Novel anti-inflammatory and analgesic agents: synthesis, molecular docking and in vivo studies.
Abstract Twelve new derivatives of benzothiazole bearing benzenesulphonamide and carboxamide were synthesised and investigated for their in vivo anti-inflammatory, analgesic and ulcerogenic activities. Molecular docking showed an excellent binding interaction of the synthesised compounds with the receptors, with 17c showing the highest binding energy (-12.50 kcal/mol). Compounds 17c and 17i inhibited carrageenan-induced rat paw oedema at 72, 76, and 80% and 64, 73, and 78% at 1 h, 2 h, and 3 h, respectively. In the analgesic activity experiment, compounds 17c, 17 g, and 17i had E...
Source: Molecular Medicine - January 28, 2018 Category: Molecular Biology Authors: Ugwu DI, Okoro UC, Ukoha PO, Gupta A, Okafor SN Tags: J Enzyme Inhib Med Chem Source Type: research

Biochemical characterisation of a Kunitz-type inhibitor from Tamarindus indica L. seeds and its efficacy in reducing plasma leptin in an experimental model of obesity.
AHA Abstract A trypsin inhibitor isolated from tamarind seed (TTI) has satietogenic effects in animals, increasing the cholecystokinin (CCK) in eutrophy and reducing leptin in obesity. We purified TTI (pTTI), characterised, and observed its effect upon CCK and leptin in obese Wistar rats. By HPLC, and after amplification of resolution, two protein fractions were observed: Fr1 and Fr2, with average mass of [M + 14H]+ = 19,594,690 Da and [M + 13H]+ = 19,578,266 Da, respectively. The protein fractions showed 54 and 53 amino acid residues with the ...
Source: Molecular Medicine - January 16, 2018 Category: Molecular Biology Authors: Medeiros AF, Costa IS, Carvalho FMC, Kiyota S, Souza BBP, Sifuentes DN, Serquiz RP, Maciel BLL, Uchôa AF, Santos EAD, Morais AHA Tags: J Enzyme Inhib Med Chem Source Type: research

Detection of Hypoxia and HIF in Paraffin-Embedded Tumor Tissues.
Abstract Hypoxia (insufficient O2 availability) is involved in various biological processes, such as tumorigenesis and inflammation. Hypoxia results in stabilization of hypoxia-inducible factors (HIFs) including HIF1α and HIF2α. Here we describe a protocol to detect mouse and human tissue hypoxia by using Hypoxyprobe and immunohistochemical staining for HIF1α and HIF2α. PMID: 29330808 [PubMed - in process] (Source: Molecular Medicine)
Source: Molecular Medicine - January 15, 2018 Category: Molecular Biology Authors: Li F, Lee KE, Simon MC Tags: Methods Mol Biol Source Type: research

Analysis of Hypoxia and the Hypoxic Response in Tumor Xenografts.
BK, Acker T Abstract Solid tumors are often characterized by insufficient oxygen supply (hypoxia), as a result of inadequate vascularization, which cannot keep up with the rapid growth rate of the tumor. Tumor hypoxia is a negative prognostic and predictive factor and is associated with a more aggressive phenotype in various tumor entities. Activation of the hypoxic response in tumors, which is centered around the hypoxia-inducible transcription factors (HIFs), has been causally linked to neovascularization, increased radio- and chemoresistance, altered cell metabolism, genomic instability, increased metastatic po...
Source: Molecular Medicine - January 15, 2018 Category: Molecular Biology Authors: Böğürcü N, Seidel S, Garvalov BK, Acker T Tags: Methods Mol Biol Source Type: research

Mol Med; +192 new citations
192 new pubmed citations were retrieved for your search. Click on the search hyperlink below to display the complete search results: Mol Med These pubmed results were generated on 2018/01/13PubMed comprises more than millions of citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites. (Source: Molecular Medicine)
Source: Molecular Medicine - January 13, 2018 Category: Molecular Biology Tags: Report Source Type: research

Forced phage uncorking: viral DNA ejection triggered by a mechanically sensitive switch.
G, Herényi L Abstract The foremost event of bacteriophage infection is the ejection of genomic material into the host bacterium after virus binding to surface receptor sites. How ejection is triggered is yet unknown. Here we show, in single mature T7 phage particles, that tapping the capsid wall with an oscillating atomic-force-microscope cantilever triggers rapid DNA ejection via the tail complex. The triggering rate increases exponentially as a function of force, following transition-state theory, across an activation barrier of 23 kcal mol-1 at 1.2 nm along the reaction coordinate. The conformation of th...
Source: Molecular Medicine - January 10, 2018 Category: Molecular Biology Authors: Kellermayer MSZ, Vörös Z, Csík G, Herényi L Tags: Nanoscale Source Type: research

