Kinase Type Research Articles

Gene Expression and Photophysiological Changes in Pocillopora acuta Coral Holobiont Following Heat Stress and Recovery.

The ability of corals to withstand changes in their surroundings is a critical survival mechanism for coping with environmental stress. While many studies have examined responses of the coral holobiont to stressful conditions, its capacity to reverse responses and recover when the stressor is removed is not well-understood. In this study, we investigated among-colony responses of Pocillopora acuta from two sites with differing distance to the mainland (Kusu (closer to the mainland) and Raffles Lighthouse (further from the mainland)) to heat stress through differential expression analysis of target genes and quantification of photophysiological metrics. We then examined how these attributes were regulated after the stressor was removed to assess the recovery potential of P. acuta. The fragments that were subjected to heat stress (2 °C above ambient levels) generally exhibited significant reduction in their endosymbiont densities, but the extent of recovery following stress removal varied depending on natal site and colony. There were minimal changes in chl a concentration and maximum quantum yield (Fv/Fm, the proportion of variable fluorescence (Fv) to maximum fluorescence (Fm)) in heat-stressed corals, suggesting that the algal endosymbionts’ Photosystem II was not severely compromised. Significant changes in gene expression levels of selected genes of interest (GOI) were observed following heat exposure and stress removal among sites and colonies, including Actin, calcium/calmodulin-dependent protein kinase type IV (Camk4), kinesin-like protein (KIF9), and small heat shock protein 16.1 (Hsp16.1). The most responsive GOIs were Actin, a major component of the cytoskeleton, and the adaptive immune-related Camk4 which both showed significant reduction following heat exposure and subsequent upregulation during the recovery phase. Our findings clearly demonstrate specific responses of P. acuta in both photophysiological attributes and gene expression levels, suggesting differential capacity of P. acuta corals to tolerate heat stress depending on the colony, so that certain colonies may be more resilient than others.

Small Molecule Inhibitors in the Treatment of Rheumatoid Arthritis and Beyond: Latest Updates and Potential Strategy for Fighting COVID-19.

The development of biological disease-modifying antirheumatic drugs (bDMARDs) and target synthetic DMARDs (tsDMARDs), also known as small molecule inhibitors, represent a breakthrough in rheumatoid arthritis (RA) treatment. The tsDMARDs are a large family of small molecules targeting mostly the several types of kinases, which are essential in downstream signaling of pro-inflammatory molecules. This review highlights current challenges associated with the treatment of RA using small molecule inhibitors targeting intracellular JAKs/MAPKs/NF-κB/SYK-BTK signaling pathways. Indeed, we have provided the latest update on development of small molecule inhibitors, their clinical efficacy and safety as a strategy for RA treatment. On the other hand, we have highlighted the risk and adverse effects of tsDMARDs administration including, among others, infections and thromboembolism. Therefore, performance of blood tests or viral infection screening should be recommended before the tsDMARDs administration. Interestingly, recent events of SARS-CoV-2 outbreak have demonstrated the potential use of small molecule inhibitors not only in RA treatment, but also in fighting COVID-19 via blocking the viral entry, preventing of hyperimmune activation and reducing cytokine storm. Thus, small molecule inhibitors, targeting wide range of pro-inflammatory singling pathways, may find wider implications not only for the management of RA but also in the controlling of COVID-19.

Schwann Cell Autocrine and Paracrine Regulatory Mechanisms, Mediated by Allopregnanolone and BDNF, Modulate PKCε in Peripheral Sensory Neurons.

