Expression Constructs Research Articles

Nerve pathology is prevented by linker proteins in mouse models for LAMA2-related muscular dystrophy.

LAMA2-related muscular dystrophy (LAMA2 MD or MDC1A) is a devastating congenital muscular dystrophy that is caused by mutations in the LAMA2 gene encoding laminin-α2, the long chain of several heterotrimeric laminins. Laminins are essential components of the extracellular matrix that interface with underlying cells. The pathology of LAMA2 MD patients is dominated by an early-onset, severe muscular dystrophy that ultimately leads to death by respiratory insufficiency. However, pathology in nonmuscle tissues has been described. Prior work in the dyW /dyW mouse model for LAMA2 MD has shown that two linker proteins, mini-agrin and αLNNd, when expressed in skeletal muscle fibers, greatly increase survival from a few months up to more than 2 years. However, the restoration of skeletal muscle function accentuates the pathology in nonmuscle tissue in dyW /dyW mice, first and foremost in the peripheral nerve resulting in paralysis of the hind limbs. We now show that the expression of the two linker proteins in all tissues ameliorates the muscular dystrophy and prevents the appearance of the hind limb paralysis. Importantly, the same ameliorating effect of the linker proteins was seen in dy3K /dy3K mice, which represent the most severe mouse model of LAMA2 MD. In summary, these data show that the two linker proteins can compensate the loss of laminin-α2 in muscle and peripheral nerve, which are the two organs most affected in LAMA2 MD. These results are of key importance for designing appropriate expression constructs for mini-agrin and αLNNd to develop a gene therapy for LAMA2 MD patients.

Expression Analysis of CoHMGS in Cornus officinalis and Subcellular Localization of the Enzyme It Encodes.

Cornus officinalis is a perennial deciduous tree or shrub. Its mature fruits are extracted and used in Traditional Chinese Medicine, called Shanzhuyu. The characteristic active components of C. officinalis include loganin and morroniside, which belong to iridoid glycosides. 3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) is a key enzyme in the cytoplasmic mevalonate pathway providing the precursor molecules isopentenyl pyrophosphate and dimethylallyl pyrophosphate for isoprenoid biosynthesis such as sterols, triterpenes, and their derivatives such as iridoid glycosides. Different concentrations of methyl jasmonate (MeJA) and ethephon (ETH) solutions were sprayed on C. officinalis seedlings, and the effect of hormones on CoHMGS gene expression was detected by real-time fluorescence quantitative PCR. The quantitative real-time PCR results showed that 750 mg/L ETH treatment had the most significant induction effect on CoHMGS gene expression. The HPLC analysis of extracts revealed that the treatment could also significantly increase the content of morroniside and loganin in the leaves of C. officinalis. By use of a CoHMGS-green fluorescent protein (GFP) fusion construct for heterologous expression in tobacco, laser scanning confocal microscopy revealed a cytoplasmic localization. This preliminary study of the CoHMGS gene could prepare the ground for more precisely elucidating the synthesis of secondary metabolite in C. officinalis.

Quality control of fluorescence polarization assay over gel-based assay.

Desmoplakin (DSP) is an integral part of the desmosome, the intercellular junction that links the intermediate filaments of adjacent cells. Specific mutations in DSP (R451G, S507F, S442F, and S299R) result in calpain hypersensitivity. Our long-term objective is to reduce this hypersensitivity by occluding the protease binding site of DSP with small molecule drugs or other molecules. To do this type of screening efficiently, we need a reliable, robust, and inexpensive DSP screening method. However, existing DSP expression constructs are low yield. Also, existing methods of observing degradation rates are done via a protein and time-intensive gel-based assay. Last, calpain is expensive. Here we describe a new fluorescence polarization assay to detect DSP degradation using both a shorter fluorescein isothiocyanate (FITC) labeled DSP construct and using trypsin as the protease. We show that this shorter construct is susceptible to degradation however the mutants degrade at a faster rate. We also show that trypsin was a reasonable substitute for calpain. Having now developed these new reagents and methods, we are well-positioned to screen for ways to rescue DSP mutants.

