Total Lysates Research Articles

Differential Proteomics Analysis of Oligodendrogliomas and Astrocytomas Using iTRAQ Quantification

Background: Astrocytoma and oligodendroglioma are two histological subtypes of primary Central Nervous System (CNS) tumors. Because of the high cytoarchitectural variability and lacking accurate diagnosis biomarkers, distinguishing astrocytomas and oligodendrogliomas clinically remains challenging. Methods: The total protein lysates from of astrocytoma and oligodendroglioma clinical specimens were analyzed by 2DLC/MS/MS and quantified via Isobaric Tags Relative and Absolute Quantitation (iTRAQ). Differentially expressed proteins were further analyzed by Ingenuity Pathway Analysis (IPA) software. Lastly, potential bio-markers’ expression levels were validated by western blot. Results: A total 1856 proteins were identified. Among them, 83 proteins were increased and 82 proteins were decreased in astroglioma specimens compared to oligodendroglioma. Our bioinformatics study showed this protein profile change in astrocytoma more likely tend to enhance cell proliferation, migration and angiogenesis. Moreover, pathway-analysis showed that protein level of Rho Family GTPases pathway components was remarkably different between astrocytoma and oligodendroglioma. Lastly, two members of Rho family of GTPases, cell division control protein 42 homolog (CDC42) and transforming protein RhoA (RHOA) were found highly expressed in astrocytoma and oligodendroglioma, respectively. Discussion: Differential-proteomic analysis was validating to distinguish between astrocytomas and oligodendrogliomas. Two members of especially the Rho family of GTPases, CDC42 and RHOA, would be potential indicators to reflect the pathological characteristics of these two diseases.

Seropositivity and identification of paramyosin for sparganosis in the Kangwon and Incheon provinces of the Republic of Korea

Sparganosis is one of the top three tissue-dwelling heterologous helminthic diseases, along with cysticercosis and paragonimiasis, in Korea. Due to a lack of effective early diagnosis and treatment methods, this parasitic disease is regarded as a public health threat. This study evaluated reactivity, against sparganum extracts, of sera from inhabitants of Cheorwon-gun, Goseong-gun and Ongjin-gun in Korea. The sera from 836 subjects were subjected to enzyme-linked immunosorbent assay and immunoblot analysis. The sera from 18 (5.8%) and 15 (5.1%) inhabitants in Cheorwon-gun (n = 312) and Goseong-gun (n = 294), respectively, exhibited highly positive reactions to the sparganum antigen, whereas only two (0.9%) inhabitants in Ongjin-gun (n = 230) showed positivity. We sought antigenic proteins for serodiagnosis of positive sera by immunoproteomic approaches. Total sparganum lysates were separated by two-dimensional electrophoresis and then subjected to immunoblot analysis with mixed sparganosis-positive sera. We found seven antigenic spots and identified paramyosin as an antigenic protein by liquid chromatography-mass spectrometry. By two-dimensional (2D)-based mass analysis and immunoblotting against sparganosis-positive sera, paramyosin was identified as a candidate antigen for serodiagnosis of sparganosis.

Toward Biomarker Development in Large Clinical Cohorts: An Integrated High-Throughput 96-Well-Plate-Based Sample Preparation Workflow for Versatile Downstream Proteomic Analyses

We describe a cheap, robust, fast, high-throughput, and flexible proteomic sample processing method based on a regular 96-well plate by acetone precipitation under low centrifuge speed (96PACS), which enables predigestion processing of 96 samples within 2 h. Tested on a complex Huh-7 total lysate, 96PACS produced comparable proteome coverage and even showed better reproducibility than FASP. Quantitative performance of 96PACS was further tested using data-independent acquisition and parallel reaction monitoring quantitation in a set of 6 benchmark samples consisting of 6 serial dilutions of BSA spiked in complex E. coli proteome background. The protocol was also successfully modified for automation and was validated in a comparative label-free proteomic study to develop serum markers for early detection of liver fibrosis and necroinflammation in patients chronically infected with hepatitis B virus.

