found that the influx of extracellular amino acids via LAT1 stimulated sensitivity to PPMX-T003. Taken together, we discovered that the amount of extracellular amino acid influx through LAT1 was the key environmental factor determining the iron dependency of ANKL cells via adjustment of their mTOR/Myc activity, which provides a good explanation for the different sensitivity to PPMX-T003 between liver- and spleen-resident ANKL cells, as the liver sinusoid contains abundant amino acids absorbed from the gut.

Long-term antibacterial activity of silver-containing hydroxyapatite coatings against Staphylococcus aureus in vitro and invivo.

BACKGROUND: The potential of silver-containing hydroxyapatite (Ag-HA) coatings to prevent orthopaedic implant-associated infection was explored previously; however, the resistance of Ag-HA coatings to late-onset orthopaedic infections is unknown. This study aimed to evaluate the long-term Ag+ elution and antibacterial properties of the Ag-HA coatings through in vitro and in vivo experiments. METHODS: Ag-HA-coated disc specimens were immersed in fetal bovine serum (FBS) for six months. Ag concentration was measured over time using inductively coupled plasma-mass spectrometry to evaluate Ag release. The hydroxyapatite (HA)- or Ag-HA-coated disc specimens were immersed in FBS for 3 months to elute Ag+ for in vitro experiments. Methicillin-resistant Staphylococcus aureus (MRSA) suspensions were inoculated onto each disc; after 48 h, the number of colonies and the biofilm volume were measured. HA- or Ag-HA-coated disc specimens were inserted under the skin of Sprague-Dawley rats for three months for in vivo experiments. In in vivo experiment 1, specimens were inoculated with MRSA and the number of colonies was counted after 48 h. In in vivo experiment 2, the specimens were inoculated with bioluminescent S. aureus Xen36 cells, and bioluminescence was measured using an in vivo imaging system. RESULTS: The Ag-HA-coated disc specimens continued to elute Ag+ after six months. The biofilm volume in the Ag-HA group was lower than in the HA group. In in vitro and in vivo experiment 1, the bacterial counts in the Ag-HA group were lower than those in the HA group. In in vivo experiment 2, the bioluminescence in the Ag-HA group was lower than that in the HA group on days 1-7 after inoculation. CONCLUSIONS: The Ag-HA-coated discs continued to elute Ag+ for a long period and exhibited antibacterial activity and inhibition of biofilm formation against S. aureus. The Ag-HA coatings have the potential to reduce late-onset orthopaedic implant-associated infections.

Targeting cis-regulatory elements of FOXO family is a novel therapeutic strategy for induction of leukemia cell differentiation.

Differentiation therapy has been proposed as a promising therapeutic strategy for acute myeloid leukemia (AML); thus, the development of more versatile methodologies that are applicable to a wide range of AML subtypes is desired. Although the FOXOs transcription factor represents a promising drug target for differentiation therapy, the efficacy of FOXO inhibitors is limited in vivo. Here, we show that pharmacological inhibition of a common cis-regulatory element of forkhead box O (FOXO) family members successfully induced cell differentiation in various AML cell lines. Through gene expression profiling and differentiation marker-based CRISPR/Cas9 screening, we identified TRIB1, a complement of the COP1 ubiquitin ligase complex, as a functional FOXO downstream gene maintaining an undifferentiated status. TRIB1 is direct target of FOXO3 and the FOXO-binding cis-regulatory element in the TRIB1 promoter, referred to as the FOXO-responsive element in the TRIB1 promoter (FRE-T), played a critical role in differentiation blockade. Thus, we designed a DNA-binding pharmacological inhibitor of the FOXO-FRE-T interface using pyrrole-imidazole polyamides (PIPs) that specifically bind to FRE-T (FRE-PIPs). The FRE-PIPs conjugated to chlorambucil (FRE-chb) inhibited transcription of TRIB1, causing differentiation in various AML cell lines. FRE-chb suppressed the formation of colonies derived from AML cell lines but not from normal counterparts. Administration of FRE-chb inhibited tumor progression in vivo without remarkable adverse effects. In conclusion, targeting cis-regulatory elements of the FOXO family is a promising therapeutic strategy that induces AML cell differentiation.