Protein kinase type C-ε (PKCε) plays important roles in the sensitization of primary afferent nociceptors, such as ion channel phosphorylation, that in turn promotes mechanical hyperalgesia and pain chronification. In these neurons, PKCε is modulated through the local release of mediators by the surrounding Schwann cells (SCs). The progesterone metabolite allopregnanolone (ALLO) is endogenously synthesized by SCs, whereas it has proven to be a crucial mediator of neuron-glia interaction in peripheral nerve fibers. Biomolecular and pharmacological studies on rat primary SCs and dorsal root ganglia (DRG) neuronal cultures were aimed at investigating the hypothesis that ALLO modulates neuronal PKCε, playing a role in peripheral nociception. We found that SCs tonically release ALLO, which, in turn, autocrinally upregulated the synthesis of the growth factor brain-derived neurotrophic factor (BDNF). Subsequently, glial BDNF paracrinally activates PKCε via trkB in DRG sensory neurons. Herein, we report a novel mechanism of SCs-neuron cross-talk in the peripheral nervous system, highlighting a key role of ALLO and BDNF in nociceptor sensitization. These findings emphasize promising targets for inhibiting the development and chronification of neuropathic pain.

Design, synthesis and anti-cancer activity of pyrrole-imidazole polyamides through target-downregulation of c-kit gene expression.

Pyrrole-imidazole polyamide (PIP) can specifically bind in the B-DNA minor groove that has been used in several biological applications, such as anti-cancer activity, gene expression and translation control, and visualization of complex genomic areas. c-kit is a family member of the Tyrosine Kinase (RTK) type III receptor and plays a vital role in tumor growth, proliferation, differentiation, and cell apoptosis; however, its mutations and overexpression induce tumor dysfunction. Here, we designed and synthesized five matched PIPs that can recognize and bind to the DNA sequence in the oncogene c-kit promoter region, and evaluated their anti-cancer activity. The RTCA assay findings revealed that the PIPs would prevent the proliferation of cancer cells A549 and SGC-7901. EMSA assay showed that the PIPs were actively interacting with the c-kit gene target DNA. RT-PCR and Western blot assays have an effect on expression levels of the c-kit gene in the presence of PIPs. Flow cytometry and wound-healing assays showed that PIPs 4, 5 would cause apoptosis of cancer cells and inhibit the migration of cells, respectively. Overall findings indicate that PIP 5 has a relatively significant intracellular and extracellular impact on the target, contributing to migration and proliferation reduction, and cancer cell apoptosis. In addition, PIP has a certain ability to evolve into c-kit gene-targeting drugs.

Abstract 438: Downregulation of Ca 2+ /calmodulin-dependent Protein Kinase Type II Delta in the Heart by Antisense Oligonucleotides Mitigates Development of Post-infarct Ventricular Arrhythmias

Pathological activation of Ca2+/calmodulin-dependent protein kinase type II delta (CaMK2d) has been implicated in the development of heart failure (HF) and arrhythmia. In preclinical models, inhibition or deletion of cardiac CaMK2d has shown a benefit to prevent HF progression or arrhythmia occurrence. We hypothesized that reduction of CaMK2d in the heart by antisense oligonucleotides (ASO) can mitigate the inducibility of MI-associated ventricular arrhythmias. Eight-week-old female C57BL6/N mice, which received permanent ligation of the left anterior descending coronary artery, were randomized to receive PBS, control ASO or two different mouse CaMK2d ASOs five days after the surgery. The ASOs were administered subcutaneously at 50 mg/kg weekly for four weeks. At the end of study, programmed electrical stimulation (PES) was performed to evaluate ventricular effective refractory period (VERP) and inducibility of ventricular arrhythmias (VA). PCR results showed 70% reduction of cardiac CaMK2d in the CaMK2d ASO groups and no changes of other CaMK2 isoforms. There is no significant change of VERP (VERP expressed as mean±SEM, PBS: 35.2±3.0 ms, control ASO: 36.2±3.8 ms, CaMK2d ASO1: 35.7±2.3 ms and CaMK2d ASO2: 33.8±1.4 ms) while the inducible arrhythmias by PES are declined after CaMK2d ASO treatment (VA inducibility index expressed as mean±SEM, PBS: 1.21±0.27, control ASO: 1.21±0.52, CaMK2d ASO1: 0.54±0.22, CaMK2d ASO2: 0.47±0.22). Echocardiography and invasive hemodynamic studies were unable to demonstrate significant functional improvements in the CaMK2d ASO treated groups, suggesting that the anti-arrhythmia effects of CaMK2d ASO treatment were not secondary to improved cardiac mechanics. In conclusion, ASO-mediated reduction of cardiac CaMK2d in a murine MI model ameliorates MI-associated ventricular arrhythmias, providing in vivo proof-of-concept support of the pathological role of CaMK2d activity in MI-related arrhythmia.

