Changes In Molecular Expression Research Articles

Serial Changes of Circulating Tumor Cells in Patients with Hepatocellular Carcinoma Treated with Atezolizumab Plus Bevacizumab.

Immune checkpoint inhibitors have promising outcomes in patients with hepatocellular carcinoma (HCC); however, there is no reliable biomarker for predicting disease progression. Circulating tumor cells (CTCs) derived from peripheral blood have attracted attention in monitoring therapeutic efficacy. In this study, CTCs were serially collected from HCC patients undergoing atezolizumab plus bevacizumab (Atezo+Bev), and changes in molecular expression and CTC numbers were analyzed to identify effective biomarkers. Changes in CTC numbers during Atezo+Bev reflected the tumor volume. Targeted RNA sequencing with next-generation sequencing (NGS) revealed that patients with elevated transforming growth factor (TGF)-β signaling molecules had a poorer response, whereas those with elevated apoptosis signaling molecules had a favorable response. In addition, compared with changes in CTC counts, changes in TGF-β signaling molecule expression in CTCs accurately and promptly predicted treatment response. Overall, NGS analysis of CTC-derived RNA showed that changes in TGF-β signaling molecules predict treatment response earlier than changes in CTC counts. These findings suggest that changes in the expression of TGF-β molecules in CTCs could serve as novel biomarkers for the early prediction of therapeutic response in patients with unresectable HCC undergoing Atezo+Bev.

Curcumin derivative NL01 induces ferroptosis in ovarian cancer cells via HCAR1/MCT1 signaling

ObjectiveCurcumin has been shown to have anti-tumor proliferative properties, but its clinical application is limited by its low bioavailability, etc. Derivatives of curcumin have been developed and tested to improve its therapeutic efficacy. Derivative NL01 could induce ferroptosis through the HCAR1/MCT1 pathway. MethodCCK-8 was used to detect curcumin and derivative IC50, crystalline violet staining was used to detect the proliferation inhibition effect of NL01 in ovarian cancer, western blot and qPCR were used to detect downstream related molecular expression changes, Transwell and survival curve assays were used to detect malignant phenotypic. ResultsNL01 inhibited cell growth of Anglne and HO8910PM ovarian cancer cells by 13 times more potent than curcumin and induced ferroptosis of these two cells. we found that NL01 was able to reduce the expression of HCAR1/MCT1 and activate the AMPK signaling pathway, which in turn induced cellular ferroptosis via SREBP1 pathway. Knock-down HCAR1 expression revealed similar phenotype and pathway alterations to NL01 treatment. HCAR1 overexpression promoted a malignant phenotype and resistance to cisplatin in both cancer cells, whereas knockdown of HCAR1 showed the opposite phenotype. Subcutaneous transplantation tumor experiments in nude mice also showed that NL01 induced iron death and inhibited ovarian cancer proliferation. Further study showed that NL01 promoted the downregulation of GPX4 expression, which is related to ferroptosis, and that addition of ferrostatin-1 partially reversed NL01-mediated inhibition of the growth of two cell lines. ConclusionNL01 exhibits better anti-tumor growth properties than curcumin, and NL01 induces ferroptosis in ovarian cancer cells.

Therapeutic effect of umbilical cord blood cells on spinal cord injury.

Spinal cord injury (SCI) is a nervous system disease characterized by sensory and motor dysfunction, axonal apoptosis, decreased vascular density, and inflammation. At present, surgical treatment, drug treatment, and cell therapy can be used. Surgical treatment can improve motor and independent function scores, and drug treatment can promote the recovery of neurons in the spinal cord, but only improve symptoms. Complete recovery of SCI has not yet been achieved. However, the differentiation of stem cells brings hope for the treatment of SCI. Umbilical cord blood cells (UCBs) are ethically readily available and can repair neuronal damage. However, it is still unclear how they can improve symptoms and repair nerve severity. In this paper, the role of UCBs in the treatment of SCI is described in detail from different aspects such as behavior, morphology, and molecular expression changes, so as to provide new ideas and theoretical directions for future research.

