Complementation Analysis Research Articles

Poly- and/or Multiculturality of Future Teachers in Foreign Language Instruction: Methodological Facet

The article considers some methodological approaches that underlie the research and study of questions connected to education and cultivation of polyculturality and multiculturality of / with future teachers in foreign language instruction in higher education. In particular, the focuses are with the study and discussion of the culturological and axiological approaches to complement synthesis and analysis, induction and deduction, etc. It is believed that it is philosophy, which seeks to act as a coordinator of interactions between others and their own - the implementation of the subjects’ understanding of their practical value, normative and cognitive behaviors in the general cultural space. To this part, philosophical thinking converges with the social action theories, where the purpose is to create a productive exchange of meanings, values ??and concepts between subjects in an interaction, in which such subjects are seen ‘engaged agents’ rather than ‘puppets’ of the society.

Species-specific accumulation of ceramides in cerebrospinal fluid from encephalomyeloradiculoneurpathy patients associated with peripheral complement activation: A pilot study

Glycolipids are now known to be rapidly converted to mediators for inflammatory reactions or to signaling molecules that control inflammatory events in the nervous system. The present study aimed to explore whether disturbed glycolipids metabolism in the nervous system is present in patients with a neuroinflammatory disorder, encephalo-myelo-radiculo-neuropathy (EMRN), because most EMRN patients have been reported to exhibit autoantibodies against neutral glycolipids. Although molecular pathogenesis of this disorder remains unknown, we tried to search the immunochemical abnormalities in this disorder. ELISA for activated peripheral C5 complement and mass spectrometry analysis of cerebrospinal fluid clearly disclosed a significant upregulation of active C5 complement, C5a levels in sera as well as a significant accumulation of species-specific ceramides but not sphingomyelin in cerebrospinal fluid from EMRN patients. Furthermore, we confirmed the occurrence of anti-neutral glycolipids antibodies in all EMRN patients.Thus, the present study might indicate the pathophysiology of this disorder is the dysregulation of glycolipids metabolism and abnormal production of autoantibodies against neutral glycolipids resulting in the abnormal complement activation, although molecular basis for these sphingolipids dysregulation and the occurrence of autoantibodies against glycolipids remains to be elucidated at present. The present study implicates a new therapeutic strategy employing anti-ceramide and/or anti-complement therapy for this disorder.

Molecular Characterization of U-box E3 Ubiquitin Ligases (TaPUB2 and TaPUB3) Involved in the Positive Regulation of Drought Stress Response in Arabidopsis.

Plant U-box E3 ubiquitin ligase (PUB) is involved in various environmental stress conditions. However, the molecular mechanism of U-box proteins in response to abiotic stress in wheat remains unknown. In this study, two U-box E3 ligase genes (TaPUB2 and TaPUB3), which are highly expressed in response to adverse abiotic stresses, were isolated from common wheat, and their cellular functions were characterized under drought stress. Transient expression assay revealed that TaPUB2 was localized in the cytoplasm and Golgi apparatus, whereas TaPUB3 was expressed only in the Golgi apparatus in wheat protoplasts. Additionally, TaPUB2 and TaPUB3 underwent self-ubiquitination. Moreover, TaPUB2/TaPUB3 heterodimer was identified in yeast and the cytoplasm of wheat protoplasts using a pull-down assay and bimolecular fluorescence complementation analysis. Heterogeneous overexpression of TaPUB2 and TaPUB3 conferred tolerance to drought stress. Taken together, these results implied that the heterodimeric form of U-box E3 ubiquitin ligases (TaPUB2/TaPUB3) responded to abiotic stress and roles as a positive regulator of drought stress tolerance.

Protein-protein interactions in fatty acid elongase complexes are important for very-long-chain fatty acid synthesis.