Design, synthesis, and biological evaluation of F-18-labelled 2, 4-diaminopyrimidine-type FAK-targeted inhibitors as potential tumour imaging agents

As a type of intracellular nonreceptor tyrosine kinase, focal adhesion kinase (FAK) can be highly expressed in most types of tumours and is thus regarded as a promising antitumour target. In this study, a series of novel 2,4-diaminopyrimidine FAK-targeted inhibitors were designed, synthesized and characterized by 1H NMR, 13C HNMR, and HRMS spectra. These compounds, with an IC50 range of 5.0–205.1 nM, showed superior inhibitory activity against FAK. Two compounds, [18F]Q-2 and [18F]Q-4, with respective IC50 values of 5.0 nM and 21.6 nM, were labelled by 18F, accompanied by evaluation of their biodistributions. For [18F]Q-2, at 30 min post-injection, promising target-to-nontarget ratios were observed, associated with tumour/blood, tumour/muscle, and tumour/bone ratios of 1.17, 2.99 and 2.19, respectively. The results indicated that [18F]Q-2 is a potential PET tracer for tumour diagnosis.

Mass spectrometry imaging and LC/MS reveal decreased cerebellar phosphoinositides in Niemann-Pick type C1-null mice

Niemann-Pick disease type C1 (NPC1) is a lipid storage disorder in which cholesterol and glycosphingolipids accumulate in late endosomal/lysosomal compartments because of mutations in the NPC1 gene. A hallmark of NPC1 is progressive neurodegeneration of the cerebellum as well as visceral organ damage; however, the mechanisms driving this disease pathology are not fully understood. Phosphoinositides are phospholipids that play distinct roles in signal transduction and vesicle trafficking. Here, we utilized a consensus spectra analysis of MS imaging data sets and orthogonal LC/MS analyses to evaluate the spatial distribution of phosphoinositides and quantify them in cerebellar tissue from Npc1-null mice. Our results suggest significant depletion of multiple phosphoinositide species, including PI, PIP, and PIP2, in the cerebellum of the Npc1-null mice in both whole-tissue lysates and myelin-enriched fractions. Additionally, we observed altered levels of the regulatory enzyme phosphatidylinositol 4-kinase type 2α in Npc1-null mice. In contrast, the levels of related kinases, phosphatases, and transfer proteins were unaltered in the Npc1-null mouse model, as observed by Western blot analysis. Our discovery of phosphoinositide lipid biomarkers for NPC1 opens new perspectives on the pathophysiology underlying this fatal neurodegenerative disease.

GSK-3β regulates the synaptic expression of NMDA receptors via phosphorylation of phosphatidylinositol 4 kinase type IIα.

Deregulation of GSK‐3β is strongly implicated in a variety of serious brain conditions, such as Alzheimer disease, bipolar disorder and schizophrenia. To understand how GSK‐3β becomes dysregulated in these conditions, it is important to understand its physiological functions in the central nervous system. In this context, GSK‐3β plays a role in the induction of NMDA receptor‐dependent long‐term depression (LTD) and several substrates for GSK‐3β have been identified in this form of synaptic plasticity, including KLC‐2, PSD‐95 and tau. Stabilization of NMDA receptors at synapses has also been shown to involve GSK‐3β, but the substrates involved are currently unknown. Recent work has identified phosphatidylinositol 4 kinase type IIα (PI4KIIα) as a neuronal GSK‐3β substrate that can potentially regulate the surface expression of AMPA receptors. In the present study, we investigated the synaptic role of PI4KIIα in organotypic rat hippocampal slices. We found that knockdown of PI4KIIα has no effect on synaptic AMPA receptor‐mediated synaptic transmission but substantially reduces NMDA receptor‐mediated synaptic transmission. Furthermore, the ability of the selective GSK‐3 inhibitor, CT99021, to reduce the amplitude of NMDA receptor‐mediated currents was occluded in shRNA‐PI4KIIα transfected neurons. The effects of knocking down PI4KIIα were fully rescued by a shRNA‐resistant wild‐type construct, but not by a mutant construct that cannot be phosphorylated by GSK‐3β. These data suggest that GSK‐3β phosphorylates PI4KIIα to stabilize NMDA receptors at the synapse.