H+-ATPase blockade reduced renal gluconeogenesis and plasma glucose in a diabetic rat model.
Abstract Vacuolar H+-adenosine triphosphatase (ATPase) plays important roles in urinary acid excretion, vesicular acidification to activate enzymes, and the membrane recycling of transporters in the kidney. As acidosis stimulates renal gluconeogenesis, we investigated the effect of blockade of H+-ATPase on renal gluconeogenesis in diabetic rats. Diabetes mellitus was induced by a single injection of streptozotocin, and a group of DM rats was treated with bafilomycin B1 intraperitoneally for 8 days. In diabetic rats, the renal expression and activity of H+-ATPase were increased with elevated urinary ammonium e...
Source: Molecular Medicine - January 9, 2018 Category: Molecular Biology Authors: Tojo A, Hatakeyama S, Nangaku M, Ishimitsu T Tags: Med Mol Morphol Source Type: research

Intraarticularly-Injected Mesenchymal Stem Cells Stimulate Anti-Inflammatory Molecules and Inhibit Pain Related Protein and Chondrolytic Enzymes in a Monoiodoacetate-Induced Rat Arthritis Model.
Abstract Persistent inflammation is well known to promote the progression of arthropathy. mesenchymal stem cells (MSCs) have been shown to possess anti-inflammatory properties and tissue differentiation potency. Although the experience so far with the intraarticular administration of mesenchymal stem cell (MSC) to induce cartilage regeneration has been disappointing, MSC implantation is now being attempted using various surgical techniques. Meanwhile, prevention of osteoarthritis (OA) progression and pain control remain important components of the treatment of early-stage OA. We prepared a shoulder arthritis model...
Source: Molecular Medicine - January 9, 2018 Category: Molecular Biology Authors: Ichiseki T, Shimazaki M, Ueda Y, Ueda S, Tsuchiya M, Souma D, Kaneuji A, Kawahara N Tags: Int J Mol Sci Source Type: research

Dual IRE1 RNase functions dictate glioblastoma development.
This study provides the first demonstration of a dual role of IRE1 downstream signaling in cancer and opens a new therapeutic window to abrogate tumor progression. PMID: 29311133 [PubMed - as supplied by publisher] (Source: Molecular Medicine)
Source: Molecular Medicine - January 8, 2018 Category: Molecular Biology Authors: Lhomond S, Avril T, Dejeans N, Voutetakis K, Doultsinos D, McMahon M, Pineau R, Obacz J, Papadodima O, Jouan F, Bourien H, Logotheti M, Jégou G, Pallares-Lupon N, Schmit K, Le Reste PJ, Etcheverry A, Mosser J, Barroso K, Vauléon E, Maurel M, Samali A, P Tags: EMBO Mol Med Source Type: research

Loss of kallikrein-related peptidase 7 exacerbates amyloid pathology in Alzheimer's disease model mice.
Abstract Deposition of amyloid-β (Aβ) as senile plaques is one of the pathological hallmarks in the brains of Alzheimer's disease (AD) patients. In addition, glial activation has been found in AD brains, although the precise pathological role of astrocytes remains unclear. Here, we identified kallikrein-related peptidase 7 (KLK7) as an astrocyte-derived Aβ degrading enzyme. Expression of KLK7 mRNA was significantly decreased in the brains of AD patients. Ablation of Klk7 exacerbated the thioflavin S-positive Aβ pathology in AD model mice. The expression of Klk7 was upregulated by Aβ treatm...
Source: Molecular Medicine - January 8, 2018 Category: Molecular Biology Authors: Kidana K, Tatebe T, Ito K, Hara N, Kakita A, Saito T, Takatori S, Ouchi Y, Ikeuchi T, Makino M, Saido TC, Akishita M, Iwatsubo T, Hori Y, Tomita T Tags: EMBO Mol Med Source Type: research

Using gene expression data to direct breast cancer therapy: evidence from a preclinical trial.
Abstract The heterogeneity both within and between breast cancers presents a significant clinical challenge for both diagnosis and therapy. This heterogeneity is present at all levels of analysis in breast cancer, ranging from genomic to metabolomic. A function of this heterogeneity is that numerous signaling networks are activated, and while treatment of one arm may be initially effective, this allows the tumor to be poised to evolve a resistance mechanism. Here we review the classification of breast cancers and discuss therapy of hormone positive, HER2 positive, and triple negative breast cancers. Model systems ...
Source: Molecular Medicine - January 8, 2018 Category: Molecular Biology Authors: Reaz S, Tamkus D, Andrechek ER Tags: J Mol Med (Berl) Source Type: research

PSA-stratified detection rates for [68Ga]THP-PSMA, a novel probe for rapid kit-based 68Ga-labeling and PET imaging, in patients with biochemical recurrence after primary therapy for prostate cancer.
FM Abstract PURPOSE: [68Ga]Tris(hydroxypyridinone)(THP)-PSMA is a novel radiopharmaceutical for one-step kit-based radiolabelling, based on direct chelation of 68Ga3+ at low concentration, room temperature and over a wide pH range, using direct elution from a 68Ge/68Ga-generator. We evaluated the clinical detection rates of [68Ga]THP-PSMA PET/CT in patients with biochemically recurrent prostate cancer after prostatectomy. METHODS: Consecutive patients (n=99) referred for evaluation of biochemical relapse of prostate cancer by [68Ga]THP-PSMA PET/CT were analyzed retrospectively. Patients underwent a standard ...
Source: Molecular Medicine - January 7, 2018 Category: Molecular Biology Authors: Derlin T, Schmuck S, Juhl C, Zörgiebel J, Schneefeld SM, Walte ACA, Hueper K, von Klot CA, Henkenberens C, Christiansen H, Thackeray JT, Ross TL, Bengel FM Tags: Eur J Nucl Med Mol Imaging Source Type: research