Olfactory neuronal cells as a promising tool to realize the "druggable genome" approach for drug discovery in neuropsychiatric disorders.

"Druggable genome" is a novel concept that emphasizes the importance of using the information of genome-wide genetic studies for drug discovery and development. Successful precedents of "druggable genome" have recently emerged for some disorders by combining genomic and gene expression profiles with medical and pharmacological knowledge. One of the key premises for the success is the good access to disease-relevant tissues from "living" patients in which we may observe molecular expression changes in association with symptomatic alteration. Thus, given brain biopsies are ethically and practically difficult, the application of the "druggable genome" approach is challenging for neuropsychiatric disorders. Here, to fill this gap, we propose the use of olfactory neuronal cells (ONCs) biopsied and established via nasal biopsy from living subjects. By using candidate genes that were proposed in a study in which genetic information, postmortem brain expression profiles, and pharmacological knowledge were considered for cognition in the general population, we addressed the utility of ONCs in the "druggable genome" approach by using the clinical and cell resources of an established psychosis cohort in our group. Through this pilot effort, we underscored the chloride voltage-gated channel 2 (CLCN2) gene as a possible druggable candidate for early-stage psychosis. The CLCN2 gene expression was associated with verbal memory, but not with other dimensions in cognition, nor psychiatric manifestations (positive and negative symptoms). The association between this candidate molecule and verbal memory was also confirmed at the protein level. By using ONCs from living subjects, we now provide more specific information regarding molecular expression and clinical phenotypes. The use of ONCs also provides the opportunity of validating the relationship not only at the RNA level but also protein level, leading to the potential of functional assays in the future. Taken together, we now provide evidence that supports the utility of ONCs as a tool for the "druggable genome" approach in translational psychiatry.

Expression changes of c-Fos and D1R/p-ERK1/2 signal pathways in nucleus accumbens of rats after ketamine abuse

Ketamine is a commonly used dissociative anesthetic in clinical applications. However, the abuse potential has posted limits to its use and the mechanism remains to be studied. We aimed to investigate the changes of dopamine D1 receptors (D1R), phosphorylation of extracellular-regulated protein kinase 1/2 (p-ERK1/2), and c-Fos expression in the nucleus accumbens (NAc) of ketamine abuse rats. Ketamine induced severe anxiety in rats, as shown by an open field test. Nissl staining demonstrated clearly different morphologies between neurons of ketamine abuse rats and normal rats. The molecular expression changes were examined using immunohistochemistry assay and western blotting. D1R, p-ERK1/2, and c-Fos were significantly highly-expressed in NAc during ketamine exposure and were decreased by D1R antagonist SCH23390 and MAPK kinases inhibitor U0126. Taken together, the results suggest that ketamine abuse may induce the overexpression of c-Fos in NAc by up-regulating the expression of D1R and p-ERK1/2.

Analysis of senescence in gingival tissues and gingival fibroblast cultures.

ObjectiveTo determine senescence‐associated changes in the gingival tissues of aged mice and gingival fibroblast cultures.Materials and MethodsThe production of senescence‐associated β‐galactosidase (SA‐β‐gal) and mRNA expression of p16, p21, interleukin (IL)‐1β, and tumor necrosis factor α (TNF‐α) were evaluated in gingival tissues, gingival fibroblasts of 10‐ and 20‐month‐old C57BL/6NCrl mice, and multiple‐passaged and hydrogen peroxide‐stimulated human gingival fibroblasts (HGFs). Changes in molecular expression in HGF cultures due to senescent cell elimination by the senolytic drug ABT‐263 (Navitoclax) were analyzed.ResultsCompared to 10‐week‐old mice, the 20‐month‐old mice had higher numbers of M1 macrophages. The proportion of cells expressing SA‐β‐gal were also higher in 20‐ month‐old mice than in 10‐week‐old‐mice. Gingival fibroblasts in 20‐month‐old mice expressed less collagen 1a1, collagen 4a1, and collagen 4a2 mRNA than those in 10‐week‐old mice. Compared to control cells, H2O2 treated HGF cells expressed higher levels of SA‐β‐gal and p16, p21, IL‐1β, and TNF‐α. Furthermore, ABT‐263 suppressed HGF cell expression of cytokines after senescence induction.ConclusionsSenescence‐associated changes were observed in the gingival tissues of aged mice and HGF cultures. In addition, the potential of senolytic drugs to modify aging‐related changes in the gingiva was shown.

