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Artykuły w czasopismach na temat "Protein Based Molecular Diseases"
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Telling, Glenn. "Protein-based PCR for prion diseases?" Nature Medicine 7, nr 7 (lipiec 2001): 778–79. http://dx.doi.org/10.1038/89895.
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Li, Yan, Yi Jia, Xiao-Lin Wang, Hai Shang i Yu Tian. "Protein-Targeted Degradation Agents Based on Natural Products". Pharmaceuticals 16, nr 1 (28.12.2022): 46. http://dx.doi.org/10.3390/ph16010046.
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Natural products are an important source of drug lead compounds, and natural products with significant biological activity are constantly being discovered and used in clinical practice. At present, natural products play an important role in the targeted therapy of cancer, cardiovascular and cerebrovascular diseases, nervous system diseases, and autoimmune diseases. Meanwhile, in recent years, the rise of protein-targeted degradation technologies, such as proteolysis-targeting chimeras (PROTACs) and molecular glues, has provided a new solution for drug resistance caused by clinical molecular-targeting drugs. It is noteworthy that natural products and their derivatives, as important components of PROTACs and molecular glues, play an important role in the development of protein-targeting drugs. Hence, this review summarized the protein-targeted degradation agents based on natural products, such as PROTACs and molecular glues. More natural products with the potential to be used in the development of PROTACs and molecular glues as targeted protein degradation agents are still being investigated.
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Yadav, Kusum, Anurag Yadav, Priyanka Vashistha, Veda P. Pandey i Upendra N. Dwivedi. "Protein Misfolding Diseases and Therapeutic Approaches". Current Protein & Peptide Science 20, nr 12 (16.12.2019): 1226–45. http://dx.doi.org/10.2174/1389203720666190610092840.
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Protein folding is the process by which a polypeptide chain acquires its functional, native 3D structure. Protein misfolding, on the other hand, is a process in which protein fails to fold into its native functional conformation. This misfolding of proteins may lead to precipitation of a number of serious diseases such as Cystic Fibrosis (CF), Alzheimer’s Disease (AD), Parkinson’s Disease (PD), and Amyotrophic Lateral Sclerosis (ALS) etc. Protein Quality-control (PQC) systems, consisting of molecular chaperones, proteases and regulatory factors, help in protein folding and prevent its aggregation. At the same time, PQC systems also do sorting and removal of improperly folded polypeptides. Among the major types of PQC systems involved in protein homeostasis are cytosolic, Endoplasmic Reticulum (ER) and mitochondrial ones. The cytosol PQC system includes a large number of component chaperones, such as Nascent-polypeptide-associated Complex (NAC), Hsp40, Hsp70, prefoldin and T Complex Protein-1 (TCP-1) Ring Complex (TRiC). Protein misfolding diseases caused due to defective cytosolic PQC system include diseases involving keratin/collagen proteins, cardiomyopathies, phenylketonuria, PD and ALS. The components of PQC system of Endoplasmic Reticulum (ER) include Binding immunoglobulin Protein (BiP), Calnexin (CNX), Calreticulin (CRT), Glucose-regulated Protein GRP94, the thiol-disulphide oxidoreductases, Protein Disulphide Isomerase (PDI) and ERp57. ER-linked misfolding diseases include CF and Familial Neurohypophyseal Diabetes Insipidus (FNDI). The components of mitochondrial PQC system include mitochondrial chaperones such as the Hsp70, the Hsp60/Hsp10 and a set of proteases having AAA+ domains similar to the proteasome that are situated in the matrix or the inner membrane. Protein misfolding diseases caused due to defective mitochondrial PQC system include medium-chain acyl-CoA dehydrogenase (MCAD)/Short-chain Acyl-CoA Dehydrogenase (SCAD) deficiency diseases, hereditary spastic paraplegia. Among therapeutic approaches towards the treatment of various protein misfolding diseases, chaperones have been suggested as potential therapeutic molecules for target based treatment. Chaperones have been advantageous because of their efficient entry and distribution inside the cells, including specific cellular compartments, in therapeutic concentrations. Based on the chemical nature of the chaperones used for therapeutic purposes, molecular, chemical and pharmacological classes of chaperones have been discussed.
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Teribele Venturin, Gianina, i Zhen Cheng. "Small Peptide and Protein-based Molecular Probes for Imaging Neurological Diseases". Current Protein & Peptide Science 17, nr 6 (15.07.2016): 543–58. http://dx.doi.org/10.2174/1389203717666160101123500.
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Chaudhuri, Tapan K., i Subhankar Paul. "Protein-misfolding diseases and chaperone-based therapeutic approaches". FEBS Journal 273, nr 7 (kwiecień 2006): 1331–49. http://dx.doi.org/10.1111/j.1742-4658.2006.05181.x.
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Lorenzo-Pouso, Alejandro I., Mario Pérez-Sayáns, Susana B. Bravo, Pía López-Jornet, María García-Vence, Manuela Alonso-Sampedro, Javier Carballo i Abel García-García. "Protein-Based Salivary Profiles as Novel Biomarkers for Oral Diseases". Disease Markers 2018 (7.11.2018): 1–22. http://dx.doi.org/10.1155/2018/6141845.
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The Global Burden of Oral Diseases affects 3.5 billion people worldwide, representing the number of people affected by the burden of untreated dental caries, severe periodontal disease, and edentulism. Thus, much more efforts in terms of diagnostics and treatments must be provided in the fight of these outcomes. In this sense, recently, the study of saliva as biological matrix has been identified as a new landmark initiative in the search of novel and useful biomarkers to prevent and diagnose these conditions. Specifically, saliva is a rich reservoir of different proteins and peptides and accessible due to recent advances in molecular biology and specially in targeted and unbiased proteomics technologies. Nonetheless, emerging barriers are an obstacle to the study of the salivary proteome in an effective way. This review aims at giving an overall perspective of salivary biomarkers identified in several oral diseases by means of molecular biology approaches.
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Pang, Yihe, i Bin Liu. "DMFpred: Predicting protein disorder molecular functions based on protein cubic language model". PLOS Computational Biology 18, nr 10 (31.10.2022): e1010668. http://dx.doi.org/10.1371/journal.pcbi.1010668.
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Intrinsically disordered proteins and regions (IDP/IDRs) are widespread in living organisms and perform various essential molecular functions. These functions are summarized as six general categories, including entropic chain, assembler, scavenger, effector, display site, and chaperone. The alteration of IDP functions is responsible for many human diseases. Therefore, identifying the function of disordered proteins is helpful for the studies of drug target discovery and rational drug design. Experimental identification of the molecular functions of IDP in the wet lab is an expensive and laborious procedure that is not applicable on a large scale. Some computational methods have been proposed and mainly focus on predicting the entropic chain function of IDRs, while the computational predictive methods for the remaining five important categories of disordered molecular functions are desired. Motivated by the growing numbers of experimental annotated functional sequences and the need to expand the coverage of disordered protein function predictors, we proposed DMFpred for disordered molecular functions prediction, covering disordered assembler, scavenger, effector, display site and chaperone. DMFpred employs the Protein Cubic Language Model (PCLM), which incorporates three protein language models for characterizing sequences, structural and functional features of proteins, and attention-based alignment for understanding the relationship among three captured features and generating a joint representation of proteins. The PCLM was pre-trained with large-scaled IDR sequences and fine-tuned with functional annotation sequences for molecular function prediction. The predictive performance evaluation on five categories of functional and multi-functional residues suggested that DMFpred provides high-quality predictions. The web-server of DMFpred can be freely accessed from http://bliulab.net/DMFpred/.
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Kovacs, Gabor G. "Molecular pathology of neurodegenerative diseases: principles and practice". Journal of Clinical Pathology 72, nr 11 (8.08.2019): 725–35. http://dx.doi.org/10.1136/jclinpath-2019-205952.
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Neurodegenerative diseases are characterised by selective dysfunction and progressive loss of synapses and neurons associated with pathologically altered proteins that deposit primarily in the human brain and spinal cord. Recent discoveries have identified a spectrum of distinct immunohistochemically and biochemically detectable proteins, which serve as a basis for protein-based disease classification. Diagnostic criteria have been updated and disease staging procedures have been proposed. These are based on novel concepts which recognise that (1) most of these proteins follow a sequential distribution pattern in the brain suggesting a seeding mechanism and cell-to-cell propagation; (2) some of the neurodegeneration-associated proteins can be detected in peripheral organs; and (3) concomitant presence of neurodegeneration-associated proteins is more the rule than the exception. These concepts, together with the fact that the clinical symptoms do not unequivocally reflect the molecular pathological background, place the neuropathological examination at the centre of requirements for an accurate diagnosis. The need for quality control in biomarker development, clinical and neuroimaging studies, and evaluation of therapy trials, as well as an increasing demand for the general public to better understand human brain disorders, underlines the importance for a renaissance of postmortem neuropathological studies at this time. This review summarises recent advances in neuropathological diagnosis and reports novel aspects of relevance for general pathological practice.
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Gul, Irfan, Amreena Hassan, Ehtishamul Haq, Syed Mudasir Ahmad, Riaz Ahmad Shah, Nazir Ahmad Ganai, Naveed Anjum Chikan, Mohamed Faizal Abdul-Careem i Nadeem Shabir. "An Investigation of the Antiviral Potential of Phytocompounds against Avian Infectious Bronchitis Virus through Template-Based Molecular Docking and Molecular Dynamics Simulation Analysis". Viruses 15, nr 4 (26.03.2023): 847. http://dx.doi.org/10.3390/v15040847.
