Expression Of Specific Proteins Research Articles

MiR-128-3p Alleviates Spinal Cord Ischemia/Reperfusion Injury Associated Neuroinflammation and Cellular Apoptosis via SP1 Suppression in Rat.

BackgroundNeuroinflammation and cellular apoptosis caused by spinal cord ischemia/reperfusion (I/R) injury result in neurological dysfunction. MicroRNAs (miRs) have crucial functions in spinal cord I/R injury pathogenesis according to previous evidences. Herein, whether miR-128-3p contributes to spinal cord I/R injury by regulating specificity protein 1 (SP1) was assessed.MethodsA rat model of spinal cord I/R injury was established by occluding the aortic arch for 14 min. Then, miR-128-3p’s interaction with SP1 was detected by dual-luciferase reporter assays. Next, miR-128-3p mimic and inhibitor, as well as adenovirus-delivered shRNA specific for SP1 were injected intrathecally for assessing the effects of miR-128-3p and SP1 on rats with spinal cord I/R injury. SP1, Bax and Bcl-2 expression levels in I/R injured spinal cord tissues were evaluated by Western blotting, while IL-1β, TNF-α, and IL-6 were quantitated by ELISA. Tarlov scores were obtained to detect hind-limb motor function. Evans blue (EB) dye extravasation was utilized to examine blood–spinal cord barrier (BSCB) permeability. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining was performed for neuronal apoptosis assessment.ResultsMiR-128-3p expression was decreased, while SP1 amounts were increased in rat spinal cord tissue specimens following I/R. SP1 was identified as a miR-128-3p target and downregulated by miR-128-3p. MiR-128-3p overexpression or SP1 silencing alleviated I/R-induced neuroinflammation and cell apoptosis, and improved Tarlov scores, whereas pretreatment with miR-128-3p inhibitor aggravated the above injuries.ConclusionOverexpression of miR-128-3p protects neurons from neuroinflammation and apoptosis during spinal cord I/R injury partially by downregulating SP1.

Antidepressant actions of ketamine engage cell-specific translation via eIF4E.

Effective pharmacotherapy for major depressive disorder remains a major challenge, as more than 30% of patients are resistant to the first line of treatment (selective serotonin reuptake inhibitors)1. Sub-anaesthetic doses of ketamine, a non-competitive N-methyl-D-aspartate receptor antagonist2,3, provide rapid and long-lasting antidepressant effects in these patients4-6, but the molecular mechanism of these effects remains unclear7,8. Ketamine has been proposed to exert its antidepressant effects through its metabolite (2R,6R)-hydroxynorketamine ((2R,6R)-HNK)9. The antidepressant effects of ketamine and (2R,6R)-HNK in rodents require activation of the mTORC1 kinase10,11. mTORC1 controls various neuronal functions12, particularly through cap-dependent initiation of mRNA translation via the phosphorylation and inactivation of eukaryotic initiation factor 4E-binding proteins (4E-BPs)13. Here we show that 4E-BP1 and 4E-BP2 are key effectors of the antidepressant activity of ketamine and (2R,6R)-HNK, and that ketamine-induced hippocampal synaptic plasticity depends on 4E-BP2 and, to a lesser extent, 4E-BP1. It has been hypothesized that ketamine activates mTORC1-4E-BP signalling in pyramidal excitatory cells of the cortex8,14. To test this hypothesis, we studied the behavioural response to ketamine and (2R,6R)-HNK in mice lacking 4E-BPs in either excitatory or inhibitory neurons. The antidepressant activity of the drugs is mediated by 4E-BP2 in excitatory neurons, and 4E-BP1 and 4E-BP2 in inhibitory neurons. Notably, genetic deletion of 4E-BP2 in inhibitory neurons induced a reduction in baseline immobility in the forced swim test, mimicking an antidepressant effect. Deletion of 4E-BP2 specifically in inhibitory neurons also prevented the ketamine-induced increase in hippocampal excitatory neurotransmission, and this effect concurred with the inability of ketamine to induce a long-lasting decrease in inhibitory neurotransmission. Overall, our data show that 4E-BPs are central to the antidepressant activity of ketamine.

Effects of energy supplements on the differentiation of skeletal muscle satellite cells.