IRES-dependent translation of the long non coding RNA meloe in melanoma cells produces the most immunogenic MELOE antigens.

MELOE-1 and MELOE-2, two highly specific melanoma antigens involved in T cell immunosurveillance are produced by IRES-dependent translation of the long « non coding » and polycistronic RNA, meloe. In the present study, we document the expression of an additional ORF, MELOE-3, located in the 5′ region of meloe. Data from in vitro translation experiments and transfection of melanoma cells with bicistronic vectors documented that MELOE-3 is exclusively translated by the classical cap-dependent pathway. Using a sensitive tandem mass spectrometry technique, we detected the presence of MELOE-3 in total lysates of both melanoma cells and normal melanocytes. This contrasts with our previous observation of the melanoma-restricted expression of MELOE-1 and MELOE-2. Furthermore, in vitro stimulation of PBMC from 6 healthy donors with overlapping peptides from MELOE-1 or MELOE-3 revealed a very scarce MELOE-3 specific T cell repertoire as compared to the abundant repertoire observed against MELOE-1. The poor immunogenicity of MELOE-3 and its expression in melanocytes is consistent with an immune tolerance towards a physiologically expressed protein. In contrast, melanoma-restricted expression of IRES-dependent MELOE-1 may explain its high immunogenicity. In conclusion, within the MELOE family, IRES-dependent antigens represent the best T cell targets for immunotherapy of melanoma.

Abstract 4423: Methylation and acetylation are novel post-translational modifications identified for the pre-apoptotic protein kinase C (PKC) delta

Abstract Members of the PKC family of diacylglycerol (DAG) receptors play a central role in cellular signaling, affecting cell proliferation, differentiation, apoptosis, etc. Their expression and/or function are often altered in cancer. Posttranslational modifications play an important role in the regulation and intracellular localization of PKCs. For example, phosphorylation of the hydrophobic and turn motifs are required for the maturation, stability and proper folding of most isoforms, and phosphorylation of S299, Y311, and Y334 in the hinge region are well characterized agonist (phorbol ester or H2O2) induced modifications of PKC delta. We have previously reported that PMA and bryostatin 1, two PKC activators having very different biology in multiple cell lines, induced a relatively complex and different pattern of PKC delta modification in LNCaP cells as detected by charge-based separation. Moreover, bryostatin 1 failed to induce Y311 phosphorylation whereas it induced S299 and T507 phosphorylation similarly to PMA. In this study, we explored by mass spectrometry the posttranslational modifications of PKC delta induced by the two drugs. GFP-tagged PKCdelta was overexpressed in LNCaP and HEK cells, and PKC delta was immunoprecipitated from treated cells (in total lysates or in cytoplasmic and nuclear fractions) using anti-GFP antibody. Using this approach, we were able to detect the previously described phosphorylations of S302/S304, S506, S645, S664 in all samples and the agonist-induced phosphorylation of S299, while detection of phosphorylated tyrosine residues was less efficient. Novel findings include detection of agonist-induced phosphorylation of S130, S359, and S647, and acetylation or methylation of multiple K or N sites, respectively. Acetylation of K275 in the C1b domain, of K319 in the hinge region, of K475 in the catalytic domain and of K649 at the C terminus was detected in almost all samples while methylation of N278, N283 and N292 in the C1b domain was detected in control LNCaP cells only. Agonist treatment induced acetylation and methylation at additional sites in the hinge region and in the C terminus of the protein. The modifications were not identical between the cytoplasmic and nuclear PKC delta after PMA treatment: phosphorylation of T50, methylation of N292 and acetylation of K382 was detected only in the cytoplasmic fraction while phosphorylation of S359 was present in the nuclear fraction only. The newly identified acetylation and methylation add to the complexity of posttranslational modifications of PKC delta that might have regulatory roles in the localization and/or activity of the protein. Citation Format: Noemi Kedei, Elliott L. Paine, Lisa M. Miller Jenkins, Peter M. Blumberg. Methylation and acetylation are novel post-translational modifications identified for the pre-apoptotic protein kinase C (PKC) delta. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4423.