Fatty acid elongase (FAE), which catalyzes the synthesis of very-long-chain fatty acids (VLCFAs), is a multiprotein complex; however, little is known about its quaternary structure. In this study, bimolecular fluorescence complementation and/or yeast two-hybrid assays showed that homo-interactions were observed in β-ketoacyl-CoA synthases (KCS2, KCS9, and KCS6), Eceriferum2-like proteins [CER2 and CER2-Like2 (C2L2)], and FAE complex proteins (KCR1, PAS2, ECR, and PAS1), except for CER2-Like1 (C2L1). Hetero-interactions were observed between KCSs (KCS2, KCS9, and KCS6), between CER2-LIKEs (CER2, C2L2, and C2L1), and between FAE complex proteins (KCR1, PAS2, ECR, and PAS1). PAS1 interacts with FAE complex proteins (KCR1, PAS2, and ECR), but not with KCSs (KCS2, KCS9, and KCS6) and CER2-LIKEs (CER2, C2L2, and C2L1). Asp308 and Arg309-Arg311 of KCS9 were essential for the homo-interactions of KCS9 and hetero-interactions between KCS9 and PAS2 or ECR. Asp339 of KCS9 is involved in its homo- and hetero-interactions with ECR. Complementation analysis of the Arabidopsis kcs9 mutant by the expression of amino acid-substituted KCS9 mutant genes showed that Asp308 and Asp339 of KCS9 are involved in the synthesis of C24 VLCFAs from C22. This study suggests that protein-protein interaction in FAE complexes is important for VLCFA synthesis and provides insight into the quaternary structure of FAE complexes for efficient synthesis of VLCFAs.

A Novel Chloroplast Protein RNA Processing 8 Is Required for the Expression of Chloroplast Genes and Chloroplast Development in Arabidopsis thaliana.

Chloroplast development involves the coordinated expression of both plastids- and nuclear-encoded genes in higher plants. However, the underlying mechanism still remains largely unknown. In this study, we isolated and characterized an Arabidopsis mutant with an albino lethality phenotype named RNA processing 8 (rp8). Genetic complementation analysis demonstrated that the gene AT4G37920 (RP8) was responsible for the mutated phenotype. The RP8 gene was strongly expressed in photosynthetic tissues at both transcription and translation protein levels. The RP8 protein is localized in the chloroplast and associated with the thylakoid. Disruption of the RP8 gene led to a defect in the accumulation of the rpoA mature transcript, which reduced the level of the RpoA protein, and affected the transcription of PEP-dependent genes. The abundance of the chloroplast rRNA, including 23S, 16S, 4.5S, and 5S rRNA, were reduced in the rp8 mutant, respectively, and the amounts of chloroplast ribosome proteins, such as, PRPS1(uS1c), PRPS5(uS5c), PRPL2 (uL2c), and PRPL4 (uL4c), were substantially decreased in the rp8 mutant, which indicated that knockout of RP8 seriously affected chloroplast translational machinery. Accordingly, the accumulation of photosynthetic proteins was seriously reduced. Taken together, these results indicate that the RP8 protein plays an important regulatory role in the rpoA transcript processing, which is required for the expression of chloroplast genes and chloroplast development in Arabidopsis.

Lupus band test can be used in combination with anti-chromatin antibodies and complement analysis to predict transition from cutaneous to systemic lupus

The lupus band test (LBT) is frequently performed for patients with lupus erythematosus (LE) but its capacity to discriminate cutaneous (C)LE from systemic (S)LE is debated, as well as its association with serum antinuclear antibodies (ANA) and complement reduction. Among 158 patients, 56 received retrospectively a diagnosis of CLE, 37 have SLE and 65 other skin disorders. Considering 29 clinical, histopathologic, LBT, and serological parameters: 5 parameters were effective in distinguishing LE from other skin disorders (e.g. skin photosensitivity, LBT positivity, basal vacuolar changes, thickening of the basement membrane, and anti-SSA-60 kDa); and 8 parameters were able to separate SLE from CLE (e.g. arthritis, lupus nephritis, hematological manifestations, Raynaud/sicca manifestations, anti-chromatin, anti-dsDNA, and low levels of C3/4). A positive LBT was further determined to be associated with systemic manifestations when associated with anti-chromatin response and complement reduction in the profile of patients evolving to a systemic form of lupus.

