The Ratio of Plasma Amyloid-ß 1-42 over Serum Albumin Can Be a Novel Biomarker Signature for Diagnosing End-Stage Renal Disease-Associated Cognitive Impairment.

BACKGROUND: Cognitive impairment (CI) is one of the major complications in chronic kidney disease patients, especially those with end-stage renal disease (ESRD). Limited biomarkers have been found that can significantly predict ESRD-associated cognitive decline. OBJECTIVE: This cohort study aimed to investigate de novo biomarkers for diagnosis of the ESRD-associated CI. METHODS: In this cohort study, qualified samples were divided into control (with an estimated glomerular filtration rate (eGFR) of≥60 mL/min and a Mini-Mental State Examination (MMSE) score of > 27), ESRD without CI (eGFR < 15 and MMSE > 27), and ESRD with CI (eGFR < 15 and MMSE < 27) groups. Levels of plasma amyloid-ß (Aß)1 - 42, serum indoxyl sulfate, and hematologic and biochemical parameters were measured. RESULTS: Compared to the control group, levels of blood urea nitrogen, creatinine, and indoxyl sulfate were elevated in ESRD patients both without and with CI. Interestingly, ESRD patients with CI had the lowest levels of serum albumin. In contrast, levels of plasma Aß1 - 42 were significantly higher in the ESRD with CI group than in the control and ESRD without CI groups. In addition, the ratio of plasma Aß1 - 42 over serum albumin was significantly higher in the ESRD with CI group than in the control or ESRD without CI groups. Importantly, the area under the receiver operating characteristic curve (AUROC) for CI in the total population by the ratio of Aß1 - 42 over albumin was 0.785 and significant (p < 0.05). CONCLUSIONS: This cohort study has shown that the ratio of plasma Aß1 - 42 over serum albumin can be a de novo biomarker for the diagnosis and prognosis of ESRD-associated cognitive decline.

Resveratrol triggers the ER stress-mediated intrinsic apoptosis of neuroblastoma cells coupled with suppression of Rho-dependent migration and consequently prolongs mouse survival.

Neuroblastoma, the most common childhood tumor, are highly malignant and fatal because neuroblastoma cells extremely defend against apoptotic targeting. Traditional treatments for neuroblastomas are usually ineffective and lead to serious side effects and poor prognoses. In this study, we investigated the molecular mechanisms of resveratrol-induced insults to neuroblastoma cells and survival extension of nude mice with neuroblastomas, especially in the endoplasmic reticular (ER) stress-intracellular reactive oxygen species (iROS) axis-mediated signals. Resveratrol specifically killed neuroblastoma cells mainly via apoptosis and autophagy rather than necrosis. As to the mechanisms, resveratrol time-dependently triggered productions of Grp78 protein and iROS in neuroblastoma cells. Attenuating the ER stress-iROS signaling axis significantly suppressed resveratrol-induced autophagy, DNA damage, and cell apoptosis. Successively, resveratrol decreased phosphorylation of retinoblastoma protein and induced cell cycle arrest at the S phase, translocation of Bak protein to mitochondria, a reduction in the mitochondrial membrane potential, cascade activation of caspases-9, -3, and -6, and DNA fragmentation. Moreover, weakening the ER stress-iROS axis concomitantly overcome resveratrol-induced decreases in translocation of Rho protein to membranes and succeeding cell migration. Interestingly, administration of resveratrol did not cause significant side effects but could protect the neuroblastoma-bearing nude mice from body weight loss and consequently extended the animal survival. In parallel, resveratrol elevated levels of Grp78 and then induced cell apoptosis in neuroblastoma tissues. This study has shown that resveratrol could kill neuroblastoma cells and extend survival of animals with neuroblastomas by triggering the ER stress-iROS-involved intrinsic apoptosis and suppression of Rho-dependent cell migration. Our results imply the potential of resveratrol as a drug candidate for chemotherapy of neuroblastoma patients.

A Non-Invasive Method for Monitoring Osteogenesis and Osseointegration Using Near-Infrared Fluorescent Imaging: A Model of Maxilla Implantation in Rats.