Relationship between antioxidant capacity, oxidative stress, and feed efficiency in beef steers1

Feed efficiency (FE) can vary between individuals but sources of variation are not well characterized. Oxidative stress is among the biological mechanisms believed to contribute to variation. The objective of this study was to evaluate the relationship between FE, antioxidant activity, and oxidative stress in feedlot steers representing phenotypic extremes for FE. Crossbred beef steers ( = 181) fed 70-d growing phase (GP) whole-shell corn-based (G-Corn) or rye baleage and soybean hull-based (G-Rough) diets in GrowSafe bunks at the University of Missouri were shipped to Iowa State University where the 12 most feed efficient (HFE) and 12 least feed efficient (LFE) steers from each diet (n = 48; 467 kg [SD 51]) were selected for evaluation. Steers received diets similar to GP diets, and 3 d after arrival, blood was sampled to evaluate antioxidant activity and oxidative stress markers for the GP following transit. Steers were transitioned to finishing phase (FP) cracked corn-based (F-Corn) or dried distillers' grains and soybean hull-based (F-Byp) diets, and on FP d 97, blood samples for the FP were collected. Data for the GP were analyzed as a 2 × 2 factorial, and data for the FP were analyzed as a 2 × 2 × 2 factorial using PROC MIXED of SAS. No GP diet × FP diet, FP diet × FE group, or 3-way interactions were noted ( ≥ 0.11) for FP measures. Steers fed the G-Rough diet had greater ( = 0.04) GP plasma protein carbonyl concentrations. During the GP, HFE steers had greater ( ≤ 0.04) protein carbonyl and ratio of oxidized:reduced blood lysate glutathione concentrations than LFE steers. There were GP diet × FE group interactions ( ≤ 0.03) during the GP and FP. During the GP, total blood lysate superoxide dismutase (SOD) activity was greater ( ≤ 0.03) in G-Rough/LFE steers than in G-Rough/HFE and G-Corn/LFE steers; G-Corn/HFE steers were intermediate. The G-Rough/LFE steers had greater ( < 0.04) glutathione peroxidase (GPX) activity than other groups and greater ( = 0.03) plasma malondialdehyde concentrations than G-Corn/LFE steers. During the FP, the G-Rough/LFE steers had greater ( ≤ 0.04) GPX activity than G-Rough/HFE and G-Corn/LFE steers; G-Corn/HFE steers were intermediate. The F-Byp diet had greater ( < 0.01) protein carbonyl than the F-Corn diet, and no other FP diet effects were noted ( ≥ 0.3) for any FP measures. The GP diet and FE groups had stronger relationships with antioxidant activity and oxidative stress markers measured for the GP than for the FP. Overall, antioxidant activity may play a role in FE as LFE steers, driven largely by G-Rough/LFE steers, had greater SOD activity and GPX activity than HFE steers, potentially using a greater proportion of energy otherwise available for tissue accretion.

Immunoregulatory effects of vitamin D3 in experimentally induced type 1 diabetes

The mechanism underlying the impairment of the function of the cellular component of the immune response and its regulation by vitamin D3 in diabetes mellitus remains incompletely characterized. The present study addresses the specific features of the functioning of the T-cell link of immune response and the humoral response to injecyion of an artificial antigen in a diabetes model and after prolonged administration of vitamin D3. Chronic hyperglycemia occurring in diabetes induced a 2.3-fold decrease of the content of the marker substance 25OHD3, the major precursor of hormonally active forms of vitamin D3, in the serum. The development of vitamin D3 deficiency is accompanied by an impairment of the proliferative activity of T cells and a change in relative numbers of regulatory (CD4+) and cytotoxic (CD8+) lymphocytes. An increase of the content of the phosphorylated p65 subunit of the nuclear factor κB and more intensive translocation of this protein to the nucleus were detected in total lysates of T lymphocytes from the spleen. Moreover, enhancement of the humoral IgG response to intraperitoneal administration of a recombinant B subunit of diphtheria toxin was demonstrated. Impairment of the cellular component of the immune response was accompanied by increased apoptotic death of splenocytes, as is evident from the increased binding of the Annexin V-GFP tag to phosphatidyl serine residues exposed on the external side of the plasmalemma during apoptosis. Prolonged administration of vitamin D3 (during 2 months; dose 20 IU) promoted the normalization of proliferative activity and the relative size of T-cell subpopulations, led to a decrease of the content of the phosphorylated p65 subunit of NF-κB, and improved the balance of the secretion of IgG targeting the artificial antigen in diabetic animals. These changes were accompanied by a decrease in the number of apoptotic events in the entire splenocyte population. The results of the present study demonstrate the important role of vitamin D3 in the regulation of the functions of the immune system in type 1 diabetes.

