Histological and immunohistochemical analyses of articular cartilage during onset and progression of pre- and early-stage osteoarthritis in a rodent model.

Early diagnosis and treatment of pre- and early-stage osteoarthritis (OA) is important. However, the cellular and cartilaginous changes occurring during these stages remain unclear. We investigated the histological and immunohistochemical changes over time between pre- and early-stage OA in a rat model of traumatic injury. Thirty-six male rats were divided into two groups, control and OA groups, based on destabilization of the medial meniscus. Histological and immunohistochemical analyses of articular cartilage were performed on days 1, 3, 7, 10, and 14 postoperatively. Cell density of proteins associated with cartilage degradation increased from postoperative day one. On postoperative day three, histological changes, including chondrocyte death, reduced matrix staining, and superficial fibrillation, were observed. Simultaneously, a compensatory increase in matrix staining was observed. The Osteoarthritis Research Society International score increased from postoperative day seven, indicating thinner cartilage. On postoperative day 10, the positive cell density decreased, whereas histological changes progressed with fissuring and matrix loss. The proteoglycan 4-positive cell density increased on postoperative day seven. These findings will help establish an experimental model and clarify the mechanism of the onset and progression of pre- and early-stage traumatic OA.

A New Strengthening Process for Carbon-Fiber-Reinforced Thermoplastic Polyphenylene Sulfide (CFRTP-PPS) Interlayered Composite by Electron Beam Irradiation to PPS Prior to Lamination Assembly and Hot Press.

Impact by hailstone, volcanic rock, bird strike, or also dropping tools can cause damage to aircraft materials. For maximum safety, the goal is to increase Charpy impact strength (auc) of a carbon-fiber-reinforced thermoplastic polyphenylene sulfide polymer (CFRTP-PPS) composite for potential application to commercial aircraft parts. The layup was three cross-weave CF plies alternating between four PPS plies, [PPS-CF-PPS-CF-PPS-CF-PPS], designated [PPS]4[CF]3. To strengthen, a new process for CFRP-PPS was employed applying homogeneous low voltage electron beam irradiation (HLEBI) to both sides of PPS plies prior to lamination assembly with untreated CF, followed by hot press under 4.0 MPa at 573 K for 8 min. Experimental results showed a 5 kGy HLEBI dose was at or near optimum, increasing auc at each accumulative probability, Pf. Optical microscopy of 5 kGy sample showed a reduction in main crack width with significantly reduced CF separation and pull-out; while, scanning electron microscopy (SEM) and electron dispersive X-ray (EDS) mapping showed PPS adhering to CF. Electron spin resonance (ESR) of a 5 kGy sample indicated lengthening of PPS chains as evidenced by a reduction in dangling bond peak. It Is assumed that 5 kGy HLEBI creates strong bonds at the interface while strengthening the PPS bulk. A model is proposed to illustrate the possible strengthening mechanism.

Strengthening Process by Electron Beam to Carbon Fiber for Impact Strength Enhancement of Interlayered Thermoplastic-Polypropylene Carbon Fiber Composite.

Strong adhesion between recyclable thermoplastic (TP) polymer and carbon fiber (CF) has always been highly sought after. Therefore, for an interlayered CF reinforced TP polypropylene (CFRTPP) composite composed of 3 sized CF plies, alternating between 4 PP sheets, designated [PP]4[CF]3, a process of activating CF plies directly on both sides with homogeneous low energy electron beam irradiation (EBI) under N2 gas, prior to lamination assembly and hot press of 4.0 MPa at 493 K for 3 min was carried out. Experimental results showed EBI dose of 43.2, 129, or 216 kGy significantly raised Charpy impact values, auc at all fracture probabilities, Pf. The 129 kGy dose appeared to be at or near optimum increasing auc 103%, 83%, and 65% at low-, median-, and high-Pf = 0.07, 0.50, and 0.93; while raising statistically lowest impact value, as at Pf = 0 calculated by 3-dimensional Weibull equation about 110%, indicating increased safety and reliability. It is assumed dangling bonds generated by the EBI rapidly form covalent bonds CF:C:O:C:PP and CF:C:C:PP at the interface, along with cross-linking in the PP near the CF. This is by charge transfer from CF to PP.

