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The identification of mutated proteins in human cancer cells-termed proteogenomics, requires several technologically independent research methodologies including DNA variant identification, RNA sequencing, and mass spectrometry. Any one of these methodologies are not optimized for identifying potential mutated proteins and any one output fails to cover completely a specific landscape.

An isogenic melanoma cell with a p53-null genotype was created by CRISPR/CAS9 system to determine how p53 gene inactivation affects mutant proteome expression. A mutant peptide reference database was developed by comparing two distinct DNA and RNA variant detection platforms using these isogenic cells. Chemically fractionated tryptic peptides from lysates were processed using a TripleTOF 5600+ mass spectrometer and their spectra were identified against this mutant reference database.

Approximately 190 mutated peptides were enriched in wt-p53 cells, 187 mutant peptides were enriched in p53-null cells, with an overlap of 147 mutated peptides. STRING analysis highlighted that the wt-p53 cell line was enriched for mutant protein pathways such as CDC5L and POLR1B, whilst the p53-null cell line was enriched for mutated proteins comprising EGF/YES, Ubiquitination, and RPL26/5 nodes.

Our study produces a well annotated p53-dependent and p53-independent mutant proteome of a common melanoma cell line model. Coupled to the application of an integrated DNA and RNA variant detection platform (CLCbio) and software for identification of proteins (ProteinPilot), this pipeline can be used to detect high confident mutant proteins in cells.

This pipeline forms a blueprint for identifying mutated proteins in diseased cell systems.

This pipeline forms a blueprint for identifying mutated proteins in diseased cell systems.

GH74 xyloglucanases are composed of two separate domains connected by two unstructured peptides. Previously, a hypothesis was made that the movement of domains may affect the enzyme mechanism of catalysis.

The molecular dynamics (MD) simulations of endo-processive xyloglucanases from Paenibacillus odorifer (PoGH74

) and Myceliophthora thermophila (MtXeg74A) were carried out.

MD simulations for both enzymes in complex with XXLG and XGXXLG oligosaccharides confirmed the possibility of domain movement. In the case of MtXeg74A, changes in the distances between C

atoms of aromatic residues involved in xyloglucan binding in -3 and+3 subsites of the active site cleft and those of selected residues on the opposite side of the cleft reached values up to 10-12Å. For PoGH74

the conformational changes were less pronounced. In MtXeg74A variants, the deletion of loop 1, which partially closes the entrance to the cleft, and the additional double mutation of two Trp residues in +3 and+5 subsites caused the enhanced mobility of the XGXXLG and also induced changes in topography of the cleft.

These findings demonstrate the possibility of existence of GH74 xyloglucanases in a more open and more closed enzyme conformation. The enzyme in an open conformation may more easily accommodate the branched polysaccharide, while its transition to the closed conformation, together with loop 1 function, should aid processivity.

Our results provide an insight into a mechanism of action of GH74 xyloglucanases and may be useful for discussing the catalytic mechanisms of glycoside hydrolases from other families.

