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Brown rice is known to improve glucose intolerance and prevent the onset of diabetes. However, the underlying mechanisms remain obscure. In the current study, we investigated the effect of brown rice and its major component, γ-oryzanol (Orz), on feeding behavior and fuel homeostasis in mice. When mice were allowed free access to a brown rice-containing chow diet (CD) and a high-fat diet (HFD), they significantly preferred CD to HFD. To reduce hypothalamic endoplasmic reticulum (ER) stress on an HFD, mice were administered with 4-phenylbutyric acid, a chemical chaperone, which caused them to prefer the CD. Notably, oral administration of Orz, a mixture of major bioactive components in brown rice, also improved glucose intolerance and attenuated hypothalamic ER stress in mice fed the HFD. In murine primary neuronal cells, Orz attenuated the tunicamycin-induced ER stress. In luciferase reporter assays in human embryonic kidney 293 cells, Orz suppressed the activation of ER stress-responsive cis-acting elements and unfolded protein response element, suggesting that Orz acts as a chemical chaperone in viable cells. Collectively, the current study is the first demonstration that brown rice and Orz improve glucose metabolism, reduce hypothalamic ER stress, and, consequently, attenuate the preference for dietary fat in mice fed an HFD. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) is the site of synthesis and folding of secretory proteins. Perturbations of ER homeostasis affect protein folding and cause ER stress. ER can sense the stress and respond to it through translational attenuation, upregulation of the genes for ER chaperones and related proteins, and degradation of unfolded proteins by a quality-control system. However, when the ER function is severely impaired, the organelle elicits apoptotic signals. ER stress has been implicated in a variety of common diseases such as diabetes, ischemia and neurodegenerative disorders. One of the components of the ER stress-mediated apoptosis pathway is C/EBP homologous protein (CHOP), also known as growth arrest- and DNA damage-inducible gene 153 (GADD153). Here, we summarize the current understanding of the roles of CHOP/GADD153 in ER stress-mediated apoptosis and in diseases including diabetes, brain ischemia and neurodegenerative disease.Cell Death and Differentiation (2004) 11, 381-389. doi:10.1038/sj.cdd.4401373 Published online 19 December 2003 [ABSTRACT FROM AUTHOR]

Study Design: Retrospective review of multicentric data., Objectives: The modified 5-item frailty index is a relatively new tool to assess the post-operative complication risks. It has been recently shown a good predictive value after posterior lumbar fusion. We aimed to compare the predictive value of the modified 5-item frailty index in cervical, thoracic and lumbar surgery., Methods: The American College of Surgeons - National Surgical Quality Improvement Program (ACS-NSQIP) Database 2015-2020 was used to identify patients who underwent elective posterior cervical, thoracic, or lumbar fusion surgeries for degenerative conditions. The mFI-5 score was calculated based on the presence of 5 co-morbidities: congestive heart failure within 30 days prior to surgery, insulin-dependent or noninsulin-dependent diabetes mellitus, chronic obstructive pulmonary disease or pneumonia, partially dependent or totally dependent functional health status at time of surgery, and hypertension requiring medication. Multivariate analysis was used to assess the independent impact of increasing mFI-5 score on the postoperative morbidity while controlling for baseline clinical characteristics., Results: 53 252 patients were included with the mean age of 64.2 ± 7.2. 7946 suffered medical complications (14.9%), 1565 had surgical complications (2.9%), and 3385 were readmitted (6.3%), 363 died (.68%) within 30 days postoperative (6.3%). The mFI-5 items score was significantly associated with higher rates of complications, readmission, and mortality in cervical, thoracic, and lumbar posterior fusion surgery., Conclusion: The modified 5-item frailty score is a reliable tool to predict complications, readmission, and mortality in patients planned for elective posterior spinal fusion surgery., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: “Each author certifies that he or she has no commercial associations (eg, consultancies, stock ownership, equity interest, patent/licensing arrangements, etc.) that might pose a conflict of interest in connection with the submitted article.”