Clinical results of periacetabular osteotomy with structural bone allograft for the treatment of severe hip dysplasia.

Aims: To clarify the mid-term results of transposition osteotomy of the acetabulum (TOA), a type of spherical periacetabular osteotomy, combined with structural allograft bone grafting for severe hip dysplasia. Methods: We reviewed patients with severe hip dysplasia, defined as Severin IVb or V (lateral centre-edge angle (LCEA) < 0°), who underwent TOA with a structural bone allograft between 1998 and 2019. A medical chart review was conducted to extract demographic data, complications related to the osteotomy, and modified Harris Hip Score (mHHS). Radiological parameters of hip dysplasia were measured on pre- and postoperative radiographs. The cumulative probability of TOA failure (progression to Tönnis grade 3 or conversion to total hip arthroplasty) was estimated using the Kaplan-Meier product-limited method, and a multivariate Cox proportional hazard model was used to identify predictors for failure. Results: A total of 64 patients (76 hips) were included in this study. The median follow-up period was ten years (interquartile range (IQR) five to 14). The median mHHS improved from 67 (IQR 56 to 80) preoperatively to 96 (IQR 85 to 97) at the latest follow-up (p < 0.001). The radiological parameters improved postoperatively (p < 0.001), with the resulting parameters falling within the normal range in 42% to 95% of hips. The survival rate was 95% at ten years and 80% at 15 years. Preoperative Tönnis grade 2 was an independent risk factor for TOA failure. Conclusion: Our findings suggest that TOA with structural bone allografting is a viable surgical option for correcting severely dysplastic acetabulum in adolescents and young adults without advanced osteoarthritis, with favourable mid-term outcomes.

Joint awareness following periacetabular osteotomy in patients with hip dysplasia.

Aims: The aims of this study were to validate the Forgotten Joint Score-12 (FJS-12) in the postoperative evaluation of periacetabular osteotomy (PAO), identify factors associated with joint awareness after PAO, and determine the FJS-12 threshold for patient-acceptable symptom state (PASS). Methods: Data from 686 patients (882 hips) with hip dysplasia who underwent transposition osteotomy of the acetabulum, a type of PAO, between 1998 and 2019 were reviewed. After screening the study included 442 patients (582 hips; response rate, 78%). Patients who completed a study questionnaire consisting of the visual analogue scale (VAS) for pain and satisfaction, FJS-12, and Hip disability and Osteoarthritis Outcome Score (HOOS) were included. The ceiling effects, internal consistency, convergent validity, and PASS thresholds of FJS-12 were investigated. Results: The median follow-up was 12 years (interquartile range 7 to 16). The ceiling effect of FJS-12 was 7.2%, the lowest of all the measures examined. FJS-12 correlated with all HOOS subscales (ρ = 0.72 to 0.77, p < 0.001) and pain and satisfaction-VAS (ρ = -0.63 and 0.56, p < 0.001), suggesting good convergent validity. Cronbach's α was 0.95 for the FJS-12, which indicated excellent internal consistency. The median FJS-12 score for preoperative Tönnis grade 0 hips (60 points) was higher than that for grade 1 (51 points) or 2 (46 points). When PASS was defined as pain-VAS < 21 and satisfaction-VAS ≥ 77, the FJS-12 threshold that maximized the sensitivity and specificity for detecting PASS was 50 points (area under the curve (AUC) = 0.85). Conclusion: Our results suggest that FJS-12 is a valid and reliable assessment tool for patients undergoing PAO, and the threshold of 50 points may be useful to determine patient satisfaction following PAO in clinical settings. Further investigation of the factors influencing postoperative joint awareness may enable improved prediction of treatment efficacy and informed decision-making regarding the indication of PAO.

Analgesic Effect of Passive Range-of-Motion Exercise on the Healthy Side for Pain after Total Knee Arthroplasty: A Prospective Randomized Trial.