To investigate the effects of the activator of AMPK and high glucose on the differentiation of mouse SMSCs, primary SMSCs were isolated from mouse extensor digitorum longus muscle and grown to near confluence (80%). Postconfluent cells were cultured in a growth medium with different inductors: AICAR, glucose, and AICAR mixed with glucose. The specific protein expressions of SMSCs, myoblasts, adipocytes, and brown adipocytes were analyzed on days 0, 3, 5, 7, and 10. The results showed treatment with AICAR in SMSCs markedly activated AMPK phosphorylation (p < .05) and increased protein expression of Pax7 and MyoD (p < .05), high concentrations of intracellular glucose upregulated UCP‐1 protein expression and enhanced lipid accumulation (p < .05), the cowork of AICAR and glucose affected a decrease on MyoD, PPARg, and UCP‐1 expression (p < .05) and an increase on Pax7. The present study indicated that the certain energy supplements influence the direction of SMSC differentiation which may contribution on the structure of muscle and meat quality, sequentially.

Teprenone ameliorates diclofenac-induced small intestinal injury via inhibiting protease activated receptors 1 and 2 activity

Objective This study aimed to investigate specific protein expression of injured intestinal mucosa induced by diclofenac, and explore the protective effects of teprenone on it. Methods Intestinal damage of Sprague Dawley male rats was gradually induced by the intragastric administration of diclofenac. After the last drug administration, the intestinal mucosa was taken off with an interval of 24 h, subsequently, its general histological injury and ultrastructure were observed and analysed by a transmission electron microscope. The expression levels of PAR1 and PAR2 protein were detected by immunohistochemistry and real-time polymerase chain reaction (PCR). Results The Reuter and Chiu scores of small intestinal damage were 5.63 ± 1.30 and 4.25 ± 0.70 respectively in the model group, which could be protected by teprenone (100 mg/kg⋅day) with the degree of 55.7% and 44%. Optical microscopy and transmission electron microscope showed that intestinal mucosa and ultrastructure were severely damaged. Distributed in the cytoplasm or aligned with the nucleus, the expression of PAR1 and PAR2 was significantly upregulated after the administration of diclofenac, while it was relieved after the treatment of teprenone. Conclusion Our study presents a new view that teprenone might protect NSAIDs-induced (diclofenac) intestinal injury via suppressing the expression of PAR1 and PAR2.

MBRS-43. ELUCIDATING HOW NOVEL EXTRACELLULAR MATRIX SUBTYPES DIFFERENTIALLY IMPACT THE SURVIVAL OF MEDULLOBLASTOMA SUBGROUPS

Medulloblastoma (MB) is the most common malignant paediatric brain tumour and frequently exhibits metastasis and chemoresistance. MBs are categorised into four molecular subgroups (WNT, Sonic hedgehog, Group 3 and Group 4), each associated with different demographics and clinical features. We have shown that the expression of specific extracellular matrix proteins in the brain tumour microenvironment differ between subgroups. A prime example is laminin (an ECM glycoprotein) the expression of which correlates with good overall survival in the SHH subgroup and poor overall survival in Group 4. Our aim is to determine the cause of this difference in survival. Candidate laminin-responsive-genes (LRGs) were identified using the Cavalli data set and RNA-Seq analysis of MB cells grown on 3D hydrogels with and without laminin. The role of laminin in the regulation of MMPs and the other LRG candidates was investigated by qRT-PCR, western blotting and zymography in 2D and long-term 3D-hydrogel assays. Thus far we have shown that in CHLA-01-R (metastatic Group 4 cell line) three of our LRGs are upregulated in response to laminin in 2D, as well as in preliminary 3D studies. Additionally, we have observed a unique MMP9 secretion profile of SHH cells grown in 3D compared to 2D, suggesting that our 3D assay allows us to observe relevant phenotypes absent in 2D culture. We are now in the process of identifying which of these LRG candidates are involved in metastasis and chemoresistance. This will enable the elucidation of novel therapeutic targets and crucially increase our understanding of MB-microenvironment interactions.

Experimental Study on Western Blot

In this paper, the expression of specific proteins in two groups of cells was studied by the methods of documentary review and experiment. The results were as follows: one group of cells expressed proteins, and the other group did not.