Familial neurohypophyseal diabetes insipidus (FNDI) is a degenerative disorder in which vasopressin-secreting neurons degenerate over time due to the production of mutant proteins. We have demonstrated therapeutic effects of chemical chaperones in an FNDI mouse model, but the complexity and length of this evaluation were problematic. In this study, we established disease-specific mouse induced pluripotent stem cells (iPSCs) from FNDI-model mice and differentiated vasopressin neurons that produced mutant proteins. Fluorescence immunostaining showed that chemical chaperones appeared to protect vasopressin neurons generated from iPSCs derived from FNDI-model mice. Although KCL stimulation released vasopressin hormone from vasopressin neurons generated from FNDI-derived iPSCs, vasopressin hormone levels did not differ significantly between baseline and chaperone-added culture. Semi-quantification of vasopressin carrier protein and mutant protein volumes in vasopressin neurons confirmed that chaperones exerted a therapeutic effect. This research provides fundamental technology for creating in vitro disease models using human iPSCs and can be applied to therapeutic evaluation of various degenerative diseases that produce abnormal proteins., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Lung cancer, primarily non-small-cell lung cancer (NSCLC), is a significant cause of cancer-related mortality worldwide. Cisplatin-based chemotherapy is a standard treatment for NSCLC; however, its effectiveness is often limited due to the development of resistance, leading to NSCLC recurrence. Thus, the identification of effective chemosensitizers for cisplatin is of paramount importance. The integrated stress response (ISR), activated by various cellular stresses and mediated by eIF2α kinases, has been implicated in drug sensitivity. ISR activation globally suppresses protein synthesis while selectively promoting the translation of ATF4 mRNA, which can induce pro-apoptotic proteins such as CHOP, ATF3, and TRIB3. To expedite and economize the development of chemosensitizers for cisplatin treatment in NSCLC, we employed a strategy to screen an FDA-approved drug library for ISR activators. In this study, we identified mifepristone as a potent ISR activator. Mifepristone activated the HRI/eIF2α/ATF4 axis, leading to the induction of pro-apoptotic factors, independent of its known role as a synthetic steroid. Our in vitro and in vivo models demonstrated mifepristone's potential to inhibit NSCLC re-proliferation following cisplatin treatment and tumor growth, respectively, via the ISR-mediated cell death pathway. These findings suggest that mifepristone, as an ISR activator, could enhance the efficacy of cisplatin-based therapy for NSCLC, highlighting the potential of drug repositioning in the search for effective chemosensitizers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Apoptosis is one of the hallmarks of MeHg-induced neuronal cell death; however, its molecular mechanism remains unclear. We previously reported that MeHg exposure induces neuron-specific ER stress in the mouse brain. Excessive ER stress contributes to apoptosis, and CHOP induction is considered to be one of the major mechanisms. CHOP is also increased by MeHg exposure in the mouse brain, suggesting that it correlates with increased apoptosis. In this study, to clarify whether CHOP mediates MeHg-induced apoptosis, we examined the effect of CHOP deletion on MeHg exposure in CHOP-knockout mice. Our data showed that CHOP deletion had no effect on MeHg exposure-induced weight loss or hindlimb impairment in mice, nor did it increase apoptosis or inhibit neuronal cell loss. Hence, CHOP plays little role in MeHg toxicity, and other apoptotic pathways coupled with ER stress may be involved in MeHg-induced cell death.

Activating transcription factor 6 (ATF6) is an endoplasmic reticulum (ER) stress-regulated transcription factor that induces expression of major molecular chaperones in the ER. We recently reported that ATF6β, a subtype of ATF6, promoted survival of hippocampal neurons exposed to ER stress and excitotoxicity, at least in part by inducing expression of calreticulin, an ER molecular chaperone with high Ca 2+ -binding capacity. In the present study, we demonstrate that ATF6β deficiency in mice also decreases calreticulin expression and increases expression of glucose-regulated protein 78, another ER molecular chaperone, in emotional brain regions such as the prefrontal cortex (PFC), hypothalamus, hippocampus, and amygdala. Comprehensive behavioral analyses revealed that Atf6b -/- mice exhibit anxiety-like behavior in the light/dark transition test and hyperactivity in the forced swim test. Consistent with these results, PFC and hypothalamic corticotropin-releasing hormone (CRH) expression was increased in Atf6b -/- mice, as was circulating corticosterone. Moreover, CRH receptor 1 antagonism alleviated anxiety-like behavior in Atf6b -/- mice. These findings suggest that ATF6β deficiency produces anxiety-like behavior and hyperactivity via a CRH receptor 1-dependent mechanism. ATF6β could play a role in psychiatric conditions in the emotional centers of the brain., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Surgical fixation of distal biceps tendon ruptures can restore supination strength and minimize biceps fatigue, resulting in high patient satisfaction rates. Surgical approaches can vary (single incision vs. double incision), and the number of fixation techniques has increased in recent years. The reported rate of postoperative complications after surgical repair of distal biceps tendon injuries is high, ranging from 15% to 35%. The purpose of this study was to assess the trends and postoperative complication profile among newly trained surgeons who performed distal biceps tendon repairs utilizing the American Board of Orthopaedic Surgery database., Methods: The American Board of Orthopaedic Surgery database was retrospectively queried for patients treated with distal biceps tendon repair by part II examination candidates between 2017 and 2020. Distal biceps tendon repairs were isolated using the Current Procedural Terminology code 24,342. Distal triceps tendon injuries were excluded with International Classification of Diseases code S46.3∗∗. Patient demographics, intraoperative data, and surgeon fellowship training were collected. Surgeon-reported postoperative 90-day complications, including general anesthetic, medical, and surgical complications, rates of readmission, and rates of reoperation were recorded. Comparisons of rates among patient groups organized by surgeon fellowship training were performed using the chi-squared test., Results: A total of 2089 distal biceps tendon repairs were included in the analysis. The average patient age was 47.5 yr, and 97.3% of patients were men. The majority of cases was performed by surgeons with fellowship training in sports medicine, hand/upper extremity, and shoulder and elbow, with 867 (41.5%) cases performed by sports medicine-trained surgeons, 740 (35.4%) by hand/upper extremity-trained surgeons, and 313 (15.0%) by shoulder and elbow-trained surgeons. In total, 608 (29.1%) patients experienced an anesthetic (0.2%), medical (1.1%), or surgical (28.2%) complication. The most common surgical complications were nerve injury (20.6%), failure of tendon repair or fixation (2.4%), and infection (1.7%). The overall reoperation rate was 2.4%. There were no significant differences in complication or reoperation rates among subspecialty training received., Discussion and Conclusion: Among newly trained surgeons, those with fellowship training in sports medicine, hand/upper extremity, and shoulder and elbow performed the most distal biceps tendon repairs, and there was no difference in complication rates among subspecialty training received. Complication rates after distal biceps tendon repair performed by newly trained surgeons were similar to those previously reported in large cohort studies, with nerve injury as the most common complication., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

