Ethanolic Leaf Extract of C. angustifolia Instigates ROS Mediated Apoptosis within Glioblastoma C6 Cells.

Glioblastoma multiforme or GBM is a destructive malignancy of the central nervous system and is accountable for leading cause of cancer related mortality. Inadequate success rate of surgical interventions and development of resistance towards the current therapeutical regime provides impetus for exploring novel therapeutical interventions against the disease. Recently, several epidemiological studies have explored the plausible utility of natural, dietary compounds in influencing the development, progression, and cancer metastasis. Recently, different phytoconstituents of Cassia angustifolia were found to be associated with anti-microbial, anti-cancer and anti-inflammatory effects. Therefore, the aim of the present study was to evaluate the anti-proliferative efficacy of ethanolic leaf extract of C. angustifolia (LCaEt-OH) against rat derived glioblastoma C6 cells. Briefly, the anti-proliferative potential of LCaEt-OH was assessed using MTT assay, quantitative estimation of ROS, and evaluation of mitochondrial membrane potential (ΔΨm). Moreover, the activity of caspases involved in intrinsic apoptotic pathways was also investigated using colorimetric kit followed by quantitative RT-PCR evaluation of modulation in gene expressions triggered due to LCaEt-OH treatment. Treatment of LCaEt-OH on C6 cells elucidated substantial dose-dependent decline in cellular viability. Furthermore, LCaEt-OH showed its efficacy in substantially enhancing intracellular ROS. LCaEt-OH also incited apoptosis in C6 cells by instigating nuclear condensation and dissipation of ΔΨm. In addition, LCaEt-OH mediated instigation of apoptosis was directly influenced by increased activity of caspases indispensable for intrinsic apoptotic pathway. These conclusive evidences indicate towards anticancer efficacy of LCaEt-OH against C6 cells.

Effects of Camk2b Overexpression and Underexpression on the Proteome of Rat Hippocampal Neurons.

Recent studies have demonstrated that Camk2b expression is modified in neuropsychiatric illnesses and potentially affects synaptic plasticity. However, the molecular events arising from Camk2b dysregulation are not fully elucidated and need to be comprehensively explored. In the present study, we first induced over-expression and under-expression of Camk2b in cultured rat hippocampal neurons through transfection with lentivirus plasmids. Then isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics followed by bioinformatics analyses were carried out to explore the impacts of Camk2b dysexpression on the proteome of the neurons. Compared with the respective controls, a total of 270 proteins in the Camk2b-overexpression group and 209 proteins in the Camk2b-underexpression group were experienced a divergence in expression. Gene ontology and pathway analyses indicated that Camk2b overexpression and under-expression respectively induced two different change profiles of protein expressions and functions, reflecting the potential differences in cellular processes and biological events. Through cross comparison, several candidate target proteins regulated directly by Camk2b were revealed. Further network and immunoblot analyses demonstrated that Mapk3 could be an important linker and Camk2b-Mapk3 might serve as a new potential pathway affecting the expression of synaptic proteins in hippocampal neurons. Collectively, the present results offer a new comprehension of the regulatory molecular mechanism of Camk2b and thereby increase our understanding of Camk2b-mediated synaptogenesis in synaptic plasticity.

Wearable knee joint fatigue estimating system based on curvature and pressure sensing.

BACKGROUND: The injury of the knee joint is found to be directly related to the fatigue caused by excessive exercise. Many previous studies used wearable devices to measure the angle of knee joint during activities, but did not pay enough attention to the load of knee joint related to the fatigue degree of it. OBJECTIVE: A wearable embedded system was designed to sense the motion state and load of knee joint and uses the sensoring data to estimate and predict the fatigue degree of knee joint during exercise in real time, so as to prevent it from being injured. METHODS: An economical wearable system is designed to measure the parameters of the knee joint during exercises. Then the warning message and recommended healthy lasting time are able to be sent to users to avoid excessive exercise. 24 healthy volunteers aged 20-25 years were involved in the experiments. Two famous evaluation scales for knee joint from Department of Orthopedics (Lysholm score and IKDC score) were adopted to evaluate the protective effect. RESULTS: After 14 days of the first stage testing, all the participants with wearable devices reported healthy knee joint state to verify the effectiveness of the system. For the second stage, the testing group equipped with wearable warning devices did not receive obvious change in the two scales. However, Lysholm score of control group dropped by at least 7.4 and IKDC score dropped by at least 11.1 which were significantly reduced. CONCLUSION: Only using human perception to prevent knee joint fatigue had a risk of failure while the designed wearable system could protect the knee successfully from injuries during exercises, such as running, badminton, table tennis and basketball. Moreover, female gender and a high BMI value may be two factors that increase the risk of knee injuries during sports.