Recent progress in understanding the mechanisms of Leydig cell differentiation.
Abstract Leydig cells in fetal and adult testes play pivotal roles in eliciting male characteristics by producing androgen. Although numerous studies of Leydig cells have been performed, the mechanisms for differentiation of the two cell types (fetal Leydig and adult Leydig cells), their developmental and functional relationship, and their differential characteristics remain largely unclear. Based on recent technical progress in genome-wide analysis and in vitro investigation, novel and fascinating observations concerning the issues above have been obtained. Focusing on fetal and adult Leydig cells, this review su...
Source: Molecular Medicine - January 5, 2018 Category: Molecular Biology Authors: Inoue M, Baba T, Morohashi KI Tags: Mol Cell Endocrinol Source Type: research

Loss of Eed leads to lineage instability and increased CD8 expression of mouse CD4+ T cells upon TGF β signaling.
Loss of Eed leads to lineage instability and increased CD8 expression of mouse CD4+ T cells upon TGFβ signaling. Mol Immunol. 2018 Jan 05;94:140-152 Authors: Naito T, Muroi S, Taniuchi I, Kondo M Abstract Tri-methylation of lysine 27 on histone H3 (H3K27me3) is a repressive epigenetic modification catalyzed by polycomb repressive complex 2 (PRC2) that is required for proper cell fate determination as well as cellular function. Numerous studies have been performed to elucidate the role of PRC2 in T-cell differentiation and function; however, its role in the regulation of T-helper (Th) subset diffe...
Source: Molecular Medicine - January 5, 2018 Category: Molecular Biology Authors: Naito T, Muroi S, Taniuchi I, Kondo M Tags: Mol Immunol Source Type: research

Deciphering cellular and molecular causes of the tumor functional heterogeneity of liver cancer.
PMID: 29303506 [PubMed - in process] (Source: Molecular Medicine)
Source: Molecular Medicine - January 5, 2018 Category: Molecular Biology Authors: Nam SW Tags: Exp Mol Med Source Type: research

T-cell immune regulator 1 enhances metastasis in hepatocellular carcinoma.
Abstract Recurrence and metastasis are major challenges in the management of hepatocellular carcinoma (HCC) patients after resection. To identify a metastasis-associated gene signature, we performed comparative gene expression analysis with recurrent HCC tissues from HCC patients who underwent partial or total hepatectomy and from non-metastatic primary HCC tissues. From this, we were able to identify genes associated with HCC recurrence. TCIRG1 (T-Cell Immune Regulator 1) was one of the aberrantly overexpressed genes in patients with recurrent HCC who had undergone total hepatectomy. The significant overexpressio...
Source: Molecular Medicine - January 5, 2018 Category: Molecular Biology Authors: Yang HD, Eun JW, Lee KB, Shen Q, Kim HS, Kim SY, Seo DW, Park WS, Lee JY, Nam SW Tags: Exp Mol Med Source Type: research

Glutamine synthetase mediates sorafenib sensitivity in β-catenin-active hepatocellular carcinoma cells.
In this study, we found that β-catenin regulates the expression of glutamine synthetase and triggers a series of metabolic changes leading to induction of autophagy in hepatocellular carcinoma cells. β-Catenin-active Hep3B and HepG2 cells exhibited higher basal levels of autophagic activity than did β-catenin wild-type cells. We also found that autophagy in β-catenin-active cells was mediated by glutamine synthetase, as silencing of glutamine synthetase significantly reduced autophagic activity. We also showed that β-catenin-active hepatocellular carcinoma cells were more sensitive to sorafenib tha...
Source: Molecular Medicine - January 5, 2018 Category: Molecular Biology Authors: Sohn BH, Park IY, Shin JH, Yim SY, Lee JS Tags: Exp Mol Med Source Type: research

Hippo pathway coactivators Yap and Taz are required to coordinate mammalian liver regeneration.
In this report, we examined the role of Yap and Taz, key components of the Hippo pathway in liver organ size regulation, both in the context of development and homeostasis. Our studies reveal that contrary to the current paradigms that Yap/Taz are not required for developmental regulation of liver size but are required for proper liver regeneration. In livers depleted of Yap and Taz, liver mass is elevated in neonates and adults. However, Yap/Taz-depleted livers exhibit profound defects in liver regeneration, including an inability to restore liver mass and to properly coordinate cell cycle entry. Taken together, our resul...
Source: Molecular Medicine - January 5, 2018 Category: Molecular Biology Authors: Lu L, Finegold MJ, Johnson RL Tags: Exp Mol Med Source Type: research

