International Journal of Molecular Sciences — Open Access Journal

Cadmium is a common environmental pollutant that causes bone damage. However, the effects of cadmium on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) and its mechanism of action in this process are unclear. Here, we determined the effects of cadmium chloride (CdCl₂) on the osteogenic differentiation of BMMSCs and the potential mechanism involved in this process. As determined in the present investigation, CdCl₂, in a concentration-dependent manner, affected the viability of BMMSCs and their cytoskeletons. Exposure to 0.1 or 0.2 µM CdCl₂ inhibited osteogenic differentiation of BMMSCs, which was reflected in the down-regulation of osteoblast-related genes (ALP, OCN, Runx2, OSX, and OPN); in suppression of the protein expression of alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2); and in decreased ALP activity and capacity for mineralization. Moreover, mRNA microarray was performed to determine the roles of these factors in BMMSCs treated with CdCl₂ in comparison to control BMMSCs. As determined with the microarrays, the Wingless-type (Wnt), mothers against decapentaplegic and the C. elegans gene Sam (SMAD), and Janus kinase-Signal Transducers and Activators of Transcription (JAK-STAT) signaling pathways were involved in the effects caused by CdCl₂. Moreover, during differentiation, the protein levels of Wnt3a, β-catenin, lymphoid enhancer factor 1 (LEF1), and T-cell factor 1 (TCF1) were reduced by CdCl₂. The current research shows that CdCl₂ suppresses the osteogenesis of BMMSCs via inhibiting the Wnt/β-catenin pathway. The results establish a previously unknown mechanism for bone injury induced by CdCl₂. Full article

X-linked retinitis pigmentosa (XLRP) is a type of severe retinal dystrophy, and female carriers of XLRP demonstrate markedly variable clinical severity. In this study, we aimed to elucidate the clinical findings of male patients with and female carriers of XLRP in a Japanese cohort and demonstrate the genetic contribution. Twelve unrelated families (13 male patients, 15 female carriers) harboring pathogenic mutations in RPGR or RP2 were included, and comprehensive ophthalmic examinations were performed. To identify potential pathogenic mutations, targeted next-generation sequencing was employed. Consequently, we identified 11 pathogenic mutations, of which five were novel. Six and five mutations were detected in RPGR and RP2, respectively. Only one mutation was detected in ORF15. Affected male patients with RP2 mutations tended to have lower visual function than those with RPGR mutations. Female carriers demonstrated varying visual acuities and visual fields. Among the female carriers, 92% had electroretinographical abnormalities and 63% had a radial autofluorescent pattern, and the carriers who had higher myopia showed worse visual acuity and more severe retinal degeneration. Our results expand the knowledge of the clinical phenotypes of male patients with and female carriers of XLRP and suggest the possibility that RP2 mutations are relatively highly prevalent in Japan. Full article

Type 2 diabetes mellitus (T2DM) is associated with pancreatic β-cell dysfunction which can be induced by oxidative stress. Deuterohemin-βAla-His-Thr-Val-Glu-Lys (DhHP-6) is a microperoxidase mimetic that can scavenge reactive oxygen species (ROS) in vivo. In our previous studies, we demonstrated an increased stability of linear peptides upon their covalent attachment to porphyrins. In this study, we assessed the utility of DhHP-6 as an oral anti-diabetic drug in vitro and in vivo. DhHP-6 showed high resistance to proteolytic degradation in vitro and in vivo. The degraded DhHP-6 product in gastrointestinal (GI) fluid retained the enzymatic activity of DhHP-6, but displayed a higher permeability coefficient. DhHP-6 protected against the cell damage induced by H₂O₂ and promoted insulin secretion in INS-1 cells. In the T2DM model, DhHP-6 reduced blood glucose levels and facilitated the recovery of blood lipid disorders. DhHP-6 also mitigated both insulin resistance and glucose tolerance. Most importantly, DhHP-6 promoted the recovery of damaged pancreas islets. These findings suggest that DhHP-6 in physiological environments has high stability against enzymatic degradation and maintains enzymatic activity. As DhHP-6 lowered the fasting blood glucose levels of T2DM mice, it thus represents a promising candidate for oral administration and clinical therapy. Full article