A preliminary comparison of dendritic cell maturation by total cellular RNA to total cellular lysate derived from breast cancer stem cells

Dendritic cells (DCs) have been widely considered as the most potent antigenpresenting cells. As such, DC-based vaccines are regarded as a promising strategy in cancer vaccination and therapy. This study compared the maturation of DCs induced by total cellular RNA and cell lysate (i.e. nucleic acid and protein). Both total RNA and cell lysate were isolated from breast cancer stem cells (BCSCs). The lysates were used to incubate with monocyte-derived immature DCs. To track the transfection efficiency, the BCSCs were stably transfected with green fluorescent protein (GFP). The maturation of DCs was evaluated by expression of costimulatory molecules including CD40, CD80, and CD86. Transfections were confirmed by evaluating GFP expression in DCs at 24 hours post transfection. The results of this study showed that GFP is expressed in DCs after both total RNA and lysate incubation. The expression of costimulatory molecules (CD40, CD80, and CD86) was significantly higher in RNA-transfected DCs than in cell lysate-primed DCs. Our findings suggest that total RNA primed BCSCs can be a suitable platform for DC-based vaccine therapy of breast cancer.

MOAP-1 Mediates Fas-Induced Apoptosis in Liver by Facilitating tBid Recruitment to Mitochondria.

Fas apoptotic signaling regulates diverse physiological processes. Acute activation of Fas signaling triggers massive apoptosis in liver. Upon Fas receptor stimulation, the BH3-only protein Bid is cleaved into the active form, tBid. Subsequent tBid recruitment to mitochondria, which is facilitated by its receptor MTCH2 at the outer mitochondrial membrane (OMM), is a critical step for commitment to apoptosis via the effector proteins Bax or Bak. MOAP-1 is a Bax-binding protein enriched at the OMM. Here, we show that MOAP-1-deficient mice are resistant to Fas-induced hepatocellular apoptosis and lethality. In the absence of MOAP-1, mitochondrial accumulation of tBid is markedly impaired. MOAP-1 binds to MTCH2, and this interaction appears necessary for MTCH2 to engage tBid. These findings reveal a role for MOAP-1 in Fas signaling in the liver by promoting MTCH2-mediated tBid recruitment to mitochondria.

Preclinical data supporting the flavone baicalein as a novel mTOR inhibitor with potent activity against endometrial cancer cells

Objectives: The mTOR pathway is a promising target for endometrial cancer therapy commonly because of the loss of PTEN expression. Metformin has been shown to inhibit endometrial cancer cell proliferation and is currently in phase II/III clinical trial testing. We have identified a natural flavone, baicalein, which markedly upregulates DNA Damage Induced Transcript 4 (DDIT4), suppresses breast and ovarian cancer cell growth, and alters mTOR pathway. We examined the activity of baicalein in endometrial cancer cells. Methods: The endometrial cancer cell line HEC1a was treated with baicalein (5–80 μM) or metformin (5–10,000 μM) and growth was assessed at 24 to 72 hours with the MTT assay. Total protein lysates were obtained for Western blot analysis to evaluate mTOR pathway mediators and DDIT4 levels. In vivo studies were performed with an HEC1a mouse model. Mice received either control or baicalein (80 mg/kg) treatment by oral gavage daily. Tumor volumes were measured over time. Results: Baicalein inhibited growth of HEC1a cell line in a dose-dependent fashion (IC50 ~10 μM). In contrast, higher millimolar concentrations (>1,000-fold) were required to inhibit growth with metformin. The observed growth inhibition with baicalein was associated with decreased PS6K1 and PS6 levels and increased DDIT4 levels. We tested baicalein (80 mg/kg) in an endometrial cancer cell mouse model. We observed significant tumor growth inhibition in the HEC1A mouse model compared with control mice. (See Fig. 1.) Conclusions: Both in vitro and in vivo studies support baicalein as a potent inhibitor of endometrial cancer cell proliferation. The inhibitory effect correlates with mTOR pathway inhibition and increase in DDIT4 expression. This preclinical study supports the use of baicalein as a novel treatment for endometrial cancer.