Loss of Pten synergizes with c-Met to promote hepatocellular carcinoma development via mTORC2 pathway.
Abstract Hepatocellular carcinoma (HCC) is a deadly malignancy with limited treatment options. Activation of the AKT/mTOR cascade is one of the most frequent events along hepatocarcinogenesis. mTOR is a serine/threonine kinase and presents in two distinct complexes: mTORC1 and mTORC2. While mTORC1 has been extensively studied in HCC, the functional contribution of mTORC2 during hepatocarcinogenesis has not been well characterized, especially in vivo. Pten expression is one of the major mechanisms leading to the aberrant activation of the AKT/mTOR signaling. Here, we show that concomitant downregulation of Pten and...
Source: Molecular Medicine - January 5, 2018 Category: Molecular Biology Authors: Xu Z, Hu J, Cao H, Pilo MG, Cigliano A, Shao Z, Xu M, Ribback S, Dombrowski F, Calvisi DF, Chen X Tags: Exp Mol Med Source Type: research

A gene expression signature of FOXM1 predicts the prognosis of hepatocellular carcinoma.
In this study, we examined an association between the gene expression signature of FOXM1 and HCC patient outcome. The co-expressed gene set of FOXM1, which is significantly associated with the prognosis of HCC patients, was identified by analyzing the gene expression profiles of 100 patients with HCC, and this gene set was validated in two independent HCC patient cohorts (n=573). In multivariate analysis, the co-expressed gene set of FOXM1 was the most significant prognostic factor for overall survival in patients with HCC (hazard ratio=1.706, 95% confidence intervals=1.176-2.475, P=0.005). We also analyzed different types...
Source: Molecular Medicine - January 5, 2018 Category: Molecular Biology Authors: Song BN, Chu IS Tags: Exp Mol Med Source Type: research

The significance of intertumor and intratumor heterogeneity in liver cancer.
Abstract Genomic analyses of primary liver cancer samples reveal a complex mutational landscape with vast intertumor and intratumor heterogeneity. Different primary liver tumors and subclones within each tumor display striking molecular and biological variations. Consequently, tumor molecular heterogeneity contributes to drug resistance and tumor relapse following therapy, which poses a substantial obstruction to improving outcomes of patients with liver cancer. There is an urgent need to the compositional and functional understanding of tumor heterogeneity. In this review, we summarize genomic and non-genomic div...
Source: Molecular Medicine - January 5, 2018 Category: Molecular Biology Authors: Liu J, Dang H, Wang XW Tags: Exp Mol Med Source Type: research

In vitro modeling of hepatocellular carcinoma molecular subtypes for anti-cancer drug assessment.
Abstract Tractable experimental model that accounts for inter-tumor molecular heterogeneity is a key element of anti-cancer drug development. Hepatocellular carcinoma is known to exhibit highly heterogeneous molecular aberrations across the tumors, including somatic genetic and epigenetic alterations. Previous studies showed that molecular tumor subtypes determined by transcriptome, as a comprehensive functional readout, are reproducibly observed across global patient populations irrespective of geographic and etiological variations. Here we demonstrate that transcriptomic hepatocellular carcinoma subtypes, S1 and...
Source: Molecular Medicine - January 5, 2018 Category: Molecular Biology Authors: Hirschfield H, Bian CB, Higashi T, Nakagawa S, Zeleke TZ, Nair VD, Fuchs BC, Hoshida Y Tags: Exp Mol Med Source Type: research

Depletion of S100A4+ stromal cells does not prevent HCC development but reduces the stem cell-like phenotype of the tumors.
In conclusion, this study demonstrated that S100A4+ cells do not contribute to HCC onset but maintain the stemness phenotype of the tumor. This study also suggests for the first time a crosstalk between inflammation and stemness. PMID: 29303514 [PubMed - in process] (Source: Molecular Medicine)
Source: Molecular Medicine - January 5, 2018 Category: Molecular Biology Authors: Jiao J, González Á, Stevenson HL, Gagea M, Sugimoto H, Kalluri R, Beretta L Tags: Exp Mol Med Source Type: research

Immunization with an adenovirus-vectored TB vaccine containing Ag85A-Mtb32 effectively alleviates allergic asthma.
In this study, we investigated the protective effects of Ad5-gsgAM, an adenovirus vector carrying two mycobacterial antigens Ag85A and Mtb32, against allergic asthma. Using an ovalbumin (OVA)-induced asthmatic mouse model, we found that Ad5-gsgAM elicited much more Th1-biased CD4+T and CD8+T cells than bacillus Calmette-Guérin (BCG). After OVA challenge, Ad5-gsgAM-immunized mice showed significantly lowered airway inflammation in comparison with mice immunized with or without BCG. Total serum immunoglobulin E and pulmonary inducible-nitric-oxide-synthase were efficiently reduced. The cytokine profiles in bronchial-a...
Source: Molecular Medicine - January 4, 2018 Category: Molecular Biology Authors: Zhang Y, Feng Y, Li L, Ye X, Wang J, Wang Q, Li P, Li N, Zheng X, Gao X, Li C, Li F, Sun B, Lai K, Su Z, Zhong N, Chen L, Feng L Tags: J Mol Med (Berl) Source Type: research