RARG S427L attenuates the DNA repair response to doxorubicin in induced pluripotent stem cell-derived cardiomyocytes.

Doxorubicin is a commonly used chemotherapeutic drug, but its use is limited by doxorubicin-induced cardiotoxicity (DIC), which can lead to irreversible heart failure and death. A missense variant rs2229774 (p.S427L) in the retinoic acid receptor gamma (RARG) gene is associated with increased susceptibility to DIC, but the precise mechanism underlying this association is incompletely understood. We performed molecular dynamic simulations to determine the effect of this variant on RARG structure and then validated these predictions using CRISPR-Cas9-genome-edited, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). We found that this variant leads to reduced activation of its target genes in response to doxorubicin, including gene pathways involved in DNA repair and consequently an inability to mediate DNA repair after exposure to doxorubicin. Our findings establish a role of RARG p.S427L in attenuating DNA repair in DIC and provide insight into the pathogenesis of this cardiotoxic effect.

Intersectional analysis of chronic mild stress-induced lncRNA-mRNA interaction networks in rat hippocampus reveals potential anti-depression/anxiety drug targets.

Despite studies providing insight into the neurobiology of chronic stress, depression and anxiety, long noncoding RNA (lncRNA)-mediated mechanisms underlying the common and distinct pathophysiology of these stress-induced disorders remain nonconclusive. In a previous study, we used the chronic mild stress paradigm to separate depression-susceptible, anxiety-susceptible and insusceptible rat subpopulations. In the current study, lncRNA and messenger RNA (mRNA) expression was comparatively profiled in the hippocampus of the three stress groups using microarray technology. Groupwise comparisons identified distinct sets of lncRNAs and mRNAs associated with the three different behavioral phenotypes of the stressed rats. To investigate the regulatory roles of the dysregulated lncRNAs upon mRNA expression, correlations between the differential lncRNAs and mRNAs were first analyzed by combined use of weighted gene coexpression network analysis and ceRNA theory-based methods. Subsequent functional analysis of strongly correlated mRNAs indicated that the dysregulated lncRNAs were involved in various biological pathways and processes to specifically induce rat susceptibility or resiliency to depression or anxiety. Further intersectional analysis of phenotype-associated and drug-associated lncRNA-mRNA networks and subnetworks assisted in identifying 16 hub lncRNAs as potential targets of anti-depression/anxiety drugs. Collectively, our study established the molecular basis for understanding the similarities and differences in pathophysiological mechanisms underlying stress-induced depression or anxiety and stress resiliency, revealing several important lncRNAs that represent potentially new therapeutic drug targets for depression and anxiety disorders.

Variation in RARG increases susceptibility to doxorubicin-induced cardiotoxicity in patient specific induced pluripotent stem cell-derived cardiomyocytes.

Doxorubicin is a potent anticancer drug used to treat a variety of cancer types. However, its use is limited by doxorubicin-induced cardiotoxicity (DIC). A missense variant in the RARG gene (S427L; rs2229774) has been implicated in susceptibility to DIC in a genome wide association study. The goal of this study was to investigate the functional role of this RARG variant in DIC. We used induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) from patients treated with doxorubicin. iPSC-CMs from individuals who experienced DIC (cases) showed significantly greater sensitivity to doxorubicin compared to iPSC-CMs from doxorubicin-treated individuals who did not develop DIC (controls) in cell viability and optical mapping experiments. Using CRISPR/Cas9, we generated isogenic cell lines that differed only at the RARG locus. Genetic correction of RARG-S427L to wild type resulted in reduced doxorubicin-induced double stranded DNA breaks, reactive oxygen species production, and cell death. Conversely, introduction of RARG-S427L increased susceptibility to doxorubicin. Finally, genetic disruption of the RARG gene resulted in protection from cell death due to doxorubicin treatment. Our findings suggest that the presence of RARG-S427L increases sensitivity to DIC, establishing a direct, causal role for this variant in DIC.

Hippocampal proteomic changes of susceptibility and resilience to depression or anxiety in a rat model of chronic mild stress.