Induction of Protective Immunity against Toxoplasmosis in BALB/c Mice Vaccinated with Toxoplasma gondii Rhoptry-1.

Toxoplasma gondii is the causative agent for toxoplasmosis. The rhoptry protein 1 (ROP1) is secreted by rhoptry, an apical secretory organelle of the parasite. ROP1 plays an important role in host cell invasion. In this study, the efficacy of ROP1 as a vaccine candidate against toxoplasmosis was evaluated through intramuscular or subcutaneous injection of BALB/c mice followed by immunological characterization (humoral- and cellular-mediated) and lethal challenge against virulent T. gondii RH strain in BALB/c mice. Briefly, a recombinant DNA plasmid (pVAX1-GFP-ROP1) was expressed in CHO cells while expression of recombinant ROP1 protein (rROP1) was carried out in Escherichia coli expression system. Immunization study involved injection of the recombinant pVAX1-ROP1 and purified rROP1 into different group of mice. Empty vector and PBS served as two different types of negative controls. Results obtained demonstrated that ROP1 is an immunogenic antigen that induced humoral immune response whereby detection of a protein band with expected size of 43 kDa was observed against vaccinated mice sera through western blot analysis. ROP1 antigen was shown to elicit cellular-mediated immunity as well whereby stimulated splenocytes with total lysate antigen (TLA) and rROP1 from pVAX1-ROP1 and rROP1-immunized mice, respectively, readily proliferated and secreted large amount of IFN-γ (712 ± 28.1 pg/ml and 1457 ± 31.19 pg/ml, respectively) and relatively low IL-4 level (94 ± 14.5 pg/ml and 186 ± 14.17 pg/ml, respectively). These phenomena suggested that Th1-favored immunity was being induced. Vaccination with ROP1 antigen was able to provide partial protection in the vaccinated mice against lethal challenge with virulent RH strain of tachyzoites. These findings proposed that the ROP1 antigen is a potential candidate for the development of vaccine against toxoplasmosis.

Activated dendritic cells delivered in tissue compatible biomatrices induce in-situ anti-tumor CTL responses leading to tumor regression.

Dendritic cell (DC) based anti-cancer immunotherapy is well tolerated in patients with advanced cancers. However, the clinical responses seen after adoptive DC therapy have been suboptimal. Several factors including scarce DC numbers in tumors and immunosuppressive tumor microenvironments contribute to the inefficacy of DCs as cellular vaccines. Hence DC based vaccines can benefit from novel methods of cell delivery that would prevent the direct exposure of immune cells to suppressive tumor microenvironments. Here we evaluated the ability of DCs harbored in biocompatible scaffolds (referred to as biomatrix entrapped DCs; beDCs) in activating specific anti-tumor immune responses against primary and post-surgery secondary tumors. Using a preclinical cervical cancer and a melanoma model in mice, we show that single treatment of primary and post-surgery secondary tumors using beDCs resulted in significant tumor growth retardation while multiple inoculations were required to achieve a significant anti-tumor effect when DCs were given in free form. Additionally, we found that, compared to the tumor specific E6/E7 peptide vaccine, total tumor lysate induced higher expression of CD80 and CD40 on DCs that induced increased levels of IFNγ production upon interaction with host lymphocytes. Remarkably, a strong immunocyte infiltration into the host-implanted DC-scaffold was observed. Importantly, the host-implanted beDCs induced the anti-tumor immune responses in the absence of any stromal cell support, and the biomatrix structure was eventually absorbed into the surrounding host tissue. Collectively, these data indicate that the scaffold-based DC delivery may provide an efficient and safe way of delivering cell-based vaccines for treatment of primary and post-surgery secondary tumors.