WNT molecules are the regulators of various biological functions, including body axis formation, organ development, and cell proliferation and differentiation. WNTs have been extensively studied as causative genes for an array of diseases. WNT10A and WNT10B , which are considered to be genes of the same origin, have been identified as causative genes for tooth deficiency in humans. However, the disrupted mutant of each gene does not show a decrease in teeth number. A negative feedback loop, interacting with several ligands based on a reaction-diffusion mechanism, was proposed to be important for the spatial patterning of tooth formation, and WNT ligands have been considered to play a pivotal role in controlling tooth patterning from mutant phenotypes of LDL receptor-related proteins (LRPs) and WNT co-receptors. The Wnt10a and Wnt10b double-mutants demonstrated severe root or enamel hypoplasia. In Wnt10a -/- and Wnt10a +/- ;Wnt10b -/- mice, changes in the feedback loop may collapse the modulation of fusion or split a sequence of tooth formation. However, in the double-knockout mutant, a decrease in the number of teeth was observed, including the upper incisor or third molar in both jaws. These findings suggest that there may be a functional redundancy between Wnt10a and Wnt10b and that the interaction between the two genes functions in conjunction with other ligands to control the spatial patterning and development of teeth.

Brown adipose tissue (BAT) is a highly specialized adipose tissue in its immobile location and size during the entire adulthood. In response to cold exposure and other β3-adrenoreceptor stimuli, BAT commits energy consumption by nonshivering thermogenesis (NST). However, the molecular machinery in controlling the BAT mass in adults is unknown. Here, we show our surprising findings that the BAT mass and functions can be manipulated in adult animals by controlling BAT adipocyte differentiation in vivo. Platelet-derived growth factor receptor α (PDGFα) expressed in BAT progenitor cells served a signaling function to avert adipose progenitor differentiation. Genetic and pharmacological loss-of-function of PDGFRα eliminated the differentiation barrier and permitted progenitor cell differentiation to mature and functional BAT adipocytes. Consequently, an enlarged BAT mass (megaBAT) was created by PDGFRα inhibition owing to increases of brown adipocyte numbers. Under cold exposure, a microRNA-485 (miR-485) was identified as a master suppressor of the PDGFRα signaling, and delivery of miR-485 also produced megaBAT in adult animals. Noticeably, megaBAT markedly improved global metabolism, insulin sensitivity, high-fat-diet (HFD)-induced obesity, and diabetes by enhancing NST. Together, our findings demonstrate that the adult BAT mass can be increased by blocking the previously unprecedented inhibitory signaling for BAT progenitor cell differentiation. Thus, blocking the PDGFRα for the generation of megaBAT provides an attractive strategy for treating obesity and type 2 diabetes mellitus (T2DM).

Background: Preoperative optimization in patients undergoing posterior spinal fusion is essential to limit the number and severity of postoperative complications. Here, we, additionally, evaluated the impact of hypoalbuminemia on morbidity and mortality after posterior spinal fusion surgery., Methods: This retrospective analysis was performed using data from a prospective multicentric database (ACSNSQIP:2015-2020) regarding patients undergoing posterior spinal fusions. Factors studied included; baseline demographics and 30-day postoperative complications (i.e., reoperations, readmissions, and mortality rates)., Results: There were 6805 patients who met the inclusion criteria. They averaged 62 years of age and had an average BMI of 30.2. Within the 30-day postoperative period, 634 (9.3%) sustained complications; 467 (6.9%) were readmitted, 263 (3.9%) required reoperations, and 37 (0.5%) expired. Although multiple preoperative risk factors were analyzed, hypoalbuminemia, severe hypoalbuminemia, and dialysis were the strongest independent risk factors associated with complications (i.e., reoperations, readmissions, and mortality)., Conclusion: Hypoalbuminemia, severe hypoalbuminemia, and dialysis were significant predictors for morbidity and mortality after posterior spinal fusion surgery., Competing Interests: There are no conflicts of interest., (Copyright: © 2022 Surgical Neurology International.)

Stress-inducible transcription factor ATF4 is essential for survival and identity of β-cell during stress conditions. However, the physiological role of ATF4 in β-cell function is not yet completely understood. To understand the role of ATF4 in glucose-stimulated insulin secretion (GSIS), β-cell-specific Atf4 knockout (βAtf4KO) mice were phenotypically characterized. Insulin secretion and mechanistic analyses were performed using islets from control Atf4f/f and βAtf4KO mice to assess key regulators for triggering and amplifying signals for GSIS. βAtf4KO mice displayed glucose intolerance due to reduced insulin secretion. Moreover, βAtf4KO islets exhibited a decrease in both the insulin content and first-phase insulin secretion. The analysis of βAtf4KO islets showed that ATF4 is required for insulin production and glucose-stimulated ATP and cAMP production. The results demonstrate that ATF4 contributes to the multifaceted regulatory process in GSIS even under stress-free conditions., Competing Interests: Declaration of interests The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors have no conflicts of interest to declare., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Perturbation of endoplasmic reticulum (ER) proteostasis is associated with impairment of cellular function in diverse diseases, especially the function of pancreatic β cells in type 2 diabetes. Restoration of ER proteostasis by small molecules shows therapeutic promise for type 2 diabetes. Here, using cell-based screening, we report identification of a chemical chaperone-like small molecule, KM04794, that alleviates ER stress. KM04794 prevented protein aggregation and cell death caused by ER stressors and a mutant insulin protein. We also found that this compound increased intracellular and secreted insulin levels in pancreatic β cells. Chemical biology and biochemical approaches revealed that the compound accumulated in the ER and interacted directly with the ER molecular chaperone BiP. Our data show that this corrector of ER proteostasis can enhance insulin storage and pancreatic β cell function., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: We analyzed the role of hypoalbuminemia, dialysis, and other risk factors that increase morbidity/ mortality following surgery for primary pyogenic spinal infections (PSIs). The American College of Surgeons' National Surgical Quality Improvement Program (ACS-NSQIP) that included 627 patients was utilized as our database., Methods: Primary spinal surgery for spondylodiscitis was evaluated in a ACS-NSQIP database involving 627 patients between 2010 and 2019. Outcome assessment included evaluation of 30-day postoperative morbidity, and mortality rates., Results: Within 30 postoperative days, complications occurred in 14.6% (92/627) of patients; 59 (9.4%) required readmission, and 39 (6.2%) required additional surgery. The most common complications were: wound infections, pneumonia, septic shock, and death (1.8%). Hypoalbuminemia (i.e., significantly associated with unplanned readmission and reoperation), and dialysis were the two major risk factors contributing to increased perioperative morbidity and mortality., Conclusion: Among 627 ACS-NSQIP patients undergoing primary surgery for PSIs, hypoalbuminemia and dialysis were associated with higher risks of major perioperative morbidity (i.e., within 30 postoperative days - mostly readmissions and reoperations) and mortality., Competing Interests: There are no conflicts of interest., (Copyright: © 2022 Surgical Neurology International.)