RETRACTED: Loss of microRNA-30e induced by extracellular vesicles from cancer-associated fibroblasts promotes breast cancer progression by binding to CTHRC1

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the authors as “some data in this study are not solid enough and need to be further explored”. The authors stated that they "found abnormalities in the re-identification of CAF-EVs, the extracted extracellular vesicles may be polluted and the elliptical structure under electron microscope is not owned by CAF-EVs. The identification of CAF-EVs by Western Blots did not refer to the definition of international society." The authors informed the journal that, after the re-experiment, they found that "there is no vesicle specific protein expression, whether the results of subsequent experiments are generated by CAF-EVs needs to be re-tested".

Inhibition of p70 isoforms of S6K1 induces anoikis to prevent transformed human hepatocyte growth

AimsThe mTOR/S6K1 signaling axis, known for cell growth regulation, is hyper-activated in multiple cancers. In this study, we have examined the mechanisms for ribosomal protein p70-S6 kinase 1 (S6K1) associated transformed human hepatocyte (THH) growth regulation. Main methodsTHH were treated with p70-S6K1 inhibitor and analyzed for cell viability, cell cycle distribution, specific marker protein expression by western blot, and tumor inhibition in a xenograft mouse model. We validated our results by knockdown of p70-S6K1 using specific siRNA. Key findingsp70-S6K1 inhibitor treatment caused impairment of in vitro hepatocyte growth, and arrested cell cycle progression at the G1 phase. Further, p70-S6K1 inhibitor treatment exhibited a decrease in FAK and Erk activation, followed by altered integrin-β1 expression, caspase 8, and PARP cleavage appeared to be anoikis like growth inhibition. p70-S6K1 inhibitor also depolymerized actin microfilaments and diminished active Rac1/Cdc42 complex formation for loss of cellular attachment. Similar results were obtained with other transformed human hepatocyte cell lines. p70-S6K1 inhibition also resulted in a reduced phospho-EGFR, Slug and Twist; implicating an inhibition of epithelial-mesenchymal transition (EMT) state. A xenograft tumor model, generated from implanted THH in nude mice, following intraperitoneal injection of S6K1 inhibitor prevented further tumor growth. SignificanceOur results suggested that p70-S6K1 inhibition alters orchestration of cell cycle progression, induces cell detachment, and sensitizes hepatocyte growth impairment. Targeting p70 isoform of S6K1 by inhibitor may prove to be a promising approach together with other therapies for hepatocellular carcinoma (HCC) treatment.

UTERINE SCAR FORMATION AND EXPRESSION OF SPECIFIC CONNECTIVE TISSUE PROTEINS

The aim of the study – to evaluate the role of expression of specific connective tissue proteins in the formation of the scar on the uterus.&#x0D; Materials and Methods. The study was performed on the basis of the regional perinatal center and KU PB No. 5 during 2017–2019. 426 women with a scar on the uterus were examined, including 115 (27.0 %) with two or more scars. The mean age of the subjects was (33.4±1.1) years. The results of sonographic studies were analyzed. For pathomorphological examination, three cases of intranatal rupture of the uterine wall along the “old” scar after cesarean section from middle-aged women with a gestational age of 33 to 40 weeks are presented. The expression of collagen types 1 and 3 and the protein of the intermediate filaments of the muscle tissue of desmin was determined using a semi-quantitative method with the calculation of H-score.&#x0D; Results and Discussion. Analysis of ultrasound data showed that the average assessment of the degree of insolvency of the scar on the uterus was in the examined women X=(1.3±0.2) points, a high level of insolvency was found in 114 (26.8 %) pregnant women. At the same time, 225 (52.8 %) pregnant women showed no signs of scar failure at all. In 87 (20.4 %) one sign of uterine scar failure was found, in 76 (17.8 %) – two signs, in 20 (4.7 %) – three signs, and in 18 (4.2 %) – four signs. Subsequently, 44 (10.3 %) women were born operatively, the rest gave birth per via naturales. Local myometrial defect after ultrasound delivery was detected in 25 of 182 (13.7 %) women who gave birth independently.&#x0D; Conclusions. A high level of scar failure risk is found in 26.8 % of pregnant women. H-score valuesfor collagen types 1 and 3 averaged (212±24) and (188±22), for desmin – (193±17). Thus, reparative processes at the site of previous surgery on the uterine wall are by incomplete regeneration (substitution) and compensatory hyperplasia of structural tissue elements.

NR4A1 counteracts JNK activation incurred by ER stress or ROS in pancreatic β-cells for protection.