β-N-Acetyl-d-hexosaminidase from Ostrinia furnacalis (OfHex1) is a new target for the design of insecticides. Although some of its inhibitors have been found, there is still no commercial drug available at present. The residence time of the ligand may be important for its pharmacodynamic effect. However, the unbinding routes of ligands from OfHex1 still remain largely unexplored. In the present study, we first simulated the six dissociation routes of N,N,N-trimethyl-d-glucosamine-chitotriomycin (TMG-chitotriomycin, a highly selective inhibitor of OfHex1) from the active pocket of OfHex1 by steered molecular dynamics simulations. By comparing the potential of mean forces (PMFs) of six routes, Route 1 was considered as the most possible route with the lowest energy barrier. Furthermore, the structures of six different states for Route 1 were snapshotted, and the key amino acid residues affecting the dissociated time were analyzed in the unbinding pathway. Moreover, we also analyzed the “open–close” mechanism of Glu368 and Trp448 and found that their conformational changes directly affected the dissociation of TMG-chitotriomycin. Our findings would be helpful to understanding and identifying novel inhibitors against OfHex1 from virtual screening or lead-optimization. Full article

ErbB2 is an oncogene receptor tyrosine kinase linked to breast cancer. It is a member of the epidermal growth factor receptor (EGFR) minifamily. ErbB2 is currently viewed as an orphan receptor since, by itself, it does not bind EGF-like ligands and can be activated only when overexpressed in malignant cells or complexed with ErbB3, another member of the EGFR minifamily. Here, we report that ErbB2 can be activated by extracellular application of mildly alkaline (pH 8–9) media to ErbB2-transfected cells. We also show that the activation of the ErbB2 receptor by alkali is dose-dependent and buffer-independent. The endogenous ErbB2 receptor of A431 cell line can also undergo alkali-dependent autophosphorylation. Thus, we describe a novel ligand-independent mechanism of ErbB2 receptor activation. Full article

Alzheimer’s disease (AD), Parkinson’s disease (PD), frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), and prion diseases have a certain degree of clinical, pathological, and molecular overlapping. Previous studies revealed that many causative mutations in AD, PD, and FTD/ALS genes could be found in clinical familial and sporadic AD. To further elucidate the missing heritability in early-onset Alzheimer’s disease (EOAD), we genetically characterized a Thai EOAD cohort by Next-Generation Sequencing (NGS) with a high depth of coverage, capturing variants in 50 previously recognized AD and other related disorders’ genes. A novel mutation, APP p.V604M, and the known causative variant, PSEN1 p.E184G, were found in two of the familiar cases. Remarkably, among 61 missense variants were additionally discovered from 21 genes out of 50 genes, six potential mutations including MAPT P513A, LRRK2 p.R1628P, TREM2 p.L211P, and CSF1R (p.P54Q and pL536V) may be considered to be probably/possibly pathogenic and risk factors for other dementia leading to neuronal degeneration. All allele frequencies of the identified missense mutations were compared to 622 control individuals. Our study provides initial evidence that AD and other neurodegenerative diseases may represent shades of the same disease spectrum, and consideration should be given to offer exactly embracing genetic testing to patients diagnosed with EOAD. Our results need to be further confirmed with a larger cohort from this area. Full article

Although Western medicine and Eastern medicine are worlds apart, there is a striking overlap in the basic principle of these types of medicine when we look at them from the perspective of energy. In both worlds, opposing forces provide the energy that flows through networks in an organism, which fuels life. In this concept, health is the ability of an organism to maintain the balance between these opposing forces, i.e., homeostasis (West) and harmony (East), which creates resilience. Moreover, strategies used to treat diseases are strikingly alike, namely adjusting the flow of energy by changing the connections in the network. The energy perspective provides a basis to integrate Eastern and Western medicine, and opens new directions for research to get the best of both worlds. Full article