The effects of imeglimin, a novel antidiabetes agent, on β-cell function remain unclear. Here, we unveiled the impact of imeglimin on β-cell survival. Treatment with imeglimin augmented mitochondrial function, enhanced insulin secretion, promoted β-cell proliferation, and improved β-cell survival in mouse islets. Imeglimin upregulated the expression of endoplasmic reticulum (ER)-related molecules, including Chop (Ddit3), Gadd34 (Ppp1r15a), Atf3, and Sdf2l1, and decreased eIF2α phosphorylation after treatment with thapsigargin and restored global protein synthesis in β-cells under ER stress. Imeglimin failed to protect against ER stress-induced β-cell apoptosis in CHOP-deficient islets or in the presence of GADD34 inhibitor. Treatment with imeglimin showed a significant decrease in the number of apoptotic β-cells and increased β-cell mass in Akita mice. Imeglimin also protected against β-cell apoptosis in both human islets and human pluripotent stem cell-derived β-like cells. Taken together, imeglimin modulates the ER homeostasis pathway, which results in the prevention of β-cell apoptosis both in vitro and in vivo., (© 2022 by the American Diabetes Association.)

Objective: Activating transcription factor 4 (ATF4) is a transcriptional regulator of the unfolded protein response and integrated stress response (ISR) that promote the restoration of normal endoplasmic reticulum (ER) function. Previous reports demonstrated that dysregulation of the ISR led to development of severe diabetes. However, the contribution of ATF4 to pancreatic β-cells remains poorly understood. In this study, we aimed to analyze the effect of ISR enhancer Sephin1 and ATF4-deficient β-cells to clarify the role of ATF4 in β-cells under ER stress conditions., Methods: To examine the role of ATF4 in vivo, ISR enhancer Sephin1 (5 mg/kg body weight, p.o.) was administered daily for 21 days to Akita mice. We also established β-cell-specific Atf4 knockout (βAtf4-KO) mice that were further crossed with Akita mice. These mice were analyzed for characteristics of diabetes, β-cell function, and morphology of the islets. To identify the downstream factors of ATF4 in β-cells, the islets of βAtf4-KO mice were subjected to cDNA microarray analyses. To examine the transcriptional regulation by ATF4, we also performed in situ PCR analysis of pancreatic sections from mice and ChIP-qPCR analysis of CT215 β-cells., Results: Administration of the ISR enhancer Sephin1 improved glucose metabolism in Akita mice. Sephin1 also increased the insulin-immunopositive area and ATF4 expression in the pancreatic islets. Akita/βAtf4-KO mice exhibited dramatically exacerbated diabetes, shown by hyperglycemia at an early age, as well as a remarkably short lifespan owing to diabetic ketoacidosis. Moreover, the islets of Akita/βAtf4-KO mice presented increased numbers of cells stained for glucagon, somatostatin, and pancreatic polypeptide and increased expression of aldehyde dehydrogenase 1 family member 3, a marker of dedifferentiation. Using microarray analysis, we identified atonal BHLH transcription factor 8 (ATOH8) as a downstream factor of ATF4. Deletion of ATF4 in β-cells showed reduced Atoh8 expression and increased expression of undifferentiated markers, Nanog and Pou5f1. Atoh8 expression was also abolished in the islets of Akita/βAtf4-KO mice., Conclusions: We conclude that transcriptional regulation by ATF4 maintains β-cell identity via ISR modulation. This mechanism provides a promising target for the treatment of diabetes., (Copyright © 2021 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

The eIF2α phosphorylation-dependent integrated stress response (ISR) is a signaling pathway that maintains homeostasis in mammalian cells exposed to various stresses. Here, ISR activation in adipocytes improves obesity and diabetes by regulating appetite in a non-cell-autonomous manner. Adipocyte-specific ISR activation using transgenic mice decreases body weight and improves glucose tolerance and obesity induced by a high-fat diet (HFD) via preferential inhibition of HFD intake. The transcriptome analysis of ISR-activated adipose tissue reveals that growth differentiation factor 15 (GDF15) expression is induced by the ISR through the direct regulation of the transcription factors ATF4 and DDIT3. Deficiency in the GDF15 receptor GFRAL abolishes the adipocyte ISR-dependent preferential inhibition of HFD intake and the anti-obesity effects. Pharmacologically, 10(E), 12(Z)-octadecadienoic acid induces ISR-dependent GDF15 expression in adipocytes and decreases the intake of the HFD. Based on our findings the specific activation of the ISR in adipocytes controls the non-cell-autonomous regulation of appetite., Competing Interests: No potential conflicts of interest relevant to this article are reported., (© 2021 The Authors.)