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Vaccination is widely used to control Infectious Bronchitis in poultry; however, the limited cross-protection and safety issues associated with these vaccines can lead to vaccination failures. Keeping these limitations in mind, the current study explored the antiviral potential of phytocompounds against the Infectious Bronchitis virus using in silico approaches. A total of 1300 phytocompounds derived from fourteen botanicals were screened for their potential ability to inhibit the main protease, papain-like protease or RNA-dependent RNA–polymerase of the virus. The study identified Methyl Rosmarinate, Cianidanol, Royleanone, and 6,7-Dehydroroyleanone as dual-target inhibitors against any two of the key proteins. At the same time, 7-alpha-Acetoxyroyleanone from Rosmarinus officinalis was found to be a multi-target protein inhibitor against all three proteins. The potential multi-target inhibitor was subjected to molecular dynamics simulations to assess the stability of the protein–ligand complexes along with the corresponding reference ligands. The findings specified stable interactions of 7-alpha-Acetoxyroyleanone with the protein targets. The results based on the in silico study indicate that the phytocompounds can potentially inhibit the essential proteins of the Infectious Bronchitis virus; however, in vitro and in vivo studies are required for validation. Nevertheless, this study is a significant step in exploring the use of botanicals in feed to control Infectious Bronchitis infections in poultry.
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Mishra i Dey. "Molecular Docking Studies of a Cyclic Octapeptide-Cyclosaplin from Sandalwood". Biomolecules 9, nr 11 (15.11.2019): 740. http://dx.doi.org/10.3390/biom9110740.
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Natural products from plants, such as chemopreventive agents, attract huge attention because of their low toxicity and high specificity. The rational drug design in combination with structure-based modeling and rapid screening methods offer significant potential for identifying and developing lead anticancer molecules. Thus, the molecular docking method plays an important role in screening a large set of molecules based on their free binding energies and proposes structural hypotheses of how the molecules can inhibit the target. Several peptide-based therapeutics have been developed to combat several health disorders, including cancers, metabolic disorders, heart-related diseases, and infectious diseases. Despite the discovery of hundreds of such therapeutic peptides however, only few peptide-based drugs have made it to the market. Moreover, the in silico activities of cyclic peptides towards molecular targets, such as protein kinases, proteases, and apoptosis related proteins have not been extensively investigated. In this study, we explored the in silico kinase and protease inhibitor potentials of cyclosaplin, and studied the interactions of cyclosaplin with other apoptosis-related proteins. Previously, the structure of cyclosaplin was elucidated by molecular modeling associated with dynamics that were used in the current study as well. Docking studies showed strong affinity of cyclosaplin towards cancer-related proteins. The binding affinity closer to 10 kcal/mol indicated efficient binding. Cyclosaplin showed strong binding affinities towards protein kinases such as EGFR, VEGFR2, PKB, and p38, indicating its potential role in protein kinase inhibition. Moreover, it displayed strong binding affinity to apoptosis-related proteins and revealed the possible role of cyclosaplin in apoptotic cell death. The protein–ligand interactions using LigPlot displayed some similar interactions between cyclosaplin and peptide-based ligands, especially in case of protein kinases and a few apoptosis related proteins. Thus, the in silico analyses gave the insights of cyclosaplin being a potential apoptosis inducer and protein kinase inhibitor.
Rozprawy doktorskie na temat "Protein Based Molecular Diseases"
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Kumari, Vandana. "Structure-Based Computer Aided Drug Design and Analysis for Different Disease Targets". The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1311612599.
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Dickerson, Matthew Thomas. "PROTEIN BASED BIOMIMETIC APPROACHS TO SURFACE HEMOCOMPATIBILITY AND BIOCOMPATIBILITY ENHANCEMENT". UKnowledge, 2012. http://uknowledge.uky.edu/cme_etds/6.
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T. pallidum can survive a primary immune response and continue growing in the host for an extended period of time. T. pallidum is thought to bind serum fibronectin (FN) through Tp0483 on the surface to obscure antigens. A Tp0483 fragment (rTp0483) was adsorbed onto functionalized self-assembled monolayers (SAMs) with FN. FN capture by adsorbed rTp0483 depended greatly on surface chemistry with COO- groups being best for FN binding. Hemocompatibility was determined by analysis of plasma protein adsorption, intrinsic pathway activation, and platelet activation. rTp0483+FN bound an equal or lesser amount of fibrinogen (Fg), human serum albumin (HSA), and factor XII (FXII) compared to rTp0483 or FN alone and adsorption of rTp0483 prior to FN greatly decreased platelet activation. Inhibition of protein binding and platelet activation suggested an attenuated hematological response. Biocompatibility of rTp0483 and FN coated surfaces was characterized by macrophage uptake of protein coated polystyrene microspheres (PSMs), macrophage adsorption onto protein coated surfaces, cytotoxic effects of adsorbed rTp0483 and FN, and TNF-α and NO2- release in macrophages stimulated with rTp0483 and FN adsorbed and in solution. Addition of FN to rTp0483 on plain and COO- PSMs reduced phagocytosis compared to rTp0483 alone and on plain PSMs compared to FN alone. On plain PSMs addition of FN to adsorbed rTp0483 decreased TNF-α generation. Adsorption of rTp0483 before FN on large, flat COO- surfaces decreased macrophage adsorption and TNF-α and NO2- generation. High concentrations of rTp0483 were mildly cytotoxic to macrophages. FN binding by Tp0483 on T. pallidum likely plays a role in antigenic disguise and rTp0483+FN coatings may potentially inhibit FN and rTp0483 specific interactions with macrophages. Molecularly imprinted polymer coatings were also examined for biomaterial development. Fouling resistant 2-methacryloyloxyethyl phosphorylcholine (MPC) was imprinted with bovine serum albumin (BSA) protein templates to facilitate BSA specific binding. The BSA template was constructed and verified and BSA specific binding quantified using quartz crystal microbalance (QCM) and enzyme linked immunosorbent assay (ELISA). BSA imprinted coatings were determined to bind significantly more BSA than nonfouling MPC controls demonstrating the feasibility of targeted protein capture.
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Drobin, Kimi. "Antibody-based bead arrays for high-throughput protein profiling in human plasma and serum". Licentiate thesis, KTH, Proteinvetenskap, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-225980.
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Affinity-based proteomics utilizes affinity binders to detect target proteins in a large-scale manner. This thesis describes a high-throughput method, which enables the search for biomarker candidates in human plasma and serum. A highly multiplexed antibody-based suspension bead array is created by coupling antibodies generated in the Human Protein Atlas project to color-coded beads. The beads are combined for parallel analysis of up to 384 analytes in patient and control samples. This provides data to compare protein levels from the different groups. In paper I osteoporosis patients are compared to healthy individuals to find disease-linked proteins. An untargeted discovery screening was conducted using 4608 antibodies in 16 cases and 6 controls. This revealed 72 unique proteins, which appeared differentially abundant. A validation screening of 91 cases and 89 controls confirmed that the protein autocrine motility factor receptor (AMFR) is decreased in the osteoporosis patients. Paper II investigates the risk proteome of inflammatory bowel disease (IBD). Antibodies targeting 209 proteins corresponding to 163 IBD genetic risk loci were selected. To find proteins related to IBD or its subgroups, sera from 49 patients with Crohn’s disease, 51 with ulcerative colitis and 50 matched controls were analyzed. From these targeted assays, the known inflammation-related marker serum amyloid protein A (SAA) was shown to be elevated in the IBD cases. In addition, the protein laccase (multi-copper oxidoreductase) domain containing 1 (LACC1) was found to be decreased in the IBD subjects. In conclusion, assays using affinity-based bead arrays were developed and applied to screen human plasma and serum samples in two disease contexts. Untargeted and targeted screening strategies were applied to discover disease-associated proteins. Upon further validation, these potential biomarker candidates could be valuable in future disease studies.QC 20180412
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Liu, Jiyun. "Structure based design of inhibitors toward disease related multivalent protein targets /". Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/8482.
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Freer, Rosie. "Molecular origins of tissue vulnerability to aberrant aggregation in protein misfolding diseases". Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/275420.
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Neurodegenerative disorders, including Alzheimer’s disease (AD) and Parkinson’s disease (PD), are increasingly common in our ageing society, are remain incurable. A major obstacle encountered by researchers in their attempts to find effective therapies is represented by the current lack of understanding of the molecular origins of these disorders. It is becoming clear that, although the aggregation of specific proteins, including amyloid β (Aβ) and tau in AD and α-synuclein in PD, hallmark these disorders, such behaviour is a consequence of a wider, system-level disruption of protein homeostasis. In order to identify the genetic factors contributing to such a disruption, the transcriptional changes that occur during neurodegenerative disease progression have received considerable scientific attention in recent years. In our approach, we considered another hallmark of these diseases - their characteristic patterns of spreading across the brain - to identify the nature of the transcriptional signature which underlies tissue vulnerability to protein aggregation. By understanding why tissues succumb in their characteristic sequential pattern in neurodegenerative diseases, and why some tissues remain almost completely resistant throughout, we hoped to obtain insight into the molecular origins of these disorders. Our results show that the AD progression can be predicted from a transcriptional signature in healthy brains related to the protein aggregation homeostasis of Aβ, tau, and the wider proteome. We highlight a relationship between a specific subproteome at high risk of aggregation (formed by supersaturated proteins), and the vulnerability to neurodegenerative diseases. We thus identify an AD-specific supersaturated set of proteins - termed the metastable subproteome, whose expression in normal brains recapitulates the staging of AD, with more vulnerable tissues having higher metastable subproteome expression. We find evidence of these vulnerability signatures transcending the tissue level of interrogation, with cellular and subcellular analysis also showing elevated levels of proteins known and predicted to predispose the aberrant aggregation of Aβ and tau. These results characterise the key protein homeostasis pathways in the inception and progression of AD, and establish an approach with the potential to be applied to other protein misfolding diseases, in the brain and beyond.