Comprehensive analysis of the RNA transcriptome expression profiles and construction of the ceRNA network in heart failure patients with sacubitril/valsartan therapeutic heterogeneity after acute myocardial infarction

Sacubitril/valsartan has a noteworthy advantage in improving ventricular remodelling, as well as reducing cardiovascular mortality and the rate of heart failure (HF) readmission. However, clinically, some patients with HF still have low sensitivity to sacubitril/valsartan, indicating sacubitril/valsartan resistance (SVR). A total of 46 patients with HF after AMI (23 SVR and 23 non-sacubitril/valsartan resistance (NSVR)) were selected. Five SVR and 5 matched NSVR samples were screened for differentially expressed ncRNAs along with mRNAs. A total of 124 differentially expressed miRNAs, 137 circRNAs, 237 lncRNAs and 50 mRNAs were screened by RNA sequencing technology. After quantitative real-time PCR (qRT‒PCR) verification of selected biomarkers in 18 pairs of samples, we found that for patients with SVR, hsa-miR-543, hsa-miR-642b-5p, hsa-miR-760, hsa_circ_0137499, ENST00000474394, ENST00000528337, E2F1, NEAT1, and YTHDF2 were upregulated, and hsa-miR-424-5p, hsa-miR-21-3p, hsa_circRNA_0003275, hsa_circRNA_0004494, hsa_circ_0093522, ENST00000467951, ENST00000558177, ACTA2, ANPEP, and CAMP were downregulated. Then, with the help of our constructed ceRNA network and functional annotation enrichment, we speculated that inflammatory pathways (such as the apelin signalling pathway) and lipid metabolism pathways (such as fatty acid metabolism) may be involved in the regulation of SVR. These discoveries lay a foundation for further mechanistic research and provide a direction for individualized drug administration.

Resolving the challenge of insoluble production of mature human growth differentiation factor 9 protein (GDF9) in E. coli using bicistronic expression with thioredoxin

Growth differentiation factor 9 (GDF9) is an oocyte-derived protein with fundamental functions in folliculogenesis. While the crucial contributions of GDF9 in follicular survival have been revealed, crystallographic studies of GDF9 structure have not yet been carried out, essentially due to the insoluble expression of GDF9 in E. coli and lack of appropriate source for structural studies. Therefore, in this study, we investigated the impact of different expression rate of bacterial thioredoxin (TrxA) using bicistronic expression constructs to induce the soluble expression of mature human GDF9 (hGDF9) driven by T7 promoter in E. coli. Our findings revealed that in BL21(DE3), the high rate of TrxA co-expression at 30 °C was sufficiently potent for the soluble expression of hGDF9 and reduction of inclusion body formation by 4 fold. We also successfully confirmed the bioactivity of the purified soluble hGDF9 protein by evaluation of follicle-stimulating hormone receptor gene expression in bovine cumulus cells derived from small follicles. This study is the first to present an effective approach for expression of bioactive form of hGDF9 using TrxA co-expression in E. coli, which may unravel the current issues regarding structural analysis of hGDF9 protein and consequently provide a better insight into hGDF9 functions and interactions.

Development of a set of novel binary expression vectors for plant gene function analysis and genetic transformation.

With the advent of multiple omics and Genome-Wide Association Studies (GWAS) technology, genome-scale functional analysis of candidate genes is to be conducted in diverse plant species. Construction of plant binary expression vectors is the prerequisite for gene function analysis. Therefore, it is of significance to develop a set of plant binary expression vectors with highly efficient, inexpensive, and convenient cloning method, and easy-to-use in screening of positive recombinant in Escherichia coli. In this study, we developed a set of plant binary expression vectors, termed pBTR vectors, based on Golden Gate cloning using BsaI restriction site. Foreign DNA fragment of interest (FDI) can be cloned into the destination pBTR by one-step digestion-ligation reaction in a single tube, and even the FDI contains internal BsaI site(s). Markedly, in one digestion-ligation reaction, multiple FDIs (exemplified by cloning four soybean Glyma.02g025400, Glyma.05g201700, Glyma.06g165700, and Glyma.17g095000 genes) can be cloned into the pBTR vector to generate multiple corresponding expression constructs (each expression vector carrying an FDI). In addition, the pBTR vectors carry the visual marker, a brightness monomeric red fluorescent protein mScarlet-I, that can be observed with the unaided eye in screening of positive recombinants without the use of additional reagents/equipment. The reliability of the pBTR vectors was validated in plants by overexpression of AtMyb75/PAP1 in tomato and GUSPlus in soybean roots via Agrobacterium rhizogenes-mediated transformation, promoter activity analysis of AtGCSpro in Arabidopsis via A. tumefaciens-mediated transformation, and protein subcellular localization of the Vitis vinifera VvCEB1opt in tobacco, respectively. These results demonstrated that the pBTR vectors can be used in analysis of gene (over)expression, promoter activity, and protein subcellular localization. These vectors will contribute to speeding up gene function analysis and the process of plant molecular breeding.