Protective roles of MITOL against myocardial senescence and ischemic injury partly via Drp1 regulation.

Abnormal mitochondrial fragmentation by dynamin-related protein1 (Drp1) is associated with the progression of aging-associated heart diseases, including heart failure and myocardial infarction (MI). Here, we report a protective role of outer mitochondrial membrane (OMM)-localized E3 ubiquitin ligase MITOL/MARCH5 against cardiac senescence and MI, partly through Drp1 clearance by OMM-associated degradation (OMMAD). Persistent Drp1 accumulation in cardiomyocyte-specific MITOL conditional-knockout mice induced mitochondrial fragmentation and dysfunction, including reduced ATP production and increased ROS generation, ultimately leading to myocardial senescence and chronic heart failure. Furthermore, ischemic stress-induced acute downregulation of MITOL, which permitted mitochondrial accumulation of Drp1, resulted in mitochondrial fragmentation. Adeno-associated virus-mediated delivery of the MITOL gene to cardiomyocytes ameliorated cardiac dysfunction induced by MI. Our findings suggest that OMMAD activation by MITOL can be a therapeutic target for aging-associated heart diseases, including heart failure and MI.

A case of autoimmune pulmonary alveolar proteinosis with severe respiratory failure treated with segmental lung lavage and oral statin therapy.

Pulmonary alveolar proteinosis (PAP) is a diffuse lung disease characterized by the accumulation of alveolar surfactants due to dysfunction of granulocyte-macrophage colony-stimulating factor-dependent cholesterol clearance. Whole-lung lavage is the current standard of care for PAP, but it can lead to the exacerbation of hypoxia. A medication targeting cholesterol homeostasis is a promising therapy for refractory PAP. We present a case of autoimmune PAP with severe hypoxia that was successfully treated with segmental lung lavage (SLL). Following SLL for disease relapse, statin treatment for dyslipidemia was started. After initiating statin treatment, the patient did not require bronchoalveolar lavage for 10 months.

Reduction of knee joint load suppresses cartilage degeneration, osteophyte formation, and synovitis in early-stage osteoarthritis using a post-traumatic rat model.

The purpose of this study was to clarify the histological effect of reducing the loading to knee on cartilage degeneration, osteophyte formation, and synovitis in early-stage osteoarthritis (OA) using a post-traumatic rat model. Ten male rats were randomly allocated into two experimental groups: OA induction by surgical destabilization of medial meniscus (DMM, OA group) and hindlimb suspension after OA induction by DMM (OAHS group). The articular cartilage, osteophyte formation, and synovial membrane in the medial tibiofemoral joint were analyzed histologically and histomorphometrically at 2 and 4 weeks after surgery. The histological scores and changes in articular cartilage and osteophyte formation were significantly milder and slower in the OAHS group than in the OA group. At 2 and 4 weeks, there were no significant differences in cartilage thickness and matrix staining intensity between both the groups, but chondrocytes density was significantly lower in the OA group. Synovitis was milder in OAHS group than in OA group at 2 weeks. Reducing knee joint loading inhibited histological OA changes in articular cartilage, osteophyte formation, and synovial inflammation. This result supports the latest clinical guidelines for OA treatment. Further studies using biochemical and mechanical analyses are necessary to elucidate the mechanism underlying delayed OA progression caused by joint-load reduction.

Identification of highest neurotoxic amyloid-ß plaque type showing reduced contact with astrocytes.