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Lewandowski, Eric Michael. "Structure Based Drug Design Targeting Bacterial Antibiotic Resistance and Alzheimer's Disease". Scholar Commons, 2015. http://scholarcommons.usf.edu/etd/5982.
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Structure based drug design is a rapidly advancing discipline that examines how protein targets structurally interact with small molecules, or known inhibitors, and then uses this information to lead inhibitor optimization efforts. In the case of novel inhibitors, protein structural information is first obtained via X-ray crystallography, NMR studies, or a combination of both approaches. Then, computational molecular docking is often used to screen, in silico, millions of small molecules and calculate the potential interactions they may have with the target protein’s binding pocket, in hopes of identifying novel low affinity inhibitors. By examining the interactions these small, low affinity, inhibitors have with the binding pocket, optimization efforts can be focused on maximizing interactions with “hot spots” within the pocket, thus leading to larger, high affinity inhibitors. A similar optimization technique can also be applied to known inhibitors. By examining the interactions of a known inhibitor with the binding site, new compounds can be designed to target “hot spots” in the binding pocket using the known inhibitors core structure as a starting point. The affinity of the newly designed compounds can then be compared to the affinity of the original inhibitor, and further rounds of optimization can be carried out. While simple in design, there are many challenges associated with structure based drug design studies, and there is no guarantee novel inhibitors will be found, but ultimately, it is an extremely powerful methodology that results in a much higher hit rate than other, similar, techniques. The work herein describes the use of structure based drug design to target several different proteins involved in bacterial antibiotic resistance, and a protein that has been implicated in the development of Alzheimer’s disease.
The goal of the first project was to design a new PBP inhibitor based upon an existing scaffold, and to better understand the binding mechanism and molecular interactions between penicillin binding proteins and their inhibitors. PBPs are a group of proteins that catalyze the last steps of bacterial cell wall formation, and are the targets of the β-lactam antibiotics. Two compounds were designed which conjugated a ferrocene or ruthenocene group to 6-aminopenicillinic acid, and their antibiotic properties were tested against a range of bacterial strains. To get a better understanding of how the 6-APA organometallic compounds interacted with the PBP active site, a CTX-M-14 β-lactamase model system was used for X-ray crystallographic studies. CTX-M-14 was chosen as its active site shares many key catalytic features with PBPs, and it easily, and reproducibly, yields crystals capable of diffracting to sub-atomic (< 1.0 Å) resolution.
I determined a 1.18 Å structure of 6-APA-Ru in complex with CTX-M-14 E166A β-lactamase and was able to gain unprecedented details of the interactions of the ruthenocene group with the CTX-M active site. This structure also revealed that the compound bound in the CTX-M active site was actually the decarboxylated and hydrolyzed product, which was the first time a decarboxylated product had been captured in the CTX-M active site. A second, 0.85 Å, structure of CTX-M in complex with 6-APA-Ru was determined and shed light on how the hydrogen bonding network in the CTX-M active site changes in response to the 6-APA-Ru product binding. A final, 1.30 Å, structure captured the carboxylated and hydrolyzed 6-APA-Ru product in complex with CTX-M, which was the first time the carboxylated product had been captured in the CTX-M active with the catalytic Ser70 residue intact. The results show the potential of the ruthenocene group in improving antibiotic potency, and help to better elucidate the changes that occur in the CTX-M active site upon inhibitor binding, while at the same time, telling us what changes could occur in the active site of PBPs.
The next project was focused on novel inhibitor discovery against several different PBPs. PBPs have been successfully inhibited by β-lactam antibiotics for decades, but the alarming rise of bacteria resistant to these antibiotics has placed increased urgency on the discovery of novel PBP inhibitors. A fragment based molecular docking approach was employed to virtually screen millions of small compounds for interactions with the targeted active sites, and then high scoring compounds were selected for visual inspection and inhibitory testing. Virtual screening was first done against Staphylococcus aureus monofunctional transglycosylase, a type of PBP. MTG provided a good binding pocket for virtual screening, but proved challenging to purify and crystallize. However, through great effort MTG crystals were eventually obtained. After repeated rounds of virtual screening against MTG, multiple compounds were selected for inhibition testing, and testing is currently ongoing. Virtual screening was also done against Pseudomonas aeruginosa PBP5 and PBP1a. Purification and crystallization of these proteins proved to be easier than MTG, and both yielded diffraction quality crystals.
The final project focused on virtual screening against a protein implicated in the development of Alzheimer’s disease, Slingshot Phosphatase 1. The brains of AD patients have been found to contain elevated levels of active Cofilin, and these elevated levels of active Cofilin may lead to the overproduction of amyloid β. Aβ overproduction, and its resulting accumulation, is believed to be one of the pathways that lead to AD symptoms. Cofilin is activated when it is dephosphorylated by SSH1, and inhibiting this activation may decrease the production of Aβ and the development of AD symptoms. There is no known structure of SSH1, so to perform virtual screening a SSH1 homology model was constructed using the homolog SSH2 as a starting point. Virtual screening was then performed using the SSH1 homology model and many compounds were selected for inhibition testing. Initial testing found several compounds that could prevent Cofilin dephosphorylation at levels > 10μM. However, three compounds were found to be exceptionally active, and could prevent Cofilin dephosphorylation at both 1 and 10 μM. One of these three compounds was tested directly against purified SSH1 and found to inhibit its activity, and reduce Aβ production. Crystallization of purified SSH1, and SSH2, was attempted in order to get complex structures with the three best compounds. SSH2 crystals were obtained which diffracted to 1.91 Å, and several initial hits were found for SSH1. Optimization of crystals for both proteins is currently ongoing. The SSH1 inhibitor, along with the two other highly active compounds, provides an excellent starting point for the development of highly potent SSH1 inhibitors.
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Hilbert, Brendan J. "Structure-based Targeting of Transcriptional Regulatory Complexes Implicated in Human Disease: A Dissertation". eScholarship@UMMS, 2013. https://escholarship.umassmed.edu/gsbs_diss/681.
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Transcriptional regulatory complexes control gene expression patterns and permit cellular responses to stimuli. Deregulation of complex components upsets target gene expression and can lead to disease. This dissertation examines proteins involved in two distinct regulatory complexes: C-terminal binding protein (CtBP) 1 and 2, and Interferon Regulatory Factors (IRF) 3 and 5. Although critical in developmental processes and injury response, CtBP transcriptional repression of cell adhesion proteins, pro-apoptotic factors, and tumor suppressors has been linked to the pathogenesis of multiple forms of cancer. IRFs function in the immune system and have been implicated in autoimmune disorders.
Understanding IRF activation is critical to treating pathogens that target IRF function or for future autoimmune disease therapies. We attempted to determine crystal structures that would provide the details of IRF activation, allowing insight into mechanisms of pathogen immune evasion and autoimmune disorders. Although no new structures were solved, we have optimized expression of C-terminal IRF-3 / co-activator complexes, as well as full-length IRF3 and IRF5 constructs. Modifying the constructs coupled with new crystal screening will soon result in structures which detail IRF activation, advancing understanding of the roles of IRF family members in disease.
Through structural and biochemical characterization we sought to identify and develop inhibitors of CtBP transcriptional regulatory functions. High concentrations of CtBP substrate, 4-Methylthio 2-oxobutyric acid (MTOB), have been shown in different cancer models to interfere with CtBP transcriptional regulation. We began the process of structure based drug design by solving crystal structures of both CtBP family members bound to MTOB. The resulting models identified critical ligand contacts and unique active site features, which were utilized in inhibitor design. Potential CtBP inhibitors were identified and co-crystallized with CtBP1. One such compound binds to CtBP more than 1000 times more tightly than does MTOB, as a result of our structure-based inclusion of a phenyl ring and a novel pattern of hydrogen bonding. This molecule provides a starting point for the development of compounds that will both bind more tightly and interfere with transcriptional signaling as we progress towards pharmacologically targeting CtBP as a therapy for specific cancers.
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Hilbert, Brendan J. "Structure-based Targeting of Transcriptional Regulatory Complexes Implicated in Human Disease: A Dissertation". eScholarship@UMMS, 2007. http://escholarship.umassmed.edu/gsbs_diss/681.
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Transcriptional regulatory complexes control gene expression patterns and permit cellular responses to stimuli. Deregulation of complex components upsets target gene expression and can lead to disease. This dissertation examines proteins involved in two distinct regulatory complexes: C-terminal binding protein (CtBP) 1 and 2, and Interferon Regulatory Factors (IRF) 3 and 5. Although critical in developmental processes and injury response, CtBP transcriptional repression of cell adhesion proteins, pro-apoptotic factors, and tumor suppressors has been linked to the pathogenesis of multiple forms of cancer. IRFs function in the immune system and have been implicated in autoimmune disorders.
Understanding IRF activation is critical to treating pathogens that target IRF function or for future autoimmune disease therapies. We attempted to determine crystal structures that would provide the details of IRF activation, allowing insight into mechanisms of pathogen immune evasion and autoimmune disorders. Although no new structures were solved, we have optimized expression of C-terminal IRF-3 / co-activator complexes, as well as full-length IRF3 and IRF5 constructs. Modifying the constructs coupled with new crystal screening will soon result in structures which detail IRF activation, advancing understanding of the roles of IRF family members in disease.
Through structural and biochemical characterization we sought to identify and develop inhibitors of CtBP transcriptional regulatory functions. High concentrations of CtBP substrate, 4-Methylthio 2-oxobutyric acid (MTOB), have been shown in different cancer models to interfere with CtBP transcriptional regulation. We began the process of structure based drug design by solving crystal structures of both CtBP family members bound to MTOB. The resulting models identified critical ligand contacts and unique active site features, which were utilized in inhibitor design. Potential CtBP inhibitors were identified and co-crystallized with CtBP1. One such compound binds to CtBP more than 1000 times more tightly than does MTOB, as a result of our structure-based inclusion of a phenyl ring and a novel pattern of hydrogen bonding. This molecule provides a starting point for the development of compounds that will both bind more tightly and interfere with transcriptional signaling as we progress towards pharmacologically targeting CtBP as a therapy for specific cancers.