Distribution, Function, and Evolution of a Gene Essential for Trichothecene Toxin Biosynthesis in Trichoderma.

Trichothecenes are terpenoid toxins produced by species in 10 fungal genera, including species of Trichoderma. The trichothecene biosynthetic gene (tri) cluster typically includes the tri5 gene, which encodes a terpene synthase that catalyzes formation of trichodiene, the parent compound of all trichothecenes. The two Trichoderma species, Trichoderma arundinaceum and T. brevicompactum, that have been examined are unique in that tri5 is located outside the tri cluster in a genomic region that does not include other known tri genes. In the current study, analysis of 35 species representing a wide range of the phylogenetic diversity of Trichoderma revealed that 22 species had tri5, but only 13 species had both tri5 and the tri cluster. tri5 was not located in the cluster in any species. Using complementation analysis of a T. arundinaceum tri5 deletion mutant, we demonstrated that some tri5 homologs from species that lack a tri cluster are functional, but others are not. Phylogenetic analyses suggest that Trichoderma tri5 was under positive selection following its divergence from homologs in other fungi but before Trichoderma species began diverging from one another. We propose two models to explain these diverse observations. One model proposes that the location of tri5 outside the tri cluster resulted from loss of tri5 from the cluster in an ancestral species followed by reacquisition via horizontal transfer. The other model proposes that in species that have a functional tri5 but lack the tri cluster, trichodiene production provides a competitive advantage.

A phosphorus-limitation induced, functionally conserved DUF506 protein is a repressor of root hair elongation in plants.

Summary Root hairs (RHs) function in nutrient and water acquisition, root metabolite exudation, soil anchorage and plant–microbe interactions. Longer or more abundant RHs are potential breeding traits for developing crops that are more resource‐use efficient and can improve soil health.While many genes are known to promote RH elongation, relatively little is known about genes and mechanisms that constrain RH growth.Here we demonstrate that a DOMAIN OF UNKNOWN FUNCTION 506 (DUF506) protein, AT3G25240, negatively regulates Arabidopsis thaliana RH growth. The AT3G25240 gene is strongly and specifically induced during phosphorus (P)‐limitation. Mutants of this gene, which we call REPRESSOR OF EXCESSIVE ROOT HAIR ELONGATION 1 (RXR1), have much longer RHs, higher phosphate content and seedling biomass, while overexpression of the gene exhibits opposite phenotypes. Co‐immunoprecipitation, pull‐down and bimolecular fluorescence complementation (BiFC) analyses reveal that RXR1 physically interacts with a RabD2c GTPase in nucleus, and a rabd2c mutant phenocopies the rxr1 mutant. Furthermore, N‐terminal variable region of RXR1 is crucial for inhibiting RH growth. Overexpression of a Brachypodium distachyon RXR1 homolog results in repression of RH elongation in Brachypodium.Taken together, our results reveal a novel DUF506‐GTPase module with a prominent role in repression of plant RH elongation especially under P stress.

The RNA Directed DNA Methylation (RdDM) Pathway Regulates Anthocyanin Biosynthesis in Crabapple (Malus cv. spp.) Leaves by Methylating the McCOP1 Promoter.

The synthesis of anthocyanin pigments in plants is known to be regulated by multiple mechanisms, including epigenetic regulation; however, the contribution of the RNA-directed DNA methylation (RdDM) pathway is not well understood. Here, we used bisulfite sequencing and Real Time (RT)-quantitative (q) PCR to analyze the methylation level of the promoter of constitutively photomorphogenic 1 (McCOP1) from Malus cv. spp, a gene involved in regulating anthocyanin biosynthesis. The CHH methylation level of the McCOP1 promoter was negatively correlated with McCOP1 RNA expression, and inhibiting DNA methylation caused decreased methylation of the McCOP1 promoter and asymmetric cytosine CHH methylation. We observed that the McCOP1 promoter was a direct target of the RdDM pathway argonaute RISC component 4 (McAGO4) protein, which bound to a McCOP1 promoter GGTTCGG site. Bimolecular fluorescence complementation (BIFC) analysis showed that RNA-directed DNA methylation (McRDM1) interacted with McAGO4 and another RdDM protein, domains rearranged methyltransferase 2 (McDRM2), to regulate the CHH methylation of the McCOP1 promoter. Detection of CHH methylation and COP1 gene expression in the Arabidopsis thaliana atago4, atdrm2 and atrdm1 mutants showed that RDM1 is the effector of the RdDM pathway. This was confirmed by silencing McRDM1 in crabapple leaves or apple fruit, which resulted in a decrease in McCOP1 CHH methylation and an increase in McCOP1 transcript levels, as well as in anthocyanin accumulation. In conclusion, these results show that the RdDM pathway is involved in regulating anthocyanin accumulation through CHH methylation of the McCOP1 promoter.