The immunoproteasome-specific inhibitor ONX 0914 reverses susceptibility to acute viral myocarditis.
Abstract Severe heart pathology upon virus infection is closely associated with the immunological equipment of the host. Since there is no specific treatment available, current research focuses on identifying new drug targets to positively modulate predisposing immune factors. Utilizing a murine model with high susceptibility to coxsackievirus B3-induced myocarditis, this study describes ONX 0914-an immunoproteasome-specific inhibitor-as highly protective during severe heart disease. Represented by reduced heart infiltration of monocytes/macrophages and diminished organ damage, ONX 0914 treatment reversed fulminan...
Source: Molecular Medicine - January 2, 2018 Category: Molecular Biology Authors: Althof N, Goetzke CC, Kespohl M, Voss K, Heuser A, Pinkert S, Kaya Z, Klingel K, Beling A Tags: EMBO Mol Med Source Type: research

Anaplastic thyroid carcinoma on 68  Ga-PSMA PET/CT: opening new frontiers.
Anaplastic thyroid carcinoma on 68 Ga-PSMA PET/CT: opening new frontiers. Eur J Nucl Med Mol Imaging. 2018 Jan 02;: Authors: Damle NA, Bal C, Singh TP, Gupta R, Reddy S, Kumar R, Tripathi M PMID: 29294167 [PubMed - as supplied by publisher] (Source: Molecular Medicine)
Source: Molecular Medicine - January 2, 2018 Category: Molecular Biology Authors: Damle NA, Bal C, Singh TP, Gupta R, Reddy S, Kumar R, Tripathi M Tags: Eur J Nucl Med Mol Imaging Source Type: research

p75NTR and TROY: Uncharted Roles of Nogo Receptor Complex in Experimental Autoimmune Encephalomyelitis.
Abstract Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), have been on the forefront of drug discovery for most of the myelin inhibitory molecules implicated in axonal regenerative process. Nogo-A along with its putative receptor NgR and co-receptor LINGO-1 has paved the way for the production of pharmaceutical agents such as monoclonal antibodies, which are already put into handful of clinical trials. On the other side, little progress has been made towards clarifying the role of neurotrophin receptor p75 (p75NTR) and TROY in disease progression, other key players of ...
Source: Molecular Medicine - January 2, 2018 Category: Molecular Biology Authors: Theotokis P, Grigoriadis N Tags: Mol Neurobiol Source Type: research

Bispecific Antibody Armed T Cells to Target Cancer Cells.
Abstract The common strategy for making bispecific antibodies (BsAbs) involves combining the variable domains of the desired monoclonal antibodies (mAbs) into a single bispecific structure. Bispecific immunotherapeutics has generated many different formats of BsAbs including chemical heteroconjugation of two complete molecules or fragments of monoclonal antibodies, quadroma, F(ab)2, diabodies, tandem diabodies, and single-chain antibodies (scFv). This chapter describes the process of generating activated T cells and arming T cells with heteroconjugated BsAbs to target cancer cells. PMID: 29264802 [PubMed - in...
Source: Molecular Medicine - December 23, 2017 Category: Molecular Biology Authors: Thakur A, Lum LG, Mittal S Tags: Methods Mol Biol Source Type: research

Medical Implications of Space Radiation Exposure Due to Low-Altitude Polar Orbits.
In this study, we have modeled the nominal and off-nominal radiation environment that a MOL-like spacecraft vehicle would be exposed to during a 30-d mission using high performance, multicore computers. RESULTS: Projected doses from a historically large SPE (e.g., the August 1972 solar event) have been analyzed in the context of the MOL orbit profile, providing an opportunity to study its impact to crew health and subsequent contingencies. DISCUSSION: It is reasonable to presume that future commercial, government, and military spaceflight missions in low-Earth orbit (LEO) will have vehicles with similar shielding...
Source: Molecular Medicine - December 14, 2017 Category: Molecular Biology Authors: Chancellor JC, Auñon-Chancellor SM, Charles J Tags: Aerosp Med Hum Perform Source Type: research

Measuring mRNA Decay in Budding Yeast Using Single Molecule FISH.
Abstract Cellular mRNA levels are determined by the rates of mRNA synthesis and mRNA decay. Typically, mRNA degradation kinetics are measured on a population of cells that are either chemically treated or genetically engineered to inhibit transcription. However, these manipulations can affect the mRNA decay process itself by inhibiting regulatory mechanisms that govern mRNA degradation, especially if they occur on short time-scales. Recently, single molecule fluorescent in situ hybridization (smFISH) approaches have been implemented to quantify mRNA decay rates in single, unperturbed cells. Here, we provide a step...
Source: Molecular Medicine - December 14, 2017 Category: Molecular Biology Authors: Trcek T, Rahman S, Zenklusen D Tags: Methods Mol Biol Source Type: research