649. Developing FGFR4 Chimeric Antigen Receptor CAR T Cell Therapy Against Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in pediatrics with an annual incidence of 4.5 cases per 1 million. Patients with high-risk metastatic disease have dismal prognosis and newer treatments are needed. We identified, fibroblast growth factor receptor 4 (FGFR4) as an overexpressed cell surface protein in RMS by mRNA expression analysis. Furthermore, activating mutations in FGFR4 are associated with metastatic disease. FGFR4 protein overexpression in RMS provides a specific target for immune-based therapy of RMS. We are developing T cells genetically modified to express chimeric antigen receptors (CARs) that target FGFR4.To verify FGFR4 RNA expression at the protein level we performed both immunohistochemistry (IHC) and electrochemillumescence (ECL) ELISA assays. Using IHC analysis we measured FGFR4 protein levels on tissue microarrays (TMA) of normal tissue and primary tumor from RMS patients, increased staining for FGFR4 protein on RMS primary tumors, compared to normal tissues was demonstrated. FGFR4 expression measured using ECL ELISA assay, shows a range of 300 - 800pg FGFR4 per 1mg of total lysate in RMS cell lines. The range for normal tissues was 30 - 40 pg/mg for all tissues with the exception of liver, which expressed 70 pg/mg. A single-chain variable fragment (scFv) cDNA library derived from a human B cells was screened, and clones that showed binding to recombinant FGFR4 extracellular domain (ECD) selected. We identified ten specific human anti-FGFR4 scFv binders. The scFvs were cloned into prokaryotic expression vector containing the human IgG1 Fc region. Anti-FGFR4 scFv-Fc were expressed in 293FT cells by transient transfection and purified using Protein A affinity chromatography. The binding of scFv-Fcs to recombinant FGFR4 ECD was verified by ELISA. scFv-Fc binders were then assayed for binding to cell surface FGFR4 on RMS cell lines using flow cytometry. Anti-FGFR4 scFv-Fc bound to 293T cells transfected to express FGFR4 but not 293T control cells. Anti-FGFR4 scFv-Fc also bound FGFR4 on three RMS cell lines. The first two anti-FGFR4 scFv binder sequences evaluated, M410 and M412, were used to make short (S, extracellular scFv only) and long format (L, scFv with a CH2CH3 domain of IgG1) CAR constructs. Activated T cells were transduced with lentiviral CAR expression vector (LV) encoding M410-L, M412-S and M412-L CAR constructs and cell surface expression of FGFR4 CAR on transduced T cells was measured using flow cytometry. The M410 and M412 FGFR4 CARs, both short and long constructs, were tested for cell-mediated cytotoxicity against RMS cell lines. M410-L showed higher cytotoxic activity compared to M412-L. M412-S showed greater cytotoxic activity compared to M412-L CAR. Thus, overall CAR structure format may be important for its functional activity. The remaining scFv will be further analyzed in various CAR formats for its functional activity including cytotoxicity and interferon-gamma production. In summary, the overexpression of FGFR4 protein in RMS versus normal cell lines demonstrates that FGFR4 may be a suitable target for immune-based therapy. FGFR4 CAR-T cell therapy offers the potential of a novel therapeutic intervention for high-risk, refractory and relapsed RMS.