Chronic stressful occurrences are documented as a vital cause of both depression and anxiety disorders. However, the stress-induced molecular mechanisms underlying the common and distinct pathophysiology of these disorders remains largely unclear. We utilized a chronic mild stress (CMS) rat model to differentiate and subgroup depression-susceptible, anxiety-susceptible, and insusceptible rats. The hippocampus was analyzed for differential proteomes by combining mass spectrometry and the isobaric tags for relative and absolute quantitation (iTRAQ) labeling technique. Out of 2593 quantified proteins, 367 were aberrantly expressed. These hippocampal protein candidates might be associated with susceptibility to stress-induced depression or anxiety and stress resilience. They provide the potential protein systems involved in various metabolic pathways as novel investigative protein targets. Further, independent immunoblot analysis identified changes in Por, Idh2 and Esd; Glo1, G6pdx, Aldh2, and Dld; Dlat, Ogdhl, Anxal, Tpp2, and Sdha that were specifically associated to depression-susceptible, anxiety-susceptible, or insusceptible groups respectively, suggesting that identical CMS differently impacted the mitochondrial and metabolic processes in the hippocampus. Collectively, the observed alterations to protein abundance profiles of the hippocampus provided significant and novel insights into the stress regulation mechanism in a CMS rat model. This might serve as the molecular basis for further studies that would contributed to a better understanding of the similarities and differences in pathophysiologic mechanisms underlying stress-induced depression or anxiety, and stress resiliency.

Ibrutinib Displays Atrial-Specific Toxicity in Human Stem Cell-Derived Cardiomyocytes.

Ibrutinib (IB) is an oral Bruton's tyrosine kinase (BTK) inhibitor that has demonstrated benefit in B cell cancers, but is associated with a dramatic increase in atrial fibrillation (AF). We employed cell-specific differentiation protocols and optical mapping to investigate the effects of IB and other tyrosine kinase inhibitors (TKIs) on the voltage and calcium transients of atrial and ventricular human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). IB demonstrated direct cell-specific effects on atrial hPSC-CMs that would be predicted to predispose to AF. Second-generation BTK inhibitors did not have the same effect. Furthermore, IB exposure was associated with differential chamber-specific regulation of a number of regulatory pathways including the receptor tyrosine kinase pathway, which may be implicated in the pathogenesis of AF. Our study is the first to demonstrate cell-type-specific toxicity in hPSC-derived atrial and ventricular cardiomyocytes, which reliably reproduces the clinical cardiotoxicity observed.

iTRAQ-Based Quantitative Proteomics Suggests Synaptic Mitochondrial Dysfunction in the Hippocampus of Rats Susceptible to Chronic Mild Stress.

Mounting studies show that hippocampal synaptic transmission and plasticity are abnormal in depression. It has been suggested that impairment of synaptic mitochondrial functions potentially occurs in the hippocampus. Thus, the synaptic mitochondria may be a crucial therapeutic target in the course of depression. Here, we investigated the potential dysregulation of synaptic mitochondrial proteins in the hippocampus of a chronic mild stress (CMS) rat model. Proteomic changes of hippocampal synaptosomes containing synaptic mitochondria were quantitatively examined using the isobaric tag for relative and absolute quantitation labeling combined with tandem mass spectrometry. 45 Proteins were identified to be differentially expressed, of which 21 were found to be putative synaptic mitochondrial proteins based on gene ontology component and SynaptomeDB analyses. Detailed investigations of protein functions and disease relevance support the importance of hippocampal synaptic mitochondria as a key substrate contributing to impairment in synaptic plasticity of stress-related disorders. Interestingly, eight synaptic mitochondrial proteins were specifically associated to the susceptible group, and might represent part of molecular basis of depression. Further analysis indicated that the synaptic mitochondrial oxidative phosphorylation (OXPHOS) system was heavily affected by CMS in the susceptible rats. The present results provide novel insights into the disease mechanism underlying the abnormal OXPHOS that is responsible for energy-demanding synaptic plasticity, and thereby increase our understanding of the role of hippocampal synaptic mitochondrial dysfunction in depression.

CRISPR/Cas9-mediated genome editing in human stem cell-derived cardiomyocytes: Applications for cardiovascular disease modelling and cardiotoxicity screening.

Cardiovascular diseases (CVDs) are leading causes of death worldwide, and drug-induced cardiotoxicity is among the most common cause of drug withdrawal from the market. Improved models of cardiac tissue are needed to study the mechanisms of CVDs and drug-induced cardiotoxicity. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) have provided a major advance to our ability to study these conditions. Combined with efficient genome editing technologies, such as CRISPR/Cas9, we now have the ability to study with greater resolution the genetic causes and underlying mechanisms of inherited and drug-induced cardiotoxicity, and to investigate new treatments. Here, we review recent advances in the use of hPSC-CMs and CRISPR/Cas9-mediated genome editing to study cardiotoxicity and model CVD.