Background: The aim of this study was to clarify the effectiveness of a new three-dimensional (3D) culture system for hepatocyte-like cells (HLCs) generated from human adipose-derived mesenchymal stem cells (ADSCs)., Methods: Human ADSCs (2 × 10 4 ) with or without 0.1 mg/mL human recombinant peptide μ-piece per well were seeded in a 96-well U-bottom plate and then our three-step differentiation protocol was applied for 21 days. At each step, cell morphology and gene expression were investigated. Mature hepatocyte functions were evaluated after HLC differentiation. These parameters were compared between 2D- and 3D-cultured HLCs, and, DNA microarray analysis was also performed. Finally, HLCs were transplanted in to CCl 4 induced acute liver failure model mice., Results: Two-dimensional-cultured HLCs at day 21 did not have a spindle shape and had formed spheroids after day 6, which gradually increased in size for 3D-cultured HLCs. Definitive endoderm, hepatoblast, and hepatocyte genes showed significantly higher expression in the 3D culture group. Three-dimensional-cultured HLCs also had higher albumin expression, CYP3A4 activity, urea synthesis, and ammonium metabolism, and much higher expression of ion transporter, blood coagulation, and cell communication genes. HLC transplantation improved serum liver function, especially in T-Bil levels, and engrafted into immunodeficient mice with HLA class I positive staining., Conclusion: Our new 3D culture protocol is effective to improve hepatocyte functions. Our HLCs might be promising for clinical cell transplantation to treat metabolic disease., (© 2021 Japanese Society of Hepato-Biliary-Pancreatic Surgery.)

While ATF6α plays a central role in the endoplasmic reticulum (ER) stress response, the function of its paralogue ATF6β remains elusive, especially in the central nervous system (CNS). Here, we demonstrate that ATF6β is highly expressed in the hippocampus of the brain, and specifically regulates the expression of calreticulin (CRT), a molecular chaperone in the ER with a high Ca 2+ -binding capacity. CRT expression was reduced to ~ 50% in the CNS of Atf6b -/- mice under both normal and ER stress conditions. Analysis using cultured hippocampal neurons revealed that ATF6β deficiency reduced Ca 2+ stores in the ER and enhanced ER stress-induced death. The higher levels of death in Atf6b -/- neurons were recovered by ATF6β and CRT overexpressions, or by treatment with Ca 2+ -modulating reagents such as BAPTA-AM and 2-APB, and with an ER stress inhibitor salubrinal. In vivo, kainate-induced neuronal death was enhanced in the hippocampi of Atf6b -/- and Calr +/- mice, and restored by administration of 2-APB and salubrinal. These results suggest that the ATF6β-CRT axis promotes neuronal survival under ER stress and excitotoxity by improving intracellular Ca 2+ homeostasis.

Background: The clinical practice of platelet-rich plasma (PRP) therapy has grown significantly in recent years in multiple medical specialties. However, comparisons of PRP studies across medical fields remain challenging because of inconsistent reporting of protocols and characterization of the PRP being administered. The purpose of this systematic review was to determine the quantity of level I/II studies within each medical specialty and compare the level of study reporting across medical fields., Methods: The Cochrane Database, PubMed, and EMBASE databases were queried for level I/II clinical studies on PRP injections across all medical specialties. From these studies, data including condition treated, PRP processing and characterization, delivery, control group, and assessed outcomes were collected., Results: A total of 132 studies met the inclusion and exclusion criteria and involved 28 different conditions across 8 specialties (cardiothoracic surgery, cosmetic, dermatology, musculoskeletal (MSK), neurology, oral maxillofacial surgery, ophthalmology, and plastic surgery). Studies on PRP for MSK injuries made up the majority of the studies (74%), with knee osteoarthritis and tendinopathy being most commonly studied. Of the 132 studies, only 44 (33%) characterized the composition of PRP used, and only 23 (17%) reported the leukocyte component. MSK studies were more likely to use patient-reported outcome measures to assess outcomes, while studies from other specialties were more likely to use clinician- or imaging-based objective outcomes. Overall, 61% of the studies found PRP to be favorable over control treatment, with no difference in favorable reporting between MSK and other medical specialties., Conclusions: The majority of level I/II clinical studies investigating PRP therapy across all medical specialties have been conducted for MSK injuries with knee osteoarthritis and tendinopathy being the most commonly studied conditions. Inconsistent reporting of PRP composition exists among all studies in medicine. Rigorous reporting in human clinical studies across all medical specialties is crucial for evaluating the effects of PRP and moving towards disease-specific and individualized treatment., Competing Interests: The authors have declared that no competing interests exist.