Genome-Wide Identification of Wheat ZIP Gene Family and Functional Characterization of the TaZIP13-B in Plants.

The ZIP (Zn-regulated, iron-regulated transporter-like protein) transporter plays an important role in regulating the uptake, transport, and accumulation of microelements in plants. Although some studies have identified ZIP genes in wheat, the significance of this family is not well understood, particularly its involvement under Fe and Zn stresses. In this study, we comprehensively characterized the wheat ZIP family at the genomic level and performed functional verification of three TaZIP genes by yeast complementary analysis and of TaZIP13-B by transgenic Arabidopsis. Totally, 58 TaZIP genes were identified based on the genome-wide search against the latest wheat reference (IWGSC_V1.1). They were then classified into three groups, based on phylogenetic analysis, and the members within the same group shared the similar exon-intron structures and conserved motif compositions. Expression pattern analysis revealed that the most of TaZIP genes were highly expressed in the roots, and nine TaZIP genes displayed high expression at grain filling stage. When exposed to ZnSO4 and FeCl3 solutions, the TaZIP genes showed differential expression patterns. Additionally, six ZIP genes responded to zinc-iron deficiency. A total of 57 miRNA-TaZIP interactions were constructed based on the target relationship, and three miRNAs were downregulated when exposed to the ZnSO4 and FeCl3 stresses. Yeast complementation analysis proved that TaZIP14-B, TaZIP13-B, and TaIRT2-A could transport Zn and Fe. Finally, overexpression of TaZIP13-B in Arabidopsis showed that the transgenic plants displayed better tolerance to Fe/Zn stresses and could enrich more metallic elements in their seeds than wild-type Arabidopsis. This study systematically analyzed the genomic organization, gene structure, expression profiles, regulatory network, and the biological function of the ZIP family in wheat, providing better understanding of the regulatory roles of TaZIPs and contributing to improve nutrient quality in wheat crops.

Knowledge Co-construction by Citizens and Researchers to Create a SNAPSHOT of the Marine Environment During and After the Covid-19 Lockdown

Lockdown measures adopted in Italy to contain the diffusion of Covid-19 altered many variables influencing the anthropogenic pressure on marine ecosystems. Public reactions included surprise at how quickly changes in human activity seemingly improved natural recovery and, at the same time, a generalized anxiety to restart economic activities. In this situation several Institutions from the Italian National Research Council (CNR) made a joint effort to quantify the effects of the unprecedented experimental conditions induced by the reduction of many anthropogenic pressures. The resulting project was conceived with a holistic, interdisciplinary approach, geared to combine scientific, economic and cultural observations to promote collective actions suitable to the governance of socio-ecological systems, reconciling respect for the environment with human activities and wellbeing, and thus grounding an ethical approach to marine resources. Alongside collecting considerable amount of scientific observations, the project is working to complement samplings and analyses with the non-formal knowledge carried by the inhabitants of a set of coastal zones, thus enriching the generated knowledge and widening inclusion and pluralism in defining the challenges at stake; simultaneously it focuses on stimulating a reflection in the research community over the process of knowledge co-construction, its meaning, role and responsibility in the societal context. After a brief contextualization of this activity, we present here the perspective adopted by researchers to build a responsible marine research plan, inclusive on the grounds of both involved actors and knowledge sources. We comment the process- and community-related features, explore limits and opportunities, and propose a set of recommendations, based on a preliminary review of our experience and oriented to promote the development of a shared Ocean ethics.