MiR-378d suppresses malignant phenotype of ESCC cells through AKT signaling

BackgroundPost-resistance progress in paclitaxel (PTX) treatment remains a major challenge in tumor treatment. A high dose of PTX was used for cell lines to analyze the changes in molecular expression. The miR-378d was sharply reduced in surviving cells, but the role of miR-378d in Esophageal squamous cell carcinoma (ESCC) remained unclear.MethodsWe analyzed the relationship between miR-378d expression and the clinicopathological features of ESCC. We constructed miR-378d silent expression cell lines to study phenotypes and molecular mechanisms.ResultsThe miR-378d expression was associated with good prognosis in patients with ESCC. miR-378d inhibition promoted chemo-resistance, monoclonal formation, EMT, migration, invasion, stemness, and metastasis of ESCC cells. miR-378d can target downregulated AKT1.ConclusionsTherefore, miR-378d expression is a good prognostic factor of patients with ESCC and regulates the malignant phenotype of tumor cells through AKT.

Spinal Nrf2 translocation may inhibit neuronal NF-κB activation and alleviate allodynia in a rat model of bone cancer pain.

Bone cancer pain (BCP) is a clinical pathology that urgently needs to be solved, but research on the mechanism of BCP has so far achieved limited success. Nuclear factor erythroid 2 (NFE2)‐related factor 2 (Nrf2) has been shown to be involved in pain, but its involvement in BCP and the specific mechanism have yet to be examined. This study aimed to test the hypothesis that BCP induces the transfer of Nrf2 from the cytoplasm to the nucleus and further promotes nuclear transcription to activate heme oxygenase‐1 (HO‐1) and inhibit the activation of nuclear factor‐kappa B (NF‐κB) signalling, ultimately regulating the neuroinflammatory response. Von‐Frey was used for behavioural analysis in rats with BCP, whereas western blotting, real‐time quantitative PCR (RT‐PCR) and enzyme‐linked immunosorbent assay (ELISA) were used to detect molecular expression changes, and immunofluorescence was used to detect cellular localization. We demonstrated that BCP induced increased Nrf2 nuclear protein expression with decreased cytoplasmic protein expression in the spinal cord. Further increases in Nrf2 nuclear protein expression can alleviate hyperalgesia and activate HO‐1 to inhibit the expression of NF‐κB nuclear protein and inflammatory factors. Strikingly, intrathecal administration of the corresponding siRNA reversed the above effects. In addition, the results of double immune labelling revealed that Nrf2 and NF‐κB were coexpressed in spinal cord neurons of rats with BCP. In summary, these findings suggest that the entry of Nrf2 into the nucleus promotes the expression of HO‐1, inhibiting activation of the NF‐κB signalling pathway, reducing neuroinflammation and ultimately exerting an anti‐nociceptive effect.

Overexpression of aquaporin 2 in renal tubular epithelial cells alleviates pyroptosis.