Development of ErbB2-Targeting Liposomes for Enhancing Drug Delivery to ErbB2-Positive Breast Cancer.

ErbB2 is a type of receptor tyrosine kinase, which is known to be involved in tumorigenesis, tumor aggressiveness, and clinical outcome. ErbB2-targeting therapy using therapeutic antibodies has been successful in breast cancer treatment. However, the need for repeated treatments and the high cost are major disadvantages with monoclonal antibody therapies. Compared with antibodies, peptides are cheap, relatively stable, and have low immunogenicity. We have developed a highly specific cancer-targeting drug delivery system using a targeting peptide to maximize the therapeutic efficiency of rapamycin and to help prevent drug resistance in ErbB2-positive breast cancer. Physicochemical characterization confirmed the successful construction of ErbB2-targeting liposomes (ErbB2Lipo). A comparison of a scrambled peptide (ScrErbB2) with the ErbB2-targeting peptide confirmed that these peptides had similar properties except for the targeting ability. The ErbB2Lipo exhibited higher delivery efficiency in ErbB2 positive BT-474 cells than non-targeting liposomes conjugated with ScrErbB2 (ScrErbB2Lipo). This peptide-targeting strategy has the potential to improve the efficacy of chemotherapy in ErbB2-positive cancers.

Identification of Four Potential Biomarkers Associated With Coronary Artery Disease in Non-diabetic Patients by Gene Co-expression Network Analysis.

BackgroundCoronary artery disease (CAD) is a type of cardiovascular disease that greatly hurts the health of human beings. Diabetic status is one of the largest clinical factors affecting CAD-associated gene expression changes. Most of the studies focus on diabetic patients, whereas few have been done for non-diabetic patients. Since the pathophysiological processes may vary among these patients, we cannot simply follow the standard based on the data from diabetic patients. Therefore, the prognostic and predictive diagnostic biomarkers for CAD in non-diabetic patient need to be fully recognized.Materials and MethodsTo screen out candidate genes associated with CAD in non-diabetic patients, weighted gene co-expression network analysis (WGCNA) was constructed to conduct an analysis of microarray expression profiling in patients with CAD. First, the microarray data GSE20680 and GSE20681 were downloaded from NCBI. We constructed co-expression modules via WGCNA after excluding the diabetic patients. As a result, 18 co-expression modules were screened out, including 1,225 differentially expressed genes (DEGs) that were obtained from 152 patients (luminal stenosis ≥50% in at least one major vessel) and 170 patients (stenosis of <50%). Subsequently, a Pearson’s correlation analysis was conducted between the modules and clinical traits. Then, a functional enrichment analysis was conducted, and we used gene network analysis to reveal hub genes. Last, we validated the hub genes with peripheral blood samples in an independent patient cohort using RT-qPCR.ResultsThe results showed that the midnight blue module and the yellow module played vital roles in the pathogenesis of CAD in non-diabetic patients. Additionally, CD40, F11R, TNRC18, and calcium/calmodulin-dependent protein kinase type II gamma (CAMK2G) were screened out and validated using enzyme-linked immunosorbent assay (ELISA) in an independent patient cohort and immunohistochemical (IHC) staining in an atherosclerosis mouse model.ConclusionOur findings demonstrate that hub genes, CD40, F11R, TNRC18, and CAMK2G, are surrogate diagnostic biomarkers and/or therapeutic targets for CAD in non-diabetic patients and require deeper validation.