Sustained hyperglycaemia and hyperlipidaemia incur endoplasmic reticulum stress (ER stress) and reactive oxygen species (ROS) overproduction in pancreatic β‐cells. ER stress or ROS causes c‐Jun N‐terminal kinase (JNK) activation, and the activated JNK triggers apoptosis in different cells. Nuclear receptor subfamily 4 group A member 1 (NR4A1) is an inducible multi‐stress response factor. The aim of this study was to explore the role of NR4A1 in counteracting JNK activation induced by ER stress or ROS and the related mechanism. qPCR, Western blotting, dual‐luciferase reporter and ChIP assays were applied to detect gene expression or regulation by NR4A1. Immunofluorescence was used to detect a specific protein expression in β‐cells. Our data showed that NR4A1 reduced the phosphorylated JNK (p‐JNK) in MIN6 cells encountering ER stress or ROS and reduced MKK4 protein in a proteasome‐dependent manner. We found that NR4A1 increased the expression of cbl‐b (an E3 ligase); knocking down cbl‐b expression increased MKK4 and p‐JNK levels under ER stress or ROS conditions. We elucidated that NR4A1 enhanced the transactivation of cbl‐b promoter by physical association. We further confirmed that cbl‐b expression in β‐cells was reduced in NR4A1‐knockout mice compared with WT mice. NR4A1 down‐regulates JNK activation by ER stress or ROS in β‐cells via enhancing cbl‐b expression.

Dual Intramolecular Electron Transfer for In Situ Coreactant‐Embedded Electrochemiluminescence Microimaging of Membrane Protein

AbstractThe demand for transporting coreactant to emitter and short lifetime of the radicals in electrochemiluminescence (ECL) emission inhibit greatly its application in cytosensing and microscopic imaging. Herein we designed a dual intramolecular electron transfer strategy and tertiary amine conjugated polymer dots (TEA‐Pdots) to develop a coreactant‐embedded ECL mechanism and microimaging system. The TEA‐Pdots could produce ECL emission at +1.2 V without need of coreactant in test solution. The superstructure and intramolecular electron transfer led to unprecedented ECL strength, which was 132 and 45 times stronger than those from the mixture of Pdots with TEA at equivalent and 62.5 times higher amounts, respectively. The ECL efficiency was even higher than that of typical [Ru(bpy)3]2+ system. Therefore, this strategy and coreactant‐embedded ECL system could be used for in situ ECL microimaging of membrane protein on single living cells without additional permeable treatment for transporting coreactant. The feasibility and validity were demonstrated by evaluating the specific protein expression on cell surface. This work opens new avenues for ECL applications in single cell analysis and dynamic study of biological events.

Dual Intramolecular Electron Transfer for In Situ Coreactant-Embedded Electrochemiluminescence Microimaging of Membrane Protein.

The demand for transporting coreactant to emitter and short lifetime of the radicals in electrochemiluminescence (ECL) emission inhibit greatly its application in cytosensing and microscopic imaging. Herein we designed a dual intramolecular electron transfer strategy and tertiary amine conjugated polymer dots (TEA-Pdots) to develop a coreactant-embedded ECL mechanism and microimaging system. The TEA-Pdots could produce ECL emission at +1.2 V without need of coreactant in test solution. The superstructure and intramolecular electron transfer led to unprecedented ECL strength, which was 132 and 45 times stronger than those from the mixture of Pdots with TEA at equivalent and 62.5 times higher amounts, respectively. The ECL efficiency was even higher than that of typical [Ru(bpy)3 ]2+ system. Therefore, this strategy and coreactant-embedded ECL system could be used for in situ ECL microimaging of membrane protein on single living cells without additional permeable treatment for transporting coreactant. The feasibility and validity were demonstrated by evaluating the specific protein expression on cell surface. This work opens new avenues for ECL applications in single cell analysis and dynamic study of biological events.

MiR‑7/SP1/TP53BP1 axis may play a pivotal role in NSCLC radiosensitivity.

