Mass spectrometrical identification of hippocampal NMDA receptor subunits NR1, NR2A-D and five novel phosphorylation sites on NR2A and NR2B

The NMDA receptor (NMDA-R) is a key element in neural transmission and mediating a vast variety of physiological and pathological processes in the nervous system. It is well-known that phosphorylation is required for functioning of the NMDA-R, and we therefore decided to study this post-translational modification in subunits NR1 and NR2A-D. Immunoprecipitation with an antibody against NR1 was carried out from rat hippocampi and SDS-PAGEs were run. Bands were punched, destained, and digested with trypsin and chymotrypsin and peptides were identified by nano-LC-ESI-MS/MS using an ion trap (HCT). Proteins were identified using specific software. Phosphorylations were verified by phosphatase treatment and reanalysis by mass spectrometry. The NMDA-R subunits NR1 and 2A-D were identified. On NR2A, a novel phosphorylation site was observed at S511, and on NR2B, four novel phosphorylation sites were revealed at S886, S917, S1303, and S1323 by mass spectrometry and verified by phosphatase treatment with mass spectrometrical reanalysis. A series of NMDA-R phosphorylations have been reported and these serve different functions as receptor activation, localization, and protein-protein interactions. Herein, findings of novel phosphorylation sites are extending knowledge on chemical characterization of the NMDA-R and warrant studying function of site-specific receptor phosphorylation in health and disease.     

Exercise decreases speedball self-administration

Aims

Epidemiological studies report that individuals who exercise are less likely to abuse drugs. Preclinical studies report that exercise, in the form of treadmill or wheel running, reliably decreases the self-administration of psychomotor stimulants and opioids. To date, preclinical studies have only examined the effects of exercise on responding maintained by individual drugs and not by combinations of multiple drugs. This limits the translational appeal of these studies because polydrug abuse is common among substance abusing populations. The purpose of this study was to examine the effects of exercise on the self-administration of speedball, a combination of cocaine and heroin that is frequently encountered in intravenous drug abusing populations.

Main methods

Female rats were obtained at weaning and assigned to sedentary or exercising conditions. Sedentary rats were housed in standard cages that permitted no exercise beyond normal cage ambulation; exercising rats were housed in similar cages with an activity wheel. After 6 weeks, rats were implanted with intravenous catheters and trained to self-administer cocaine, heroin, and dose combinations of cocaine and heroin (i.e., speedball) on a progressive ratio schedule of reinforcement.

Key findings

Doses of speedball maintained greater levels of responding than corresponding doses of cocaine and heroin alone. Importantly, responding maintained by cocaine, heroin, and speedball was lower in exercising rats than sedentary rats.

Significance

These data indicate that exercise decreases the self-administration of speedball and suggest that exercise may reduce the abuse of drug combinations that have traditionally been resistant to treatment.     

Comparative Evaluation of Cardiac Markers in Differentiated Cells from Menstrual Blood and Bone Marrow-Derived Stem Cells In Vitro

In recent years, menstrual blood-derived stem cells (MenSCs) have been introduced as easily accessible and refreshing stem cell source without ethical considerations in the field of regenerative medicine. The aim of this study was to investigate in vitro cardiac differentiation capacity of MenSCs compared to bone marrow-derived stem cells (BMSCs) under two protocols using 5-aza-2′-deoxycytidine (5-aza) and basic fibroblast growth factor (bFGF). Our data revealed that differentiated MenSCs and BMSCs acquired some features of cardiomyocytes; however, degree of differentiation was dependent on the protocol. In a similar manner with BMSCs, differentiated MenSCs showed upper levels of mRNA/protein of late-stage cardiac markers under 5-aza stimulation and continuous treatment with bFGF (protocol 2) compared to those induced by 5-aza alone (protocol 1) evidencing the key role of bFGF in cardiac development of stem cells. Compared to corresponding undifferentiated cells differentiated MenSCs under protocol 2 showed remarkable expression of connexin-43 and TNNT2 at both gene and protein levels, whereas developed BMSCs under the same condition only expressed connextin-43 at the higher level. Superiority of protocol 2 over protocol 1 was confirmed by assessment of LDH and cTnI production by differentiated cells. Based on the accumulative data, our study provided convincing evidence that MenSCs have relatively higher capability to be differentiated toward cardiomyocyte compared with BMSCs. Furthermore, usage of bFGF and 5-aza to induce in vitro cardiac differentiation of MenSCs is highly recommended.     