Currently, computed tomography and conventional X-ray radiography usually generate a micro-artifact around metal implants. This metal artifact frequently causes false positive or negative diagnoses of bone maturation or pathological peri-implantitis around implants. In an attempt to repair the artifacts, a highly specific nanoprobe, an osteogenic biomarker, and nano-Au-Pamidronate were designed to monitor the osteogenesis. In total, 12 Sprague Dawley rats were included in the study and could be chategorized in 3 groups: 4 rats in the X-ray and CT group, 4 rats in the NIRF group, and 4 rats in the sham group. A titanium alloy screw was implanted in the anterior hard palate. The X-ray, CT, and NIRF images were taken 28 days after implantation. The X-ray showed that the tissue surrounded the implant tightly; however, a gap of metal artifacts was noted around the interface between dental implants and palatal bone. Compared to the CT image, a fluorescence image was noted around the implant site in the NIRF group. Furthermore, the histological implant-bone tissue also exhibited a significant NIRF signal. In conclusion, this novel NIRF molecular imaging system precisely identifies the image loss caused by metal artifacts and can be applied to monitoring bone maturation around orthopedic implants. In addition, by observing the new bone formation, a new principle and timetable for an implant osseointegrated with bone can be established and a new type of implant fixture or surface treatment can be evaluated using this system.

Enzalutamide Induces Apoptotic Insults to Human Drug-Resistant and -Sensitive Glioblastoma Cells via an Intrinsic Bax-Mitochondrion-Cytochrome C Caspase Cascade Activation Pathway.

Glioblastoma multiforme (GBM) is the most common and malignant brain tumor. Temozolomide (TMZ) is the first-line chemotherapeutic drug for treating GBM. However, drug resistance is still a challenging issue in GBM therapy. Our preliminary results showed upregulation of androgen receptor (AR) gene expression in human GBM tissues. This study was designed to evaluate the effects of enzalutamide, a specific inhibitor of the AR, on killing drug-resistant and -sensitive glioblastoma cells and the possible mechanisms. Data mining from The Cancer Genome Atlas (TCGA) database revealed upregulation of AR messenger (m)RNA and protein expressions in human GBM tissues, especially in male patients, compared to normal human brains. In addition, expressions of AR mRNA and protein in human TMZ-sensitive U87 MG and -resistant U87 MG-R glioblastoma cells were elevated compared to normal human astrocytes. Exposure of human U87 MG and U87 MG-R cells to enzalutamide concentration- and time-dependently decreased cell viability. As to the mechanism, enzalutamide killed these two types of glioblastoma cells via an apoptotic mechanism. Specifically, exposure to enzalutamide augmented enzyme activities of caspase-9 rather than those of caspase-8. Moreover, enzalutamide successively triggered an elevation in levels of the proapoptotic Bax protein, a reduction in the mitochondrial membrane potential, release of cytochrome c, cascade activation of caspases-3 and -6, DNA fragmentation, and cell apoptosis in human TMZ-sensitive and -resistant glioblastoma cells. Pretreatment with Z-VEID-FMK, an inhibitor of caspase-6, caused significant attenuations in enzalutamide-induced morphological shrinkage, DNA damage, and apoptotic death. Taken together, this study showed that enzalutamide could significantly induce apoptotic insults to human drug-resistant and -sensitive glioblastoma cells via an intrinsic Bax-mitochondrion-cytochrome c-caspase cascade activation pathway. Enzalutamide has the potential to be a drug candidate for treating GBM by targeting the AR signaling axis.

Contribution of the Testosterone Androgen Receptor-PARD3B Signaling Axis to Tumorigenesis and Malignance of Glioblastoma Multiforme through Stimulating Cell Proliferation and Colony Formation.

BACKGROUND: Glioblastoma multiforme (GBM) is the most common and malignant brain tumor with very poor prognoses. After surgical resection of the primary tumor, rapid proliferation of residual glioblastoma cells is a critical cause explaining tumor malignance and recurrence. In this study, we evaluated de novo roles of the testosterone androgen receptor (AR)-PARD3B signaling axis in the tumorigenesis and malignance of human GBM and the possible related mechanisms. METHODS: AR and PARD3B gene expressions and their correlations were mined from The Cancer Genome Atlas (TCGA) database and analyzed using the UALCAN system. Analyses using a real-time PCR, cell proliferation, and colony formation and a loss-of-function strategy by suppressing AR activity with its specific inhibitor, enzalutamide, were then carried out to determine roles of the testosterone AR-PARD3B signaling axis in tumor malignance. RESULTS: Expressions of AR, PARD3B mRNA, and proteins in human GBM tissues were upregulated compared to normal human brain tissues. In contrast, levels of AR and PARD3B mRNA in most TCGA pan-cancer types were downregulated compared to their respective normal tissues. Interestingly, a highly positive correlation between AR and PARD3B gene expressions in human GBM was identified. The results of a bioinformatics search further showed that there were five AR-specific DNA-binding elements predicted in the 5' promoter of the PARD3B gene. Regarding the mechanisms, exposure of human glioblastoma cells to testosterone induced AR and PARD3B gene expressions and successively stimulated cell proliferation and colony formation. Suppressing AR activity concurrently resulted in significant attenuations of testosterone-induced PARD3B gene expression, cell proliferation, and colony formation in human glioblastoma cells. CONCLUSIONS: This study showed the contribution of the testosterone AR-PARD3B signaling axis to the tumorigenesis and malignance of human GBM through stimulating cell proliferation and colony formation. Therefore, the AR-PARD3B signaling axis could be targeted for potential therapy for human GBM.