Amyloid-ß (Aß) plaques are strongly associated with the development of Alzheimer's disease (AD). However, it remains unclear how morphological differences in Aß plaques determine the pathogenesis of Aß. Here, we categorized Aß plaques into four types based on the macroscopic features of the dense core, and found that the Aß-plaque subtype containing a larger dense core showed the strongest association with neuritic dystrophy. Astrocytes dominantly accumulated toward these expanded/dense-core-containing Aß plaques. Previously, we indicated that deletion of the mitochondrial ubiquitin ligase MITOL/MARCH5 triggers mitochondrial impairments and exacerbates cognitive decline in a mouse model with AD-related Aß pathology. In this study, MITOL deficiency accelerated the formation of expanded/dense-core-containing Aß plaques, which showed reduced contacts with astrocytes, but not microglia. Our findings suggest that expanded/dense-core-containing Aß-plaque formation enhanced by the alteration of mitochondrial function robustly contributes to the exacerbation of Aß neuropathology, at least in part, through the reduced contacts between Aß plaques and astrocytes.

MITOL promotes cell survival by degrading Parkin during mitophagy.

Parkin promotes cell survival by removing damaged mitochondria via mitophagy. However, although some studies have suggested that Parkin induces cell death, the regulatory mechanism underlying the dual role of Parkin remains unknown. Herein, we report that mitochondrial ubiquitin ligase (MITOL/MARCH5) regulates Parkin-mediated cell death through the FKBP38-dependent dynamic translocation from the mitochondria to the ER during mitophagy. Mechanistically, MITOL mediates ubiquitination of Parkin at lysine 220 residue, which promotes its proteasomal degradation, and thereby fine-tunes mitophagy by controlling the quantity of Parkin. Deletion of MITOL leads to accumulation of the phosphorylated active form of Parkin in the ER, resulting in FKBP38 degradation and enhanced cell death. Thus, we have shown that MITOL blocks Parkin-induced cell death, at least partially, by protecting FKBP38 from Parkin. Our findings unveil the regulation of the dual function of Parkin and provide a novel perspective on the pathogenesis of PD.

Mitochondrial ubiquitin ligase alleviates Alzheimer's disease pathology via blocking the toxic amyloid-ß oligomer generation.

Mitochondrial pathophysiology is implicated in the development of Alzheimer's disease (AD). An integrative database of gene dysregulation suggests that the mitochondrial ubiquitin ligase MITOL/MARCH5, a fine-tuner of mitochondrial dynamics and functions, is downregulated in patients with AD. Here, we report that the perturbation of mitochondrial dynamics by MITOL deletion triggers mitochondrial impairments and exacerbates cognitive decline in a mouse model with AD-related Aß pathology. Notably, MITOL deletion in the brain enhanced the seeding effect of Aß fibrils, but not the spontaneous formation of Aß fibrils and plaques, leading to excessive secondary generation of toxic and dispersible Aß oligomers. Consistent with this, MITOL-deficient mice with Aß etiology exhibited worsening cognitive decline depending on Aß oligomers rather than Aß plaques themselves. Our findings suggest that alteration in mitochondrial morphology might be a key factor in AD due to directing the production of Aß form, oligomers or plaques, responsible for disease development.

Significance of PSA Screening in Niigata, Japan: Survey of Actual Status of New Cases of Prostate Cancer.

PURPOSE: This study aims to investigate the utility of prostate-specific antigen (PSA) screening by conducting an all-case survey of newly diagnosed prostate cancer patients at Niigata Prefecture, Japan. PATIENTS AND METHODS: Depending on whether patients were subjected to screening, information was prospectively collected on all prostate cancer patients newly diagnosed between October 1, 2019, and September 30, 2020, at all institutions in Niigata Prefecture where urologists performing prostate biopsy routinely work and differences in clinical parameters were investigated. RESULTS: PSA was measured in 478 out of 1332 patients (35.8%) as part of a community health screening. The rate of metastatic carcinoma (M1) in all patients was 14.9%. When patients were divided into three categories of population-based screening (community health screening and workplace health screening), opportunistic screening (PSA measurements at complete medical check-ups or on patient request), and testing triggered by clinical symptoms or findings, the proportion of metastatic cancer was 4.5%, 3.7%, and 30.6%, respectively, demonstrating that the number of distant metastases was significantly lesser in all patients who underwent screening. CONCLUSION: The one-year all-case survey of newly diagnosed prostate cancer patients demonstrated that PSA screening significantly contributed to the early diagnosis of current prostate cancer in Japan.