Background: Although the toxic effects of bupivacaine on chondrocyte monolayer culture have been well described, its cellular and mechanical effects on native and engineered articular cartilage remain unclear. For the repair of articular cartilage defects, fresh autologous and allogenic cartilage grafts are commonly used, and engineered cell-based therapies are emerging. The outcome of grafting therapies aimed at repairing damaged cartilage relies largely on maintaining proper viability and mechanical suitability of the donor tissues., Purpose: To investigate the in vitro effects of single bupivacaine exposure on the viability and mechanics of 2 cartilage graft types: native articular cartilage and engineered neocartilage., Study Design: Controlled laboratory study., Methods: Articular cartilage explants were harvested from the bovine stifle femoral condyles, and neocartilage constructs were engineered from bovine stifle chondrocytes using the self-assembling process, a scaffold-free approach to engineer cartilage tissue. Both explants and neocartilage were exposed to chondrogenic medium containing a clinically applicable bolus of 0.5%, 0.25%, or 0% (control) bupivacaine for 1 hour, followed by fresh medium wash and exchange. Cell viability and matrix content (collagen and glycosaminoglycan) were assessed at t = 24 hours after treatment, and compressive mechanical properties were assessed with creep indentation testing at t = 5 to 6 days after treatment., Results: Single bupivacaine exposure was chondrotoxic in both explants and neocartilage, with 0.5% bupivacaine causing a significant decrease in chondrocyte viability compared with the control condition (55.0% ± 13.4% vs 71.9% ± 13.5%; P < .001). Bupivacaine had no significant effect on matrix content for either tissue type. There was significant weakening of the mechanical properties in the neocartilage when treated with 0.5% bupivacaine compared with control, with decreased aggregate modulus (415.8 ± 155.1 vs 660.3 ± 145.8 kPa; P = .003), decreased shear modulus (143.2 ± 14.0 vs 266.5 ± 89.2 kPa; P = .002), and increased permeability (14.7 ± 8.1 vs 6.6 ± 1.7 × 10 -15 m 4 /Ns; P = .009). Bupivacaine exposure did not have a significant effect on the mechanical properties of native cartilage explants., Conclusion: Single bupivacaine exposure resulted in significant chondrotoxicity in native explants and neocartilage and significant weakening of mechanical properties of neocartilage. The presence of abundant extracellular matrix does not appear to confer any additional resistance to the toxic effects of bupivacaine., Clinical Relevance: Clinicians should be judicious regarding the use of intra-articular bupivacaine in the setting of articular cartilage repair.

UTX/KDM6A encodes a major histone H3 lysine 27 (H3K27) demethylase, and is frequently mutated in various types of human cancers. Although UTX appears to play a crucial role in oncogenesis, the mechanisms involved are still largely unknown. Here we show that a specific pharmacological inhibitor of H3K27 demethylases, GSK-J4, induces the expression of transcription activating factor 4 (ATF4) protein as well as the ATF4 target genes (e.g. PCK2, CHOP, REDD1, CHAC1 and TRIB3). ATF4 induction by GSK-J4 was due to neither transcriptional nor post-translational regulation. In support of this view, the ATF4 induction was almost exclusively dependent on the heme-regulated eIF2α kinase (HRI) in mouse embryonic fibroblasts (MEFs). Gene expression profiles with UTX disruption by CRISPR-Cas9 editing and the following stable re-expression of UTX showed that UTX specifically suppresses the expression of the ATF4 target genes, suggesting that UTX inhibition is at least partially responsible for the ATF4 induction. Apoptosis induction by GSK-J4 was partially and cell-type specifically correlated with the activation of ATF4-CHOP. These findings highlight that the anti-cancer drug candidate GSK-J4 strongly induces ATF4 and its target genes via HRI activation and raise a possibility that UTX might modulate cancer formation by regulating the HRI-ATF4 axis.

CRISPR/Cas9-mediated gene editing often generates founder generation (F0) mice that exhibit somatic mosaicism in the targeted gene(s). It has been known that Fibroblast growth factor 10 (Fgf10)-null mice exhibit limbless and lungless phenotypes, while intermediate limb phenotypes (variable defective limbs) are observed in the Fgf10-CRISPR F0 mice. However, how the lung phenotype in the Fgf10-mosaic mutants is related to the limb phenotype and genotype has not been investigated. In this study, we examined variable lung phenotypes in the Fgf10-targeted F0 mice to determine if the lung phenotype was correlated with percentage of functional Fgf10 genotypes. Firstly, according to a previous report, Fgf10-CRISPR F0 embryos on embryonic day 16.5 (E16.5) were classified into three types: type I, no limb; type II, limb defect; and type III, normal limbs. Cartilage and bone staining showed that limb truncations were observed in the girdle, (type I), stylopodial, or zeugopodial region (type II). Deep sequencing of the Fgf10-mutant genomes revealed that the mean proportion of codons that encode putative functional FGF10 was 8.3 ± 6.2% in type I, 25.3 ± 2.7% in type II, and 54.3 ± 9.5% in type III (mean ± standard error of the mean) mutants at E16.5. Histological studies showed that almost all lung lobes were absent in type I embryos. The accessory lung lobe was often absent in type II embryos with other lobes dysplastic. All lung lobes formed in type III embryos. The number of terminal tubules was significantly lower in type I and II embryos, but unchanged in type III embryos. To identify alveolar type 2 epithelial (AECII) cells, known to be reduced in the Fgf10-heterozygous mutant, immunostaining using anti-surfactant protein C (SPC) antibody was performed: In the E18.5 lungs, the number of AECII was correlated to the percentage of functional Fgf10 genotypes. These data suggest the Fgf10 gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung. Since dysfunction of AECII cells has been implicated in the pathogenesis of parenchymal lung diseases, the Fgf10-CRISPR F0 mouse would present an ideal experimental system to explore it., Competing Interests: Supported by an academic grant from Pfizer Japan, Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Dickkopf 3 (Dkk3) is a secreted protein belonging to the Dkk family and encoded by the orthologous gene of REIC. Dkk3/REIC is expressed by mouse and human adrenal glands, but the understanding of its roles in this organ is still limited. To determine the functions of Dkk3 in the mouse adrenal gland, we first identified that the mouse Dkk3 protein is N-glycosylated in the adrenal gland as well as in the brain. We performed proteome analysis on adrenal glands from Dkk3-null mice, in which exons 5 and 6 of the Dkk3 gene are deleted. Twodimensional polyacrylamide gel electrophoresis of adrenal proteins from wild-type and Dkk3-null mice revealed 5 protein spots whose intensities were altered between the 2 genotypes. Mass spectrometry analysis of these spots identified binding immunoglobulin protein (BiP), an endoplasmic reticulum (ER) chaperone. To determine whether mouse Dkk3 is involved in the unfolded protein response (UPR), we carried out a reporter assay using ER-stress responsive elements. Forced expression of Dkk3 resulted in the induction of distinct levels of reporter expression, showing the UPR initiated by the ER membrane proteins of activating transcription factor 6 (ATF6) and inositol-requring enzyme 1 (IRE1). Thus, it is possible that Dkk3 is a physiological ER stressor in the mouse adrenal gland., Competing Interests: No potential conflict of interest relevant to this article was reported.