BackgroundSevere renal ischemia-reperfusion injury results in worse outcomes of kidney transplantation. Compared to the collecting duct, the proximal tubule is more likely to exhibit severe pyroptosis and damage during renal ischemia-reperfusion. Aquaporins were reported of having regulatory roles in pyroptosis. We explored whether aquaporin 2 overexpression in proximal tubular cells could alleviate ischemia-reperfusion injury related pyroptosis.MethodsA renal ischemia-reperfusion model of mice was established, and human kidney 2 cells were treated with hypoxia-reoxygenation. Aquaporin 2 overexpression was achieved in human kidney 2 cells transfected with lentivirus, which were then cultured with murine cells. Renal tissues and serum of the mice, and human kidney 2 cells were subjected to histological, molecular, and biochemical examinations.ResultsCompared with the sham group, the renal function of the ischemia-reperfusion group was significantly decreased, and the tissue injury was severe and accompanied by more nuclear dissolved and necrosis. Besides, the expression of aquaporin 1-5 decreased significantly, while the expression of Toll-like receptor 4, caspase-1, kim-1 and interleukin 1β and 18 increased significantly in ischemia-reperfusion group. Similar results were observed in the human kidney 2 cells test. Overexpression of aquaporin 2 partially reversed the cell damage, pyroptosis, and molecular expression changes of human kidney 2 cells induced by hypoxia-reoxygenation.ConclusionsOur findings suggest that aquaporin 2 overexpression can potentially reduce pyroptosis in proximal tubular cells, and thus might be a novel target for relieving pyroptosis and injury in renal ischemia-reperfusion injury.

Treatment Results for Recurrent Glioblastoma and Alteration of Programmed Death-Ligand 1 Expression After Recurrence

This study was designed to analyze the results of recurrent glioblastoma (GBM) treatment, investigate the changes in molecular expression on paired primary and recurrent tumor specimens of GBM, and evaluate the effect of these changes on patient survival. A total of 170 adult patients were diagnosed with recurrent GBM at a single institution between 2005 and 2015. Patients were divided into the reoperation and nonoperation groups. In addition, we evaluated the expression of immunologic markers of 43 paired surgical specimens from the first and second operations. The median overall survival (OS) after recurrence in the reoperation group was significantly longer than that in the nonoperation group (median, 9.1 months vs. 5.6 months; P= 0.024). The groups differed in characteristics such as age, performance scale, and progression-free survival. In the reoperation group, higher performance scale at recurrence, better extent of resection, and adjuvant treatment were related to longer overall survival. Among 43 paired surgical specimens, programmed death-ligand 1 (PD-L1) was positively expressed in 17 (39.5%) and 6 (13.9%) patients after the first and second operations, respectively. PD-L1 expression after recurrence showed an increase, decrease, and no change in 6 (13.9%), 14 (32.5%), and 23 (53.4%) patients, respectively. Changes in PD-L1 expression after recurrence did not affect survival after recurrence during progression. The extent of resection and adjuvant treatment was important for prolonged survival. Reoperation without adjuvant treatment was not effective for prolonged survival. Initial and follow-up PD-L1 expression from both operations did not influence patient survival.

Proteomics and Transcriptomics Analysis Reveals Clues into the Mechanism of the Beneficial Effect of Electrical Stimulation on Rat Denervated Gastrocnemius Muscle.

The aim of this study was to confirm the beneficial effects of electrical stimulation on denervated skeletal muscle and explore a novel underlying mechanism. Morphological and contractile analyses were performed on rats allocated to three groups: sham operation (SHAM), denervated (DN), and denervated and electrical stimulation (DN-SM). Proteomics, transcriptomics, bioinformatics, and skeletal muscle functional modules analysis were conducted to determine the changes in molecular expression resulting from electrical stimulation. Rats in the DN-SM group maintained a greater muscle mass, muscle fiber diameter, and contractile properties than those of the DN group. A total of 66 proteins and 402 mRNAs were differentially expressed between groups. Bioinformatics analysis suggested that the FoxO and p53 signaling pathways play significant roles in structural protection. Skeletal muscle function modules analysis suggested that anti-apoptosis proteins (KCNA7, KCNJ11), muscle fiber type related proteins (TNNI1, TNNT1, ACTN2, MYOZ2, MYLK2, and MYOM2), M-line structural protein (MYOM2), dystrophin combined with glycoprotein complex proteins (SGCB, SGCD, and DTNA), and anti-fibrosis-related proteins (POSTN, COL1A1, COL1A2, COL6A1, COL6A2, COL6A3, FN1, and LUM), may be related to the effects of electrical stimulation. Electrical stimulation can maintain the denervated muscle morphology and function. Anti-apoptosis, inhibition of muscle fiber type differentiation, protection against dystrophin-associated-glycoprotein complex mutation, and anti-fibrosis are potential mechanisms of the beneficial effects of electrical stimulation.