Article of Significant Interest in This Issue

The intracellular membrane domain (IMD) is a metabolically active membrane compartment of mycobacteria associated with cell growth. The IMD proteome is not yet fully established. Rokicki et al. (e00419-21) took advantage of the Mycobacterial Systems Resource, a database that includes fluorescence microscopy of 1,153 proteins highly conserved among mycobacteria, to complement and cross-validate proteomic analysis of the IMD. They confirmed the subcellular localization of 20 previously identified IMD proteins while discovering three new ones, demonstrating the utility of this novel strategy in overcoming some of the limitations of conventional proteomics.

Complement multiplex testing: Concept, promises and pitfalls

Complement is a complex system. This complexity becomes more obvious when looking at complement analysis in health and disease, where one presentation can require a number of measurements to understand the full role of this cascade in the disease. The current state of clinical testing requires multiple tests to cover the whole of the complement cascade. There is a clear potential for multiplex testing to help address this need for comprehensive analysis of the state of complement deficiency, activation or inhibition. Fortunately, there are a number of potential methods for multiplex analysis, each with advantages and disadvantages that need to be considered in light of the intricacy of the complement cascade and its interconnection to other systems. Despite the complexities of such methods several groups have started utilizing multiplex analysis for research and even for diagnostic testing. The potential methods, current successes, and the type of testing that needs to be streamlined are reviewed in this text.

DNA binding and gene regulatory functions of MSMEG_2295, a repressor encoded by the dinB2 operon of Mycobacterium smegmatis.

MSMEG_2295 is a TetR family protein encoded by the first gene of a Mycobacterium smegmatis (Msm) operon that expresses the gene for DinB2 (MSMEG_2294), a translesion DNA repair enzyme. We have carried out investigations to understand its function by performing DNA binding studies and gene knockout experiments. We found that the protein binds to a conserved inverted repeat sequence located upstream of the dinB2 operon and several other genes. Using a knockout of MSMEG_2295, we show that MSMEG_2295 controls the expression of at least five genes, the products of which could potentially influence carbohydrate and fatty acid metabolism as well as antibiotic and oxidative stress resistance. We have demonstrated that MSMEG_2295 is a repressor by performing complementation analysis. Knocking out of MSMEG_2295 had a significant impact on pyruvate metabolism. Pyruvate dehydrogenase activity was virtually undetectable in ΔMSMEG_2295, although in the complemented strain, it was high. We also show that knocking out of MSMEG_2295 causes resistance to H2O2, reversed in the complemented strain. We have further found that the mycobacterial growth inhibitor plumbagin, a compound of plant origin, acts as an inducer of MSMEG_2295 regulated genes. We, therefore, establish that MSMEG_2295 functions by exerting its role as a repressor of multiple Msm genes and that by doing so, it plays a vital role in controlling pyruvate metabolism and response to oxidative stress.

Antiviral Activity of Canine RIG-I against Canine Influenza Virus and Interactions between Canine RIG-I and CIV.

RIG-I functions as a virus sensor that induces a cellular antiviral response. Although it has been investigated in other species, there have been no further studies to date on canine RIG-I against canine influenza virus (CIV). In the present study, we cloned the RIG-I gene of beagle dogs and characterized its expression, subcellular localization, antiviral response, and interactions with CIV proteins. RIG-I was highly expressed and mainly localized in the cytoplasm, with low levels detected in the nucleus. The results revealed that overexpression of the CARD domain of RIG-I and knockdown of RIG-I showed its ability to activate the RLR pathway and induced the expression of downstream interferon-stimulated genes. Moreover, overexpression of canine RIG-I suppressed the replication of CIV. The association between RIG-I and CIV was evaluated with the luciferase assay and by indirect immunofluorescence and bimolecular fluorescence complementation analyses. The results showed that CIV nonstructural protein 1 (NS1) can strongly suppress the RIG-I–mediated innate immune response, and the novel interactions between CIV matrix proteins (M1 and M2) and canine RIG-I were disclosed. These findings provide a basis for investigating the antiviral mechanism of canine RIG-I against CIV, which can lead to effective strategies for preventing CIV infection in dogs.