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Lau, Kin-chong, i 劉健莊. "Microarray-based investigations of genetic diseases". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B45894760.
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Hall, David. "An XML-based Database of Molecular Pathways". Thesis, Linköping University, Department of Computer and Information Science, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-3717.
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Research of protein-protein interactions produce vast quantities of data and there exists a large number of databases with data from this research. Many of these databases offers the data for download on the web in a number of different formats, many of them XML-based.
With the arrival of these XML-based formats, and especially the standardized formats such as PSI-MI, SBML and BioPAX, there is a need for searching in data represented in XML. We wanted to investigate the capabilities of XML query tools when it comes to searching in this data. Due to the large datasets we concentrated on native XML database systems that in addition to search in XML data also offers storage and indexing specially suited for XML documents.
A number of queries were tested on data exported from the databases IntAct and Reactome using the XQuery language. There were both simple and advanced queries performed. The simpler queries consisted of queries such as listing information on a specified protein or counting the number of reactions.
One central issue with protein-protein interactions is to find pathways, i.e. series of interconnected chemical reactions between proteins. This problem involve graph searches and since we suspected that the complex queries it required would be slow we also developed a C++ program using a graph toolkit.
The simpler queries were performed relatively fast. Pathway searches in the native XML databases took long time even for short searches while the C++ program achieved much faster pathway searches.
Książki na temat "Protein Based Molecular Diseases"
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Protein chaperones and protection from neurodegenerative diseases. Hoboken: John Wiley & Sons, 2011.
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Protein evolution. Oxford [England]: Blackwell Science, 1999.
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Henrik, Bohr, i Brunak Søren, red. Protein folds: A distance-based approach. Boca Raton: CRC Press, 1996.
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Coleman, Thomas F. Parallel continuation-based global optimization for molecular conformation and protein folding. Ithaca, N.Y: Cornell Theory Center, Cornell University, 1994.
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Protein evolution. Wyd. 2. Oxford: Blackwell Science, 2007.
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service), SpringerLink (Online, red. Osteoimmunopathology: Evidence-Based Perspectives from Molecular Biology to Systems Biology. New York, NY: Springer Science+Business Media, LLC, 2011.
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Crichton, Robert R. Metal-based neurodegeneration: From molecular mechanisms to therapeutic strategies. Wyd. 2. Chichester, West Sussex, U.K: John Wiley & Sons, 2013.
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J, Ward Roberta, red. Metal-based neurodegeneration: From molecular mechanisms to therapeutic strategies. Chichester: J. Wiley & Sons, 2006.
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Schwarz, Siegfried. Molecules of life & mutations: Understanding diseases by understanding proteins. Basel: Karger, 2002.
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Schwarz, Siegfried. Molecules of life & mutations: Understanding diseases by understanding proteins. Basel: Karger, 2002.
Znajdź pełny tekst źródłaCzęści książek na temat "Protein Based Molecular Diseases"
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Herrero-Hernandez, Pablo, Atze J. Bergsma i W. W. M. Pim Pijnappel. "Generation of Human iPSC-Derived Myotubes to Investigate RNA-Based Therapies In Vitro". W Methods in Molecular Biology, 235–43. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2010-6_15.
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AbstractAlternative pre-mRNAsplicing can be cell-type specific and results in the generation of different protein isoforms from a single gene. Deregulation of canonical pre-mRNAsplicing by disease-associated variants can result in genetic disorders. Antisense oligonucleotides (AONs) offer an attractive solution to modulate endogenous gene expression through alteration of pre-mRNAsplicing events. Relevant in vitro models are crucial for appropriate evaluation of splicing modifying drugs. In this chapter, we describe how to investigate the splicing modulating activity of AONs in an in vitro skeletal muscle model, applied to Pompe disease. We also provide a detailed description of methods to visualize and analyze gene expression in differentiated skeletal muscle cells for the analysis of muscledifferentiation and splicing outcome. The methodology described here is relevant to develop treatment options using AONs for other genetic muscle diseases as well, including Duchenne muscular dystrophy, myotonic dystrophy, and facioscapulohumeral muscular dystrophy.
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Davtyan, Hayk, Irina Petrushina i Anahit Ghochikyan. "Immunotherapy for Alzheimer’s Disease: DNA- and Protein-Based Epitope Vaccines". W Methods in Molecular Biology, 259–81. New York, NY: Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4939-0410-5_16.
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Dutta, Naibedya, Suvranil Ghosh i Mahadeb Pal. "Neurodegenerative Diseases and Small Molecule Protein Chaperone Activator of Natural Origin". W Evidence Based Validation of Traditional Medicines, 117–27. Singapore: Springer Singapore, 2021. http://dx.doi.org/10.1007/978-981-15-8127-4_5.
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Wang, Hong, i Samir Hanash. "Intact-Protein Analysis System for Discovery of Serum-Based Disease Biomarkers". W Methods in Molecular Biology, 69–85. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-068-3_4.
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Yan, Bin, Panwen Wang, Junwen Wang i Kenneth R. Boheler. "Discovery of Surface Target Proteins Linking Drugs, Molecular Markers, Gene Regulation, Protein Networks, and Disease by Using a Web-Based Platform Targets-search". W Methods in Molecular Biology, 331–44. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-7553-2_19.
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Suárez-Herrera, Nuria, Tomasz Z. Tomkiewicz, Alejandro Garanto i Rob W. J. Collin. "Development and Use of Cellular Systems to Assess and Correct Splicing Defects". W Methods in Molecular Biology, 145–65. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2010-6_9.
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AbstractA significant proportion of mutations underlying genetic disorders affect pre-mRNA splicing, generally causing partial or total skipping of exons, and/or inclusion of pseudoexons. These changes often lead to the formation of aberrant transcripts that can induce nonsense-mediated decay, and a subsequent lack of functional protein. For some genetic disorders, including inherited retinal diseases (IRDs), reproducing splicing dynamics in vitro is a challenge due to the specific environment provided by, e.g. the retinal tissue, cells of which cannot be easily obtained and/or cultured. Here, we describe how to engineer splicing vectors, validate the reliability and reproducibility of alternative cellular systems, assess pre-mRNA splicing defects involved in IRD, and finally correct those by using antisense oligonucleotide-based strategies.
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Chakraborty, Kausik, Florian Georgescauld, Manajit Hayer-Hartl i F. Ulrich Hartl. "Role of Molecular Chaperones in Protein Folding". W Protein Misfolding Diseases, 47–72. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2010. http://dx.doi.org/10.1002/9780470572702.ch3.
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Tessier, Peter M., i Susan Lindquist. "Unraveling Molecular Mechanisms and Structures of Self-Perpetuating Prions". W Protein Misfolding Diseases, 145–74. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2010. http://dx.doi.org/10.1002/9780470572702.ch8.
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Solís-Fernández, Guillermo, Ana Montero-Calle, Miren Alonso-Navarro, Miguel Ángel Fernandez-Torres, Victoria Eugenia Lledó, María Garranzo-Asensio, Rodrigo Barderas i Ana Guzman-Aranguez. "Protein Microarrays for Ocular Diseases". W Methods in Molecular Biology, 239–65. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1562-1_17.
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Esposito, Gennaro, i Vittorio Bellotti. "Emerging Molecular Targets in the Therapy of Dialysis-Related Amyloidosis". W Protein Misfolding Diseases, 843–65. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2010. http://dx.doi.org/10.1002/9780470572702.ch38.
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Faria, Gustavo Hugo de Souza. "The impact of epigenetics on the development of neurodegenerative diseases". W XIII Congresso Paulista de Neurologia. Zeppelini Editorial e Comunicação, 2021. http://dx.doi.org/10.5327/1516-3180.654.
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Introduction: Neurodegenerative diseases affect thousands of people in Brazil and have been increasing in frequency with the aging population. However, little is known about the molecular mechanisms and biomarkers of these diseases, which leads to a medical approach based on symptomatic and unresolving characteristics. Epigenetics, including DNA methylation, histone modifications, and changes in regulatory RNAs, emerges as a tool for prevention of neurodegenerative diseases. Objectives: To review studies that discuss the role of epigenetics in the development of neurodegenerative diseases. Methodology: This study involved an integrative review of papers published from 2016 to 2021 by searching PubMed and Scopus. Results: The studies showed that there is evidence that epigenetic mechanisms interfere with the development of major neurodegenerative diseases. Huntington’s disease presents an altered gene from birth, but transcriptional dysregulation is characteristic of the pathology that may be correlated to the age of disease onset in the cortex. In Parkinson’s disease dysregulation of expression of a specific protein is believed to play a central role in the disease and occurs through aberrant methylation that controls activation or suppression. In relation to Alzheimer’s disease, it has been found that deregulated DNA methylation and demethylation is linked to the onset and progression of the disease. In addition, these epigenetic factors are interfered with by diet, aging, and exercise. Conclusions: Investment in epigenetic studies is needed to understand possible markers of neurodegenerative diseases, for early diagnosis and the formation of epidrugs with the ability to treat.
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Wijeratne, Shalini. "A Comparative Analysis of Nanoluc Luciferase and Alkaline Phosphatase as Reporter Proteins for Phage-based Pathogen Detection". W 2022 AOCS Annual Meeting & Expo. American Oil Chemists' Society (AOCS), 2022. http://dx.doi.org/10.21748/iibu6123.