Identifying miRNA Targets Using AGO-RIPseq.
Abstract microRNAs (miRNA) are small, noncoding RNAs that bind to messenger RNAs (mRNAs) and regulate their activity. They are, therefore, important posttranscriptional regulators. In recent years it has become clear that miRNAs regulate large genetic networks, rather than single genes, and that one gene can be targeted by several miRNAs. To understand the role of miRNAs in cells or tissues, it is therefore important to analyze the targetome of miRNAs. Here, we present a technique called Argonaute-RNA Immunoprecipitation (AGO-RIP) which takes advantages of the fact that miRNAs and their targets are directly bound ...
Source: Molecular Medicine - December 14, 2017 Category: Molecular Biology Authors: Petri R, Jakobsson J Tags: Methods Mol Biol Source Type: research

Post-Bisulfite Adaptor Tagging for PCR-Free Whole-Genome Bisulfite Sequencing.
Abstract Post-bisulfite adaptor tagging (PBAT) is a highly efficient procedure to construct libraries for whole-genome bisulfite sequencing (WGBS). PBAT attaches adaptors to bisulfite-converted genomic DNA to circumvent bisulfite-induced degradation of library DNA inherent to conventional WGBS protocols. Consequently, it enables PCR-free WGBS from nanogram quantities of mammalian DNA, thereby serving as an invaluable tool for methylomics. PMID: 29224142 [PubMed - in process] (Source: Molecular Medicine)
Source: Molecular Medicine - December 12, 2017 Category: Molecular Biology Authors: Miura F, Ito T Tags: Methods Mol Biol Source Type: research

Expression and Purification of Rat Glucose Transporter 1 in Pichia pastoris.
Abstract Large amounts of pure and homogenous protein are a prerequisite for several biochemical and biophysical analyses, and in particular if aiming at resolving the three-dimensional protein structure. Here we describe the production of the rat glucose transporter 1 (GLUT1), a membrane protein facilitating the transport of glucose in cells. The protein is recombinantly expressed in the yeast Pichia pastoris. It is easily maintained and large-scale protein production in shaker flasks, as commonly performed in academic research laboratories, results in relatively high yields of membrane protein. The purifica...
Source: Molecular Medicine - December 10, 2017 Category: Molecular Biology Authors: Venskutonytė R, Elbing K, Lindkvist-Petersson K Tags: Methods Mol Biol Source Type: research

Glucose Transport Activity Measured in Giant Vesicles.
Abstract Incorporation of membrane proteins and internal reporter systems directly into giant vesicles, during their formation from a hydrogel surface, has emerged as a promising new concept in membrane protein characterization. Here, we provide the detailed protocol for a glucose transporter activity assay based on giant vesicles containing a fluorescent enzyme-linked reporter system internally. This assay is applicable for the functional analysis of a variety of hexose-transporting proteins. We furthermore believe that it can aid in the development of drugs targeting hexose transporters. PMID: 29218519 [Pub...
Source: Molecular Medicine - December 10, 2017 Category: Molecular Biology Authors: Hansen JS, Lindkvist-Petersson K Tags: Methods Mol Biol Source Type: research

Total Internal Reflection Fluorescence Microscopy to Study GLUT4 Trafficking.
la KG Abstract Total internal reflection fluorescence (TIRF) microscopy is a powerful method that allows examination of plasma membrane close events in real time. The last decade, the method has successfully been used to explore GLUT4 translocation in adipocytes. Here, we describe the procedure for studying GLUT4 trafficking using TIRF microscopy in isolated primary adipocytes. PMID: 29218524 [PubMed - in process] (Source: Molecular Medicine)
Source: Molecular Medicine - December 10, 2017 Category: Molecular Biology Authors: Wasserstrom S, Morén B, Stenkula KG Tags: Methods Mol Biol Source Type: research

Quantitative Proteomic Analysis of Human Aqueous Humor Using iTRAQ 4plex Labeling.
Abstract iTRAQ 4plex method enables multiplexing of up to four samples for simultaneous quantitation to improve sensitivity and scope of proteomic analysis. Here, we describe iTRAQ 4plex labeling of human aqueous humor specimens followed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and data analysis for peptide identification and quantitation using Proteome Discoverer software. The protocol can be applied for other animals as well; however, pooling of specimens may be required to obtain sufficient amount of protein for labeling. PMID: 29190021 [PubMed - in process] (S...
Source: Molecular Medicine - December 2, 2017 Category: Molecular Biology Authors: Trzeciecka A, Pattabiraman P, Piqueras MC, Toris C, Bhattacharya SK Tags: Methods Mol Biol Source Type: research

Shotgun Sphingolipid Analysis of Human Aqueous Humor.
We describe the Bligh and Dyer crude lipid extraction method and electrospray ionization tandem mass spectrometry (ESI-MS/MS) coupled with MZmine 2.21 data processing for identification and ratiometric quantitation of sphingosine, sphingosine-1-phosphate, sphingomyelin, and ceramide. PMID: 29190022 [PubMed - in process] (Source: Molecular Medicine)
Source: Molecular Medicine - December 2, 2017 Category: Molecular Biology Authors: Trzeciecka A, Arbelo U, Barron A, Edwards G, Sampathkumar S, Toris C, Bhattacharya SK Tags: Methods Mol Biol Source Type: research