Exosomal mediated signal transduction through artificial microRNA (amiRNA): A potential target for inhibition of SARS-CoV-2.

Exosome trans-membrane signals provide cellular communication between the cells through transport and/or receiving the signal by molecule, change the functional metabolism, and stimulate and/or inhibit receptor signal complexes. COVID19 genetic transformations are varied in different geographic positions, and single nucleotide polymorphic lineages were reported in the second waves due to the fast mutational rate and adaptation. Several vaccines were developed and in treatment practice, but effective control has yet to reach in cent presence. It was initially a narrow immune-modulating protein target. Controlling these diverse viral strains may inhibit their transuding mechanisms primarily to target RNA genes responsible for COVID19 transcription. Exosomal miRNAs are the main sources of transmembrane signals, and trans-located miRNAs can directly target COVID19 mRNA transcription. This review discussed targeted viral transcription by delivering the artificial miRNA (amiRNA) mediated exosomes in the infected cells and significant resources of exosome and their efficacy.

Dissecting the Transcriptomes of Multiple Metronidazole-Resistant and Sensitive Trichomonas vaginalis Strains Identified Distinct Genes and Pathways Associated with Drug Resistance and Cell Death.

Trichomonas vaginalis is the causative agent of trichomoniasis, the most prevalent non-viral sexually transmitted infection worldwide. Metronidazole (MTZ) is the mainstay of anti-trichomonal chemotherapy; however, drug resistance has become an increasingly worrying issue. Additionally, the molecular events of MTZ-induced cell death in T. vaginalis remain elusive. To gain insight into the differential expression of genes related to MTZ resistance and cell death, we conducted RNA-sequencing of three paired MTZ-resistant (MTZ-R) and MTZ-sensitive (MTZ-S) T. vaginalis strains treated with or without MTZ. Comparative transcriptomes analysis identified that several putative drug-resistant genes were exclusively upregulated in different MTZ-R strains, such as ATP-binding cassette (ABC) transporters and multidrug resistance pumps. Additionally, several shared upregulated genes among all the MTZ-R transcriptomes were not previously identified in T. vaginalis, such as 5'-nucleotidase surE and Na+-driven multidrug efflux pump, which are a potential stress response protein and a multidrug and toxic compound extrusion (MATE)-like protein, respectively. Functional enrichment analysis revealed that purine and pyrimidine metabolisms were suppressed in MTZ-S parasites upon drug treatment, whereas the endoplasmic reticulum-associated degradation (ERAD) pathway, proteasome, and ubiquitin-mediated proteolysis were strikingly activated, highlighting the novel pathways responsible for drug-induced stress. Our work presents the most detailed analysis of the transcriptional changes and the regulatory networks associated with MTZ resistance and MTZ-induced signaling, providing insights into MTZ resistance and cell death mechanisms in trichomonads.

Naringin Improves Osteoblast Mineralization and Bone Healing and Strength through Regulating Estrogen Receptor Alpha-Dependent Alkaline Phosphatase Gene Expression.

Phytoestrogens are strongly recommended for treating osteoporosis. Our previous study showed that naringin, a citrus flavonoid, can enhance the bone mass in ovariectomized rats. In this study, we further elucidated the mechanisms of naringin-induced osteoblast maturation and bone healing. Treatment of human osteoblasts with naringin increased cell viability and proliferation. In parallel, exposure to naringin enhanced translocation of estrogen receptor alpha (ERα) to nuclei and its transactivation activity. Sequentially, naringin induced alkaline phosphatase (ALP) mRNA and protein expression and its enzyme activity. Pretreatment with methylpiperidinopyrazole (MPP), a specific inhibitor of ERα, attenuated naringin-induced augmentations in ERα transactivation activity, ALP gene expression, and cell mineralization. The beneficial effects of naringin were also confirmed in mouse MC3T3-E1 cells. Moreover, administration of mice with a bone defect with naringin increased levels of ERα and ALP in damaged sites and simultaneously enhanced the healing rate and bone strength. Nevertheless, treatment with MPP weakened naringin-triggered expression of ERα and ALP and improved bone healing and mass. Therefore, naringin could improve osteoblast mineralization and bone healing via regulating ERα-dependent ALP gene expression. Naringin can be clinically applied for treatment of osteoporosis-related bone diseases.

