Identification of suitable reference genes for expression studies in pomegranate under different biotic and abiotic stress conditions

Pomegranate (Punica granatum L.) is an important economic fruit crop, facing many biotic and abiotic challenges during cultivation. Several research programs are in progress to understand both biotic and abiotic stress factors and mitigate these challenges using gene expression studies based on the qPCR approach. However, research publications are not available yet to select the standard reference gene for normalizing target gene expression values in pomegranate. The most suitable candidate reference gene is required to ensure precise and reliable results for qPCR analysis. Eight candidate reference genes' stability was evaluated under different stress conditions using different algorithms such as ?Ct, geNorm, BestKeeper, NormFinder, and RefFinder. The various algorithms revealed that EFA1 and 18S rRNA were common and most stable reference genes (RGs) under abiotic and wilt stress. Whereas comprehensive ranking by RefFinder showed GAPDH and CYPF were the most stable RGs under combined biotic (pooled samples of all biotic stress) and bacterial blight samples. For normalizing target gene expression under wilt, nematode, bacterial blight, and abiotic stress conditions both GAPDH and CYPFreference genes are adequate for qPCR. The above data provide comprehensive details for the selection of a candidate reference gene in various stresses in pomegranate

     

Proteomic profiling of S-acylated macrophage proteins identifies a role for palmitoylation in mitochondrial targeting of phospholipid scramblase 3

S-Palmitoylation, the reversible post-translational acylation of specific cysteine residues with the fatty acid palmitate, promotes the membrane tethering and subcellular localization of proteins in several biological pathways. Although inhibiting palmitoylation holds promise as a means for manipulating protein targeting, advances in the field have been hampered by limited understanding of palmitoylation enzymology and consensus motifs. In order to define the complement of S-acylated proteins in the macrophage, we treated RAW 264.7 macrophage membranes with hydroxylamine to cleave acyl thioesters, followed by biotinylation of newly exposed sulfhydryls and streptavidin-agarose affinity chromatography. Among proteins identified by LC-MS/MS, S-acylation status was established by spectral counting to assess enrichment under hydroxylamine versus mock treatment conditions. Of 1183 proteins identified in four independent experiments, 80 proteins were significant for S-acylation at false discovery rate = 0.05, and 101 significant at false discovery rate = 0.10. Candidate S-acylproteins were identified from several functional categories, including membrane trafficking, signaling, transporters, and receptors. Among these were 29 proteins previously biochemically confirmed as palmitoylated, 45 previously reported as putative S-acylproteins in proteomic screens, 24 not previously associated with palmitoylation, and three presumed false-positives. Nearly half of the candidates were previously identified by us in macrophage detergent-resistant membranes, suggesting that palmitoylation promotes lipid raft-localization of proteins in the macrophage. Among the candidate novel S-acylproteins was phospholipid scramblase 3 (Plscr3), a protein that regulates apoptosis through remodeling the mitochondrial membrane. Palmitoylation of Plscr3 was confirmed through (3)H-palmitate labeling. Moreover, site-directed mutagenesis of a cluster of five cysteines (Cys159-161-163-164-166) abolished palmitoylation, caused Plscr3 mislocalization from mitochondrion to nucleus, and reduced macrophage apoptosis in response to etoposide, together suggesting a role for palmitoylation at this site for mitochondrial targeting and pro-apoptotic function of Plscr3. Taken together, we propose that manipulation of protein palmitoylation carries great potential for intervention in macrophage biology via reprogramming of protein localization.     

Myocardial oxygen consumption change predicts left ventricular relaxation improvement in obese humans after weight loss

Obesity adversely affects myocardial metabolism, efficiency, and diastolic function. Our objective was to determine whether weight loss can ameliorate obesity-related myocardial metabolism and efficiency derangements and that these improvements directly relate to improved diastolic function in humans. We studied 30 obese (BMI >30 kg/m2) subjects with positron emission tomography (PET) (myocardial metabolism, blood flow) and echocardiography (structure, function) before and after marked weight loss from gastric bypass surgery (N = 10) or moderate weight loss from diet (N = 20). Baseline BMI, insulin resistance, hemodynamics, left ventricular (LV) mass, systolic function, myocardial oxygen consumption (MVO2), and fatty acid (FA) metabolism were similar between the groups. MVO2/g decreased after diet-induced weight loss (P = 0.009). Total MVO2 decreased after dietary (P = 0.02) and surgical weight loss (P = 0.0006) and was related to decreased BMI (P = 0.006). Total myocardial FA utilization decreased (P = 0.03), and FA oxidation trended lower (P = 0.06) only after surgery. FA esterification and LV efficiency were unchanged. After surgical weight loss, LV mass decreased by 23% (Doppler-derived) E/E' by 33%, and relaxation increased (improved) by 28%. Improved LV relaxation related significantly to decreased BMI, insulin resistance, total MVO2, and LV mass but not FA utilization. Decreased total MVO(2) predicted LV relaxation improvement independent of BMI change (P = 0.02). Weight loss can ameliorate the obesity-related derangements in myocardial metabolism and LV structure and diastolic function. Decreased total MVO2 independently predicted improved LV relaxation, suggesting that myocardial oxygen metabolism may be mechanistically important in determining cardiac relaxation.     