Iron-dependent post transcriptional control of mitochondrial aconitase expression.

Iron regulatory proteins (IRPs) control the translation of animal cell mRNAs encoding proteins with diverse roles. This includes the iron storage protein ferritin and the tricarboxylic cycle (TCA) enzyme mitochondrial aconitase (ACO2) through iron-dependent binding of IRP to the iron responsive element (IRE) in the 5' untranslated region (UTR). To further elucidate the mechanisms allowing IRPs to control translation of 5' IRE-containing mRNA differentially, we focused on Aco2 mRNA, which is weakly controlled versus the ferritins. Rat liver contains two classes of Aco2 mRNAs, with and without an IRE, due to alterations in the transcription start site. Structural analysis showed that the Aco2 IRE adopts the canonical IRE structure but lacks the dynamic internal loop/bulge five base pairs 5' of the CAGUG(U/C) terminal loop in the ferritin IREs. Unlike ferritin mRNAs, the Aco2 IRE lacks an extensive base-paired flanking region. Using a full-length Aco2 mRNA expression construct, iron controlled ACO2 expression in an IRE-dependent and IRE-independent manner, the latter of which was eliminated with the ACO23C3S mutant that cannot bind the FeS cluster. Iron regulation of ACO23C3S encoded by the full-length mRNA was completely IRE-dependent. Replacement of the Aco23C3S 5' UTR with the Fth1 IRE with base-paired flanking sequences substantially improved iron responsiveness, as did fusing of the Fth1 base-paired flanking sequences to the native IRE in the Aco3C3S construct. Our studies further define the mechanisms underlying the IRP-dependent translational regulatory hierarchy and reveal that Aco2 mRNA species lacking the IRE contribute to the expression of this TCA cycle enzyme.

Probing protein misfolding and dissociation with an infrared free-electron laser.

Misfolding is observed in the mutant proteins that are causative for neurodegenerative disorders such as polyglutamine diseases. These proteins are prone to aggregate in the cytoplasm and nucleus of cells. To reproduce cells with the aggregated proteins, gene expression system is usually applied, in which the expression construct having the mutated DNA sequence of the interest is transfected into cells. The transfected DNA is finally converted into the mutant protein, which is gradually aggregated in the cells. In addition, a simple method to prepare the cells having aggregates inside has been recently applied. Peptides were first aggregated by incubating them in water. The aggregates are spontaneously taken up by cells because aggregated proteins generally transfer between cells. Peptides with different degrees of aggregation can be made by changing the incubation times and temperatures, which enables to examine contribution of aggregation to the toxicity to the recipient cells. Moreover, such cells can be used for therapeutic researches of diseases in which aggregates are involved. In this chapter, we show methods to induce aggregation of peptides. The functional analyses of the cells with aggregates are also described. Then, experimental dissociation of the aggregates produced using this method by mid infrared free electron laser irradiation and its theoretical support by molecular dynamics simulation are introduced as the therapeutic research for neurodegenerative disorders.