Histone deacetylase 6 acts upstream of DNA damage response activation to support the survival of glioblastoma cells.

DNA repair promotes the progression and recurrence of glioblastoma (GBM). However, there remain no effective therapies for targeting the DNA damage response and repair (DDR) pathway in the clinical setting. Thus, we aimed to conduct a comprehensive analysis of DDR genes in GBM specimens to understand the molecular mechanisms underlying treatment resistance. Herein, transcriptomic analysis of 177 well-defined DDR genes was performed with normal and GBM specimens (n = 137) from The Cancer Genome Atlas and further integrated with the expression profiling of histone deacetylase 6 (HDAC6) inhibition in temozolomide (TMZ)-resistant GBM cells and patient-derived tumor cells. The effects of HDAC6 inhibition on DDR signaling were examined both in vitro and intracranial mouse models. We found that the expression of DDR genes, involved in repair pathways for DNA double-strand breaks, was upregulated in highly malignant primary and recurrent brain tumors, and their expression was related to abnormal clinical features. However, a potent HDAC6 inhibitor, MPT0B291, attenuated the expression of these genes, including RAD51 and CHEK1, and was more effective in blocking homologous recombination repair in GBM cells. Interestingly, it resulted in lower cytotoxicity in primary glial cells than other HDAC6 inhibitors. MPT0B291 reduced the growth of both TMZ-sensitive and TMZ-resistant tumor cells and prolonged survival in mouse models of GBM. We verified that HDAC6 regulated DDR genes by affecting Sp1 expression, which abolished MPT0B291-induced DNA damage. Our findings uncover a regulatory network among HDAC6, Sp1, and DDR genes for drug resistance and survival of GBM cells. Furthermore, MPT0B291 may serve as a potential lead compound for GBM therapy.

The Role of Plasma Neurofilament Light Protein for Assessing Cognitive Impairment in Patients With End-Stage Renal Disease.

Introduction: End-stage renal disease (ESRD) is defined as the irreversible loss of renal function, necessitating renal replacement therapy. Patients with ESRD tend to have more risk factors for cognitive impairment than the general population, including hypertension, accumulative uremic toxin, anemia, and old age. The association between these risk factors and the pathologic protein was lacking. Blood-based assays for detecting pathologic protein, such as amyloid beta (Aß), total tau protein, and neurofilament light chain (NfL), have the advantages of being less invasive and more cost-effective for diagnosing patients with cognitive impairment. The aim of the study is to validate if the common neurologic biomarkers were different in ESRD patients and to differentiate if the specific biomarkers could correlate with specific correctable risk factors. Methods: In total, 67 participants aged >45 years were enrolled. The definition of ESRD was receiving maintenance hemodialysis for >3 months. Cognitive impairment was defined as a Mini-Mental State Examination score of <24. The participants were divided into groups for ESRD with and without cognitive impairment. The blood-based biomarkers (tau protein, Aß1/40, Aß1/42, and NfL) were analyzed through immunomagnetic reduction assay. Other biochemical and hematologic data were obtained simultaneously. Summary of results: The study enrolled 43 patients with ESRD who did not have cognitive impairment and 24 patients with ESRD who had cognitive impairment [Mini-Mental State Examination (MMSE): 27.60 ± 1.80 vs. 16.84 ± 6.40, p < 0.05]. Among the blood-based biomarkers, NfL was marginally higher in the ESRD with cognitive impairment group than in the ESRD without cognitive impairment group (10.41 ± 3.26 vs. 8.74 ± 2.81 pg/mL, p = 0.037). The concentrations of tau protein, amyloid ß 1/42, and amyloid ß 1/40 (p = 0.504, 0.393, and 0.952, respectively) were similar between the two groups. The area under the curve of NfL to distinguish cognitively impaired and unimpaired ESRD patients was 0.687 (95% confidence interval: 0.548-0.825, p = 0.034). There was no correlation between the concentration of NfL and MMSE among total population (r = -0.153, p = 0.277), patients with (r = 0.137, p = 0.583) or without cognitive impairment (r = 0.155, p = 0.333). Conclusion: Patients with ESRD who had cognitive impairment had marginally higher plasma NfL concentrations. NfL concentration was not correlated with the biochemical parameters, total MMSE among total population or individual groups with or without cognitive impairment. The concentrations of Aß1/40, Aß1/42, and tau were similar between the groups.