Point Mutations in the Glycoprotein Ectodomain of Field Rabies Viruses Mediate Cell Culture Adaptation through Improved Virus Release in a Host Cell Dependent and Independent Manner.

Molecular details of field rabies virus (RABV) adaptation to cell culture replication are insufficiently understood. A better understanding of adaptation may not only reveal requirements for efficient RABV replication in cell lines, but may also provide novel insights into RABV biology and adaptation-related loss of virulence and pathogenicity. Using two recombinant field rabies virus clones (rRABV Dog and rRABV Fox), we performed virus passages in three different cell lines to identify cell culture adaptive mutations. Ten passages were sufficient for the acquisition of adaptive mutations in the glycoprotein G and in the C-terminus of phosphoprotein P. Apart from the insertion of a glycosylation sequon via the mutation D247N in either virus, both acquired additional and cell line-specific mutations after passages on BHK (K425N) and MDCK-II (R346S or R350G) cells. As determined by virus replication kinetics, complementation, and immunofluorescence analysis, the major bottleneck in cell culture replication was the intracellular accumulation of field virus G protein, which was overcome after the acquisition of the adaptive mutations. Our data indicate that limited release of extracellular infectious virus at the plasma membrane is a defined characteristic of highly virulent field rabies viruses and we hypothesize that the observed suboptimal release of infectious virions is due to the inverse correlation of virus release and virulence in vivo.

In silico characterization of the GH5-cellulase family from uncultured microorganisms: physicochemical and structural studies

BackgroundHydrolysis of cellulose-based biomass by cellulases produce fermented sugar for making biofuels, such as bioethanol. Cellulases hydrolyze the β-1,4-glycosidic linkage of cellulose and can be obtained from cultured and uncultured microorganisms. Uncultured microorganisms are a source for exploring novel cellulase genes through the metagenomic approach. Metagenomics concerns the extraction, cloning, and analysis of the entire genetic complement of a habitat without cultivating microbes. The glycoside hydrolase 5 family (GH5) is a cellulase family, as the largest group of glycoside hydrolases. Numerous variants of GH5-cellulase family have been identified through the metagenomic approach, including CelGH5 in this study. University-CoE-Research Center for Biomolecule Engineering, Universitas Airlangga successfully isolated CelGH5 from waste decomposition of oil palm empty fruit bunches (OPEFB) soil by metagenomics approach. The properties and structural characteristics of GH5-cellulases from uncultured microorganisms can be studied using computational tools and software. ResultsThe GH5-cellulase family from uncultured microorganisms was characterized using standard computational-based tools. The amino acid sequences and 3D-protein structures were retrieved from the GenBank Database and Protein Data Bank. The physicochemical analysis revealed the sequence length was roughly 332–751 amino acids, with the molecular weight range around 37–83 kDa, dominantly negative charges with pI values below 7. Alanine was the most abundant amino acid making up the GH5-cellulase family and the percentage of hydrophobic amino acids was more than hydrophilic. Interestingly, ten endopeptidases with the highest average number of cleavage sites were found. Another uniqueness demonstrated that there was also a difference in stability between in silico and wet lab. The II values indicated CelGH5 and ACA61162.1 as unstable enzymes, while the wet lab showed they were stable at broad pH range. The program of SOPMA, PDBsum, ProSA, and SAVES provided the secondary and tertiary structure analysis. The predominant secondary structure was the random coil, and tertiary structure has fulfilled the structure quality of QMEAN4, ERRAT, Ramachandran plot, and Z score. ConclusionThis study can afford the new insights about the physicochemical and structural properties of the GH5-cellulase family from uncultured microorganisms. Furthermore, in silico analysis could be valuable in selecting a highly efficient cellulases for enhanced enzyme production.