The innate immune system drives the initiation of inflammation and progression to chronic inflammation in two important chronic inflammatory lung diseases involving the small airways, chronic obstructive pulmonary disease (COPD) and bronchiolitis obliterans syndrome (BOS), following lung transplantation. Recently natural killer T cell like (NKT-like) cells, which represent a bridge between the innate and adaptive immune response as well as the innate natural killer cell (NK) cells, have been shown to be important cells in these two chronic lung diseases. Importantly these cells have been shown to be resistant to commonly used anti-inflammatory drugs such as glucocorticoids and as such their inflammatory nature has been difficult to suppress. Mechanisms leading to steroid resistance in both diseases has recently been shown. Glucocorticoids switch off inflammatory genes by first entering the cell and binding to glucocorticoid receptors (GCRs). The steroid-GCR complex must then be chaperoned into the nucleus via several heat shock proteins, where they engage histone deacetylase 2 to switch off pro-inflammatory gene transcription. Many of these mechanisms are altered in NK and NKT-like cells in COPD and BOS requiring novel intervention using combinations of currently available drugs. Evidence will be presented to show how these drugs can overcome these mechanisms of drug resistance ex vivo advising novel therapeutic strategies for the treatment these two important chronic inflammatory lung diseases. Full article

Intrauterine growth restriction (IUGR) is a serious pathological complication associated with compromised fetal development during pregnancy. The aim of the study was to broaden knowledge about the transcriptomic complexity of the human placenta by identifying genes potentially involved in IUGR pathophysiology. RNA-Seq data were used to profile protein-coding genes, detect alternative splicing events (AS), single nucleotide variant (SNV) calling, and RNA editing sites prediction in IUGR-affected placental transcriptome. The applied methodology enabled detection of 37,501 transcriptionally active regions and the selection of 28 differentially-expressed genes (DEGs), among them 10 were upregulated and 18 downregulated in IUGR-affected placentas. Functional enrichment annotation indicated that most of the DEGs were implicated in the processes of inflammation and immune disorders related to IUGR and preeclampsia. Additionally, we revealed that some genes (S100A13, GPR126, CTRP1, and TFPI) involved in the alternation of splicing events were mainly implicated in angiogenic-related processes. Significant SNVs were overlapped with 6533 transcripts and assigned to 2386 coding sequence (CDS), 1528 introns, 345 5’ untranslated region (UTR), 1260 3’UTR, 918 non-coding RNA (ncRNA), and 10 intergenic regions. Within CDS regions, 543 missense substitutions with functional effects were recognized. Two known mutations (rs4575, synonymous; rs3817, on the downstream region) were detected within the range of AS and DEG candidates: PA28β and PINLYP, respectively. Novel genes that are dysregulated in IUGR were detected in the current research. Investigating genes underlying the IUGR is crucial for identification of mechanisms regulating placental development during a complicated pregnancy. Full article

Telomerase has cellular functions beyond telomere stabilization, including a role in mitochondria. The function of the catalytic component—TERT—in mitochondria is still unknown, but it seems to play a role in the response to oxidative stress. Here, we interrogated the role of the subcellular localization of TERT to the response to hydrogen peroxide (H₂O₂) treatment. Using normal human fibroblasts (NHF) expressing non-tagged wild type (WT) human TERT (hTERT) or nuclear localization and function (_nuchTERT), a mutant that we previously described as being competent in telomere elongation, while not being able to localize to mitochondria, we found the differential activation of autophagy as a function of hTERT’s subcellular localization. Specifically, we found that only cells expressing the mutant had significant increases in autophagy markers as a response to H₂O₂ challenge. Either the reintroduction of the mitochondrial pool of hTERT or the expression of mitochondrially-targeted catalase in mutant cells blunted the autophagic response under oxidative stress. Interestingly, autophagy activation was also associated with decreased levels of mitochondrial DNA damage. Taken together, these results suggest that the loss of hTERT in mitochondria initiates a signaling cascade that allows for cells to adapt to and cope with the lack of mitochondrial telomerase. Such effects also influence the cellular response to oxidative damage. Full article

Tomentosin is a natural sesquiterpene lactone extracted from various plants and is widely used as a medicine because it exhibits essential therapeutic properties. In this study, we investigated the anti-carcinogenic effects of tomentosin in human osteosarcoma MG-63 cells by performing cell migration/viability/proliferation, apoptosis, and reactive oxygen species (ROS) analysis assays. MG-63 cells were treated with various doses of tomentosin. After treatment with tomentosin, MG-63 cells were analyzed using the MTT assay, colony formation assay, cell counting assay, wound healing assay, Boyden chamber assay, zymography assay, cell cycle analysis, FITC Annexin V apoptosis assay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, western blot analysis, and ROS detection analysis. Our results indicated that tomentosin decreased cell viability and migration ability in MG-63 cells. Moreover, tomentosin induced apoptosis, cell cycle arrest, DNA damage, and ROS production in MG-63 cells. Furthermore, tomentosin-induced intracellular ROS decreased cell viability and induced apoptosis, cell cycle arrest, and DNA damage in MG-63 cells. Taken together, our results suggested that tomentosin exerted anti-carcinogenic effects in MG-63 cells by induction of intracellular ROS. Full article