Background: Exercise can reduce the pain threshold momentarily and induce analgesia, which is called exercise-induced hypoalgesia (EIH). Exercise therapy for inducing EIH may be an effective treatment option for pain. We aimed at investigating whether continuous passive motion (CPM) on both healthy and affected sides could induce EIH and reduce pain in the operated knee in patients after unilateral total knee arthroplasty (TKA). Patients and Methods. In this prospective randomized controlled trial, participants were randomly assigned to two groups: a bilateral group that received bilateral exercise on the operated and healthy sides and a unilateral group that received exercise therapy only on the affected side. We enrolled 40 patients aged ≥60 years who were scheduled to undergo unilateral TKA. Visual analogue scale (VAS) scores and range of motion (ROM) on the operated side were measured immediately before and after CPM on postoperative days 2, 4, 7, and 14. The primary outcome was the difference in the VAS scores before and after CPM on postoperative day 14. The secondary outcome was the difference in the ROM before and after CPM on postoperative day 14. Results: Comparison of VAS scores before and after CPM showed no significant intergroup differences on all measurement dates. However, there was a significant difference in values on day 14 (P < 0.05). Both groups showed an increase in ROM after CPM, with significant increments observed on days 2 and 4 in the bilateral group and on day 14 in the unilateral group. There was no significant difference in values on postoperative day 14. Conclusion: Post-TKA pain was reduced by performing the same exercise on the healthy knee during CPM therapy. This could be due to EIH, and the results indicated that EIH can also influence postoperative pain immediately after surgery.

Nupr1 deficiency downregulates HtrA1, enhances SMAD1 signaling, and suppresses age-related bone loss in male mice.

Nuclear protein 1 (NUPR1) is a stress-induced protein activated by various stresses, such as inflammation and oxidative stress. We previously reported that Nupr1 deficiency increased bone volume by enhancing bone formation in 11-week-old mice. Analysis of differentially expressed genes between wild-type (WT) and Nupr1-knockout (Nupr1-KO) osteocytes revealed that high temperature requirement A 1 (HTRA1), a serine protease implicated in osteogenesis and transforming growth factor-ß signaling was markedly downregulated in Nupr1-KO osteocytes. Nupr1 deficiency also markedly reduced HtrA1 expression, but enhanced SMAD1 signaling in in vitro-cultured primary osteoblasts. In contrast, Nupr1 overexpression enhanced HtrA1 expression in osteoblasts, suggesting that Nupr1 regulates HtrA1 expression, thereby suppressing osteoblastogenesis. Since HtrA1 is also involved in cellular senescence and age-related diseases, we analyzed aging-related bone loss in Nupr1-KO mice. Significant spine trabecular bone loss was noted in WT male and female mice during 6-19 months of age, whereas aging-related trabecular bone loss was attenuated, especially in Nupr1-KO male mice. Moreover, cellular senescence-related markers were upregulated in the osteocytes of 6-19-month-old WT male mice but markedly downregulated in the osteocytes of 19-month-old Nupr1-KO male mice. Oxidative stress-induced cellular senescence stimulated Nupr1 and HtrA1 expression in in vitro-cultured primary osteoblasts, and Nupr1 overexpression enhanced p16ink4a expression in osteoblasts. Finally, NUPR1 expression in osteocytes isolated from the bones of patients with osteoarthritis was correlated with age. Collectively, these results indicate that Nupr1 regulates HtrA1-mediated osteoblast differentiation and senescence. Our findings unveil a novel Nupr1/HtrA1 axis, which may play pivotal roles in bone formation and age-related bone loss.

Case Series of Silver Oxide-Containing Hydroxyapatite Coating in Antibacterial Cementless Total Hip Arthroplasty: Clinical Results of 50 Cases at 5-Year Follow-Up.