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Rapid and specific detection of pathogenic bacteria in food and water sources can be crucial to disease diagnosis and prevention. Genetically modified bacteriophage-based assays are a promising alternative over standard overnight culture-based assays as they can provide comparatively rapid detection. Bacteriophage (phage) viruses specifically infect live bacterial cells for the rapid replication of their viral genome. Scientists exploit this in-built molecular amplification system by genetically modifying phage genes to express certain reporter proteins during an infection. The expression of reporter proteins is confirmed through enzymatic and/or sensory assays, indicating the presence of the pathogenic bacteria. The sensitivity of the reporter phage assays depend on the ability of the genetically engineered phage to successfully express its reporter protein with conserved activity. In this study, we compared two enzymes, alkaline phosphatase (ALP) and nanoluc luciferase (NLuc) as reporter proteins in the context of the above criteria. We genetically modified T7 phage genome to overexpress these enzymes upon infecting BL21 E. coli cells. The reporter proteins were quantified and detected by measuring its luminescence activities. NLuc phage was significantly better at its gene expression in comparison to ALP phage, averaging at 9.8 × 105 molecules of protein/CFU of E. coli and providing a limit of detection at 107 CFU of E. coli/mL. On the other hand, ALP phage was only able to produce 8.6 × 104 molecules of protein/CFU of E. coli and provide a limit of detection at 109 CFU of E. coli/mL. These findings will allow researchers to select their choice of reporter proteins to improve phage-based assays and continue their progress in the field of pathogen detection.
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Uzel, Sebastien, i Markus J. Buehler. "Molecular and Mesoscale Mechanisms of Osteogenesis Imperfecta Disease". W ASME 2010 First Global Congress on NanoEngineering for Medicine and Biology. ASMEDC, 2010. http://dx.doi.org/10.1115/nemb2010-13160.
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Collagen is a crucial structural protein material, formed through a hierarchical assembly of tropocollagen molecules, arranged in collagen fibrils that constitute the basis for larger-scale fibrils and fibers. Osteogenesis imperfecta is a genetic disorder in collagen characterized by mechanically weakened tendon, fragile bones, skeletal deformities and in severe cases prenatal death. Even though many studies have attempted to associate specific mutation types with phenotypic severity, the mechanisms by which a single point mutation influences the mechanical behavior of tissues at multiple length-scales remain unknown. In this study, we report a series of systematic molecular scale based bottom-up computational experiments focused on pure collagenous tissue, carried out using atomistic-level molecular dynamics (MD), adaptive Poison-Boltzmann solver (APBS) calculations, and a mesoscale molecular model of collagen fibrils.
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Kemp, Regina, Kevin Fraser, Kyoko Fujita, Douglas MacFarlane i Gloria Elliott. "Biocompatible Ionic Liquids: A New Approach for Stabilizing Proteins in Liquid Formulation". W ASME 2008 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2008. http://dx.doi.org/10.1115/sbc2008-192986.
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The stabilization of proteins is a priority for several important fields, most notably the pharmaceutical industry. Protein-based therapeutic drugs have demonstrated significant efficacy in controlling and curing disease. Unlike traditional small molecule-based drug therapies, a major hurdle in the development of protein drugs is the challenge of maintaining the protein in the folded state throughout processing and also during storage at the end point-of-use. When a protein is taken from its native environment, it is often unstable and unfolds. Because the protein’s 3-dimensional structure is responsible for its functional activity, much work has been dedicated to finding excipients that will stabilize proteins outside of their native environment.
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Kuznetsov, A. V., A. A. Avramenko i D. G. Blinov. "Simulation of Traffic Jam Formation in Fast Axonal Transport". W ASME 2009 Heat Transfer Summer Conference collocated with the InterPACK09 and 3rd Energy Sustainability Conferences. ASMEDC, 2009. http://dx.doi.org/10.1115/ht2009-88345.
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Many neurodegenerative diseases, such as Alzheimer’s disease, are linked to swellings occurring in long arms of neurons. Many scientists believe that these swellings result from traffic jams caused by the failure of intracellular machinery responsible for fast axonal transport; such traffic jam can plug an axon and prevent the sufficient amount of organelles to be delivered toward the synapse of the axon. Mechanistic explanation of the formation of traffic jams in axons induced by overexpression of tau protein is based on the hypothesis that the traffic jam is caused not by the failure of molecular motors to transport organelles along individual microtubules but rather by the disruption of the microtubule system in an axon, by the formation of a swirl of disoriented microtubules at a certain location in the axon. This paper investigates whether a microtubule swirl itself, without introducing into the model microtubule discontinuities in the traffic jam region, is capable of capturing the traffic jam formation. The answer to this question can provide important insight into the mechanics of the formation of traffic jams in axons.
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Szabo, T. "FRAGMENTATION OF CERULOPLASMIN BY THROMBINF". W XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644663.
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Copper containing proteolytic fragments of ceruloplasmin (CP), a 135 KDα2 -glycoprotein, have been shown to induce angiogenesis in the rabbit cornea assay.Neovascularisation plays an important role in different biological phenomena including chronic inflammation, wound healing, recanalisation of occluded blood vessels, tumor growth etc. Most of these events are associated with elevated CP levels. Neovascularisation occurs in diabetic macro- and microangiopathies as well. Serum CP concentrations in 92 diabetics were measured and compored to 50 healthy blood donors and 50 unselected hospitalized patients without malignancies. A marked CP elevation was observed in diabetics, especially in those with vascular complications. There was no correlation between the CP and the actual blood glucose concentrations, duration of the disease of the type of the treatment. Activation of the haemostatic processes has been found in the majority of diabetic patients with vascular diseases. In case CP is a substrate for thrombin, the generation of this specific serine protease may lead to the release of angiogenic peptides from CP on the site of vascular occlusion. For this reason, purified human CP was incubated with thrombin at pH 7.4, 37×C, and samples were removed at 0, 30, 60, 120, 240, min., and\after 2k hours for CP oxidase activity measurements and for SDS PAGE. Thrombin treatment did not affect the enzymic activity of CP. On SDS PAGE the band corresponding to the parent molecule eventually disappeared, and a Mr ll6 KD fragment together with three smaller peptides has been produced, with molecular masses 61, 4l and 20 KD, respectively. Based on these experiments, CP is to be considered as a new protein substrate for thrombin. The physiological relevance of this phenomenon needs further examination.
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Breton, Michael E., i Monica B. Patel. "Decline in ERG Maximum a-wave and b-wave Amplitudes with Age". W Vision Science and its Applications. Washington, D.C.: Optica Publishing Group, 1995. http://dx.doi.org/10.1364/vsia.1995.tub1.
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Growth in the population over age 60 has increased the clinical importance of diseases of the retina associated with aging. The electroretinogram (ERG), long used as a test of retinal function, has potential for providing important clinical insight for retinal diseases of aging patients. However, interpretation of the ERG is complicated by the well documented, but less well understood, decline in response amplitude as a function of increasing age. Insight into factors leading to ERG amplitude decline with age may be provided by study of receptoral changes, reflected in the a-wave component, compared to changes in inner retinal function, reflected in the b-wave component (Pearlman, 1983). Breton et al (1994) and others (Hood and Birch, 1994) have developed a method of ERG a-wave analysis that yields parameters interpretable in terms of total rod dark current (amax), a constant of transduction amplification (A), and a brief delay associated with cascade molecular interactions (t'eff). This analysis is based on a quantitative model of the G-protein mediated phototransduction cascade proposed by Lamb and Pugh (1992). For purposes of this study, an important feature of the Breton et al (1994) procedure and analysis is the recording of rod response at high stimulus intensities where saturated a-wave and b-wave component amplitudes (amax and bmax) can be effectively measured with minimal algebraic interference with one another (Breton and Montzka, 1992). Based on this approach, changes in ERG amplitudes during development and aging can be used to infer changes in underlying retinal mechanisms. We use this approach to measure maximum a-wave and b-wave amplitudes and receptor transduction response as a function of age in human infants, toddlers and adults from several days up to 80 years.
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Bandeira, Jonathan, Mêuser Valença i Renan Alencar. "Using GANs and MLP Artificial Neural Networks to support early diagnosis of Alzheimer’s disease: a study on the potential of artificial data expansion". W Congresso Brasileiro de Inteligência Computacional. SBIC, 2021. http://dx.doi.org/10.21528/cbic2021-23.
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The life expectancy of the population in the most developed countries is growing every day and, consequently, there is an increase in various age-related diseases. In Brazil, just over 1.1 million people have Alzheimer’s disease (AD). In 2019, according to the World Health Organization, Alzheimer’s disease and other dementias were the third leading cause of mortality in the Americas and Europe. Despite being a degenerative and irreversible disease, if diagnosed early, treatments can be performed to slow the progression of symptoms and ensure a better quality of life for the patient. Most papers that study Computational Intelligence solutions to support diagnosis follow an approach based on neuroimaging evidence. In addition to this, another approach that has been gaining prominence is biochemical and molecular analysis. Following this approach, Ray et al., Ravetti & Moscato and Dantas & Valença carried out studies with classifiers from statistics or Computational Intelligence to support the early diagnosis of the disease. The work was carried out from a dataset with values of 120 blood proteins. Through this, they were able to classify whether or not the patient could be diagnosed with AD. As a result, Ray et al., Ravetti & Moscato and Dantas & Valença obtained an average accuracy rate of 89%, 93% and 94.34%, respectively. Thus, this work aims to use a traditional approach with a proposed Multilayer Perceptron Artificial Neural Network model to perform the early diagnosis of a patient with or without AD and compare the results obtained with the results of the related works cited. In addition, this work has as main objective to evaluate the potential of using synthetic data generated using a Generative Adversarial Network in the training and tests of the proposed classification model.
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Tenchov, B., S. Zaharinova, S. Abarova, L. Traikov i R. Koynova. "Thermodynamics of protein fibrillization. Relation to molecular basis of diseases". W 10th Jubilee International Conference of the Balkan Physical Union. Author(s), 2019. http://dx.doi.org/10.1063/1.5091366.