MSX1 is a causative gene for oligodontia in humans. Although conventional Msx1-deficient mice die neonatally, a mutant mouse lacking the C-terminus MH6 domain of MSX1 (Msx1 ΔMH6/ΔMH6 ) showed two different phenotypes; newborn homozygotes with cleft palates died neonatally, whereas those with thin palates remained alive and had craniofacial dysplasia and growth retardation compared with wild-type mice, with most mice dying by the age of 4-5 weeks. In a previously reported case of human oligodontia caused by a heterozygous defect of the Msx1 MH6 domain, a small foramen was observed on the occipital bone. The aim of this study was to test the hypothesis that the Msx1 MH6 domain is involved in bone formation in vivo. In Msx1 ΔMH6/ΔMH6 mice, cranial suture fusion was delayed at embryonic day 18.5, and the anteroposterior cranial diameter was smaller and long bone length was decreased at 3 weeks of age. The femoral epiphysis showed no change in the trabecular number, but decreased bone mass, bone density, and trabecular width in Msx1 ΔMH6/ΔMH6 mice. In addition, cancellous bone mass was reduced and the cartilage layer in the growth plate was thinner in Msx1 ΔMH6/ΔMH6 mice. The mRNA expression levels of major osteoblast and chondrocyte differentiation marker genes were decreased in Msx1 ΔMH6/ΔMH6 mice compared with wild-type mice. These findings suggest that the C-terminal region including the MH6 domain of MSX1 plays important roles not only in tooth development and palatal fusion, but also in postnatal bone formation., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interest., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Patients with adult T-cell leukemia (ATL) exhibit a poor prognosis and overall survival rate when treated with standard chemotherapy, highlighting the continued requirement for the development of novel safe and effective therapies for human T-cell leukemia virus type 1 (HTLV-1)-related diseases. In this study, we demonstrated that MK-2048, a second-generation HIV-1 integrase (IN) inhibitor, potently and selectively kills HTLV-1-infected cells. Differential transcriptome profiling revealed significantly elevated levels of gene expression of the unfolded protein response (UPR) PKR-like ER kinase (PERK) signaling pathway in ATL cell lines following MK-2048 treatment. We also identified a significant downregulation in glucose regulated protein 78 (GRP78), a master regulator of the UPR in the CD4+CADM1+ HTLV-1-infected cell population of primary HTLV-1 carrier peripheral blood mononuclear cells (PBMCs) (n = 9), suggesting that HTLV-1-infected cells are hypersensitive to endoplasmic reticulum (ER) stress-mediated apoptosis. MK-2048 efficiently reduced proviral loads in primary HTLV-1 carrier PBMCs (n = 4), but had no effect on the total numbers of these cells, indicating that MK-2048 does not affect the proliferation of HTLV-1-uninfected PBMCs. MK-2048 specifically activated the ER stress-related proapoptotic gene, DNA damage-inducible transcript 3 protein (DDIT3), also known as C/EBP homologous protein (CHOP), in HTLV-1-infected but not uninfected cells of HTLV-1-carrier PBMCs. Our findings demonstrated that MK-2048 selectively induces HTLV-1-infected cell apoptosis via the activation of the UPR. This novel regulatory mechanism of the HIV IN inhibitor MK-2048 in HTLV-1-infected cells provides a promising prophylactic and therapeutic target for HTLV-1-related diseases including ATL., (© 2020 by The American Society of Hematology.)

The integrated stress response (ISR) is one of the most important cytoprotective mechanisms and is integrated by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α). Four eIF2α kinases, heme-regulated inhibitor (HRI), double-stranded RNA-dependent protein kinase (PKR), PKR-like endoplasmic reticulum kinase (PERK), and general control nonderepressible 2 (GCN2), are activated in response to several stress conditions. We previously reported that nanosecond pulsed electric fields (nsPEFs) are a potential therapeutic tool for ISR activation. In this study, we examined which eIF2α kinase is activated by nsPEF treatment. To assess the responsible eIF2α kinase, we used previously established eIF2α kinase quadruple knockout (4KO) and single eIF2α kinase-rescued 4KO mouse embryonic fibroblast (MEF) cells. nsPEFs 70 ns in duration with 30 kV/cm electric fields caused eIF2α phosphorylation in wild-type (WT) MEF cells. On the other hand, nsPEF-induced eIF2α phosphorylation was completely abolished in 4KO MEF cells and was recovered by HRI overexpression. CM-H2DCFDA staining showed that nsPEFs generated reactive oxygen species (ROS), which activated HRI. nsPEF-induced eIF2α phosphorylation was blocked by treatment with the ROS scavenger N-acetyl-L-cysteine (NAC). Our results indicate that the eIF2α kinase HRI is responsible for nsPEF-induced ISR activation and is activated by nsPEF-generated ROS., Competing Interests: The authors have declared that no competing interests exist.