Background: Prosthetic joint infection, which is caused by implant treatment, is a severe complication. Consequently, silver-containing hydroxyapatite (Ag-HA)-coated implants have been developed to prevent prosthetic joint infection by combining Ag with HA. The Ag-HA-coated total hip prosthesis, which combines the antibacterial activity of Ag and the osteoconductivity of HA, is the first antibacterial cementless total hip prosthesis worldwide. This study aimed to evaluate the short-term outcomes of total hip arthroplasty (THA) with Ag-HA-coated implants. Methods: Overall, 50 hips with various disabling hip diseases and postoperative infection risks that underwent a primary THA using an Ag-HA total hip prosthesis were enrolled. The patients included 37 women (41 hips) and 8 men (9 hips), and the mean age at the time of surgery was 77 years. The clinical outcomes and hip function before and at 5 years postoperatively were measured using the Japanese Orthopaedic Association hip score. Implant stability was assessed, and postoperative complications were also examined. Results: The Japanese Orthopaedic Association score increased in all cases and improved from 41 to 86 points after the THA (P < .001). Radiography revealed no implant failure. Dislocation and deep vein thrombosis also occurred in 1 case each. However, there were no adverse reactions associated with Ag, and argyria was not observed in any case. Additionally, none of the patients experienced infection following the surgery. Conclusions: Silver-containing hydroxyapatite-coated implants significantly enhanced patients' daily activities without any adverse effects on the human body attributed to Ag, and they are expected to reduce postoperative infections.

Comparison of the clinical outcomes following total knee arthroplasty in osseous ankylosed and non-ankylosed knees using propensity-score matching.

OBJECTIVES: Few studies have compared the clinical outcomes and complications of total knee arthroplasty (TKA) in patients with and without osseous ankylosed knees. Thus, we investigated the clinical outcomes and complications of TKA in patients with osseous ankylosed knees, using a propensity-score matching method. METHODS: Thirteen knees in the osseous ankylosed-knees group and 13 knees in the non-ankylosed-knees group were included after excluding those with less than two years of follow-up or a lack of data and after propensity-score matching. The American Knee Society Score-knee (AKSS-knee), American Knee Society Score-function (AKSS-function), knee-flexion angle, knee-extension angle, knee range of motion (ROM) before and after TKA, and the number of knees with postoperative complications were evaluated as primary outcomes. RESULTS: The AKSS-knee, AKSS-function, knee-flexion angle, and knee ROM in the osseous ankylosed-knees group after TKA were significantly lower than those in the non-ankylosed-knees group. The knee-extension angle after TKA and number of knees with postoperative complications within two years were not significantly different between the two groups. CONCLUSIONS: The clinical results of TKA in patients with osseous ankylosed knees were inferior to those in patients with non-ankylosed knees.

Differential dynamics of bone graft transplantation and mesenchymal stem cell therapy during bone defect healing in a murine critical size defect.

Background: A critical size bone defect is a clinical scenario in which bone is lost or excised due to trauma, infection, tumor, or other causes, and cannot completely heal spontaneously. The most common treatment for this condition is autologous bone grafting to the defect site. However, autologous bone graft is often insufficient in quantity or quality for transplantation to these large defects. Recently, tissue engineering methods using mesenchymal stem cells (MSCs) have been proposed as an alternative treatment. However, the underlying biological principles and optimal techniques for tissue regeneration of bone using stem cell therapy have not been completely elucidated. Methods: In this study, we compare the early cellular dynamics of healing between bone graft transplantation and MSC therapy in a murine chronic femoral critical-size bone defect. We employ high-dimensional mass cytometry to provide a comprehensive view of the differences in cell composition, stem cell functionality, and immunomodulatory activity between these two treatment methods one week after transplantation. Results: We reveal distinct cell compositions among tissues from bone defect sites compared with original bone graft, show active recruitment of MSCs to the bone defect sites, and demonstrate the phenotypic diversity of macrophages and T cells in each group that may affect the clinical outcome. Conclusion: Our results provide critical data and future directions on the use of MSCs for treating critical size defects to regenerate bone.Translational Potential of this article: This study showed systematic comparisons of the cellular and immunomodulatory profiles among different interventions to improve the healing of the critical-size bone defect. The results provided potential strategies for designing robust therapeutic interventions for the unmet clinical need of treating critical-size bone defects.