There is an urgent need to develop novel drugs for osteoporosis which occurs due to estrogen deficiency. Phytoestrogens derived from medicinal plants would be the best alternative to chemical drugs with harmful side effects. The main purpose of the present study was to investigate the effect of ferutinin compared to 17β-estradiol (E2) on bone mineralization of zebrafish larvae. Regarding the lack of publications, the histology analysis was performed after exposure to E2 to find effective treatment on bone mineralization of developing zebrafish larvae. Then, the larvae were exposed to four concentrations of ferutinin at three time points to assess the mortality, the expression of some related genes and histology of the ceratohyal and hyomandibular of treated larvae. The RT-PCR result of the treatment groups demonstrated the similar expression pattern in the larvae which were exposed to 1.25 μg/mL of ferutinin and 2 µM of E2 at 2 dpf, which confirmed the result of histology analysis. In addition, RT-qPCR of high concentration of ferutinin and E2 demonstrated that bmp2a/b and esr1 were downregulated and upregulated when the larvae were exposed to 5 μg/mL of ferutinin and 10 µM of E2, respectively. Full article

An inverse association exists between physical activity and breast cancer incidence and outcomes. An objective indicator of an individual’s recent physical activity exposure is aerobic capacity. We took advantage of the fact that there is an inherited as well as inducible component of aerobic capacity to show that experimentally induced mammary cancer is inversely related to inherent aerobic capacity (IAC). The objective of this study was to determine whether cell signaling pathways involved in the development of mammary cancer differed in rats with low inherent aerobic capacity (LIAC, n = 55) versus high inherent aerobic capacity (HIAC, n = 57). Cancer burden was 0.21 ± 0.16 g/rat in HIAC versus 1.14 ± 0.45 in LIAC, p < 0.001. Based on protein expression, cancer in LIAC animals was associated with upregulated glucose utilization, and protein and fatty acid synthesis. Signaling in cancers from HIAC rats was associated with energy sensing, fatty acid oxidation and cell cycle arrest. These findings support the thesis that pro-glycolytic, metabolic inflexibility in LIAC favors not only insulin resistance and obesity but also tumor development and growth. This provides an unappreciated framework for understanding how obesity and low aerobic fitness, hallmarks of physical inactivity, are associated with higher cancer risk and poorer prognosis. Full article

Secretion from adipose tissue of adipokines or adipocytokines, comprising of bioactive peptides or proteins, immune molecules and inflammatory mediators, exert critical roles in inflammatory arthritis and obesity. This review considers the evidence generated over the last decade regarding the effects of several adipokines including leptin, adiponectin, visfatin, resistin, chemerin and apelin, in cartilage and bone homeostasis in the pathogenesis of rheumatoid arthritis and osteoarthritis, which has important implications for obesity. Full article

Na-amino acid co-transporters (NaAAcT) are uniquely affected in rabbit intestinal villus cell brush border membrane (BBM) during chronic intestinal inflammation. Specifically, Na-alanine co-transport (ASCT1) is inhibited secondary to a reduction in the affinity of the co-transporter for alanine, whereas Na-glutamine co-transport (B0AT1) is inhibited secondary to a reduction in BBM co-transporter numbers. During chronic intestinal inflammation, there is abundant production of the potent oxidant peroxynitrite (OONO). However, whether OONO mediates the unique alteration in NaAAcT in intestinal epithelial cells during chronic intestinal inflammation is unknown. In this study, ASCT1 and B0AT1 were inhibited by OONO in vitro. The mechanism of inhibition of ASCT1 by OONO was secondary to a reduction in the affinity of the co-transporter for alanine, and secondary to a reduction in the number of co-transporters for B0AT1, which were further confirmed by Western blot analyses. In conclusion, peroxynitrite inhibited both BBM ASCT1 and B0AT1 in intestinal epithelial cells but by different mechanisms. These alterations in the villus cells are similar to those seen in the rabbit model of chronic enteritis. Therefore, this study indicates that peroxynitrite may mediate the inhibition of ASCT1 and B0AT1 during inflammation, when OONO levels are known to be elevated in the mucosa. Full article