Heterologous Production of Unnatural Flavipucine Family Products Provides Insights into Flavipucines Biosynthesis.

Heterologous expression of the flavipucine biosynthetic gene cluster in Aspergillusnidulans led to the production of flavipucine (1) and dihydroisoflavipucine (3), as well as six unusual flavipucine related products containing three classes of heterocycles. This combined with gene inactivation, chemical complementation, and transcriptome analysis demonstrated unprecedented ways to form 2-pyridone and 2-pyrone structures by the oxidative rearrangements of pyrrolinone precursors as well as provided insights into the biosynthesis of this important class of natural products.

Oxidized LDL but not angiotensin II induces cardiomyocyte hypertrophic responses through the interaction between LOX-1 and AT1 receptors

It is well known that lectin-like oxidized low-density lipoprotein (ox-LDL) and its receptor LOX-1, angiotensin II (AngII) and its type 1 receptor (AT1-R) play an important role in the development of cardiac hypertrophy. However, the molecular mechanism is not clear. In this study, we found that ox-LDL-induced cardiac hypertrophy was suppressed by inhibition of LOX-1 or AT1-R but not by AngII inhibition. These results suggest that the receptors LOX-1 and AT1-R, rather than AngII, play a key role in the role of ox-LDL. The same results were obtained in mice lacking endogenous AngII and their isolated cardiomyocytes. Ox-LDL but not AngII could induce the binding of LOX-1 and AT1-R; inhibition of LOX-1 or AT1-R but not AngII could abolish the binding of these two receptors. Overexpression of wild type LOX-1 with AT1-R enhanced ox-LDL-induced binding of two receptors and phosphorylation of ERKs, however, transfection of LOX-1 dominant negative mutant (lys266ala / lys267ala) or an AT1-R mutant (glu257ala) not only reduced the binding of two receptors but also inhibited the ERKs phosphorylation. Phosphorylation of ERKs induced by ox-LDL in LOX-1 and AT1-R-overexpression cells was abrogated by an inhibitor of Gq protein rather than Jak2, Rac1 or RhoA. Genetically, an AT1-R mutant lacking Gq protein coupling ability inhibited ox-LDL induced ERKs phosphorylation. Furthermore, through bimolecular fluorescence complementation analysis, we confirmed that ox-LDL rather than AngII stimulation induced the direct binding of LOX-1 and AT1-R. We conclude that direct binding of LOX-1 and AT1-R and the activation of downstream Gq protein are important mechanisms of ox-LDL-induced cardiomyocyte hypertrophy.

Addressing the issue of bias in observational studies: Using instrumental variables and a quasi-randomization trial in an ESME research project.

PurposeObservational studies using routinely collected data are faced with a number of potential shortcomings that can bias their results. Many methods rely on controlling for measured and unmeasured confounders. In this work, we investigate the use of instrumental variables (IV) and quasi-trial analysis to control for unmeasured confounders in the context of a study based on the retrospective Epidemiological Strategy and Medical Economics (ESME) database, which compared overall survival (OS) with paclitaxel plus bevacizumab or paclitaxel alone as first-line treatment in patients with HER2-negative metastatic breast cancer (MBC).Patients and methodsCausal interpretations and estimates can be made from observation data using IV and quasi-trial analysis. Quasi-trial analysis has the same conceptual basis as IV, however, instead of using IV in the analysis, a “superficial” or “pseudo” randomized trial is used in a Cox model. For instance, in a multicenter trial, instead of using the treatment variable, quasi-trial analysis can consider the treatment preference in each center, which can be informative, and then comparisons of results between centers or clinicians can be informative.ResultsIn the original analysis, the OS adjusted for major factors was significantly longer with paclitaxel and bevacizumab than with paclitaxel alone. Using the center-treatment preference as an instrument yielded to concordant results. For the quasi-trial analysis, a Cox model was used, adjusted on all factors initially used. The results consolidate those obtained with a conventional multivariate Cox model.ConclusionUnmeasured confounding is a major concern in observational studies, and IV or quasi-trial analysis can be helpful to complement analysis of studies of this nature.