Loss of C9 ORF 72 impairs autophagy and synergizes with polyQ Ataxin‐2 to induce motor neuron dysfunction and cell death

An intronic expansion of GGGGCC repeats within the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). Ataxin-2 with intermediate length of polyglutamine expansions (Ataxin-2 Q30x) is a genetic modifier of the disease. Here, we found that C9ORF72 forms a complex with the WDR41 and SMCR8 proteins to act as a GDP/GTP exchange factor for RAB8a and RAB39b and to thereby control autophagic flux. Depletion of C9orf72 in neurons partly impairs autophagy and leads to accumulation of aggregates of TDP-43 and P62 proteins, which are histopathological hallmarks of ALS-FTD SMCR8 is phosphorylated by TBK1 and depletion of TBK1 can be rescued by phosphomimetic mutants of SMCR8 or by constitutively active RAB39b, suggesting that TBK1, SMCR8, C9ORF72, and RAB39b belong to a common pathway regulating autophagy. While depletion of C9ORF72 only has a partial deleterious effect on neuron survival, it synergizes with Ataxin-2 Q30x toxicity to induce motor neuron dysfunction and neuronal cell death. These results indicate that partial loss of function of C9ORF72 is not deleterious by itself but synergizes with Ataxin-2 toxicity, suggesting a double-hit pathological mechanism in ALS-FTD.

Silencing of microRNA-132 reduces renal fibrosis by selectively inhibiting myofibroblast proliferation

Chronic kidney disease is associated with progressive renal fibrosis, where perivascular cells give rise to the majority of α-smooth muscle actin (α-SMA) positive myofibroblasts. Here we sought to identify pericytic miRNAs that could serve as a target to decrease myofibroblast formation. Kidney fibrosis was induced in FoxD1-GC;Z/Red-mice by unilateral ureteral obstruction followed by FACS sorting of dsRed-positive FoxD1-derivative cells and miRNA profiling. MiR-132 selectively increased 21-fold during pericyte-to-myofibroblast formation, whereas miR-132 was only 2.5-fold up in total kidney lysates (both in obstructive and ischemia-reperfusion injury). MiR-132 silencing during obstruction decreased collagen deposition (35%) and tubular apoptosis. Immunohistochemistry, Western blot, and qRT-PCR confirmed a similar decrease in interstitial α-SMA(+) cells. Pathway analysis identified a rate-limiting role for miR-132 in myofibroblast proliferation that was confirmed invitro. Indeed, antagomir-132-treated mice displayed a reduction in the number of proliferating Ki67(+) interstitial myofibroblasts. Interestingly, this was selective for the interstitial compartment and did not impair the reparative proliferation of tubular epithelial cells, as evidenced by an increase in Ki67(+) epithelial cells, as well as increased phospho-RB1, Cyclin-A and decreased RASA1, p21 levels in kidney lysates. Additional pathway and gene expression analyses suggest miR-132 coordinately regulates genes involved in TGF-β signaling (Smad2/Smad3), STAT3/ERK pathways, and cell proliferation (Foxo3/p300). Thus, silencing miR-132 counteracts the progression of renal fibrosis by selectively decreasing myofibroblast proliferation and could potentially serve as a novel antifibrotic therapy.

An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client

An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client

IN SILICO DETERMINATION AND VALIDATION OF OMR STRUCTURE AND LIGAND BINDING SITE AS A VACCINE CANDIDATE IN ACINETOBACTER BAUMANNII

Acinetobacterbaumannii is an emerging nosocomial pathogen, causes multi-drug and pan-drug resistant infections. This phenomenon necessitates the development of new treatment options and vaccines against this pathogen. Iron acquisition systems are important factors for the virulence of pathogenic organisms. Antibodies against iron Regulated Outer Membrane Proteins (IROMPs) showed bactericidal and opsonizing activities against A. baumanniiin vitro . Data obtained from proteomic studies could be valuable for vaccine design. Comparative proteomic analysis of total lysate and outer membrane fractions isolated from A. baumannii ATCC 19606 cells cultured under iron-rich and chelated conditions resulted in the identification of protein spots differentially produced. Among iron-regulated A. baumannii OMPs, carried out a primary screening based on hydrophilicity, flexibility, beta-turns, solubility and overall antigenic probability. Bioinformatic tools are imperative for analysis of these data as well as to design novel proteins bearing desired properties. This study undertakes in silico identification of the OMR 3D structure in A.baumannii which can be employed as vaccine candidate.