Targets of cGMP/cGKI in Cardiac Myocytes.

The 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase type I (cGKI aka PKGI) is a major cardiac effector acting downstream of nitric oxide (NO)-sensitive soluble guanylyl cyclase and natriuretic peptides (NPs), which signal through transmembrane guanylyl cyclases. Consistent with the wide distribution of the cGMP-generating guanylyl cyclases, cGKI, which usually elicits its cellular effects by direct phosphorylation of its targets, is present in multiple cardiac cell types including cardiomyocytes (CMs). Although numerous targets of cGMP/cGKI in heart were identified in the past, neither their exact patho-/physiological functions nor cell-type specific roles are clear. Herein, we inform about the current knowledge on the signal transduction downstream of CM cGKI. We believe that better insights into the specific actions of cGMP and cGKI in these cells will help to guide future studies in the search for predictive biomarkers for the response to pharmacological cGMP pathway modulation. In addition, targets downstream of cGMP/cGKI may be exploited for refined and optimized diagnostic and therapeutic strategies in different types of heart disease and their causes. Importantly, key functions of these proteins and particularly sites of regulatory phosphorylation by cGKI should, at least in principle, remain intact, although upstream signaling through the second messenger cGMP is impaired or dysregulated in a stressed or diseased heart state.

Relationship between Sphk1/S1P and microRNAs in human cancers.

Sphingosine kinases type 1 (SphK1) is a key enzyme in the phosphorylation of sphingosine to sphingosine 1-phosphate (S1P). Different abnormalities in SphK1 functions may correspond with poor prognosis in various cancers. Additionally, upregulated SphK1/S1P could promote cancer cell proliferation, angiogenesis, mobility, invasion, and metastasis. MicroRNAs as conserved small noncoding RNAs play major roles in cancer initiation, progression, metastasis, etc. Their posttranscriptionally mechanisms could affect the development of cancer growth or tumorigenesis suppression. The growing number of studies has described that various microRNAs can be regulated by SphK1, and its expression level can also be regulated by microRNAs. In this review, the relationship of SphK1 and microRNA functions and their interaction in human malignancies have been discussed. Based on them novel treatment strategies can be introduced.

Anterior thalamic nucleus stimulation protects hippocampal neurons by activating autophagy in epileptic monkeys.

Deep brain stimulation of the anterior nucleus of the thalamus (ANT-DBS) is effective in treating temporal lobe epilepsy (TLE) and protects hippocampal neurons. Autophagy plays an essential role in epileptogenesis; however, the underlying effect of autophagy on ANT-DBS-mediated neuroprotection remains unclear. A monkey model of epilepsy was established by injecting kainic acid into the hippocampus and amygdala using a robot-assisted system. ANT-DBS was delivered in the chronic stage of the epileptic model and continued for 8 weeks. We found that ANT-DBS reduced the frequency of seizures and exerted neuroprotective effects via activating autophagy in hippocampal neurons. ANT-DBS increased light chain 3 (LC3) II level and co-localization of LC3 and lysosomal-associated membrane protein-1, accompanied by decreased expression of the autophagy substrate ubiquitin-binding protein p62, suggesting increased autophagosome formation. Most importantly, brain-derived neurotrophic factor (BDNF) –tropomyosin-related kinase type B (TrkB) pathway were involved in the regulation of autophagy. Both protein levels were reduced by ANT-DBS, and there was less phosphorylation of downstream regulators, extracellular signal-regulated kinase and Akt, followed by inactivation of mammalian target of rapamycin complex 1. Taken together, chronic ANT-DBS exerts neuroprotective effects on hippocampal neurons through inducing autophagy via suppressing the BDNF–TrkB pathway in a TLE monkey model.