RHEB is a potential therapeutic target in T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of immature T lymphocytes. Although various therapeutic approaches have been developed, refractoriness of chemotherapy and relapse cause a poor prognosis of the disease and further therapeutic strategies are required. Here, we report that Ras homolog enriched in brain (RHEB), a critical regulator of mTOR complex 1 activity, is a potential target for T-ALL therapy. In this study, we established an sgRNA library that comprehensively targeted mTOR upstream and downstream pathways, including autophagy. CRISPR/Cas9 dropout screening revealed critical roles of mTOR-related molecules in T-ALL cell survival. Among the regulators, we focused on RHEB because we previously found that it is dispensable for normal hematopoiesis in mice. Transcriptome and metabolic analyses revealed that RHEB deficiency suppressed de novo nucleotide biosynthesis, leading to human T-ALL cell death. Importantly, RHEB deficiency suppressed tumor growth in both mouse and xenograft models. Our data provide a potential strategy for efficient therapy of T-ALL by RHEB-specific inhibition.

Therapeutic advantage of targeting lysosomal membrane integrity supported by lysophagy in malignant glioma.

Lysosomes function as the digestive system of a cell and are involved in macromolecular recycling, vesicle trafficking, metabolic reprogramming, and progrowth signaling. Although quality control of lysosome biogenesis is thought to be a potential target for cancer therapy, practical strategies have not been established. Here, we show that lysosomal membrane integrity supported by lysophagy, a selective autophagy for damaged lysosomes, is a promising therapeutic target for glioblastoma (GBM). In this study, we found that ifenprodil, an FDA-approved drug with neuromodulatory activities, efficiently inhibited spheroid formation of patient-derived GBM cells in a combination with autophagy inhibition. Ifenprodil increased intracellular Ca2+ level, resulting in mitochondrial reactive oxygen species-mediated cytotoxicity. The ifenprodil-induced Ca2+ elevation was due to Ca2+ release from lysosomes, but not endoplasmic reticulum, associated with galectin-3 punctation as an indicator of lysosomal membrane damage. As the Ca2+ release was enhanced by ATG5 deficiency, autophagy protected against lysosomal membrane damage. By comparative analysis of 765 FDA-approved compounds, we identified another clinically available drug for central nervous system (CNS) diseases, amoxapine, in addition to ifenprodil. Both compounds promoted degradation of lysosomal membrane proteins, indicating a critical role of lysophagy in quality control of lysosomal membrane integrity. Importantly, a synergistic inhibitory effect of ifenprodil and chloroquine, a clinically available autophagy inhibitor, on spheroid formation was remarkable in GBM cells, but not in nontransformed neural progenitor cells. Finally, chloroquine dramatically enhanced effects of the compounds inducing lysosomal membrane damage in a patient-derived xenograft model. These data demonstrate a therapeutic advantage of targeting lysosomal membrane integrity in GBM.

Leukemia/lymphoma-related factor (LRF) or osteoclast zinc finger protein (OCZF) overexpression promotes osteoclast survival by increasing Bcl-xl mRNA: A novel regulatory mechanism mediated by the RNA binding protein SAM68.

RANKL induces NFATc1, a key transcriptional factor to induce osteoclast-specific genes such as cathepsin K, whereas transcriptional control of osteoclast survival is not fully understood. Leukemia/lymphoma-related factor (LRF) in mouse and osteoclast zinc finger protein (OCZF) in rat are zinc finger and BTB domain-containing protein (zBTB) family of transcriptional regulators, and are critical regulators of hematopoiesis. We have previously shown that differentiation and survival were enhanced in osteoclasts from OCZF-Transgenic (Tg) mice. In the present study, we show a possible mechanism of osteoclast survival regulated by LRF/OCZF and the role of OCZF overexpression in pathological bone loss. In the in vitro cultures, LRF was highly colocalized with NFATc1 in cells of early stage in osteoclastogenesis, but only LRF expression persisted after differentiation into mature osteoclasts. LRF expression was further enhanced in resorbing osteoclasts formed on dentin slices. Osteoclast survival inhibitor such as alendronate, a bisphosphonate reduced LRF expression. Micro CT evaluation revealed that femurs of OCZF-Tg mice showed significantly lower bone volume compared to that of WT mice. Furthermore, OCZF overexpression markedly promoted bone loss in ovariectomy-induced osteolytic mouse model. The expression of anti-apoptotic Bcl-xl mRNA, which is formed by alternative splicing, was enhanced in the cultures in which osteoclasts are formed from OCZF-Tg mice. In contrast, the expression of pro-apoptotic Bcl-xs mRNA was lost in the culture derived from OCZF-Tg mice. We found that the expression levels of RNA binding splicing regulator, Src substrate associated in mitosis of 68 kDa (Sam68) protein were markedly decreased in OCZF-Tg mice-derived osteoclasts. In addition, shRNA-mediated knockdown of Sam68 expression increased the expression of Bcl-xl mRNA, suggesting that SAM68 regulates the expression of Bcl-xl. These results indicate that OCZF overexpression reduces protein levels of Sam68, thereby promotes osteoclast survival, and suggest that LRF/OCZF is a promising target for regulating pathological bone loss.