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Novak, Tyler, Adam Griebel i Corey P. Neu. "Strains in Magnetically Aligned Collagen Scaffolds as Determined by Displacement-Encoded MRI". W ASME 2012 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2012. http://dx.doi.org/10.1115/sbc2012-80897.
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Collagen is the most abundant structural protein in a broad range of tissues, including ligament, cartilage, and bone. Collagen-based tissue constructs, including those with defined fibril alignment, have shown considerable potential for repair and regeneration of diseased collagen-rich tissues [1,2], where fibril ultrastructure of constructs emulate the native tissue counterpart. Magnetic alignment of collagen is an established method to control fiber orientation without undesirable changes in molecular stability and structure [5]. To date, studies concerning the magnetic alignment of collagen have shown the ability of fibril alignment to drive preferential cell growth characteristics [6]. While the bulk mechanical characteristics due to alignment has been investigated in our laboratory (data not shown), the mechanical strain throughout the interior of the scaffolds has not been investigated. Knowledge of internal strains is required to determine the degree that fiber alignment affects spatiotemporal mechanics of microenvironments within the collagen structures. Spatially-dependent mechanics, which could be tailored based on unique fabrication methods that control fibril alignment in three dimensions, would be expected to directly influence local cell-matrix interactions.
Raporty organizacyjne na temat "Protein Based Molecular Diseases"
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Gafny, Ron, A. L. N. Rao i Edna Tanne. Etiology of the Rugose Wood Disease of Grapevine and Molecular Study of the Associated Trichoviruses. United States Department of Agriculture, wrzesień 2000. http://dx.doi.org/10.32747/2000.7575269.bard.
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Rugose wood is a complex disease of grapevines, characterized by modification of the woody cylinder of affected vines. The control of rugose wood is based on the production of healthy propagation material. Detection of rugose wood in grapevines is difficult and expensive: budwood from tested plants is grafted onto sensitive Vitis indicators and the appearance of symptoms is monitored for 3 years. The etiology of rugose wood is complex and has not yet been elucidated. Several elongated clostero-like viruses are consistently found in affected vines; one of them, grapevine virus A (GVA), is closely associated with Kober stem grooving, a component of the rugose wood complex. GVA has a single-stranded RNA genome of 7349 nucleotides, excluding a polyA tail at the 3' terminus. The GVA genome includes five open reading frames (ORFs 1-5). ORF 4, which encodes for the coat protein of GVA, is the only ORF for which the function was determined experimentally. The original objectives of this research were: 1- To produce antisera to the structural and non-structural proteins of GVA and GVB and to use these antibodies to establish an effective detection method. 2- Develop full length infectious cDNA clones of GVA and GVB. 3- Study the roll of GVA and GVB in the etiology of the grapevine rugose wood disease. 4- Determine the function of Trichovirus (now called Vitivirus) encoded genes in the virus life cycle. Each of the ORFs 2, 3, 4 and 5 genes of GVA were cloned and expressed in E. coli and used to produce antisera. Both the CP (ORF 4) and the putative MP (ORF 3) were detected with their corresponding antisera in-GVA infected N. benthamiana and grapevine. The MP was first detected at an early stage of the infection, 6-12 h after inoculation, and the CP 2-3 days after inoculation. The MP could be detected in GVA-infected grapevines that tested negative for CP, both with CP antiserum and with a commercially available ELISA kit. Antisera to ORF 2 and 5 encoded proteins could react with the recombinant proteins but failed to detect both proteins in GVA infected plants. A full-length cDNA clone of grapevine virus A (GVA) was constructed downstream from the bacteriophage T7 RNA polymerase promoter. Capped in vitro transcribed RNA was infectious in N. benthamiana and N. clevelandii plants. Symptoms induced by the RNA transcripts or by the parental virus were indistinguishable. The infectivity of the in vitro-transcribed RNA was confirmed by serological detection of the virus coat and movement proteins and by observation of virions by electron microscopy. The full-length clone was modified to include a gus reporter gene and gus activity was detected in inoculated and systemic leaves of infected plants. Studies of GVA mutants suggests that the coat protein (ORF 4) is essential for cell to cell movement, the putative movement protein (ORF 3) indeed functions as a movement protein and that ORF 2 is not required for virus replication, cell to cell or systemic movement. Attempts to infect grapevines by in-vitro transcripts, by inoculation of cDNA construct in which the virus is derived by the CaMV 35S promoter or by approach grafting with infected N. benthamiana, have so far failed. Studies of the subcellular distribution of GFP fusion with each of ORF 2, 3 and 4 encoded protein showed that the CP fusion protein accumulated as a soluble cytoplasmatic protein. The ORF 2 fusion protein accumulated in cytoplasmatic aggregates. The MP-GFP fusion protein accumulated in a large number of small aggregates in the cytoplasm and could not move from cell to cell. However, in conditions that allowed movement of the fusion protein from cell to cell (expression by a PVX vector or in young immature leaves) the protein did not form cytoplasmatic aggregates but accumulated in the plasmodesmata.
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Matthews, Lisa, Guanming Wu, Robin Haw, Timothy Brunson, Nasim Sanati, Solomon Shorser, Deidre Beavers, Patrick Conley, Lincoln Stein i Peter D'Eustachio. Illuminating Dark Proteins using Reactome Pathways. Reactome, październik 2022. http://dx.doi.org/10.3180/poster/20221027matthews.
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Diseases are often the consequence of proteins or protein complexes that are non-functional or that function improperly. An active area of research has focused on the identification of molecules that can interact with defective proteins and restore their function. While 22% percent of human proteins are estimated to be druggable, less than fifteen percent are targeted by FDA-approved drugs, and the vast majority of untargeted proteins are understudied or so-called "dark" proteins. Elucidation of the function of these dark proteins, particularly those in commonly drug-targeted protein families, may offer therapeutic opportunities for many diseases. Reactome is the most comprehensive, open-access pathway knowledgebase covering 2585 pathways and including 14246 reactions, 11088 proteins, 13984 complexes, and 1093 drugs. Placing dark proteins in the context of Reactome pathways provides a framework of reference for these proteins facilitating the generation of hypotheses for experimental biologists to develop targeted experiments, unravel the potential functions of these proteins, and then design drugs to manipulate them. To this end, we have trained a random forest with 106 protein/gene pairwise features collected from multiple resources to predict functional interactions between dark proteins and proteins annotated in Reactome and then developed three scores to measure the interactions between dark proteins and Reactome pathways based on enrichment analysis and fuzzy logic simulations. Literature evidence via manual checking and systematic NLP-based analysis support predicted interacting pathways for dark proteins. To visualize dark proteins in the context of Reactome pathways, we have also developed a new website, idg.reactome.org, by extending the Reactome web application with new features illustrating these proteins together with tissue-specific protein and gene expression levels and drug interactions.
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Bar-Joseph, Moshe, William O. Dawson i Munir Mawassi. Role of Defective RNAs in Citrus Tristeza Virus Diseases. United States Department of Agriculture, wrzesień 2000. http://dx.doi.org/10.32747/2000.7575279.bard.
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This program focused on citrus tristeza virus (CTV), the largest and one of the most complex RNA-plant-viruses. The economic importance of this virus to the US and Israeli citrus industries, its uniqueness among RNA viruses and the possibility to tame the virus and eventually turn it into a useful tool for the protection and genetic improvement of citrus trees justify these continued efforts. Although the overall goal of this project was to study the role(s) of CTV associated defective (d)-RNAs in CTV-induced diseases, considerable research efforts had to be devoted to the engineering of the helper virus which provides the machinery to allow dRNA replication. Considerable progress was made through three main lines of complementary studies. For the first time, the generation of an engineered CTV genetic system that is capable of infecting citrus plants with in vitro modified virus was achieved. Considering that this RNA virus consists of a 20 kb genome, much larger than any other previously developed similar genetic system, completing this goal was an extremely difficult task that was accomplished by the effective collaboration and complementarity of both partners. Other full-length genomic CTV isolates were sequenced and populations examined, resulting in a new level of understanding of population complexities and dynamics in the US and Israel. In addition, this project has now considerably advanced our understanding and ability to manipulate dRNAs, a new class of genetic elements of closteroviruses, which were first found in the Israeli VT isolate and later shown to be omnipresent in CTV populations. We have characterized additional natural dRNAs and have shown that production of subgenomic mRNAs can be involved in the generation of dRNAs. We have molecularly cloned natural dRNAs and directly inoculated citrus plants with 35S-cDNA constructs and have shown that specific dRNAs are correlated with specific disease symptoms. Systems to examine dRNA replication in protoplasts were developed and the requirements for dRNA replication were defined. Several artificial dRNAs that replicate efficiently with a helper virus were created from infectious full-genomic cDNAs. Elements that allow the specific replication of dRNAs by heterologous helper viruses also were defined. The T36-derived dRNAs were replicated efficiently by a range of different wild CTV isolates and hybrid dRNAs with heterologous termini are efficiently replicated with T36 as helper. In addition we found: 1) All CTV genes except of the p6 gene product from the conserved signature block of the Closteroviridae are obligate for assembly, infectivity, and serial protoplast passage; 2) The p20 protein is a major component of the amorphous inclusion bodies of infected cells; and 3) Novel 5'-Co-terminal RNAs in CTV infected cells were characterized. These results have considerably advanced our basic understanding of the molecular biology of CTV and CTV-dRNAs and form the platform for the future manipulation of this complicated virus. As a result of these developments, the way is now open to turn constructs of this viral plant pathogen into new tools for protecting citrus against severe CTV terms and development of virus-based expression vectors for other citrus improvement needs. In conclusion, this research program has accomplished two main interconnected missions, the collection of basic information on the molecular and biological characteristics of the virus and its associated dRNAs toward development of management strategies against severe diseases caused by the virus and building of novel research tools to improve citrus varieties. Reaching these goals will allow us to advance this project to a new phase of turning the virus from a pathogen to an ally.