Abstract 4854: Targeting ribonucleotide reductase M2 using didox causes inhibition of estrogen receptor-negative, inflammatory breast cancer cell proliferation and tumor emboli formation in culture

Abstract Ribonucleotide reductase (RR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides during the rate-limiting step of DNA synthesis. Given its significant role in cell cycle regulation, RR and its catalytic subunits are significant molecular targets for inhibiting tumor cell growth and proliferation. Didox (3,4-dihydroxybenzohydroxamic acid) is a known inhibitor of the catalytic RR subunit of RRM2 and overall DNA synthesis. Previous meta-analyses have revealed that RRM2 is highly expressed in estrogen receptor-negative (ER-) breast cancer tumors. As ER negativity is frequent in inflammatory breast cancer (IBC), a distinct and the most aggressive breast cancer subtype, we proposed to evaluate efficacy of didox in IBC cell models. ER- inflammatory breast cancer (IBC) cell lines SUM149 (ER-, PR-, EGFR-activated), lapatinib-resistant SUM149 (rSUM149), and non-IBC BT474M1 metastatic subline of BT474 (ER+, PR+, HER2+) were compared for the cytotoxic and cytostatic effects of didox using cell viability (trypan blue exclusion) and proliferation (MTT) assays. Protein profiling was performed to evaluate changes in molecular expression of key proteins involved in cellular signaling pathways for apoptosis and proliferation. Only ER- cell lines exhibited decreased dose-dependent decrease in viability following didox treatment. In addition, didox inhibited tumor emboli formation in a novel ex vivo tumor emboli in culture assay that mimics the clinicopathologic hallmark of IBC disease in patients. Moreover, didox decreased proliferation in lapatinib-resistant rSUM149, suggesting that the drug may help circumvent lapatinib resistance. Studies are ongoing to evaluate the mechanism of didox in overcoming lapatinib resistance and evaluation in an in vivo IBC invasion model. IBC is rare but accounts for 10% of breast cancer deaths, and despite multimodal therapy the 5-yr survival is only 25-55%. Further, there is a need to identify new therapies that can inhibit IBC tumor emboli formation and migration. Didox has demonstrated minimal toxicity in human phase I/II cancer clinical trials and therefore is an attractive strategy for inflammatory breast cancer therapy. Support in part from the Development funds (GRD) of the Duke Cancer Institute as part of the P30 Cancer Center Support Grant NIH CA014236 and Department of Defense Breakthrough Level 2 W81XWH-17-1-0297 (GRD). Citation Format: Pranalee Patel, Ben Mirman, Renzhi Zhan, Gayathri R. Devi. Targeting ribonucleotide reductase M2 using didox causes inhibition of estrogen receptor-negative, inflammatory breast cancer cell proliferation and tumor emboli formation in culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4854.

Precise spatial restriction of BMP signaling in developing joints is perturbed upon loss of embryo movement.

Dynamic mechanical loading of synovial joints is necessary for normal joint development, as evidenced in certain clinical conditions, congenital disorders and animal models where dynamic muscle contractions are reduced or absent. Although the importance of mechanical forces on joint development is unequivocal, little is known about the molecular mechanisms involved. Here, using chick and mouse embryos, we observed that molecular changes in expression of multiple genes analyzed in the absence of mechanical stimulation are consistent across species. Our results suggest that abnormal joint development in immobilized embryos involves inappropriate regulation of Wnt and BMP signaling during definition of the emerging joint territories, i.e. reduced β-catenin activation and concomitant upregulation of pSMAD1/5/8 signaling. Moreover, dynamic mechanical loading of the developing knee joint activates Smurf1 expression; our data suggest that Smurf1 insulates the joint region from pSMAD1/5/8 signaling and is essential for maintenance of joint progenitor cell fate.