Application Model Construction of Emotional Expression and Propagation Path of Deep Learning in National Vocal Music

Application Model Construction of Emotional Expression and Propagation Path of Deep Learning in National Vocal Music

Using metaphorical associative images for improving efficiency of english language classes in higher education institutions

The relevance of using metaphorical associative images (MAI) in English language classes at higher education institutions is due to the need to ensure effective communication in the context of accomplishing communicative tasks and providing conditions for the development of students’ creative potential. The aim of the article is to determine the expediency of using metaphorical associative images as a means of improving efficiency of English language classes taking into account methodological, didactic, linguistic and psychological factors. The tasks consist in the study of MAI as a universal psychological tool, highlighting some aspects concerning visual aids in teaching foreign languages in connection with the use of MAI and the development of communicative activities based on the MAI use for teaching students in English language classes at higher education institutions. There have been analyzed the results of research in the field of psychological science regarding MAI as a universal psychological tool that allows specialists to work with both emotional and cognitive spheres of the personality through the interpretation of images. MAI application is based on a metaphor, appeals to the human subconscious and is used for diagnosis, psychocorrection, psychotherapy as well as the development of a person, revealing his or her creative abilities. From the angle of foreign language teaching, visual aids are considered as one of the effective methods that contribute to the construction of speech expressions. The idea of the possibility of using MAI in foreign language teaching for the formation of students’ speaking skills based on the activation of their cognitive mental processes and appeal to the flow of associations has been substantiated. Career-oriented English language activities on the topic “Digital media projects” with the use of MAI for students studying under the educational programme “Content producing of digital media projects” have been presented. The expediency of using the MAI method as a means of improving efficiency of English language classes in terms of implementation of methodological, didactic, linguistic and psychological aspects has been determined.

Secure Content Based Image Retrieval Scheme Based on Deep Hashing and Searchable Encryption

To solve the problem that the existing ciphertext domain image retrieval system is challenging to balance security, retrieval efficiency, and retrieval accuracy. This research suggests a searchable encryption and deep hashing-based secure image retrieval technique that extracts more expressive image features and constructs a secure, searchable encryption scheme. First, a deep learning framework based on residual network and transfer learning model is designed to extract more representative image deep features. Secondly, the central similarity is used to quantify and construct the deep hash sequence of features. The Paillier homomorphic encryption encrypts the deep hash sequence to build a high-security and low-complexity searchable index. Finally, according to the additive homomorphic property of Paillier homomorphic encryption, a similarity measurement method suitable for computing in the retrieval system’s security is ensured by the encrypted domain. The experimental results, which were obtained on Web Image Database from the National University of Singapore (NUS-WIDE), Microsoft Common Objects in Context (MS COCO), and ImageNet data sets, demonstrate the system’s robust security and precise retrieval, the proposed scheme can achieve efficient image retrieval without revealing user privacy. The retrieval accuracy is improved by at least 37% compared to traditional hashing schemes. At the same time, the retrieval time is saved by at least 9.7% compared to the latest deep hashing schemes.

Исследование по динамике рамных конструкций

Determining the natural and forced oscillations of frame structures simulated by the rods with distributed masses (an infinite number of degrees of freedom) is quite difficult. Therefore, in the article, the frame model is endued with a finite number of degrees of freedom: the mass is placed in a certain number of nodes that elastically interact with rods that have no mass. The rods work only for bending. Longitudinal displacements are not taken into account, since the frequency of longitudinal oscillations is two orders of magnitude higher than the frequency of bending ones. Such a model leads to the construction of expressions of the kinetic and potential energy, which then allows using the Lagrange equations of second kind to obtain a system of differential oscillation equations of the structure. The problem of free oscillations of the G-shaped frame was solved in the article using Green's functions, matrices of stiffness, masses, malleability, etc. The obtained approximate results were compared with little-known exact results and demonstrated good convergence, especially with an increase in the number of degrees of freedom (the number of concentrated masses simulating the distributed mass of the rods of the G-shaped frame).

Suppression of P-cadherin expression as a key regulatory element for embryonic stem cell stemness.