Overview of Mitochondrial E3 Ubiquitin Ligase MITOL/MARCH5 from Molecular Mechanisms to Diseases.

The molecular pathology of diseases seen from the mitochondrial axis has become more complex with the progression of research. A variety of factors, including the failure of mitochondrial dynamics and quality control, have made it extremely difficult to narrow down drug discovery targets. We have identified MITOL (mitochondrial ubiquitin ligase: also known as MARCH5) localized on the mitochondrial outer membrane and previously reported that it is an important regulator of mitochondrial dynamics and mitochondrial quality control. In this review, we describe the pathological aspects of MITOL revealed through functional analysis and its potential as a drug discovery target.

MITOL dysfunction causes dwarfism with anterior pituitary hypoplasia.

In mitochondrial disorders, short stature and growth failure are common symptoms, but their underlying mechanism remains unknown. In this study, we examined the cause of growth failure of mice induced by nestin promoter-driven knockout of the mitochondrial ubiquitin ligase MITOL (MARCH5), a key regulator of mitochondrial function. MITOL-knockout mice have congenital hypoplasia of the anterior pituitary caused by decreased expression of pituitary transcript factor 1 (Pit1). Consistently, both mRNA levels of growth hormone (GH) and prolactin levels were markedly decreased in the anterior pituitary of mutant mice. Growth failure of mutant mice was partly rescued by hypodermic injection of recombinant GH. To clarify whether this abnormality was induced by the primary effect of MITOL knockdown in the anterior pituitary or a secondary effect of other lesions, we performed lentiviral-mediated knockdown of MITOL on cultured rat pituitary GH3 cells, which secrete GH. GH production was severely compromised in MITOL-knockdown GH3 cells. In conclusion, MITOL plays a critical role in the development of the anterior pituitary; therefore, mice with MITOL dysfunction exhibited pituitary dwarfism caused by anterior pituitary hypoplasia. Our findings suggest that mitochondrial dysfunction is commonly involved in the unknown pathogenesis of pituitary dwarfism.

Mitochondrial Dynamics Regulation in Skin Fibroblasts from Mitochondrial Disease Patients.

Mitochondria are highly dynamic organelles that constantly fuse, divide, and move, and their function is regulated and maintained by their morphologic changes. Mitochondrial disease (MD) comprises a group of disorders involving mitochondrial dysfunction. However, it is not clear whether changes in mitochondrial morphology are related to MD. In this study, we examined mitochondrial morphology in fibroblasts from patients with MD (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and Leigh syndrome). We observed that MD fibroblasts exhibited significant mitochondrial fragmentation by upregulation of Drp1, which is responsible for mitochondrial fission. Interestingly, the inhibition of mitochondrial fragmentation by Drp1 knockdown enhanced cellular toxicity and led to cell death in MD fibroblasts. These results suggest that mitochondrial fission plays a critical role in the attenuation of mitochondrial damage in MD fibroblasts.

Daily and short-term application of joint movement for the prevention of infrapatellar fat pad atrophy due to immobilization.