Measuring Innate Immune Responses to Bacterial Viability.
Abstract The innate immune system directly senses microbial viability via the detection of a special class of viability-associated pathogen-associated molecular patterns (vita-PAMPs), such as prokaryotic messenger RNA. In the case of Gram-negative bacteria, detection of bacterial viability by phagocytes leads to a unique activation of inflammasome and type I interferon pathways, resulting in a robust pro-inflammatory innate response and a vigorous adaptive immune response. This protocol describes the methods required to study activation of both inflammasome and type I interferon pathways after stimulation of mouse...
Source: Molecular Medicine - November 29, 2017 Category: Molecular Biology Authors: Moretti J, Vabret N, Blander JM Tags: Methods Mol Biol Source Type: research

High Content Screening of Mammalian Primary Cortical Neurons.
Abstract High Content Screening (HCS) can be used to analyze the morphology of neuronal primary cultures on a large scale. When used in the field of neuronal regeneration this approach allows the screening of hundreds or thousands of perturbagens, such as miRNAs, cDNAs, or compounds, for their ability to induce neuronal growth. One of the most important steps while designing these kinds of experiments is the choice of the correct neuronal model. Testing the correct neuronal type is critical to obtain results that are biologically significant and that can later be translated to a clinical setting. For example, if t...
Source: Molecular Medicine - October 31, 2017 Category: Molecular Biology Authors: Motti D, Blackmore M, Bixby JL, Lemmon VP Tags: Methods Mol Biol Source Type: research

Flow Cytometric Analysis of Erythroblast Enucleation.
Abstract Enucleation is the final step in mammalian erythropoiesis. In this process, the nucleus is extruded by budding off from the erythroblast, forming the reticulocyte. Herein, we describe the flow cytometry-based assays for enucleation assessment. The separation of nucleated erythroblasts, reticulocytes, and extruded nuclei by flow cytometry is based on DNA staining, surface expression of erythrocyte specific markers, or forward scatter (FSC). The enucleation of murine erythroblasts is assessed by the surface expression of murine erythrocyte marker Ter119 and DNA staining. Three discrete populations that repr...
Source: Molecular Medicine - October 28, 2017 Category: Molecular Biology Authors: An X, Chen L Tags: Methods Mol Biol Source Type: research

Stopped-Flow Fluorometric Ion Flux Assay for Ligand-Gated Ion Channel Studies.
Abstract Quantitative investigations into functional properties of purified ion channel proteins using standard electrophysiological methods are challenging, in particular for the determination of average ion channel behavior following rapid changes in experimental conditions (e.g., ligand concentration). Here, we describe a method for determining the functional activity of liposome-reconstituted K(+) channels using a stopped-flow fluorometric ion flux assay. Channel activity is quantified by measuring the rate of fluorescence decrease of a liposome-encapsulated fluorophore, specifically quenched by thallium ions ...
Source: Molecular Medicine - October 24, 2017 Category: Molecular Biology Authors: Posson DJ, Rusinova R, Andersen OS, Nimigean CM Tags: Methods Mol Biol Source Type: research

cCLIP-Seq: Retrieval of Chimeric Reads from HITS-CLIP (CLIP-Seq) Libraries.
Abstract HITS-CLIP (High-Throughput Sequencing after in vivo Crosslinking and Immunoprecipitation, CLIP-Seq) libraries contain fragments of the RNA sequences bound in vivo by an RNA binding protein (RBP). Such fragments, especially if they represent RNA duplexes bound in vivo by the RBP, can occasionally be ligated together to form chimeric CLIP tags. Chimeric CLIP tags from Argonaute CLIP libraries can provide the exact base pairing profiles of small RNAs with their target RNA sequences, thus solving a critical problem in the field of post-transcriptional regulation. We recently reported an analysis of chimeric r...
Source: Molecular Medicine - October 17, 2017 Category: Molecular Biology Authors: Alexiou P, Maragkakis M, Mourelatos Z, Vourekas A Tags: Methods Mol Biol Source Type: research

A Versatile Assay for Detection of Aberrant DNA Methylation in Bladder Cancer.
Abstract Urothelial carcinoma of the bladder is one of the most common malignancies in the industrialized world, mainly caused by smoking and occupational exposure to chemicals. The favorable prognosis of early stage bladder cancer underscores the importance of early detection for the treatment of this disease. The high recurrence rate of this malignancy also highlights the need for close post-diagnosis monitoring of bladder cancer patients. As for other malignancies, aberrant DNA methylation has been shown to play a crucial role in the initiation and progression of bladder cancer, and thus holds great promise as ...
Source: Molecular Medicine - September 11, 2017 Category: Molecular Biology Authors: Tommasi S, Besaratinia A Tags: Methods Mol Biol Source Type: research

Acquisition and adaptation of the airway microbiota in the early life of cystic fibrosis patients.
The objective of this mini-review is to give an update on the current knowledge about the development of the microbiota in the early life of CF patients. Microbial acquisition in the human airways can be described by the island model: Microbes found in the lower airways of CF patients represent "islands" that are at first populated from the upper airways reflecting the "mainland." Colonization can be modeled following the neutral theory in which the most abundant bacteria in the mainland are also frequently found in the lower airways initially. At later times, however, the colonization process of the lo...
Source: Molecular Medicine - January 19, 2017 Category: Molecular Biology Authors: Boutin S, Dalpke AH Tags: Mol Cell Pediatr Source Type: research