In embryonic stem (ES) cell colonies, a small subpopulation that changes cell shape and loses pluripotency often appears in two-dimensional (2D) cultures, even in the presence of a stemness factor. We have previously shown that membrane translocation of the syntaxin4, t-SNARE protein contributes to this phenomenon. Here, we show that ES cells in three-dimensional (3D) aggregates do not succumb to extruded syntaxin4 owing to suppressed expression of P-cadherin protein. While extracellular expression of syntaxin4 led to the striking upregulation of P-cadherin mRNA in both 2D and 3D-ES cells, morphological changes and appreciable expression of P-cadherin protein were detected only in 2D-ES cells. Importantly, the introduction of an expression cassette for P-cadherin practically reproduced the effects induced by extracellular syntaxin4, where the transgene product was clearly detected in 2D-, but not 3D-ES cells. An expression construct for P-cadherin-Venus harboring an in-frame insertion of the P2A sequence at the joint region gave fluorescent signals only in the cytoplasm of 2D-ES cells, demonstrating translational regulation of P-cadherin. These results provide the mechanistic insight into the uncontrollable differentiation in 2D-ES cells and shed light on the validity of the "embryoid body protocol commonly used for ES cell handling" for directional differentiation.Key words: differentiation, embryoid body, ES cells, P-cadherin, syntaxin4.

The large extracellular loop is important for recruiting CD63 to exosomes.

Exosomes are small extracellular vesicles that are secreted from cells. To characterize exosome fraction marker proteins of the tetraspanin family in particular, CD9, CD63, and CD81 are routinely used. CD63 expression constructs were employed to investigate the influence of the large extracellular loop (LEL) of CD63 on sorting into exosomes. When the LEL of CD63 fused with mCherry was deleted, the protein was no longer found in the purified exosome fraction. This finding demonstrates the importance of the LEL sequence for the recruitment of CD63 into exosomes.

Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors.

CRISPR interference (CRISPRi) enables programmable, reversible, and titratable repression of gene expression (knockdown) in mammalian cells. Initial CRISPRi-mediated genetic screens have showcased the potential to address basic questions in cell biology, genetics, and biotechnology, but wider deployment of CRISPRi screening has been constrained by the large size of single guide RNA (sgRNA) libraries and challenges in generating cell models with consistent CRISPRi-mediated knockdown. Here, we present next-generation CRISPRi sgRNA libraries and effector expression constructs that enable strong and consistent knockdown across mammalian cell models. First, we combine empirical sgRNA selection with a dual-sgRNA library design to generate an ultra-compact (1-3 elements per gene), highly active CRISPRi sgRNA library. Next, we compare CRISPRi effectors to show that the recently published Zim3-dCas9 provides an excellent balance between strong on-target knockdown and minimal non-specific effects on cell growth or the transcriptome. Finally, we engineer a suite of cell lines with stable expression of Zim3-dCas9 and robust on-target knockdown. Our results and publicly available reagents establish best practices for CRISPRi genetic screening.

Landscape as Heritage in the province of Chañaral, Atacama region, Chile

Memorial site. “Animita”, 2017 - photo by Sebastian Palacios To understand the landscape as heritage is to recognize it in a synthesizing condition, as a complex system of ecological, historical and social legacies. This approach allows an articulation and reading of the territory not as a sum of particularities but rather as a particular system, where the landscape, as a collective construction of society, is not only understood as a material construction, but also as a construction of memory and cultural expressions, which in the Atacama Desert have become evident with a clear mining emphasis, temporally since pre-Columbian times and spatially throughout the transversality, longitudinality and altitudinal levels of the territory.

AN APPROACH TO ROMANIAN LANGUAGE ANGLICISMS

English language is spoken all over the world, and today, anglicisms represent a linguistic unit from the point of view of the expression, meaning or grammatical construction in Romanian, as well as the spelling and pronunciation from British or American English. The present paper aims at studying Romanian language anglicisms and at establishing whether they are necessary or luxury loanwords. Nonetheless, the paper studies the presence of these terms in the sectors they appear most often, starting from various articles published in Capital newspaper.