Hypoxia Induced by Cobalt Chloride Triggers Autophagic Apoptosis of Human and Mouse Drug-Resistant Glioblastoma Cells through Targeting the PI3K-AKT-mTOR Signaling Pathway.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor. Drug resistance mainly drives GBM patients to poor prognoses because drug-resistant glioblastoma cells highly defend against apoptotic insults. This study was designed to evaluate the effects of cobalt chloride (CoCl2) on hypoxic stress, autophagy, and resulting apoptosis of human and mouse drug-resistant glioblastoma cells. Treatment of drug-resistant glioblastoma cells with CoCl2 increased levels of hypoxia-inducible factor- (HIF-) 1α and triggered hypoxic stress. In parallel, the CoCl2-induced hypoxia decreased mitochondrial ATP synthesis, cell proliferation, and survival in chemoresistant glioblastoma cells. Interestingly, CoCl2 elevated the ratio of light chain (LC)3-II over LC3-I in TMZ-resistant glioblastoma cells and subsequently induced cell autophagy. Analyses by loss- and gain-of-function strategies further confirmed the effects of the CoCl2-induced hypoxia on autophagy of drug-resistant glioblastoma cells. Furthermore, knocking down HIF-1α concurrently lessened CoCl2-induced cell autophagy. As to the mechanisms, the CoCl2-induced hypoxia decreased levels of phosphoinositide 3-kinase (PI3K) and successive phosphorylations of AKT and mammalian target of rapamycin (mTOR) in TMZ-resistant glioblastoma cells. Interestingly, long-term exposure of human chemoresistant glioblastoma cells to CoCl2 sequentially triggered activation of caspases-3 and -6, DNA fragmentation, and cell apoptosis. However, pretreatment with 3-methyladenine, an inhibitor of autophagy, significantly attenuated the CoCl2-induced autophagy and subsequent apoptotic insults. Taken together, this study showed that long-term treatment with CoCl2 can induce hypoxia and subsequent autophagic apoptosis of drug-resistant glioblastoma cells via targeting the PI3K-AKT-mTOR pathway. Thus, combined with traditional prescriptions, CoCl2-induced autophagic apoptosis can be clinically applied as a de novo strategy for therapy of drug-resistant GBM patients.

Genistein Triggers Translocation of Estrogen Receptor-Alpha in Mitochondria to Induce Expressions of ATP Synthesis-Associated Genes and Improves Energy Production and Osteoblast Maturation.

Our previous study showed that estrogen can induce mitochondrial adenosine triphosphate (ATP) synthesis-associated gene expressions and osteoblast maturation. Genistein, a phytoestrogenic isoflavone that is widely found in various foods and traditional herb products, is beneficial for osteogenesis by selectively triggering estrogen receptor alpha (ER[Formula: see text] expression. In this study, we further investigated the mechanisms of genistein-induced energy production and osteoblast activation. Exposure of rat calvarial osteoblasts and human U-2 OS cells to genistein triggered osteoblast activation without affecting cell survival. Treatment with genistein time-dependently induced ER[Formula: see text] mRNA and protein expressions in rat calvarial osteoblasts. Analyses by confocal microscopy and immunoblotting showed that genistein stimulated translocation of ER[Formula: see text] from the cytoplasm to mitochondria. Subsequently, expressions of mitochondrial cytochrome c oxidase (COX) I and II mRNAs and proteins in primary rat osteoblasts were induced after exposure to genistein. Knocking-down ER[Formula: see text] concurrently inhibited genistein-induced COX I and II mRNA expressions. In addition, mitochondrial complex enzyme activities, the mitochondrial membrane potential, and cellular ATP levels in rat calvarial osteoblasts were time-dependently augmented by genistein. Suppressing ER[Formula: see text] expression instantaneously lowered genistein-induced enhancements of mitochondrial energy production and osteoblast activation. Effects of genistein on ER[Formula: see text] translocation, COX I and II mRNA expressions, ATP synthesis, and osteoblast activation were further confirmed in human U-2 OS cells. This study showed that genistein can stimulate energy production and consequent osteoblast activation via inducing ER[Formula: see text]-mediated mitochondrial ATP synthesis-linked gene expressions.

Histone deacetylase inhibitor MPT0B291 suppresses Glioma Growth in vitro and in vivo partially through acetylation of p53.