Digital Transformation Will Change Medical Education and Rehabilitation in Spine Surgery.

The concept of minimally invasive spine therapy (MIST) has been proposed as a treatment strategy to reduce the need for overall patient care, including not only minimally invasive spine surgery (MISS) but also conservative treatment and rehabilitation. To maximize the effectiveness of patient care in spine surgery, the educational needs of medical students, residents, and patient rehabilitation can be enhanced by digital transformation (DX), including virtual reality (VR), augmented reality (AR), mixed reality (MR), and extended reality (XR), three-dimensional (3D) medical images and holograms; wearable sensors, high-performance video cameras, fifth-generation wireless system (5G) and wireless fidelity (Wi-Fi), artificial intelligence, and head-mounted displays (HMDs). Furthermore, to comply with the guidelines for social distancing due to the unexpected COVID-19 pandemic, the use of DX to maintain healthcare and education is becoming more innovative than ever before. In medical education, with the evolution of science and technology, it has become mandatory to provide a highly interactive educational environment and experience using DX technology for residents and medical students, known as digital natives. This study describes an approach to pre- and intraoperative medical education and postoperative rehabilitation using DX in the field of spine surgery that was implemented during the COVID-19 pandemic and will be utilized thereafter.

Ageing attenuates bone healing by mesenchymal stem cells in a microribbon hydrogel with a murine long bone critical-size defect model.

BACKGROUND: Despite the high incidence of fractures and pseudoarthrosis in the aged population, a potential role for the use of mesenchymal stem cells (MSCs) in the treatment of bone defects in elderly patients has not been elucidated. Inflammation and the innate immune system, including macrophages, play crucial roles in the differentiation and activation of MSCs. We have developed lentivirus-transduced interleukin 4 (IL4) over-expressing MSCs (IL4-MSCs) to polarize macrophages to an M2 phenotype to promote bone healing in an established young murine critical size bone defect model. In the current study, we explore the potential of IL4-MSCs in aged mice. METHODS: A 2 mm femoral diaphyseal bone defect was created and fixed with an external fixation device in 15- to 17-month-old male and female BALB/c mice. Microribbon (µRB) scaffolds (Sc) with or without encapsulation of MSCs were implanted in the defect sites. Accordingly, the mice were divided into three treatment groups: Sc-only, Sc + MSCs, and Sc + IL4-MSCs. Mice were euthanized six weeks after the surgery; subsequently, MicroCT (µCT), histochemical and immunohistochemical analyses were performed. RESULTS: µCT analysis revealed that bone formation was markedly enhanced in the IL4-MSC group. Compared with the Sc-only, the amount of new bone increased in the Sc + MSCs and Sc + IL4-MSC groups. However, no bridging of bone was observed in all groups. H&E staining showed fibrous tissue within the defect in all groups. Alkaline phosphatase (ALP) staining was increased in the Sc + IL4-MSC group. The Sc + IL4-MSCs group showed a decrease in the number of M1 macrophages and an increase in the number of M2 macrophages, with a significant increase in the M2/M1 ratio. DISCUSSION: IL4 promotes macrophage polarization to an M2 phenotype, facilitating osteogenesis and vasculogenesis. The addition of IL4-MSCs in the µRB scaffold polarized macrophages to an M2 phenotype and increased bone formation; however, complete bone bridging was not observed in any specimens. These results suggest that IL4-MSCs are insufficient to heal a critical size bone defect in aged mice, as opposed to younger animals. Additional therapeutic strategies are needed in this challenging clinical scenario.