The Emory‐Sage‐SGC TREAT‐AD Center: Tool and probe development for emerging targets in Alzheimer’s Disease

AbstractBackgroundAlzheimer’s disease is the most common form of dementia and there is currently no effective therapy for either treatment or prevention. With repeated failure in DRUG DEVELOPMENT across industry and demonstrated heterogeneity in biological and genetic components of the disease, there is a need to evaluate a broad range of therapeutic hypotheses for target prioritization. The Emory‐Sage‐SGC TREAT‐AD Center (https://treatad.org) is generating and openly distributing validated experimental tools necessary to test target predictions generated through sequence‐based characterization of human disease state. We believe that these tools and reagents, including chemical and biological probes that target the multifaceted dysregulation in the brains of AD patients will advance the discovery of potential drug targets for AD.MethodThe Emory‐Sage‐SGC TREAT‐AD Center uses integrated computational approaches to identify target predictions from a set of prioritized therapeutic hypotheses. Emerging AD targets derived from systems biology studies within the Accelerating Medicines Partnership in AD (AMP‐AD) consortium and additional NIA‐supported AD consortia were mapped to 15 biological domains (BDs) and prioritized based on an unbiased bioinformatic assessment across multiple lines of evidence for overall AD‐risk. Targets are then evaluated to identify a set of experimental reagents necessary for hypothesis testing, termed a “target enablement package (TEP).ResultWithin these 15 biological domains, we prioritized more than 30 targets for target enabling package (TEP) development. TEPs include expression constructs, knockout cell lines, assays, antibody validation, and crystal structures. All reagents are developed to meet established quality criteria. For a subset of tractable targets (MSN, SYK, SFRP1, SDC4 and CAPN2), chemical probe development is underway to provide tools to test these therapeutic hypotheses in cellular and animal systems. TREAT‐AD investigators place all data, knowledge, reagents, and tools into the open domain with no intellectual property claims.ConclusionThe open drug discovery approach of TREAT‐AD is aimed to de‐risk potential AD therapeutics to catalyze robust and independent evaluation of a diverse portfolio of promising yet untested AD therapeutic hypotheses. All data, protocols, reagent sets, and chemical tools will be made widely available on the AD Knowledge Portal. For more information see www.treatad.org.

Insulin/IGF signaling regulates presynaptic glutamate release in aversive olfactory learning.

Insulin/insulin-like growth factor (IGF) receptor signaling (IIS) supports context-dependent learning in vertebrates and invertebrates. Here, we identify cell-specific mechanisms of IIS that integrate sensory information with food context to drive synaptic plasticity and learning. In the nematode Caenorhabditis elegans, pairing food deprivation with an odor such as butanone suppresses attraction to that odor. We find that aversive olfactory learning requires the insulin receptor substrate (IRS) protein IST-1 and atypical signaling through the insulin/IGF-1 receptor DAF-2. Cell-specific knockout and rescue demonstrate that DAF-2 acts in the AWCON sensory neuron, which detects butanone, and that learning preferentially depends upon the axonally localized DAF-2c isoform. Acute food deprivation increases DAF-2 levels in AWCON post-transcriptionally through an insulin- and insulin receptor substrate-1 (ist-1)-dependent process. Aversive learning alters the synaptic output of AWCON by suppressing odor-regulated glutamate release in wild-type animals, but not in ist-1 mutants, suggesting that axonal insulin signaling regulates synaptic transmission to support aversive memory.

A novel biallelic loss-of-function variant in DAND5 causes heterotaxy syndrome.

The majority of heterotaxy cases do not obtain a molecular diagnosis, although pathogenic variants in more than 50 genes are known to cause heterotaxy. A heterozygous missense variant in DAND5, a nodal inhibitor, which functions in early development for establishment of right-left patterning, has been implicated in heterotaxy. Recently, the first case was reported of a DAND5 biallelic loss-of-function (LoF) variant in an individual with heterotaxy. Here, we describe a second unrelated individual with heterotaxy syndrome and a homozygous frameshift variant in DAND5 (NM_152654.2:c.197del [p.Leu66ArgfsTer22]). Using an in vitro assay, we demonstrate that the DAND5 c.197del variant is unable to inhibit nodal signaling when compared with the wild-type expression construct. This work strengthens the genetic and functional evidence for biallelic LoF variants in DAND5 causing an autosomal recessive heterotaxy syndrome.