Background: Histone deacetylase (HDAC) inhibitors have emerged as a new class of anti-tumor agents for various types of tumors, including glioblastoma. Methods and results: We found that a novel HDAC inhibitor, MPT0B291, significantly reduced the cell viability and increased cell death of human and rat glioma cell lines, but not in normal astrocytes. We also demonstrated that MPT0B291 suppressed proliferation by inducing G1 phase cell cycle arrest and increased apoptosis in human and rat glioma cell lines by flow cytometry and immunocytochemistry. We further investigated the anti-tumor effects of MPT0B291 in xenograft (mouse) and allograft (rat) models. The IVIS200 images and histological analysis indicated MPT0B291 (25 mg/kg, p. o.) reduced tumor volume. Mechanistically, MPT0B291 increased phosphorylation and acetylation/activation of p53 and increased mRNA levels of the apoptosis related genes PUMA, Bax, and Apaf1 as well as increased protein level of PUMA, Apaf1 in C6 cell line. The expression of cell cycle related gene p21 was also increased and Cdk2, Cdk4 were decreased by MPT0B291. Conclusion: Our study highlights the anti-tumor efficacy of a novel compound MPT0B291 on glioma growth.

Genistein Improves Bone Healing via Triggering Estrogen Receptor Alpha-Mediated Expressions of Osteogenesis-Associated Genes and Consequent Maturation of Osteoblasts.

Osteoporosis-associated fractures may cause higher morbidity and mortality. Our previous study showed the effects of genistein, a phytoestrogen, on the induction of estrogen receptor alpha (ERα) gene expression and stimulation of osteoblast mineralization. In this study, rat calvarial osteoblasts and an animal bone defect model were used to investigate the effects of genistein on bone healing. Treatment with genistein caused a time-dependent increase in alkaline phosphatase (ALP) activity in rat osteoblasts. Levels of cytosolic and nuclear ERα significantly augmented following exposure to genistein. Subsequently, genistein elevated levels of ALP mRNA and protein in rat osteoblasts. Moreover, genistein induced other osteogenesis-associated osteocalcin and Runx2 mRNA and protein expressions. Knocking-down ERα using RNA interference concurrently inhibited genistein-induced Runx2, osteocalcin, and ALP mRNA expression. Attractively, administration of ICR mice suffering bone defects with genistein caused significant increases in the callus width, chondrocyte proliferation, and ALP synthesis. Results of microcomputed tomography revealed that administration of genistein increased trabecular bone numbers and improved the bone thickness and volume. This study showed that genistein can improve bone healing via triggering ERα-mediated osteogenesis-associated gene expressions and subsequent osteoblast maturation.

Inhibition of the estrogen receptor alpha signaling delays bone regeneration and alters osteoblast maturation, energy metabolism, and angiogenesis.

AIMS: The estrogen-ERα axis participates in osteoblast maturation. This study was designed to further evaluated the roles of the estrogen-ERα axis in bone healing and the possible mechanisms. MAIN METHODS: Female ICR mice were created a metaphyseal bone defect in the left femurs and administered with methylpiperidinopyrazole (MPP), an inhibitor of ERα. Bone healing was evaluated using micro-computed tomography. Colocalization of ERα with alkaline phosphatase (ALP) and ERα translocation to mitochondria were determined. Levels of ERα, ERß, PECAM-1, VEGF, and ß-actin were immunodetected. Expression of chromosomal Runx2, ALP, and osteocalcin mRNAs and mitochondrial cytochrome c oxidase (COX) I and COXII mRNAs were quantified. Angiogenesis was measured with immunohistochemistry. KEY FINDINGS: Following surgery, the bone mass was time-dependently augmented in the bone-defect area. Simultaneously, levels of ERα were specifically upregulated and positively correlated with bone healing. Administration of MPP to mice consistently decreased levels of ERα and bone healing. As to the mechanisms, osteogenesis was enhanced in bone healing, but MPP attenuated osteoblast maturation. In parallel, expressions of osteogenesis-related ALP, Runx2, and osteocalcin mRNAs were induced in the injured zone. Treatment with MPP led to significant inhibition of the alp, runx2, and osteocalcin gene expressions. Remarkably, administration of MPP lessened translocation of ERα to mitochondria and expressions of mitochondrial energy production-related coxI and coxII genes. Furthermore, exposure to MPP decreased levels of PECAM-1 and VEGF in the bone-defect area. SIGNIFICANCE: The present study showed the contributions of the estrogen-ERα axis to bone healing through stimulation of energy production, osteoblast maturation, and angiogenesis.