[Purpose] To mobilize the knee joint during cast fixation and to determine whether infrapatellar fat pad changes can be prevented. [Materials and Methods] We randomly allocated Wistar rats into 3 groups as follows: normal group, raised in normal conditions (n=5); contracture group, immobilized with cast fixation (n=5); and prevention group, treated with joint movement during immobilization (n=5). We immobilized the right hindlimb using cast fixation. Joint movement in the prevention group was accomplished by repeatedly pulling the right hindlimb caudally and then returning the leg to the bent position for 10 minutes every day for 2 weeks. We used a metronome to maintain a constant speed, with one set lasting 2 seconds (1-second traction and 1-second return). [Results] The contracture group had adipose cells of various sizes and fibrosis in the infrapatellar fat pad. These changes were also found in milder forms in the prevention group. We found significant differences in the cross section of adipose cells and in knee extension restriction between the groups. [Conclusion] Promoting joint movement may not only have a therapeutic effect on adipose cells but also a preventative effect.

Critical role of CRAG, a splicing variant of centaurin-γ3/AGAP3, in ELK1-dependent SRF activation at PML bodies.

CRMP-5-associated GTPase (CRAG), a short splicing variant of centaurin-γ3/AGAP3, is predominantly expressed in the developing brain. We previously demonstrated that CRAG, but not centaurin-γ3, translocates to the nucleus and activates the serum response factor (SRF)-c-Fos pathway in cultured neuronal cells. However, the physiological relevance of CRAG in vivo is unknown. Here, we found that CRAG/centaurin-γ3-knockout mice showed intensively suppressed kainic acid-induced c-fos expression in the hippocampus. Analyses of molecular mechanisms underlying CRAG-mediated SRF activation revealed that CRAG has an essential role in GTPase activity, interacts with ELK1 (a co-activator of SRF), and activates SRF in an ELK1-dependent manner. Furthermore, CRAG and ELK1 interact with promyelocytic leukaemia bodies through SUMO-interacting motifs, which is required for SRF activation. These results suggest that CRAG plays a critical role in ELK1-dependent SRF-c-fos activation at promyelocytic leukaemia bodies in the developing brain.

Achieving PSA < 0.2 ng/ml before Radiation Therapy Is a Strong Predictor of Treatment Success in Patients with High-Risk Locally Advanced Prostate Cancer.

BACKGROUND: To predict long-term treatment outcome of radiation therapy (RT) plus androgen deprivation therapy (ADT) for high-risk locally advanced prostate cancer. METHODS: In total, 204 patients with the National Comprehensive Cancer Network (NCCN) high risk locally advanced prostate cancer (PSA > 20 ng/ml, Gleason score ≧ 8, clinical T stage ≧ 3a) were treated with definitive RT with ADT. Median follow up period was 113 months (IQR: 95-128). Median neoadjuvant ADT and total ADT duration were 7 months (IQR: 6-10) and 27 months (IQR: 14-38), respectively. RESULTS: PSA recurrence-free survival (PSA-RFS), cancer specific survival (CSS), and overall survival (OS) rates at 5 years were 84.1%, 98.5%, and 93.6%, respectively, and 67.9%, 91.2%, and 78.1%, respectively, at 10 years. Pre-RT PSA less than 0.2 ng/ml was associated with superior outcomes of PSA-RFS (HR = 0.42, 95% CI: 0.25-0.70, p = 0.001), CSS (HR = 0.27, 95% CI: 0.09-0.82, p = 0.013), and OS (HR = 0.48, 95% CI: 0.26-0.91, p = 0.021). On multivariate analysis, age (≥70 y.o.) and pre-RT PSA (≥0.2 ng/ml) were factors predictive of poorer OS (p = 0.032) , but iPSA, T stage, Gleason score, number of NCCN high-risk criteria, a combination with anti-androgen therapy and neoadjuvant ADT duration were not predictive of treatment outcome. CONCLUSION: In patient with high-risk prostate cancer, RT plus ADT achieved good oncologic outcomes. PSA < 0.2 ng/ml before radiation therapy is a strong independent predictor for long overall survival.

Mitochondrial retrograde signaling to the endoplasmic-reticulum regulates unfolded protein responses.