The incidence of hepatocellular carcinoma deriving from metabolic dysfunctions has increased in the last years. Sirtuin- (SIRT-3), phospho-mammalian target of rapamycin (p-mTOR) and hypoxia-inducible factor- (HIF-1α) are involved in metabolism and cancer. However, their role in hepatocellular carcinoma (HCC) metabolism, drug resistance and progression remains unclear. This study aimed to better clarify the biological and clinical function of these markers in HCC patients, in relation to the presence of metabolic alterations, metformin therapy and clinical outcome. A total of 70 HCC patients were enrolled: 48 and 22 of whom were in early stage and advanced stage, respectively. The expression levels of the three markers were assessed by immunohistochemistry and summarized using descriptive statistics. SIRT-3 expression was higher in diabetic than non-diabetic patients, and in metformin-treated than insulin-treated patients. Interestingly, p-mTOR was higher in patients with metabolic syndrome than those with different etiology, and, similar to SIRT-3, in metformin-treated than insulin-treated patients. Moreover, our results describe a slight, albeit not significant, benefit of high SIRT-3 and a significant benefit of high nuclear HIF-1α expression in early-stage patients, whereas high levels of p-mTOR correlated with worse prognosis in advanced-stage patients. Our study highlighted the involvement of SIRT-3 and p-mTOR in metabolic dysfunctions that occur in HCC patients, and suggested SIRT-3 and HIF-1α as predictors of prognosis in early-stage HCC patients, and p-mTOR as target for the treatment of advanced-stage HCC. Full article

An environmentally friendly technique was used to produce levan-capped silver nanoparticles of about 30 nm (with a loading of 30%) that showed bactericide effect, for E. coli and B. subtilis. That effect was mathematically studied with a dose-response model (lethal dose of 12.4 ppm and 6.8 ppm respectively). These silver nanoparticles were subsequently introduced in a gel to create a silver release system with bacteria inhibition activity. Silver release from the gel and its bactericidal activity was theoretically studied to develop a unique model that is able to predict accurately both silver release and lethal dose for any type of bacteria. This model will be useful for performing predictions for future silver in gel applications. Full article

Metabolic inhibition is a common condition observed during ischemic heart disease and heart failure. It is usually accompanied by a reduction in L-type Ca²⁺ channel (LTCC) activity. In this study, however, we show that metabolic inhibition results in a biphasic effect on LTCC current (I_CaL) in human and rat cardiac myocytes: an initial increase of I_CaL is observed in the early phase of metabolic inhibition which is followed by the more classical and strong inhibition. We studied the mechanism of the initial increase of I_CaL in cardiac myocytes during β-adrenergic stimulation by isoprenaline, a non-selective agonist of β-adrenergic receptors. The whole-cell patch–clamp technique was used to record the I_CaL in single cardiac myocytes. The initial increase of I_CaL was induced by a wide range of metabolic inhibitors (FCCP, 2,4-DNP, rotenone, antimycin A). In rat cardiomyocytes, the initial increase of I_CaL was eliminated when the cells were pre-treated with thapsigargin leading to the depletion of Ca²⁺ from the sarcoplasmic reticulum (SR). Similar results were obtained when Ca²⁺ release from the SR was blocked with ryanodine. These data suggest that the increase of I_CaL in the early phase of metabolic inhibition is due to a reduced calcium dependent inactivation (CDI) of LTCCs. This was further confirmed in human atrial myocytes where FCCP failed to induce the initial stimulation of I_CaL when Ca²⁺ was replaced by Ba²⁺, eliminating CDI of LTCCs. We conclude that the initial increase in I_CaL observed during the metabolic inhibition in human and rat cardiomyocytes is a consequence of an acute reduction of Ca²⁺ release from SR resulting in reduced CDI of LTCCs. Full article