Role of PCSK1N and GPR83 in salt resistance

Previous studies from our laboratory show that in mice increased expression/function of the orphan receptor GPR83 is associated with salt resistance. GPR83 is expressed in proximal and distal convoluted tubules of the mouse kidney. Global deletion or renal‐specific silencing of the GPR83 gene increases systolic blood pressure (SBP) and increased G protein‐coupled receptor kinase type 4 (GRK4) expression in the kidney. Recent evidence indicated that peptides derived from the Proprotein Convertase Subtilisin/Kexin Type 1 Inhibitor (PCSK1N) may be the endogenous ligands of GPR83. We hypothesized that these peptides are implicated in the regulation of blood pressure. Renal specific siRNA‐mediated silencing of PCSK1N in mice resulted in an increase in SBP (130±4 vs control wild‐type 100±1 mmHg, n=3/group; P&lt;0.05); this was associated with decreased urinary Na+ excretion (P&lt;0.05) and decreased renal cortex expression of GPR83, but increased NHE3 and Na+/K+‐ATPase expressions (P&lt;0.05 all). The peptides PEN (proline, glutamic acid, and asparagine) and SAAS (serine, alanine, alanine, and serine) are derived from enzymatic breakdown of Pro‐SAAS. Renal restricted infusion of PEN (1 μg/day; 7 days) did not affect SBP, slightly decreased Na+ excretion and increased GPR83, GRK4, and NHE3 expressions (P&lt;0.05 all) but did not affect Na+/K+‐ATPase expression. By contrast, renal‐restricted infusion of SAAS (1 μg/day; 7 days) in mice decreased SBP (82± vs 100± mmHg; P&lt;0.05), Na+ excretion did not change but decreased the renal cortex expression of GRK4, GPR83, NHE3 and Na+/K+‐ATPAse (P&lt;0.05 all). Furthermore, PCSK1N expression was increased by a high salt diet (4%NaCl) in non‐transgenic mice but was decreased in the salt‐sensitive GRK4 486V mice on the same diet. Our result suggested that Pro‐SAAS peptides may be involved in the development of salt sensitivity through gene‐gene interactions among PCSK1N, GPR83, and GRK4.Support or Funding InformationThis work was supported by NIH grant t R01DK039308 (P.A.J.);NIH grant R37HL023081(P.A.J.); NIH grant P01HL074940 (P.A.J.); NIH grant P01HL068686 (P.A.J.); NIH grant R01HL092196 (P.A.J., I.A.); NIH grant R01DK09098 (I.A.)

Baicalin alleviates bleomycin‑induced pulmonary fibrosis and fibroblast proliferation in rats via the PI3K/AKT signaling pathway.

Baicalin is an important flavonoid compound THAT is isolated from the Scutellaria baicalensis Georgi Chinese herb and plays a critical role in anti-oxidative, anti-inflammatory, anti-infection and anti-tumor functions. Although baicalin can suppress the proliferation of tumor cells, the underlying mechanisms of baicalin in bleomycin (BLM)-induced pulmonary fibrosis remain to be elucidated. Thus, the aim of the present study was to determine the role of baicalin in pulmonary fibrosis and fibroblast proliferation in rats. Hematoxylin and eosin (H&E) and Masson staining were used to measure the morphology of pulmonary fibrosis, ELIASA kits were used to test the ROS and inflammation, and western blotting and TUNEL were performed to study the apoptosis proteins. In vitro, MTT assay, flow cytometry, western blotting and immunofluorescence were performed to investigate the effects of baicalin on proliferation of fibroblasts. The most significantly fibrotic changes were identified in the lungs of model rats at day 28. Baicalin (50 mg/kg) attenuated the degree of pulmonary fibrosis, and the hydroxyproline content of the lung tissues was decreased in the baicalin group, compared with the BLM group. Further investigation revealed that baicalin significantly increased glutathione peroxidase (GSH-px), total-superoxide dismutase (T-SOD) and glutathione (GSH) levels, whilst decreasing that of serum malondialdehyde (MDA). TUNEL-positive cells were significantly decreased in rats treated with baicalin group, compared with the model group. Furthermore, it was found that BLM promoted fibroblasts viability in a dose-dependent manner in vivo, which was restricted following treatment with different concentrations of baicalin. Moreover, BLM promoted the expression levels of cyclin A, D and E, proliferating cell nuclear antigen, phosphorylated (p)-AKT and p-calcium/calmodulin-dependent protein kinase type. BLM also promoted the transition of cells from the G0/G1 phase to the G2/M and S phases, and increased the intracellular Ca2+ concentration, which was subsequently suppressed by baicalin. Collectively, the results of the present study suggested that baicalin exerted a suppressive effect on BLM-induced pulmonary fibrosis and fibroblast proliferation.