TRANSGENIC MICE OVEREXPRESSING DSC2 DEVELOP BIVENTRICULAR CARDIOMYOPATHY ASSOCIATED WITH FIBROSIS AND NECROSIS

Arrhythmogenic cardiomyopathy (AC) is an inherited heart disease associated with arrhythmias and right or biventricular dilation often leading to sudden cardiac death or heart failure. AC is mainly caused by mutations in five genes encoding cardiac desmosomal proteins (JUP, DSP, PKP2, DSG2 and DSC2). The cardiac desmosomes are cell-cell junctions coupling the cardiomyocytes. Desmocollin-2 (DSC2) and desmoglein-2 (DSG2) are members of the cadherin family and mediate the Ca2+-dependent cardiomyocyte adhesion. Plakophilin-2 (PKP2), plakoglobin (JUP) and desmoplakin (DSP) are linker proteins connecting the cytoplasmic domains of these cadherins to the intermediate filament system. However, the molecular and cellular pathomechanisms induced by mutations in these genes are widely unknown limiting the efficient development of therapeutic therapies. So far, no Dsc2 mouse model mimicking an AC is described. The aim of this project was therefore to establish and characterize an adequate mouse model to understand the underlying pathomechanisms in vivo and ex vivo. We developed and characterized a transgenic mouse model with a cardiac-specific overexpression of DSC2. Echocardiography was used to characterize the heart function and (immuno)histology was used to characterize the structural defects the transgenic mice. Western-blot analysis and qRT-PCR was used to characterize the molecular expression changes of the desmosomal genes. Transgenic mice with a cardiac specific overexpression of DSC2 developed a severe cardiomyopathy shortly after birth with significantly reduced fractional shortening and ejection fractions. Time-dependent functional analysis revealed that the phenotype is deteriorating leading to heart failure and cardiac death after 12 weeks. The myocardial tissue of the septum as well as of both ventricular walls was significantly replaced by fibrosis and was associated with calcification and necrosis in these areas. The established cardiac specific overexpressing DSC2 mice are viable but develop a progressive biventricular cardiomyopathy mimicking the clinical phenotype of patients with arrhythmogenic cardiomyopathy. In the future this novel mouse model could contribute to further understanding of the molecular and cellular pathomechanisms leading to AC.

Single-cell mass spectrometry reveals small molecules that affect cell fates in the 16-cell embryo

Spatial and temporal changes in molecular expression are essential to embryonic development, and their characterization is critical to understand mechanisms by which cells acquire different phenotypes. Although technological advances have made it possible to quantify expression of large molecules during embryogenesis, little information is available on metabolites, the ultimate indicator of physiological activity of the cell. Here, we demonstrate that single-cell capillary electrophoresis-electrospray ionization mass spectrometry is able to test whether differential expression of the genome translates to the domain of metabolites between single embryonic cells. Dissection of three different cell types with distinct tissue fates from 16-cell embryos of the South African clawed frog (Xenopus laevis) and microextraction of their metabolomes enabled the identification of 40 metabolites that anchored interconnected central metabolic networks. Relative quantitation revealed that several metabolites were differentially active between the cell types in the wild-type, unperturbed embryos. Altering postfertilization cytoplasmic movements that perturb dorsal development confirmed that these three cells have characteristic small-molecular activity already at cleavage stages as a result of cell type and not differences in pigmentation, yolk content, cell size, or position in the embryo. Changing the metabolite concentration caused changes in cell movements at gastrulation that also altered the tissue fates of these cells, demonstrating that the metabolome affects cell phenotypes in the embryo.