Unfolded protein response (UPRs) directs adaption or apoptosis depending on the severity of endoplasmic-reticulum (ER) stress. We found that apoptotic signaling by inositol requiring enzyme 1α (IRE1α), a transducer of UPRs, is suppressed by mitochondrial ubiquitin ligase MITOL/MARCH5 on ER-mitochondria contacts, suggesting that mitochondria regulate cell fate under ER stress.

MITOL deletion in the brain impairs mitochondrial structure and ER tethering leading to oxidative stress.

Mitochondrial abnormalities are associated with developmental disorders, although a causal relationship remains largely unknown. Here, we report that increased oxidative stress in neurons by deletion of mitochondrial ubiquitin ligase MITOL causes a potential neuroinflammation including aberrant astrogliosis and microglial activation, indicating that mitochondrial abnormalities might confer a risk for inflammatory diseases in brain such as psychiatric disorders. A role of MITOL in both mitochondrial dynamics and ER-mitochondria tethering prompted us to characterize three-dimensional structures of mitochondria in vivo. In MITOL-deficient neurons, we observed a significant reduction in the ER-mitochondria contact sites, which might lead to perturbation of phospholipids transfer, consequently reduce cardiolipin biogenesis. We also found that branched large mitochondria disappeared by deletion of MITOL. These morphological abnormalities of mitochondria resulted in enhanced oxidative stress in brain, which led to astrogliosis and microglial activation partly causing abnormal behavior. In conclusion, the reduced ER-mitochondria tethering and excessive mitochondrial fission may trigger neuroinflammation through oxidative stress.

MITOL prevents ER stress-induced apoptosis by IRE1α ubiquitylation at ER-mitochondria contact sites.

Unresolved endoplasmic reticulum (ER) stress shifts the unfolded protein response signaling from cell survival to cell death, although the switching mechanism remains unclear. Here, we report that mitochondrial ubiquitin ligase (MITOL/MARCH5) inhibits ER stress-induced apoptosis through ubiquitylation of IRE1α at the mitochondria-associated ER membrane (MAM). MITOL promotes K63-linked chain ubiquitination of IRE1α at lysine 481 (K481), thereby preventing hyper-oligomerization of IRE1α and regulated IRE1α-dependent decay (RIDD). Therefore, under ER stress, MITOL depletion or the IRE1α mutant (K481R) allows for IRE1α hyper-oligomerization and enhances RIDD activity, resulting in apoptosis. Similarly, in the spinal cord of MITOL-deficient mice, ER stress enhances RIDD activity and subsequent apoptosis. Notably, unresolved ER stress attenuates IRE1α ubiquitylation, suggesting that this directs the apoptotic switch of IRE1α signaling. Our findings suggest that mitochondria regulate cell fate under ER stress through IRE1α ubiquitylation by MITOL at the MAM.

[SMALL CELL PROSTATE CANCER PRODUCING SYNDROME OF INAPPROPRIATE SECRETION OF ANTIDIURETIC HORMONE; A CASE REPORT].

A 73-year-old man with progressive prostate cancer visited our hospital after prostate biopsy performed at another hospital. His serum PSA level was 29.02 ng/ml. CT revealed invasion of the bladder, bilateral ureters, and rectum. Otherwise, there was no evidence of metastasis. Pathological findings showed a poorly differentiated adenocarcinoma (Gleason score 4+5) and small cell carcinoma component. Two months after administering combined androgen blockade therapy, he was admitted due to severe hyponatremia caused by the inappropriate secretion of antidiuretic hormone. Furthermore, CT revealed right ureter metastasis, although the PSA levels remained low. Therefore, the patient was put on fluid restriction and sodium administration. After the patient recovered from hyponatremia, chemotherapy, including VP-16 and CDDP, was initiated. However, CT after two chemotherapy cycles revealed disease progression, with multiple bone metastases. Second-line chemotherapy, including CPT-11 and CDDP, was less effective, and the patient died 9 months after the diagnosis.