Label-Free Identification and Quantification of SUMO Target Proteins.

Mass spectrometry-based approaches are utilized with increasing frequency to facilitate identification of novel SUMO target proteins and to elucidate the dynamics of SUMOylation in response to cellular stresses. Here, we describe a robust method for the identification of SUMO target proteins, and the relative quantification of SUMOylation dynamics, using a label-free approach. The method relies on a decahistidine (His10)-tagged SUMO, which is expressed at a low level in a mammalian cell line or model organism. The His10-tag allows for a single-step, high-yield, and high-purity enrichment of SUMOylated proteins, which are then digested and analyzed by high-resolution mass spectrometry. Matching between runs and label-free quantification integrated in the freely available MaxQuant software allow for a high rate and accuracy of quantification, providing a strong alternative to laborious sample or cell labeling techniques. The method described here allows for identification of >1000 SUMO target proteins, and characterization of their SUMOylation dynamics, without requiring sample fractionation. The purification procedure, starting from total lysate, can be performed in ~4 days.

Expression of ARE-binding proteins AUF1 and HuR in follicular adenoma and carcinoma of thyroid gland.

Both adenylate-uridylate rich elements binding proteins AUF1 and HuR may participate in thyroid carcinoma progression. In this study we investigated the expression of both factors on aprotein level with aspecial focus on follicular adenoma and follicular thyroid carcinoma. By employment of immunofluorescence and western blot on 68 thyroid tissues including 7 goiter, 16 follicular adenoma (4 adenomatous hyperplasia), 19 follicular thyroid carcinomas, 13 papillary thyroid carcinomas and 14 undifferentiated thyroid carcinomas we investigated protein expression of AUF1 and HuR. In addition to previous results we demonstrated that AUF1 and HuR are significantly up-regulated in carcinoma tissues as compared with follicular adenoma or goiter tissues. Furthermore, by evaluation of AUF1 or HuR expression, or combination of both proteins on total tissue lysates, we were able to demonstrate asignificant difference between follicular adenoma and follicular thyroid carcinoma. Overexpression of AUF1 and HuR is acommon finding observed in thyroid malignancy. Analysis of the tissues obtained by surgical resection as demonstrated in this study is comparable to afine needle aspiration and in combination with AUF1/HuR immuno-analysis may support the conventional immunohistological investigations. The promising results of this study were performed on relatively small collective, but justify future development of aquick thyroid diagnostic test on larger cohort of the patients, especially for thyroid samples which are inadequate for histological examinations.

Inhaled nitric oxide decreases pulmonary endothelial nitric oxide synthase expression and activity in normal newborn rat lungs.

Inhaled nitric oxide (iNO) is commonly used in the treatment of very ill pre-term newborns. Previous studies showed that exogenous NO could affect endothelial NO synthase (eNOS) activity and expression in vascular endothelial cell cultures or adult rat models, but this has never been fully described in newborn rat lungs. We therefore aimed to assess the effects of iNO on eNOS expression and activity in newborn rats.Rat pups, post-natal day (P) 0 to P7, and their dams were placed in a chamber containing NO at 5 ppm (iNO-5 ppm group) or 20 ppm (iNO-20 ppm group), or in room air (control group). Rat pups were sacrificed at P7 and P14 for evaluation of lung eNOS expression and activity.At P7, eNOS protein expression in total lung lysates, in bronchial and arterial sections, was significantly decreased in the iNO-20 ppm versus control group. At P14, eNOS expression was comparable among all three groups. The amounts of eNOS mRNA significantly differed at P7 between the iNO-20 ppm and control groups. NOS activity decreased in the iNO-20 ppm group at P7 and returned to normal levels at P14. There was an imbalance between superoxide dismutase and NOS activities in the iNO-20 ppm group at P7.Inhalation of NO at 20 ppm early after birth decreases eNOS gene transcription, protein expression and enzyme activity. This decrease might account for the rebound phenomenon observed in patients treated with iNO.