Is miR-144 an effective inhibitor of PTEN mRNA: a controversy in breast cancer

Breast cancer is the first common cancer among women worldwide. One of the major signaling pathways playing a role in the onset and progression of this disease is PI3K/Akt/mTOR, which can be inhibited by PTEN. miRNAs are small non-coding molecules that regulate the expression of their targets by inhibition or suppression, and thus, their dysregulated expression results in the development of cancer. Using various software applications predicting miRNAs and evaluating GEO microarray data, miR-144 was selected as an inhibitor of PTEN. The expression of miR-144 and PTEN was evaluated in 18 triple negative breast cancer (TNBC) clinical samples and cell lines including 4T1, MDA-MB-231, MDA-MB-468, SK-BR-3, and MCF-7 in comparison with normal cells. PTEN and miR-144 expression analysis revealed their elevated expression in MCF-7 cells. MDA-MB-468, SK-BR-3, and MDA-MB-231 cells showed decreased levels of PTEN and increased levels of miR-144. In contrast, 4T1 cells had an increased expression of PTEN and decreased expression of miR-144. In clinical samples, miR-144 was up-regulated in 22% of the cases and PTEN was down-regulated in 78% of the cases. The results showed that the expression of PTEN and miR-144 was inversely correlated in metastatic breast cancer cell lines. However, in TNBC clinical samples, there was no correlation between the expression of miR-144 and PTEN. Literature shows that there are other influencing factors affecting the expression of miRNAs. Therefore, care should be taken in interpreting the results of gene expression studies and its relation with cancer diagnosis/prognosis.     

Flavonoid Baicalein Modulates H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Mitogen-Activated Protein Kinases Activation and Cell Death in SK-N-MC Cells

It is believed that ROS-induced oxidative stress triggers numerous signaling pathways which are involved in neurodegenerative diseases, including Alzheimer’s disease. To find the effective drugs for neurodegenerative diseases, the deep delve into molecular mechanisms underlie these diseases is necessary. In the current study, we investigated the effects of flavonoid baicalein on H₂O₂-induced oxidative stress and cell death in SK-N-MC cells. Our results revealed that the treatment of SK-N-MC cells with H₂O₂ led to a decrease in cell viability through phosphorylation and activation of extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs) pathways followed by increase in Bax/Bcl2 ratio and initiation of caspase-dependent apoptotic pathways. In addition, our results showed that the exposure of SK-N-MC cells to H₂O₂ ended up in reduction of glutathione (GSH) levels of SK-N-MC cells via JNK/ERK-mediated down-regulation of γ-glutamyl-cysteine synthetase (γ-GCS) expression. Our results demonstrated that flavonoid baicalein protected against H₂O₂-induced cell death by inhibition of JNK/ERK pathways activation and other key molecules in apoptotic pathways, including blockage of Bax and caspase-9 activation, induction of Bcl-2 expression and prevention of cell death. Baicalein supported intracellular defense mechanisms through maintaining GSH levels in SK-N-MC cells by the removal of inhibition effects of JNK/ERK pathways from γ-GCS expression. In addition, baicalein attenuated lipid and protein peroxidation and intracellular reactive oxygen species in SK-N-MC cells. In accordance with these observations, baicalein can be a promising candidate in antioxidant therapy and designing of natural-based drug for ROS-induced neurodegenerative disorders.     

Incorporation of SPION-casein core-shells into silk-fibroin nanofibers for cardiac tissue engineering

Mimicking the structure of extracellular matrix (ECM) of myocardium is necessary for fabrication of functional cardiac tissue. The superparamagnetic iron oxide nanoparticles (SPIONs, Fe₃O₄), as new generation of magnetic nanoparticles (NPs), are highly intended in biomedical studies. Here, SPION NPs (1 wt%) were synthesized and incorporated into silk-fibroin (SF) electrospun nanofibers to enhance mechanical properties and topography of the scaffolds. Then, the mouse embryonic cardiac cells (ECCs) were seeded on the scaffolds for in vitro studies. The SPION NPs were studied by scanning electron microscope (SEM), X-ray diffraction (XRD), and transmission electron microscope (TEM). SF nanofibers were characterized after incorporation of SPIONs by SEM, TEM, water contact angle measurement, and tensile test. Furthermore, cytocompatibility of scaffolds was confirmed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. SEM images showed that ECCs attached to the scaffolds with elongated morphologies. Also, the real-time PCR and immunostaining studies approved upregulation of cardiac functional genes in ECCs seeded on the SF/SPION-casein scaffolds including GATA-4, cardiac troponin T, Nkx 2.5, and alpha-myosin heavy chain, compared with the ones in SF. In conclusion, incorporation of core-shells in SF supports cardiac differentiation, while has no negative impact on ECCs' proliferation and self-renewal capacity.     

Intra-lymph Node Injection of Biodegradable Polymer Particles

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these foreign molecules to T and B lymphocytes. Lymph nodes have thus become critical targets for new vaccines and immunotherapies. A recent strategy for targeting these tissues is direct lymph node injection of soluble vaccine components, and clinical trials involving this technique have been promising. Several biomaterial strategies have also been investigated to improve lymph node targeting, for example, tuning particle size for optimal drainage of biomaterial vaccine particles. In this paper we present a new method that combines direct lymph node injection with biodegradable polymer particles that can be laden with antigen, adjuvant, or other vaccine components. In this method polymeric microparticles or nanoparticles are synthesized by a modified double emulsion protocol incorporating lipid stabilizers. Particle properties (e.g. size, cargo loading) are confirmed by laser diffraction and fluorescent microscopy, respectively. Mouse lymph nodes are then identified by peripheral injection of a nontoxic tracer dye that allows visualization of the target injection site and subsequent deposition of polymer particles in lymph nodes. This technique allows direct control over the doses and combinations of biomaterials and vaccine components delivered to lymph nodes and could be harnessed in the development of new biomaterial-based vaccines.