Laser-capture microdissection for analysis of cell type-specific gene expression of muscarinic receptor subtypes in the rat bladder with cyclophosphamide-induced cystitis.

This study examined whether the laser-capture microdissection (LCM) method can achieve separation of urothelial cells from detrusor cells or superficial urothelial cells from intermediate/basal urothelial cells, using α-smooth muscle actin (SMA) and cytokeratin 20 (CK20). In addition, we investigated the changes in expression of muscarinic receptors in laser-captured urothelial and detrusor cells in rats with chronic cystitis. Female SD rats were injected with cyclophosphamide (75 mg/kg) intraperitoneally at day 1, 4, 7 and 10 to induce chronic cystitis. Saline was injected in the same protocol for controls. Bladder specimens were cut at 8 μm thickness, fixed in 70% ethanol and lightly stained by hematoxylin and eosin, and then superficial urothelium, intermediate/basal urothelium and detrusor muscles were laser-captured separately. Real-time PCR was performed to examine expressions of α-SMA, CK20, muscarinic 2 receptors (M2R) and muscarinic 3 receptors (M3R). The expression of α-SMA mRNA in detrusor muscle cells was 200 times higher than that in urothelial cells in controls. CK20 mRNA expression in apical urothelial cells was 55 times more than that in detrusor muscle and four times more than that in intermediate/basal urothelial cells. Expressions of M2R and M3R mRNA were increased in urothelial cells and decreased in detrusor muscles following chronic cystitis. The LCM could be useful for tissue collection of detrusor muscle and different layers of urothelial cells with minimal contamination of other cell types, and cell type-specific changes in molecular expression could accurately be analyzed. Increased expression of urothelial MR might enhance urothelial-afferent interactions to induce bladder overactivity/pain conditions associated with bladder inflammation.

Response of various conduit arteries in tachycardia- and volume overload-induced heart failure.

Although hemodynamics changes occur in heart failure (HF) and generally influence vascular function, it is not clear whether various HF models will affect the conduit vessels differentially or whether local hemodynamic forces or systemic factors are more important determinants of vascular response in HF. Here, we studied the hemodynamic changes in tachycardia or volume-overload HF swine model (created by either high rate pacing or distal abdominal aortic-vena cava fistula, respectively) on carotid, femoral, and renal arteries function and molecular expression. The ejection fraction was reduced by 50% or 30% in tachycardia or volume-overload model in four weeks, respectively. The LV end diastolic volume was increased from 65±22 to 115±78 ml in tachycardia and 67±19 to 148±68 ml in volume-overload model. Flow reversal was observed in diastolic phase in carotid artery of both models and femoral artery in volume-overload model. The endothelial function was also significantly impaired in carotid and renal arteries of tachycardia and volume-overload animals. The endothelial dysfunction was observed in femoral artery of volume-overload animals but not tachycardia animals. The adrenergic receptor-dependent contractility decreased in carotid and femoral arteries of tachycardia animals. The protein expressions of NADPH oxidase subunits increased in the three arteries and both animal models while expression of MnSOD decreased in carotid artery of tachycardia and volume-overload model. In conclusion, different HF models lead to variable arterial hemodynamic changes but similar vascular and molecular expression changes that reflect the role of both local hemodynamics as well as systemic changes in HF.

The Impact of ICAM1 and VCAM1 Gene Polymorphisms on Chronic Allograft Nephropathy and Transplanted Kidney Function