Methylpiperidinopyrazole Attenuates Estrogen-Induced Mitochondrial Energy Production and Subsequent Osteoblast Maturation via an Estrogen Receptor Alpha-Dependent Mechanism.

An estrogen deficiency is the main cause of osteoporosis in postmenopausal women. In bone remodeling, estrogen receptors (ERs) can mediate estrogen-transducing signals. Methylpiperidinopyrazole (MPP) is a highly specific antagonist of ER-alpha (ERα). This study was designed to evaluate the effects of MPP on estrogen-induced energy production, subsequent osteoblast maturation, and the possible mechanisms. Exposure of primary osteoblasts isolated from neonatal rat calvarias to MPP did not affect cell morphology or survival. Estradiol can induce translocation of ERα into mitochondria from the cytoplasm. Interestingly, pretreatment of rat calvarial osteoblasts with MPP lowered estrogen-induced ERα translocation. Sequentially, estrogen-triggered expressions of mitochondrial energy production-linked cytochrome c oxidase (COX) I and COX II messenger (m)RNAs were inhibited following pretreatment with MPP. Consequently, MPP caused decreases in estrogen-triggered augmentation of the activities of mitochondrial respiratory complex enzymes and levels of cellular adenosine phosphate (ATP). During progression of osteoblast maturation, estrogen induced bone morphogenetic protein (BMP)-6 and type I collagen mRNA expressions, but MPP treatment inhibited such induction. Consequently, estrogen-induced osteoblast activation and mineralization were attenuated after exposure to MPP. Taken together, MPP suppressed estrogen-induced osteoblast maturation through decreasing chromosomal osteogenesis-related BMP-6 and type I collagen mRNA expressions and mitochondrial ATP synthesis due to inhibiting energy production-linked COX I and II mRNA expressions. MPP can appropriately be applied to evaluate estrogen-involved bioenergetics and osteoblast maturation.

Increased activation of HDAC1/2/6 and Sp1 underlies therapeutic resistance and tumor growth in glioblastoma.

BACKGROUND: Glioblastoma is associated with poor prognosis and high mortality. Although the use of first-line temozolomide can reduce tumor growth, therapy-induced stress drives stem cells out of quiescence, leading to chemoresistance and glioblastoma recurrence. The specificity protein 1 (Sp1) transcription factor is known to protect glioblastoma cells against temozolomide; however, how tumor cells hijack this factor to gain resistance to therapy is not known. METHODS: Sp1 acetylation in temozolomide-resistant cells and stemlike tumorspheres was analyzed by immunoprecipitation and immunoblotting experiments. Effects of the histone deacetylase (HDAC)/Sp1 axis on malignant growth were examined using cell proliferation-related assays and in vivo experiments. Furthermore, integrative analysis of gene expression with chromatin immunoprecipitation sequencing and the recurrent glioblastoma omics data were also used to further determine the target genes of the HDAC/Sp1 axis. RESULTS: We identified Sp1 as a novel substrate of HDAC6, and observed that the HDAC1/2/6/Sp1 pathway promotes self-renewal of malignancy by upregulating B cell-specific Mo-MLV integration site 1 (BMI1) and human telomerase reverse transcriptase (hTERT), as well as by regulating G2/M progression and DNA repair via alteration of the transcription of various genes. Importantly, HDAC1/2/6/Sp1 activation is associated with poor clinical outcome in both glioblastoma and low-grade gliomas. However, treatment with azaindolyl sulfonamide, a potent HDAC6 inhibitor with partial efficacy against HDAC1/2, induced G2/M arrest and senescence in both temozolomide-resistant cells and stemlike tumorspheres. CONCLUSION: Our study uncovers a previously unknown regulatory mechanism in which the HDAC6/Sp1 axis induces cell division and maintains the stem cell population to fuel tumor growth and therapeutic resistance.

The Role of Vitamin D in Modulating Mesenchymal Stem Cells and Endothelial Progenitor Cells for Vascular Calcification.