MicroRNA-7 (miR-7) has been identified as a tumor suppressor in non-small cell lung cancer (NSCLC) and a radiosensitivity regulator. Numerous studies have revealed that specific protein 1 (SP1) plays a critical role in the tumorigenesis of various types of cancers and regulates radiosensitivity and tumor suppressor p53-binding protein 1 (TP53BP1), which plays an essential role in DNA repair. However, it is not clear whether miR-7 has a regulatory effect on SP1 and TP53BP1 in NSCLC. In the present study it was revealed that miR-7 directly binds to the 3′UTR of SP1, thereby suppressing SP1 expression to regulate radiosensitivity. Overexpression of miR-7 and SP1 and knockdown of miR-7 and SP1 were performed using lentiviral transfection. Protein and mRNA abundance of SP1 and TP53BP1 were determined using western blotting and RT-qPCR, respectively, while miR-7 binding to SP1 was validated using a luciferase reporter assay. Biological function analysis indicated that miR-7 negatively regulated SP1 and inhibited cell proliferation, migration, and invasion when combined with radiation. It was also revealed that the expression of TP53BP1 was positively regulated by SP1 or negatively regulated by miR-7. In conclusion, SP1 was a target of miR-7, and the decreased expression of SP1 resulting from miR-7 overexpression in NSCLC was vital for improving radiosensitivity in NSCLC cells. Moreover, SP1 expression was detected in 95 paired NSCLC and adjacent normal tissues, and it was determined that SP1 was significantly upregulated in NSCLC tissues and that its upregulation was correlated with the degree of tissue differentiation. Thus, SP1 and/or miR-7 may be potential molecular targets in NSCLC radiotherapy.

Is there a relationship between morphological and functional platelet changes and depressive disorder?

Blood platelets, due to shared biochemical and functional properties with presynaptic serotonergic neurons, constituted, over the years, an attractive peripheral biomarker of neuronal activity. Therefore, the literature strongly focused on the investigation of eventual structural and functional platelet abnormalities in neuropsychiatric disorders, particularly in depressive disorder. Given their impact in biological psychiatry, the goal of the present paper was to review and critically analyze studies exploring platelet activity, functionality, and morpho-structure in subjects with depressive disorder. According to the PRISMA guidelines, we performed a systematic review through the PubMed database up to March 2020 with the search terms: (1) platelets in depression [Title/Abstract]"; (2) "(platelets[Title]) AND depressive disorder[Title/Abstract]"; (3) "(Platelet[Title]) AND major depressive disorder[Title]"; (4) (platelets[Title]) AND depressed[Title]"; (5) (platelets[Title]) AND depressive episode[Title]"; (6) (platelets[Title]) AND major depression[Title]"; (7) platelet activation in depression[All fields]"; and (8) platelet reactivity in depression[All fields]." After a detailed screening analysis and the application of specific selection criteria, we included in our review a total of 106 for qualitative synthesis. The studies were classified into various subparagraphs according to platelet characteristics analyzed: serotonergic system (5-HT2A receptors, SERT activity, and 5-HT content), adrenergic system, MAO activity, biomarkers of activation, responsivity, morphological changes, and other molecular pathways. Despite the large amount of the literature examined, nonunivocal and, occasionally, conflicting results emerged. However, the findings on structural and metabolic alterations, modifications in the expression of specific proteins, changes in the aggregability, or in the responsivity to different pro-activating stimuli, may be suggestive of potential platelet dysfunctions in depressed subjects, which would result in a kind of hyperreactive state. This condition could potentially lead to an increased cardiovascular risk. In line with this hypothesis, we speculated that antidepressant treatments would seem to reduce this hyperreactivity while representing a potential tool for reducing cardiovascular risk in depressed patients and, maybe, in other neuropsychiatric conditions. However, the problem of the specificity of platelet biomarkers is still at issue and would deserve to be deepened in future studies.

BV-2 Microglial Cells Overexpressing C9orf72 Hexanucleotide Repeat Expansion Produce DPR Proteins and Show Normal Functionality but No RNA Foci.

Hexanucleotide repeat expansion (HRE) in the chromosome 9 open-reading frame 72 (C9orf72) gene is the most common genetic cause underpinning frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). It leads to the accumulation of toxic RNA foci and various dipeptide repeat (DPR) proteins into cells. These C9orf72 HRE-specific hallmarks are abundant in neurons. So far, the role of microglia, the immune cells of the brain, in C9orf72 HRE-associated FTLD/ALS is unclear. In this study, we overexpressed C9orf72 HRE of a pathological length in the BV-2 microglial cell line and used biochemical methods and fluorescence imaging to investigate its effects on their phenotype, viability, and functionality. We found that BV-2 cells expressing the C9orf72 HRE presented strong expression of specific DPR proteins but no sense RNA foci. Transiently increased levels of cytoplasmic TAR DNA-binding protein 43 (TDP-43), slightly altered levels of p62 and lysosome-associated membrane protein (LAMP) 2A, and reduced levels of polyubiquitinylated proteins, but no signs of cell death were detected in HRE overexpressing cells. Overexpression of the C9orf72 HRE did not affect BV-2 cell phagocytic activity or response to an inflammatory stimulus, nor did it shift their RNA profile toward disease-associated microglia. These findings suggest that DPR proteins do not affect microglial cell viability or functionality in BV-2 cells. However, additional studies in other models are required to further elucidate the role of C9orf72 HRE in microglia.