Calcium-calmodulin-dependent protein kinase type 2 induces apoptosis of hepatocytes after liver transplantation.

The warm ischemia-reperfusion injury confines the prevalence of allografts. To improve the success rate of allotransplantation, we designed experiments to study the mechanism of calcium-calmodulin-dependent protein kinase type 2 (CaMK II) in ischemia-reperfusion (I/R) injury. We established the I/R model in SD rats and performed the liver transplantation (LT). As a result, the expression of CaMK II in tissues was detected. CaMK II was interfered with and overexpressed by the transference of the lentivirus vector, and the hepatocyte apoptosis and viability were inspected. At the same time, the content of cytochrome c and apoptosis-inducing factor (AIF) were determined. The measurement of mitochondrial membrane potential and detection of intercellular calcium levels were performed. The expression of CaMK II significantly increased and is highly corresponded with the duration of warm ischemia. In BRL-3A cells and liver tissues, increased cellular apoptosis and less viability had been observed in the CaMK II overexpression group. Cytochrome c and AIF were also largely increased compared to the interfered group. Moreover, apparent mitochondrial membrane potential loss has also been detected in the CaMK II overexpression group. It suggested that CaMK II induces cell apoptosis. Our findings may give a novel indication that inhibition of CaMK II could be a new way for the therapy of warm ischemia-reperfusion injury after LT in future clinical practice.

Assessment of the Phenolic Profiles, Hypoglycemic Activity, and Molecular Mechanism of Different Highland Barley (Hordeum vulgare L.) Varieties.

The phenolic profiles, hypoglycemic activity, and molecular mechanism of the effect on type 2 diabetes mellitus (T2DM) of four highland barley varieties were investigated in the present study. The fundamental phenolics in highland barley were ferulic acid, naringin, and catechin, which mainly existed in bound form. These varieties showed favorable hypoglycemic activity via inhibition of α-glucosidase and α-amylase activities, enhancement of glucose consumption, glycogen accumulation and glycogen synthase 2 (GYS2) activity, and down-regulation of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) activities. Specifically, ZQ320 variety exhibited the strongest hypoglycemic activity compared to the other varieties. Highland barley phenolics could inhibit gluconeogenesis and motivate glycogen synthesis via down-regulating the gene expression of G6Pase, PEPCK, and glycogen synthase kinase 3β (GSK3β), while activating the expression of insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3 kinase (PI3K), serine/threonine kinase (Akt), GYS2, and glucose transporter type 4 (GLUT4). Therefore, phenolics from highland barley could be served as suitable candidates for therapeutic agent in T2DM to improve human health.

Rational Drug Design of Axl Tyrosine Kinase Type I Inhibitors as Promising Candidates Against Cancer.

The high level of Axl tyrosine kinase expression in various cancer cell lines makes it an attractive target for the development of anti-cancer drugs. In this study, we carried out several sets of in silico screening for the ATP-competitive Axl kinase inhibitors based on different molecular docking protocols. The best drug-like candidates were identified, after parental structure modifications, by their highest affinity to the target protein. We found that our newly designed compound R5, a derivative of the R428 patented analog, is the most promising inhibitor of the Axl kinase according to the three molecular docking algorithms applied in the study. The molecular docking results are in agreement with the molecular dynamics simulations using the MM-PBSA/GBSA implicit solvation models, which confirm the high affinity of R5 toward the protein receptor. Additionally, the selectivity test against other kinases also reveals a high affinity of R5 toward ABL1 and Tyro3 kinases, emphasizing its promising potential for the treatment of malignant tumors.