Vascular calcification, which involves the deposition of calcifying particles within the arterial wall, is mediated by atherosclerosis, vascular smooth muscle cell osteoblastic changes, adventitial mesenchymal stem cell osteoblastic differentiation, and insufficiency of the calcification inhibitors. Recent observations implied a role for mesenchymal stem cells and endothelial progenitor cells in vascular calcification. Mesenchymal stem cells reside in the bone marrow and the adventitial layer of arteries. Endothelial progenitor cells that originate from the bone marrow are an important mechanism for repairing injured endothelial cells. Mesenchymal stem cells may differentiate osteogenically by inflammation or by specific stimuli, which can activate calcification. However, the bioactive substances secreted from mesenchymal stem cells have been shown to mitigate vascular calcification by suppressing inflammation, bone morphogenetic protein 2, and the Wingless-INT signal. Vitamin D deficiency may contribute to vascular calcification. Vitamin D supplement has been used to modulate the osteoblastic differentiation of mesenchymal stem cells and to lessen vascular injury by stimulating adhesion and migration of endothelial progenitor cells. This narrative review clarifies the role of mesenchymal stem cells and the possible role of vitamin D in the mechanisms of vascular calcification.

Tc-99m TRODAT-1 SPECT is a Potential Biomarker for Restless Leg Syndrome in Patients with End-Stage.

Rationales: Restless leg syndrome (RLS) is a common complication in patients with end-stage renal disease (ESRD). However, there is a lack of biomarkers linking uremic RLS to dopaminergic neurons. Previous studies demonstrated that Tc-99m TRODAT-1 SPECT was a biomarker for RLS but the correlation between the physiologic parameter was lacking. METHODS: Overall, 32 patients were enrolled in the study and divided into the following 3 groups: (1) control (n = 13), (2) ESRD without RLS (n = 8) and (3) ESRD with RLS (n = 11). All patients had a clinical diagnosis of RLS and received Tc-99m TRODAT-1 SPECT. A subgroup analysis was performed to compare differences between the control and ESRD with RLS groups. Tc-99m TRODAT-1 SPECT was performed and activities in the striatum and occipital areas were measured using manually delineated regions of interest (ROIs) by an experienced nuclear medicine radiologist who was blinded to clinical data. RESULTS: The total ratio of Tc-99m TRODAT SPECT was lower in the ESRD with RLS group (p = 0.046). The uptake ratio of TRODAT negatively correlated with serum parathyroid hormone (r = -0.577, p = 0.015) and ferritin (r = -0.464, p = 0.039) concentrations. However, the uptake positively correlated with the hemoglobin concentration (r = 0.531, p = 0.011). The sensitivity and specificity of the total TRODAT ratio for predicting RLS in the overall population were 95.0% and 67.7%, respectively, at a cutoff value of 0.980 (area under the curve of receiver operating characteristic curve was 0.767, p = 0.024). CONCLUSION: In patients with ESRD and RLS, Tc-99m TRODAT might be a potential biomarker. Dysregulated hemoglobin, serum parathyroid hormone and serum ferritin concentrations might influence the uptake of the TRODAT ratio.

Major Contribution of Caspase-9 to Honokiol-Induced Apoptotic Insults to Human Drug-Resistant Glioblastoma Cells.

Temozolomide (TMZ)-induced chemoresistance to human glioblastomas is a critical challenge now. Our previous studies showed that honokiol, a major bioactive constituent of Magnolia officinalis (Houpo), can kill human glioblastoma cells and suppresses glioblastoma growth. This study was further aimed to evaluate the effects of honokiol on human drug-resistant glioblastoma cells and the possible mechanisms. The results by data mining in the cancer genome atlas (TCGA) database and immunohistochemistry displayed that expression of caspase-9 mRNA and protein in human glioblastomas was induced. Human TMZ-resistant U87-MG-R9 glioblastoma cells were selected and prepared from human drug-sensitive U87-MG cells. Compared to human drug-sensitive U87-MG cells, TMZ did not affect viability of U87-MG-R9 glioblastoma cells. Interestingly, treatment with honokiol suppressed proliferation and survival of human drug-resistant glioblastoma cells in concentration- and time-dependent manners. Compared to caspase-8 activation, honokiol chiefly increased activity of caspase-9 in U87-MG-R9 cells. Successively, levels of cleaved caspase-3 and activities of caspase-3 and caspase-6 in human TMZ-tolerant glioblastoma cells were augmented following honokiol administration. In parallel, honokiol triggered DNA fragmentation of U87-MG-R9 cells. Accordingly, treatment of human TMZ-resistant glioblastoma cells with honokiol induced cell apoptosis but did not affect cell necrosis. Fascinatingly, suppressing caspase-9 activity using its specific inhibitors repressed honokiol-induced caspase-6 activation, DNA fragmentation, and cell apoptosis. Taken together, this study has shown the major roles of caspase-9 in transducing honokiol-induced mitochondria-dependent apoptosis in human drug-resistant glioblastoma cells. Thus, honokiol may be clinically applied as a drug candidate for treatment of glioblastoma patients with chemoresistance.