Mitochondria play a central role in the function of brown adipocytes (BAs). Although mitochondrial biogenesis, which is indispensable for thermogenesis, is regulated by coordination between nuclear DNA transcription and mitochondrial DNA transcription, the molecular mechanisms of mitochondrial development during BA differentiation are largely unknown. Here, we show the importance of the ER-resident sensor PKR-like ER kinase (PERK) in the mitochondrial thermogenesis of brown adipose tissue. During BA differentiation, PERK is physiologically phosphorylated independently of the ER stress. This PERK phosphorylation induces transcriptional activation by GA-binding protein transcription factor α subunit (GABPα), which is required for mitochondrial inner membrane protein biogenesis, and this novel role of PERK is involved in maintaining the body temperatures of mice during cold exposure. Our findings demonstrate that mitochondrial development regulated by the PERK-GABPα axis is indispensable for thermogenesis in brown adipose tissue., (© 2020 Kato et al.)

Atovaquone, a US Food and Drug Administration-approved antiparasitic drug previously shown to reduce interleukin-6/STAT3 signaling in myeloma cells, is well tolerated, and plasma concentrations of 40 to 80 µM have been achieved with pediatric and adult dosing. We conducted preclinical testing of atovaquone with acute myeloid leukemia (AML) cell lines and pediatric patient samples. Atovaquone induced apoptosis with an EC50 <30 µM for most AML lines and primary pediatric AML specimens. In NSG mice xenografted with luciferase-expressing THP-1 cells and in those receiving a patient-derived xenograft, atovaquone-treated mice demonstrated decreased disease burden and prolonged survival. To gain a better understanding of the mechanism of atovaquone, we performed an integrated analysis of gene expression changes occurring in cancer cell lines after atovaquone exposure. Atovaquone promoted phosphorylation of eIF2α, a key component of the integrated stress response and master regulator of protein translation. Increased levels of phosphorylated eIF2α led to greater abundance of the transcription factor ATF4 and its target genes, including proapoptotic CHOP and CHAC1. Furthermore, atovaquone upregulated REDD1, an ATF4 target gene and negative regulator of the mechanistic target of rapamycin (mTOR), and caused REDD1-mediated inhibition of mTOR activity with similar efficacy as rapamycin. Additionally, atovaquone suppressed the oxygen consumption rate of AML cells, which has specific implications for chemotherapy-resistant AML blasts that rely on oxidative phosphorylation for survival. Our results provide insight into the complex biological effects of atovaquone, highlighting its potential as an anticancer therapy with novel and diverse mechanisms of action, and support further clinical evaluation of atovaquone for pediatric and adult AML., (© 2019 by The American Society of Hematology.)

The endoplasmic reticulum (ER) is responsible for folding secretory and membrane proteins, but disturbed ER proteostasis may lead to protein aggregation and subsequent cellular and clinical pathologies. Chemical chaperones have recently emerged as a potential therapeutic approach for ER stress-related diseases. Here, we identified 2-phenylimidazo[2,1- b ]benzothiazole derivatives (IBTs) as chemical chaperones in a cell-based high-throughput screen. Biochemical and chemical biology approaches revealed that IBT21 directly binds to unfolded or misfolded proteins and inhibits protein aggregation. Finally, IBT21 prevented cell death caused by chemically induced ER stress and by a proteotoxin, an aggression-prone prion protein. Taken together, our data show the promise of IBTs as potent chemical chaperones that can ameliorate diseases resulting from protein aggregation under ER stress., Competing Interests: KK, ST, EK, YH, MM, MO, HK, HK, TK, SY, TH, SO No competing interests declared, (© 2019, Kitakaze et al.)

: Carnosic acid (CA) is a phytochemical found in some dietary herbs, such as Rosmarinus officinalis L., and possesses antioxidative and anti-microbial properties. We previously demonstrated that CA functions as an activator of nuclear factor, erythroid 2 (NF-E2)-related factor 2 (Nrf2), an oxidative stress-responsive transcription factor in human and rodent cells. CA enhances the expression of nerve growth factor (NGF) and antioxidant genes, such as HO-1 in an Nrf2-dependent manner in U373MG human astrocytoma cells. However, CA also induces NGF gene expression in an Nrf2-independent manner, since 50 μM of CA administration showed striking NGF gene induction compared with the classical Nrf2 inducer tert -butylhydroquinone (tBHQ) in U373MG cells. By comparative transcriptome analysis, we found that CA activates activating transcription factor 4 (ATF4) in addition to Nrf2 at high doses. CA activated ATF4 in phospho-eIF2α- and heme-regulated inhibitor kinase (HRI)-dependent manners, indicating that CA activates ATF4 through the integrated stress response (ISR) pathway. Furthermore, CA activated Nrf2 and ATF4 cooperatively enhanced the expression of NGF and many antioxidant genes while acting independently to certain client genes. Taken together, these results represent a novel mechanism of CA-mediated gene regulation evoked by Nrf2 and ATF4 cooperation., Competing Interests: The authors declare no conflict of interest. Nagase & Co. Ltd. did not provide funding for this study.