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Ohad, Nir, i Robert Fischer. Regulation of plant development by polycomb group proteins. United States Department of Agriculture, styczeń 2008. http://dx.doi.org/10.32747/2008.7695858.bard.
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Our genetic and molecular studies have indicated that FIE a WD-repeat Polycomb group (PcG) protein takes part in multi-component protein complexes. We have shown that FIE PcG protein represses inappropriate programs of development during the reproductive and vegetative phases of the Arabidopsis life cycle. Moreover, we have shown that FIE represses the expression of key regulatory genes that promote flowering (AG and LFY), embryogenesis (LEC1), and shoot formation (KNAT1). These results suggest that the FIE PcG protein participates in the formation of distinct PcG complexes that repress inappropriate gene expression at different stages of plant development. PcG complexes modulate chromatin compactness by modifying histones and thereby regulate gene expression and imprinting. The main goals of our original project were to elucidate the biological functions of PcG proteins, and to understand the molecular mechanisms used by FIE PcG complexes to repress the expression of its gene targets. Our results show that the PcG complex acts within the central cell of the female gametophyte to maintain silencing of MEA paternal allele. Further more we uncovered a novel example of self-imprinting mechanism by the PgG complex. Based on results obtained in the cures of our research program we extended our proposed goals and elucidated the role of DME in regulating plant gene imprinting. We discovered that in addition to MEA,DME also imprints two other genes, FWA and FIS2. Activation of FWA and FIS2 coincides with a reduction in 5-methylcytosine in their respective promoters. Since endosperm is a terminally differentiated tissue, the methylation status in the FWA and FIS2 promoters does not need to be reestablished in the following generation. We proposed a “One-Way Control” model to highlight differences between plant and animal genomic imprinting. Thus we conclude that DEMETER is a master regulator of plant gene imprinting. Future studies of DME function will elucidate its role in processes and disease where DNA methylation has a key regulatory role both in plants and animals. Such information will provide valuable insight into developing novel strategies to control and improve agricultural traits and overcome particular human diseases.
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Sessa, Guido, i Gregory Martin. MAP kinase cascades activated by SlMAPKKKε and their involvement in tomato resistance to bacterial pathogens. United States Department of Agriculture, styczeń 2012. http://dx.doi.org/10.32747/2012.7699834.bard.
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The research problem: Pseudomonas syringae pv. tomato (Pst) and Xanthomonas campestrispv. vesicatoria (Xcv) are the causal agents of tomato bacterial speck and spot diseases, respectively. These pathogens colonize the aerial parts of the plant and cause economically important losses to tomato yield worldwide. Control of speck and spot diseases by cultural practices or chemicals is not effective and genetic sources of resistance are very limited. In previous research supported by BARD, by gene expression profiling we identified signaling components involved in resistance to Xcvstrains. Follow up experiments revealed that a tomato gene encoding a MAP kinase kinase kinase (MAPKKKe) is required for resistance to Xcvand Pststrains. Goals: Central goal of this research was to investigate the molecular mechanisms by which MAPKKKεand associated MAP kinase cascades regulate host resistance. Specific objectives were to: 1. Determine whether MAPKKKεplays a broad role in defense signaling in plants; 2. Identify components of MAP kinase cascades acting downstream of MAPKKKε; 3. Determine the role of phosphorylation-related events in the function of MAPKKKε; 4. Isolate proteins directly activated by MAPKKKε-associatedMAPK modules. Our main achievements during this research program are in the following major areas: 1. Characterization of MAPKKKεas a positive regulator of cell death and dissection of downstream MAP kinase cascades (Melech-Bonfil et al., 2010; Melech-Bonfil and Sessa, 2011). The MAPKKKεgene was found to be required for tomato resistance to Xcvand Pstbacterial strains and for hypersensitive response cell death triggered by different R gene/effector gene pairs. In addition, overexpression analysis demonstrated that MAPKKKεis a positive regulator of cell death, whose activity depends on an intact kinase catalytic domain. Epistatic experiments delineated a signaling cascade downstream of MAPKKKεand identified SIPKK as a negative regulator of MAPKKKε-mediated cell death. Finally, genes encoding MAP kinase components downstream of MAPKKKεwere shown to contribute to tomato resistance to Xcv. 2. Identification of tomato proteins that interact with MAPKKKεand play a role in plant immunity (Oh et al., 2011). We identified proteins that interact with MAPKKKε. Among them, the 14-3-3 protein TFT7 was required for cell death mediated by several R proteins. In addition, TFT7 interacted with the MAPKK SlMKK2 and formed homodimersin vivo. Thus, TFT7 is proposed to recruit SlMKK2 and MAPKKK client proteins for efficient signal transfer. 3. Development of a chemical genetic approach to identify substrates of MAPKKKε-activated MAP kinase cascades (Salomon et al., 2009, 2011). This approach is based on engineering the kinase of interest to accept unnatural ATP analogs. For its implementation to identify substrates of MAPKKKε-activated MAP kinase modules, we sensitized the tomato MAP kinase SlMPK3 to ATP analogs and verified its ability to use them as phosphodonors. By using the sensitized SlMPK3 and radiolabeled N6(benzyl)ATP it should be possible to tag direct substrates of this kinase. 4. Development of methods to study immunity triggered by pathogen-associated molecular patterns (PAMPs) in tomato and N. benthamiana plants (Kim et al., 2009; Nguyen et al. 2010). We developed protocols for measuring various PTI-associatedphenotypes, including bacterial populations after pretreatment of leaves with PAMPs, induction of reporter genes, callose deposition at the cell wall, activation of MAP kinases, and a luciferase-based reporter system for use in protoplasts. Scientific and agricultural significance: Our research activities discovered and characterized a signal transduction pathway mediating plant immunity to bacterial pathogens. Increased understanding of molecular mechanisms of immunity will allow them to be manipulated by both molecular breeding and genetic engineering to produce plants with enhanced natural defense against disease. In addition, we successfully developed new biochemical and molecular methods that can be implemented in the study of plant immunity and other aspects of plant biology.
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Ehrlich, Marcelo, John S. Parker i Terence S. Dermody. Development of a Plasmid-Based Reverse Genetics System for the Bluetongue and Epizootic Hemorrhagic Disease Viruses to Allow a Comparative Characterization of the Function of the NS3 Viroporin in Viral Egress. United States Department of Agriculture, wrzesień 2013. http://dx.doi.org/10.32747/2013.7699840.bard.
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Project Title: "Development of a plasmid-based reverse genetics system for the Bluetongue and Epizootic Hemorrhagic Disease viruses to allow comparative characterization of the function of the NS3 viroporin in viral egress". Project details: No - IS-4192-09; Participants – Ehrlich M. (Tel Aviv University), Parker J.S. (Cornell University), DermodyT.S. (Vanderbilt University); Period - 2009-2013. Orbiviruses are insect-borne infectious agents of ruminants that cause diseases with considerable economical impact in Israel and the United States. The recent outbreaks of BTV in Europe and of Epizootic Hemorrhagic Disease Virus (EHDV) in Israel, underscore the need for: (i) a better comprehension of the infection process of orbiviruses, (ii) the identification of unique vs. common traits among different orbiviruses, (iii) the development of novel diagnosis and treatment techniques and approaches; all aimed at the achievement of more effective control and treatment measures. It is the context of these broad goals that the present project was carried out. To fulfill our long-term goal of identifying specific viral determinants of virulence, growth, and transmission of the orbiviruses, we proposed to: (i) develop reverse genetics systems for BTV and EHDV2-Ibaraki; and (ii) identify the molecular determinants of the NS3 nonstructural protein related to viroporin/viral egress activities. The first objective was pursued with a two-pronged approach: (i) development of a plasmid-based reverse genetics system for BTV-17, and (ii) development of an "in-vitro" transcription-based reverse genetics system for EHDV2-Ibaraki. Both approaches encountered technical problems that hampered their achievement. However, dissection of the possible causes of the failure to achieve viral spread of EHDV2-Ibaraki, following the transfection of in-vitro transcribed genomic segments of the virus, revealed a novel characteristic of EHDV2-Ibaraki infection: an uncharacteristically low fold increase in titer upon infection of different cell models. To address the function and regulation of NS3 we employed the following approaches: (i) development (together with Anima Cell Metrology) of a novel technique (based on the transfection of fluorescently-labeledtRNAs) that allows for the detection of the levels of synthesis of individual viral proteins (i.e. NS3) in single cells; (ii) development of a siRNA-mediated knockdown approach for the reduction in levels of expression of NS3 in EHDV2-Ibaraki infected cells; (iii) biochemical and microscopy-based analysis of the localization, levels and post-translational modifications of NS3 in infected cells. In addition, we identified the altered regulation and spatial compartmentalization of protein synthesis in cells infected with EHDV2-Ibaraki or the mammalian reovirus. In EHDV2-Ibaraki-infected cells such altered regulation in protein synthesis occurs in the context of a cell stress reponse that includes the induction of apoptosis, autophagy and activation of the stressrelated kinase c-Jun N-terminal Kinase (JNK). Interestingly, inhibition of such stress-related cellular processes diminishes the production of infectious virions, suggesting that EHDV usurps these responses for the benefit of efficient infection. Taken together, while the present project fell short of the generation of novel reverse genetics systems for orbiviruses, the development of novel experimental approaches and techniques, and their employment in the analysis of EHDV-infected cells, yielded novel insights in the interactions of orbiviruses with mammalian cells.