ICAM-1 and VCAM-1 adhesion molecules play important roles in the immune response and emergence of chronic allograft nephropathy (CAN). The several polymorphisms of ICAM1 and VCAM1 genes are associated with changes in molecular expression therefore affecting allograft function and immune responses after kidney transplantation. The aim of this study was to examine the impact of polymorphisms in ICAM1 and VCAM1 genes on biopsy-proven CAN and renal allograft function. The 270 Caucasian renal transplant recipients (166 men and 104 women) were genotyped for the rs5498 ICAM1 and rs1041163 and rs3170794 VCAM1 gene polymorphisms using real-time polymerase chain reaction. There was no correlation between polymorphisms and CAN. Creatinine concentrations in the first month after transplantation differed between the rs5498 ICAM1 genotypes (P = .095), being higher for GG carriers (AA + AG vs GG, P =.07) albeit not with statistical significance. Creatinine concentrations at 12, 24, and 36 months after transplantation differed significantly among rs5498 ICAM1 genotypes (P = .0046, P =.016, and P = .02) and were higher among GG carriers (AA + AG vs GG, P = .001, P = .004, and P = .006). Rs5498 ICAM1 GG genotype and receipient male gender were independent factors associated with higher creatinine concentrations. These results suggest that the rs5498 ICAM1 GG genotype may be associated with long-term allograft function.

Quantitative proteomic analysis of pregnancy-related proteins from peripheral blood mononuclear cells during pregnancy in pigs

Information obtained from peripheral blood could help us understand the underlying mechanisms in autoimmune diseases, cancer, pregnancy, and other conditions. In this paper, we present the protein map of porcine peripheral blood mononuclear cells (PBMC) to better understand the molecular expression changes that occur during pregnancy using proteomic analysis. We detected 94 differentially expressed proteins in pregnant vs. non-pregnant (NP) pigs, and a representative set of the proteins was subjected to LC–MS/MS analysis. Furthermore, the identified proteins were categorized according to their biological process and molecular function. By classifying the proteins according to their functions, a large number of differentially regulated proteins involved in anti-oxidant, detoxification and stress response pathways were found, including peroxiredoxin (PRX) 1, 2, and 6, glutathione-S-transferase (GST), annexin A2, and A6, and heat shock protein 27 (HSP 27) during pregnancy (pregnancy d of E40, embryonic day 40; E70, embryonic day 70; and E93, embryonic day 93) compared with non-pregnancy. In this study, a proteomic approach utilizing 2-DE and LC–MS/MS was applied to evaluate specific molecular expression changes during pregnancy compared with non-pregnancy. Together, these data offer new information about the proteome map and factors that are differentially regulated during maintenance of normal pregnancy.

Tbr2 Deficiency in Mitral and Tufted Cells Disrupts Excitatory-Inhibitory Balance of Neural Circuitry in the Mouse Olfactory Bulb

The olfactory bulb (OB) is the first relay station in the brain where odor information from the olfactory epithelium is integrated, processed through its intrinsic neural circuitry, and conveyed to higher olfactory centers. Compared with profound mechanistic insights into olfactory axon wiring from the nose to the OB, little is known about the molecular mechanisms underlying the formation of functional neural circuitry among various types of neurons inside the OB. T-box transcription factor Tbr2 is expressed in various types of glutamatergic excitatory neurons in the brain including the OB projection neurons, mitral and tufted cells. Here we generated conditional knockout mice in which the Tbr2 gene is inactivated specifically in mitral and tufted cells from late embryonic stages. Tbr2 deficiency caused cell-autonomous changes in molecular expression including a compensatory increase of another T-box member, Tbr1, and a concomitant shift of vesicular glutamate transporter (VGluT) subtypes from VGluT1 to VGluT2. Tbr2-deficient mitral and tufted cells also exhibited anatomical abnormalities in their dendritic morphology and projection patterns. Additionally, several non-cell-autonomous phenotypes were observed in parvalbumin-, calbindin-, and 5T4-positive GABAergic interneurons. Furthermore, the number of dendrodendritic reciprocal synapses between mitral/tufted cells and GABAergic interneurons was significantly reduced. Upon stimulation with odorants, larger numbers of mitral and tufted cells were activated in Tbr2 conditional knockout mice. These results suggest that Tbr2 is required for not only the proper differentiation of mitral and tufted cells, but also for the establishment of functional neuronal circuitry in the OB and maintenance of excitatory-inhibitory balance crucial for odor information processing.