A Calcium-Dependent Plasticity Rule for HCN Channels Maintains Activity Homeostasis and Stable Synaptic Learning

Theoretical and computational frameworks for synaptic plasticity and learning have a long and cherished history, with few parallels within the well-established literature for plasticity of voltage-gated ion channels. In this study, we derive rules for plasticity in the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, and assess the synergy between synaptic and HCN channel plasticity in establishing stability during synaptic learning. To do this, we employ a conductance-based model for the hippocampal pyramidal neuron, and incorporate synaptic plasticity through the well-established Bienenstock-Cooper-Munro (BCM)-like rule for synaptic plasticity, wherein the direction and strength of the plasticity is dependent on the concentration of calcium influx. Under this framework, we derive a rule for HCN channel plasticity to establish homeostasis in synaptically-driven firing rate, and incorporate such plasticity into our model. In demonstrating that this rule for HCN channel plasticity helps maintain firing rate homeostasis after bidirectional synaptic plasticity, we observe a linear relationship between synaptic plasticity and HCN channel plasticity for maintaining firing rate homeostasis. Motivated by this linear relationship, we derive a calcium-dependent rule for HCN-channel plasticity, and demonstrate that firing rate homeostasis is maintained in the face of synaptic plasticity when moderate and high levels of cytosolic calcium influx induced depression and potentiation of the HCN-channel conductance, respectively. Additionally, we show that such synergy between synaptic and HCN-channel plasticity enhances the stability of synaptic learning through metaplasticity in the BCM-like synaptic plasticity profile. Finally, we demonstrate that the synergistic interaction between synaptic and HCN-channel plasticity preserves robustness of information transfer across the neuron under a rate-coding schema. Our results establish specific physiological roles for experimentally observed plasticity in HCN channels accompanying synaptic plasticity in hippocampal neurons, and uncover potential links between HCN-channel plasticity and calcium influx, dynamic gain control and stable synaptic learning.     

Meta-Analysis Identifies NF-κB as a Therapeutic Target in Renal Cancer

Objective

To determine the expression patterns of NF-κB regulators and target genes in clear cell renal cell carcinoma (ccRCC), their correlation with von Hippel Lindau (VHL) mutational status, and their association with survival outcomes.

Methods

Meta-analyses were carried out on published ccRCC gene expression datasets by RankProd, a non-parametric statistical method. DEGs with a False Discovery Rate of < 0.05 by this method were considered significant, and intersected with a curated list of NF-κB regulators and targets to determine the nature and extent of NF-κB deregulation in ccRCC.

Results

A highly-disproportionate fraction (~40%; p < 0.001) of NF-κB regulators and target genes were found to be up-regulated in ccRCC, indicative of elevated NF-κB activity in this cancer. A subset of these genes, comprising a key NF-κB regulator (IKBKB) and established mediators of the NF-κB cell-survival and pro-inflammatory responses (MMP9, PSMB9, and SOD2), correlated with higher relative risk, poorer prognosis, and reduced overall patient survival. Surprisingly, levels of several interferon regulatory factors (IRFs) and interferon target genes were also elevated in ccRCC, indicating that an ‘interferon signature’ may represent a novel feature of this disease. Loss of VHL gene expression correlated strongly with the appearance of NF-κB- and interferon gene signatures in both familial and sporadic cases of ccRCC. As NF-κB controls expression of key interferon signaling nodes, our results suggest a causal link between VHL loss, elevated NF-κB activity, and the appearance of an interferon signature during ccRCC tumorigenesis.

Conclusions

These findings identify NF-κB and interferon signatures as clinical features of ccRCC, provide strong rationale for the incorporation of NF-κB inhibitors and/or and the exploitation of interferon signaling in the treatment of ccRCC, and supply new NF-κB targets for potential therapeutic intervention in this currently-incurable malignancy.     

Effects of insecticides on behavior of adult Bactericera cockerelli (Hemiptera: Triozidae) and transmission of Candidatus Liberibacter psyllaurous

The potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), is a serious pest of potatoes (Solanum tuberosum L.) that can cause yield loss by direct feeding on crop plants and by vectoring a bacterial pathogen, Candidatus Liberibacer psyllaurous. Current pest management practices rely on the use of insecticides to control the potato psyllid to lower disease incidences and increase yields. Although many studies have focused on the mortality that insecticides can cause on potato psyllid populations, little is known regarding the behavioral responses of the potato psyllid to insecticides or whether insecticides can decrease pathogen transmission. Thus, the objectives of this study were to determine the effects of insecticides on adult potato psyllid behaviors, the residual effects of insecticides on potato psyllid behaviors over time, and effects of these insecticides on Ca. L. psyllaurous transmission. Insecticides tested included imidacloprid, kaolin particle film, horticultural spray oil, abamectin, and pymetrozine. All insecticides significantly reduced probing durations and increased the amount of time adult psyllids spent off the leaflets, suggesting that these chemicals may be deterrents to feeding as well as repellents. Nonfeeding behaviors such as tasting, resting, and cleaning showed variable relationships with the different insecticide treatments over time. The insecticides imidacloprid and abamectin significantly lowered transmission of Ca. L. psyllaurous compared with untreated controls. The implications of our results for the selection of insecticides useful for an integrated pest management program for potato psyllid control are discussed.     

Evaluation of serum proteome from Indian psoriasis patients

Psoriasis is a polygenic chronic skin condition, associated with many systemic disorders. Though it is most studied dermatological condition, molecular mechanism leading to its pathogenesis is still unclear. An insight into its proteome may help unrevealing some biomarkers and therapeutic targets. In this study, we carried out mass spectrometry based quantitative proteomic analysis of serum from psoriasis patients by employing Tandem Mass Tags (TMT) approach. We identified 861,887 MS/MS spectra corresponding to 493 proteins. These dysregulated proteins were further classified by Gene Ontology and protein-protein interaction of dys-regulated proteins revealed networks in psoriasis patients.