Effective lock-in strategy for proteomic analysis of corona complexes bound to amino-free ligands of gold nanoparticles.

For specific applications, gold nanoparticles (GNPs) are commonly functionalized with various biological ligands, including amino-free ligands such as amino acids, peptides, proteins, and nucleic acids. Upon entering a biological fluid, the protein corona that forms around GNPs can conceal the targeting ligands and sterically hinder the functional properties. The protein corona is routinely prepared by standard centrifugation or sucrose cushion centrifugation. However, such methodologies are not applicable to the exclusive analysis of a ligand-binding protein corona. In this study, we first proposed a lock-in strategy based on a combination of rapid crosslinking and stringent washing. Cysteine was used as a model of amino-free ligands and attached to GNPs. After corona formation in the human plasma, GNP cysteine and corona proteins were quickly fixed by 5 s of crosslinking with 7.5% formaldehyde. After stringent washing using SDS buffer with sonication, the cysteine-bound proteins were effectively separated from unbound proteins. Qualitative and quantitative analyses using a mass spectrometry-based proteomics approach indicated that the protein composition of the cysteine-binding corona from the new method was significantly different from the composition of the whole corona from the two conventional methods. Furthermore, network and formaldehyde-linked site analyses of cysteine-binding proteins provided useful information toward a better knowledge of the behavior of protein-ligand and protein-protein interactions. Collectively, our new strategy has the capability to particularly characterize the protein composition of a cysteine-binding corona. The presented methodology in principal provides a generic way to analyze a nanoparticle corona bound to amino-free ligands and has the potential to decipher corona-masked ligand functions.

Effectively Measuring Respiratory Flow With Portable Pressure Data Using Back Propagation Neural Network.

Continuous respiratory monitoring is an important tool for clinical monitoring. The most widely used flow measure device is nasal cannulae connected to a pressure transducer. However, most of these devices are not easy to carry and continue working in uncontrolled environments which is also a problem. For portable breathing equipment, due to the volume limit, the pressure signals acquired by using the airway tube may be too weak and contain some noise, leading to huge errors in respiratory flow measures. In this paper, a cost-effective portable pressure sensor-based respiratory measure device is designed. This device has a new airway tube design, which enables the pressure drop efficiently after the air flowing through the airway tube. Also, a new back propagation (BP) neural network-based algorithm is proposed to stabilize the device calibration and remove pressure signal noise. For improving the reliability and accuracy of proposed respiratory device, a through experimental evaluation and a case study of the proposed BP neural network algorithm have been carried out. The results show that giving proper parameters setting, the proposed BP neural network algorithm is capable of efficiently improving the reliability of newly designed respiratory device.

Quantitative Proteomic Analysis Reveals Synaptic Dysfunction in the Amygdala of Rats Susceptible to Chronic Mild Stress.

The amygdala plays a key role in the pathophysiology of depression, but the molecular mechanisms underlying amygdalar hyperactivity in depression remain unclear. In this study, we used a chronic mild stress (CMS) protocol to separate susceptible and insusceptible rat subgroups. Proteomes in the amygdalae were analyzed differentially across subgroups based on labeling with isobaric tags for relative and absolute quantitation (iTRAQ) combined with mass spectrometry. Of 2562 quantified proteins, 102 were differentially expressed. Several proteins that might be associated with the stress insusceptibility/susceptibility difference, including synapse-related proteins, were identified in the amygdala. Immunoblot analysis identified changes in VGluT1, NMDA GluN2A and GluN2B and AMPA GluA1 receptors, and PSD-95, suggesting that CMS perturbs glutamatergic transmission in the amygdala. Changes in these regulatory and structural proteins provide insight into the molecular mechanisms underlying the abnormal synaptic morphological and functional plasticity in the amygdalae of stress-susceptible rats. Interestingly, the expression level of CaMKIIß, potentially involved in regulation of glutamatergic transmission, was significantly increased in the susceptible group. Subsequent in vitro experimentation showed that overexpression of CaMKIIß increased the expression of PSD-95 and GluA1 in cultured hippocampal neurons. This result suggested that CaMKIIß functions upstream from PSD-95 and GluA1 to affect LTP-based postsynaptic functional plasticity in the amygdalae of susceptible rats. Therefore, amygdalar CaMKIIß is a potential antidepressant target. Collectively, our findings contribute to a better understanding of amygdalar synaptic plasticity in depression.