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Manulis, Shulamit, Christine D. Smart, Isaac Barash, Guido Sessa i Harvey C. Hoch. Molecular Interactions of Clavibacter michiganensis subsp. michiganensis with Tomato. United States Department of Agriculture, styczeń 2011. http://dx.doi.org/10.32747/2011.7697113.bard.
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Clavibacter michiganensis subsp. michiganensis (Cmm), the causal agent of bacterial wilt and canker of tomato, is the most destructive bacterial disease of tomato causing substantial economic losses in Israel, the U.S.A. and worldwide. The molecular strategies that allow Cmm, a Gram-positive bacterium, to develop a successful infection in tomato plants are largely unknown. The goal of the project was to elucidate the molecular interactions between Cmmand tomato. The first objective was to analyze gene expression profiles of susceptible tomato plants infected with pathogenic and endophytic Cmmstrains. Microarray analysis identified 122 genes that were differentially expressed during early stages of infection. Cmm activated typical basal defense responses in the host including induction of defense-related genes, production of scavenging of free oxygen radicals, enhanced protein turnover and hormone synthesis. Proteomic investigation of the Cmm-tomato interaction was performed with Multi-Dimensional Protein Identification Technology (MudPIT) and mass spectroscopy. A wide range of enzymes secreted by Cmm382, including cell-wall degrading enzymes and a large group of serine proteases from different families were identified in the xylem sap of infected tomato. Based on proteomic results, the expression pattern of selected bacterial virulence genes and plant defense genes were examined by qRT-PCR. Expression of the plasmid-borne cellulase (celA), serine protease (pat-1) and serine proteases residing on the chp/tomA pathogenicity island (chpCandppaA), were significantly induced within 96 hr after inoculation. Transcription of chromosomal genes involved in cell wall degradation (i.e., pelA1, celB, xysA and xysB) was also induced in early infection stages. The second objective was to identify by VIGS technology host genes affecting Cmm multiplication and appearance of disease symptoms in plant. VIGS screening showed that out of 160 tomato genes, which could be involved in defense-related signaling, suppression of 14 genes led to increase host susceptibility. Noteworthy are the genes Snakin-2 (inhibitor of Cmm growth) and extensin-like protein (ELP) involved in cell wall fortification. To further test the significance of Snakin -2 and ELP in resistance towards Cmm, transgenic tomato plants over-expressing the two genes were generated. These plants showed partial resistance to Cmm resulting in a significant delay of the wilt symptoms and reduction in size of canker lesion compared to control. Furthermore, colonization of the transgenic plants was significantly lower. The third objective was to assess the involvement of ethylene (ET), jasmonate (JA) and salicylic acid (SA) in Cmm infection. Microarray and proteomic studies showed the induction of enzymes involved in ET and JA biosynthesis. Cmm promoted ET production 8 days after inoculation and SIACO, a key enzyme of ET biosynthesis, was upregulated. Inoculation of the tomato mutants Never ripe (Nr) impaired in ET perception and transgenic plants with reduced ET synthesis significantly delayed wilt symptoms as compared to the wild-type plants. The retarded wilting in Nr plants was shown to be a specific effect of ET insensitivity and was not due to altered expression of defense related genes, reduced bacterial population or decrease in ethylene biosynthesis . In contrast, infection of various tomato mutants impaired in JA biosynthesis (e.g., def1, acx1) and JA insensitive mutant (jai1) yielded unequivocal results. The fourth objective was to determine the role of cell wall degrading enzymes produced by Cmm in xylem colonization and symptoms development. A significance increase (2 to 7 fold) in expression of cellulases (CelA, CelB), pectate lyases (PelA1, PelA2), polygalacturonase and xylanases (XylA, XylB) was detected by qRT-PCR and by proteomic analysis of the xylem sap. However, with the exception of CelA, whose inactivation led to reduced wilt symptoms, inactivation of any of the other cell wall degrading enzymes did not lead to reduced virulence. Results achieved emphasized the complexity involved in Cmm-tomato interactions. Nevertheless they provide the basis for additional research which will unravel the mechanism of Cmm pathogenicity and formulating disease control measures.
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Vakharia, Vikram, Shoshana Arad, Yonathan Zohar, Yacob Weinstein, Shamila Yusuff i Arun Ammayappan. Development of Fish Edible Vaccines on the Yeast and Redmicroalgae Platforms. United States Department of Agriculture, luty 2013. http://dx.doi.org/10.32747/2013.7699839.bard.
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Betanodaviruses are causative agents of viral nervous necrosis (VNN), a devastating disease of cultured marine fish worldwide. Betanodavirus (BTN) genome is composed of two single-stranded, positive-sense RNA molecules. The larger genomic segment, RNA1 (3.1 kb), encodes the RNA-dependent RNA polymerase, while the smaller genomic segment, RNA 2 (1.4kb), encodes the coat protein. This structural protein is the host-protective antigen of VNN which assembles to form virus-like particles (VLPs). BTNs are classified into four genotypes, designated red-spotted grouper nervous necrosis virus (RGNNV), barfin flounder nervous necrosis virus (BFNNV), tiger puffer nervous necrosis virus (TPNNV), and striped jack nervous necrosis virus (SJNNV), based on phylogenetic analysis of the coat protein sequences. RGNNV type is quite important as it has a broad host-range, infecting warm-water fish species. At present, there is no commercial vaccine available to prevent VNN in fish. The general goal of this research was to develop oral fish vaccines in yeast and red microalgae (Porphyridium sp.) against the RGNNV genotype. To achieve this, we planned to clone and sequence the coat protein gene of RGNNV, express the coat protein gene of RGNNV in yeast and red microalgae and evaluate the immune response in fish fed with recombinantVLPs antigens produced in yeast and algae. The collaboration between the Israeli group and the US group, having wide experience in red microalgae biochemistry, molecular genetics and large-scale cultivation, and the development of viral vaccines and eukaryotic protein expression systems, respectively, was synergistic to produce a vaccine for fish that would be cost-effective and efficacious against the betanodavirus infection.
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Citovsky, Vitaly, i Yedidya Gafni. Viral and Host Cell Determinants of Nuclear Import and Export of the Tomato Yellow Leaf Curl Virus in Tomato Plants. United States Department of Agriculture, sierpień 2002. http://dx.doi.org/10.32747/2002.7585200.bard.
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Tomato yellow leaf curl geminivirus (TYLCV) is a major pathogen of cultivated tomato, causing up to 100% crop loss in many parts of the world. In Israel, where TYLCV epidemics have been recorded since the 1960' s, this viral disease is well known and has been of economic significance ever since. In recent years, TYLCV outbreaks also occurred in the "New World" - Cuba, The Dominican Republic, and in the USA, in Florida, Georgia and Louisiana. Thus, TYLCV substantially hinders tomato growth throughout the world. Surprisingly, however, little is known about the molecular mechanisms of TYLCV interaction with the host tomato cells. The present proposal, a continuation of the project supported by BARD from 1994, expanded our understanding of the molecular mechanisms by which TYLCV enters the host cell nucleus for replication and transcription and exits it for the subsequent cell-to-cell spread. Our project sought two objectives: I. To study the roles of the viral capsid protein (CP) and host cell factors in TYLCV nuclear import. II. To study the roles of CP and host cell factors in TYLCV nuclear export. Our research toward these goals have produced the following major achievements: . Developed a one-hybrid assay for protein nuclear export and import (#3 in the List of Publications). . Identified a functional nuclear export signal (NES) in the capsid protein (CP) of TYLCV (#3 in the List of Publications). . Discovered homotypic interactions between intact TYLCV CP molecules and analyzed these interactions using deletion mutagenesis of TYLCV CP (#5 in the List of Publications). . Showed developmental and tissue-specific expression of the host factor required for nuclear import of TYLCV CP, tomato karyopherin alpha 1, in transgenic tomato plants (#14 in the List of Publications). . By analogy to nuclear import of TYLCV ,identified an Arabidopsis VIPI protein that participates in nuclear import of Agrobacterium T -complexes via the karyopherin alpha pathway (#4,6, and 8 in the List of Publications). These research findings provided significant insights into (i) the molecular pathway of TYLCV entry into the host cell nucleus, and (ii) the mechanism by which TYLCV is exported from the nucleus for the cell-to-cell spread of infection. Furthermore, the obtained knowledge will help to develop specific strategies to attenuate TYLCV infection, for example, by blocking viral entry into and/or exit out of the host cell nucleus. Also, as much of our findings is relevant to all geminiviruses, new anti- TYLCV approaches developed based on the results of our research will be useful to combat other members of the Geminivirus family. Finally, in addition to the study of TYLCV nuclear import and export, our research contributed to our understanding of general mechanisms for nucleocytoplasmic shuttling of proteins and nucleic acids in plant cells.
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Young, Erin, Cem Kuscu, Christine Watkins i Murat Dogan. Using CRISPR Gene Editing to Prevent Accumulation of Lipids in Hepatocytes. University of Tennessee Health Science Center, styczeń 2022. http://dx.doi.org/10.21007/com.lsp.2022.0007.
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CRISPR gene editing is a molecular technology that can be used to silence gene expression. In this experiment, genes that are known to play a role in lipid accumulation in hepatocytes were targeted. Specifically, levels of fatty acid transport proteins 2 and 5 (FATP2 & 5) have been shown to be elevated in cases of non-alcoholic fatty liver disease. The goal of this experiment was to reduce expression of these genes by using a dead Cas9 (dCas9) protein with an attached inhibitory domain (KRAB) that acts on the promotor region. When measuring the mRNA expression, it was determined that the levels of the CRISPR-modified gene products were significantly reduced compared to the control. However, the same extent of inhibition was not consistently observed when conducting flow cytometry. Current work is aimed at discovering why lipid accumulation is not inhibited to the expected degree based on the results of mRNA expression.