Bacterial Proteasomes: Mechanistic and Functional Insights.
Abstract Regulated proteolysis is essential for the normal physiology of all organisms. While all eukaryotes and archaea use proteasomes for protein degradation, only certain orders of bacteria have proteasomes, whose functions are likely as diverse as the species that use them. In this review, we discuss the most recent developments in the understanding of how proteins are targeted to proteasomes for degradation, including ATP-dependent and -independent mechanisms, and the roles of proteasome-dependent degradation in protein quality control and the regulation of cellular physiology. Furthermore, we explore newly ...
Source: Molecular Medicine - December 17, 2016 Category: Molecular Biology Authors: Becker SH, Darwin KH Tags: Microbiol Mol Biol Rev Source Type: research

Hepatitis B Virus Infection of HepaRG Cells, HepaRG-hNTCP Cells, and Primary Human Hepatocytes.
Abstract Investigations of virus-host interactions rely on suitable in vitro cell culture systems that efficiently support virus infection. Such systems should ideally provide conditions that resemble those of natural host cells, e.g., the cell-type specific signaling and metabolic pathways. For HBV infection, primary human hepatocytes (PHHs) are the most faithful system fulfilling these requirements but access to these cells is limited. Moreover, the reproducibility of experimental results depends on many factors including the preparation method or variability of the donors. The human liver cell line HepaRG, afte...
Source: Molecular Medicine - December 17, 2016 Category: Molecular Biology Authors: Ni Y, Urban S Tags: Methods Mol Biol Source Type: research

Live Cell Imaging Confocal Microscopy Analysis of HBV Myr-PreS1 Peptide Binding and Uptake in NTCP-GFP Expressing HepG2 Cells.
e D Abstract To obtain basic knowledge about specific molecular mechanisms involved in the entry of pathogens into cells is the basis for establishing pharmacologic substances blocking initial viral binding, infection, and subsequent viral spread. Lack of information about key cellular factors involved in the initial steps of HBV infection has hampered the characterization of HBV binding and entry for decades. However, recently, the liver-specific sodium-dependent taurocholate cotransporting polypeptide (NTCP) has been discovered as a functional receptor for HBV and HDV, thus opening the field for new concepts of ...
Source: Molecular Medicine - December 17, 2016 Category: Molecular Biology Authors: König A, Glebe D Tags: Methods Mol Biol Source Type: research

Studying HBV Infection and Therapy in Immune-Deficient NOD-Rag1-/-IL2RgammaC-null (NRG) Fumarylacetoacetate Hydrolase (Fah) Knockout Mice Transplanted with Human Hepatocytes.
Abstract Chimeric mouse models with a humanized liver provide a unique tool to study hepatic virus diseases, including viral infection, viral pathogenesis, and antiviral therapy. Here we describe a detailed protocol for studying hepatitis B infection in NRG-derived fumarylacetoacetate hydrolase (Fah) knockout mice repopulated with human hepatocytes. The procedures include (1) maintenance and genotyping of the homozygous NRG-fah/fah mutant mice (NRG/F), (2) intrasplenic injection of human hepatocytes, (3) NTBC drug reduction cycling to improve human hepatocyte repopulation, (4) human albumin detection, and (5) HBV ...
Source: Molecular Medicine - December 17, 2016 Category: Molecular Biology Authors: Li F, Nio K, Yasui F, Murphy CM, Su L Tags: Methods Mol Biol Source Type: research

Monitoring Synapses Via Trans-Synaptic GFP Complementation.
Abstract Over the last years, the analysis of synaptic connectivity in the mammalian brain has been accelerated by the use of techniques combining electrophysiology, light microscopy, viral tracing, and genetic manipulations in animal models. Of particular interest are methods that aim to label synapses by tethering complementary split GFP fragments in opposing sites of the synaptic cleft. Here, I describe SynView, a method for monitoring synapse formation based on GFP complementation, and provide a detailed protocol for use in neuronal cultures from mouse hippocampus. PMID: 27943182 [PubMed - in process] (So...
Source: Molecular Medicine - December 14, 2016 Category: Molecular Biology Authors: Tsetsenis T Tags: Methods Mol Biol Source Type: research

Quantifying Tensile Force and ERK Phosphorylation on Actin Stress Fibers.
Abstract ERK associates with the actin cytoskeleton, and the actin-associated pool of ERK can be activated (phosphorylated in the activation loop) to induce specific cell responses. Increasing evidence has shown that mechanical conditions of cells significantly affect ERK activation. In particular, tension developed in the actin cytoskeleton has been implicated as a critical mechanism driving ERK signaling. However, a quantitative study of the relationship between actin tension and ERK phosphorylation is missing. In this chapter, we describe our novel methods to quantify tensile force and ERK phosphorylation on in...
Source: Molecular Medicine - December 9, 2016 Category: Molecular Biology Authors: Hirata H, Gupta M, Vedula SR, Lim CT, Ladoux B, Sokabe M Tags: Methods Mol Biol Source Type: research