Associations of Sedentary Patterns with Cardiometabolic Biomarkers in Physically Active Young Males.

ABSTRACTPurposeSitting time (ST) is a serious global health issue and positively associated with cardiometabolic disease. The present study investigated associations between objectively measured ST, sedentary patterns, and cardiometabolic biomarkers in physically active young males.MethodsCross-sectional analysis was completed in 94 males 18–35 yr of age. Total ST, prolonged sedentary bouts (≥30 min with no interruption), and sedentary breaks (transitions from sitting/lying to standing/stepping) were assessed using activPAL. Lipids, insulin, C-peptide, C-reactive protein (CRP), vascular cellular adhesion molecule-1, intercellular adhesion molecule 1, E-selectin, P-selectin, leptin, resistin, and adiponectin were measured using assay kits. The expression of specific proteins related to endothelial dysfunction was determined using quantitative real-time polymerase chain reaction. Associations between total ST, prolonged sedentary bouts, and sedentary breaks with cardiometabolic biomarkers and total ST and levels of gene expression were assessed using generalized linear models.ResultsTotal ST was significantly associated with triglycerides (B = 1.814), insulin (B = 2.117), homeostasis model assessment of insulin resistance (B = 0.071), and E-selectin (B = 2.052). Leptin (B = 0.086), E-selectin (B = 1.623), and P-selectin (B = 2.519) were significantly associated with prolonged sedentary bouts, whereas leptin (B = −0.017) and CRP (B = −0.016) were associated with sedentary breaks. After adjustment for moderate to vigorous physical activity, the associations between triglycerides (B = 2.048) and total ST, and between CRP (B = −0.016) and sedentary breaks, remained significant. E-selectin mRNA levels (B = 0.0002) were positively associated with ST with or without adjustment for moderate to vigorous physical activity.ConclusionsTotal ST and prolonged sedentary bouts were positively associated with several cardiometabolic biomarkers, with interruptions in ST potentially contributing to reduced cardiometabolic risk in physically active young male adults.

Role of the CXCR4/ALK5/Smad3 Signaling Pathway in Cancer-Induced Bone Pain.

PurposeThe chemokine receptor, CXCR4, and the transforming growth factor-beta receptor, ALK5, both contribute to various processes associated with the sensation of pain. However, the relationship between CXCR4 and ALK5 and the possible mechanisms promoted by ALK5 in the development of pain have not been evaluated.Materials and MethodsTumor cell implantation (TCI) technology was used to generate a model of cancer-induced bone pain (CIBP) in rats; intrathecal (i.t.) injections of small interfering (si) RNAs targeting CXCR4 and the ALK5-specific inhibitor, RepSox, were performed. Behavioral outcomes, Western blotting, and immunofluorescence techniques were used to evaluate the expression of the aforementioned specific target proteins in the CIBP model.ResultsThe results revealed that i.t. administration of siRNAs targeting CXCR4 resulted in significant reductions in both mechanical and thermal hyperalgesia in rats with CIBP and likewise significantly reduced the expression of ALK5 in the spinal cord. Similarly, i.t. administration of RepSox also resulted in significant reductions in mechanical and thermal hyperalgesia in rats with CIBP together with diminished levels of spinal p-Smad3.ConclusionTaken together, our results suggest that CXCR4 expression in the spinal cord may be a critical mediator of CIBP via its capacity to activate ALK5 and downstream signaling pathways.