Remifentanil protects neurological function of rats with cerebral ischemia-reperfusion injury via NR2B/CaMKIIα signaling pathway.

The effects of remifentanil were studied on cerebral ischemia-reperfusion injury (IRI) in rats, and its regulatory effect was explored on the N-methyl D-aspartate receptor subtype 2B (NR2B)/calcium/calmodulin-dependent protein kinase type II subunit alpha (CaMKII) signaling pathway in cerebral tissues. A total of 40 Sprague-Dawley rats were randomly assigned into sham group, model group, low-dose group (remifentanil injected into the caudal veins at 2 μg/kg) and high-dose group (remifentanil injected into the caudal veins at 10 μg/kg). Then, in the model, low-dose and high-dose groups, the rat model of cerebral IRI was established through middle cerebral artery occlusion (namely, ischemia for 1 h and reperfusion for 2 h), while no thread was inserted into the rats in the sham group. Neurological function of rats in each group was evaluated, and the cerebral infarct size was measured using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Moreover, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was performed to determine the neuronal apoptosis level, and mitochondrial membrane potential was measured via JC-1 assay. Moreover, the reactive oxygen species (ROS) level in neurons and the malondialdehyde (MDA) content in cerebral tissues were determined using 2',7'-dichlorofluorescin diacetate (DCFH-DA) assay and enzyme-linked immunosorbent assay (ELISA), respectively. Finally, the expression levels of apoptosis-associated proteins and the NR2B/CaMKIIα signaling pathway-associated proteins in cerebral tissues were measured by Western blotting. Remifentanil preconditioning substantially decreased the neurological score of rats (p<0.01), cerebral infarct size (p<0.01), neuronal apoptosis level (p<0.01), ROS level in neurons (p<0.01), MDA content (p<0.01) and expression levels of cysteinyl aspartate specific proteinase-3 (Caspase-3), NR2B, phosphorylated CaMKIIα (p-CaMKIIα) and p-cAMP responsive element binding protein (p-CREB) (p<0.01), but it increased the mitochondrial membrane potential (p<0.01) and B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein (Bax) ratio (p<0.01). Remifentanil can repress the NR2B/ CaMKIIα signaling pathway in the neurons of rats with cerebral IRI to decrease the p-CREB expression, ROS level and MDA content in neurons, neuronal apoptosis level and cerebral infarct size, and increase the mitochondrial membrane potential, thereby protecting the neurological function.

Experimental Study of Hepatocellular Carcinoma Treatment by Shikonin Through Regulating PKM2.

ObjectiveShikonin is a natural product with many activities, including anti-cancer effects. Pyruvate kinase type M2 (PKM2) plays a crucial role in the growth of tumor cells. However, the effect of shikonin on PKM2 in hepatocellular carcinoma (HCC) is unclear.MethodsCell viability, apoptosis level, glucose uptake, and lactate production were detected in HCC cells. Lentivirus-overexpressed and –shRNA of PKM2 were used to verify the key target of shikonin. A xenograft mouse model was used to detect the efficacy of shikonin and its combination with sorafenib in vivo.ResultsShikonin inhibited proliferation and glycolysis and induced apoptosis in HCC cells. Either PKM2-overexpressed or PKM2-shRNA alleviated or enhanced this effect. The results of CCK-8 showed that shikonin significantly inhibited cell viability of HCC cells. The levels of glucose uptake and lactate production were dramatically decreased by shikonin-treated. Results of flow cytometry and Western blot showed that the levels of apoptosis of HCC cells were significantly increased in a dose-dependent manner after shikonin treatment. In addition, shikonin enhanced the anti-cancer effect of sorafenib in vitro and in vivo. Our results showed that SK combined with sorafenib markedly inhibits tumor growth in HCC-transplanted nude mice compared to SK or sorafenib alone.ConclusionBy inhibiting PKM2, shikonin inhibited proliferation and glycolysis and induced cell apoptosis in HCC cells. The effect of shikonin on tumor cell proliferation, apoptosis and glycolsis will make it promising drug for HCC patients.