Wharton's jelly derived mesenchymal stem cells differentiate into oocyte like cells in vitro by follicular fluid and cumulus cells conditioned medium

Wharton's jelly derived-mesenchymal stem cells (WJ-MSCs) have a same developmental origin with primordial germ cells. WJ-MSCs perhaps differentiate into oocyte and germ like-cells (OLCs/GLCs) in the presence of appropriate inducers. Human follicular fluid (FF) and cumulus cells conditioned medium (CCM) are naturally rich sources for oocyte development. The aim of this study was to evaluate WJ-MSCs potential for differentiating into OLCs and GLCs exposed to FF and CCM. WJ-MSCs were cultured in two different induction media (10% FF, 10% CCM) for 21 days. Morphological changes and expression of developmental genes were evaluated on days 0, 7, 14 and 21 of culture. Also, on 21st day of culture, the expression of oocyte and germ cell proteins investigated using immunofluorescence staining. Appearance of round shaped cells from 7th day onwards indicated that WJ-MSCs can differentiate into OLCs when exposed to FF and CCM. The size of produced OLCs and expression of oocyte specific genes and proteins were increased more positively in FF group rather than CCM group. Although, WJ-MSCs could differentiate into OLCs by FF and CCM, however, the induction potential of FF for producing OLCs was better than CCM.

Downregulation of T7 RNA polymerase transcription enhances pET-based recombinant protein production in Escherichia coli BL21 (DE3) by suppressing autolysis.

Escherichia coli BL21 (DE3) is an excellent and widely used host for recombinant protein production. Many variant hosts were developed from BL21 (DE3), but improving the expression of specific proteins remains a major challenge in biotechnology. In this study, we found that when BL21 (DE3) overexpressed glucose dehydrogenase (GDH), a significant industrial enzyme, severe cell autolysis was induced. Subsequently, we observed this phenomenon in the expression of 10 other recombinant proteins. This precludes a further increase of the produced enzyme activity by extending the fermentation time, which is not conducive to the reduction of industrial enzyme production costs. Analysis of membranestructure and messenger RNA expression analysis showed that cells could underwent a form of programmed cell death (PCD) during the autolysis period. However, blocking three known PCD pathways in BL21 (DE3) did not completely alleviate autolysis completely. Consequently, we attempted to develop a strong expression host resistant to autolysis by controlling the speed of recombinant protein expression. To find a more suitable protein expression rate, the high- and low-strength promoter lacUV5 and lac were shuffled and recombined to yield the promoter variants lacUV5-1A and lac-1G. The results showed that only one base in lac promoter needs to be changed, and the A at the+1 position was changed to a G, resulting in the improved host BL21 (DE3-lac1G), which resistant to autolysis. As a consequence, the GDH activity at 43 h was greatly increased from 37.5 to 452.0 U/ml. In scale-up fermentation, the new host was able to produce the model enzyme with a high rate of 89.55 U/ml/h at 43 h, compared to only 3 U/ml/h achieved using BL21 (DE3). Importantly, BL21 (DE3-lac1G) also successfully improved the production of 10 other enzymes. The engineered E. coli strain constructed in this study conveniently optimizes recombinant protein overexpression by suppressing cell autolysis, and shows great potential for industrial applications.

An MRTF-A-Sp1-PDE5 Axis Mediates Angiotensin-II-Induced Cardiomyocyte Hypertrophy.

Cardiac hypertrophy is a critical intermediate step in the pathogenesis of heart failure. A myriad of signaling networks converge on cardiomyocytes to elicit hypertrophic growth in response to various injurious stimuli. In the present study, we investigated the cardiomyocyte-specific role of myocardin-related transcription factor A (MRTF-A) in angiotensin-II (Ang-II)-induced cardiac hypertrophy and the underlying mechanism. We report that conditional MRTF-A deletion in cardiomyocytes attenuated Ang-II-induced cardiac hypertrophy in mice. Similarly, MRTF-A knockdown or inhibition suppressed Ang-II-induced prohypertrophic response in cultured cardiomyocytes. Of note, Ang II treatment upregulated expression of phosphodiesterase 5 (PDE5), a known mediator of cardiac hypertrophy and heart failure, in cardiomyocytes, which was blocked by MRTF-A depletion or inhibition. Mechanistically, MRTF-A activated expression of specificity protein 1 (Sp1), which in turn bound to the PDE5 promoter and upregulated PDE5 transcription to promote hypertrophy of cardiomyocytes in response to Ang II stimulation. Therefore, our data unveil a novel MRTF-A–Sp1–PDE5 axis that mediates Ang-II-induced hypertrophic response in cardiomyocytes. Targeting this newly identified MRTF-A–Sp1–PDE5 axis may yield novel interventional solutions against heart failure.