Our results provide an insight into a mechanism of action of GH74 xyloglucanases and may be useful for discussing the catalytic mechanisms of glycoside hydrolases from other families.The combination of hypomethylating agents with the selective Bcl-2 inhibitor venetoclax (HMA-VEN) has emerged as a highly active regimen in patients with acute myelogenous leukemia (AML) in both the upfront and relapsed/refractory (r/r) settings. We report our early experience with a cohort of patients who were able to proceed to allogeneic hematopoietic cell transplantation (alloHCT) after HMA-VEN therapy. Thirty-two patients with AML (19 r/r and 13 de novo) with a median age of 62 years underwent alloHCT after HMA-VEN therapy. Twenty-two (68.8%) were in complete remission (CR)/CR with incomplete count recovery at time of HCT. With a median follow up of 14.4 months, the 1-year overall survival (OS) was 62.5%, and disease-free survival was 43.8%. The 1-year nonrelapse mortality rate was 18.8%, and the cumulative incidence of relapse was 37.5%. Among patients who underwent alloHCT in CR, the 1-year OS was 77.3%, and the cumulative incidence of nonrelapse mortality was 9.1%. The cumulative incidence of grade II-IV acute graft-versus-host disease was 43.8%. We conclude that alloHCT after HMA-VEN is therapy associated with favorable allogeneic HCT outcomes in newly diagnosed older patients with AML, as well as those with r/r AML.Organ transplantation remains the gold standard therapeutic option for patients with end-stage organ failure. However, there have been few improvements in the management of post-transplant immunosuppression. As the long-term use of immunosuppressive agents (ISAs) may result in off-target systemic toxicity and complications, minimizing the ISA dosage while preserving the pharmacological efficacy could be a promising solution to address these challenges. Here, we present the design and application of self-assembled prodrug nanoparticles based on chemically derived mycophenolate mofetil, which further provide a hydrophobic core to noncovalently encapsulate additional ISAs such as tacrolimus. selleck inhibitor The resulting immunosuppressant cocktail nanoparticles are further refined by PEGylation with amphiphilic polymers to form colloidally stable self-assembled immunosuppressant cocktails (SAICs) that are suitable for preclinical studies. In a rat model of allogeneic orthotopic liver transplantation (OLT), administration of SAICs markedly extends graft/recipient survival, retards weight loss and attenuates allograft damage. Furthermore, SAICs significantly abolish intragraft inflammatory cell infiltration and proinflammatory cytokine profiles as well as improve liver graft function. This study demonstrates the superiority of SAICs over traditional ISAs in the treatment of allograft rejection and may support the emerging application of the SAIC platform in clinical settings.One of the most promising approaches for the treatment of colorectal cancer is targeting epidermal growth factor receptor (EGFR). Comprehensive research has led to significant clinical outcomes using EGFR-targeted anticancer drugs; however, the response to these drugs still largely varies among individuals. The current diagnostic platform provides limited information that does not enable successful prediction of the anticancer performance of EGFR-targeted drugs. Here, we developed a EGFR-targeted activatable probe for predicting therapeutic efficacy of EGFR-targeted doxorubicin prodrug in colorectal cancer therapy. The EGF-conjugated fluorescence-activatable probe (EGF-probe) and EGF-conjugated doxorubicin prodrug (EGF-prodrug) were both fabricated using peptide substrates that can be dissociated by lysosomal enzymes, and thus share an intracellular mechanism of action. We demonstrated that after EGFR-mediated endocytosis, lysosomal enzymes de-quench the fluorescence of EGF-probe and activate the cytotoxicity of EGF-prodrug. When evaluated in vivo, EGF-probe yielded an outstanding cancer-specific imaging ability with reduced background signals. EGF-prodrug also successfully targeted the tumor and promoted cancer cell death. We tested different colorectal cancer cell types to investigate the correlation between the fluorescence recovery efficiency of EGF-probe and the cytotoxicity of EGF-prodrug. Strong correlations were observed both in vitro and in vivo. The actions of EGF-probe and EGF-prodrug were dependent on the inherent lysosomal activity of the cell type rather than its EGFR expression level. Our proposed approach using EGF-probe and EGF-prodrug may overcome the major drawback of the conventional theranostic platform and provide great opportunity for successful personalized cancer therapy.Nanogels as a versatile vehicle for doxorubicin have attracted great attention during the last decade. Since a nanogel composite device transport encapsulated drugs to the site of action and release them in a desirable time-frame, it could provide higher therapeutic effect. By implementation of different polymers, polymer/inorganic NPs and various crosslinking chemistry, it is possible to fabricate novel composite nanogel systems with favorable characteristics such as smart intelligent systems or multipurpose platforms. Due to high stability, good drug loading capacity for hydrophobic and hydrophilic agents, nanogels introduce great opportunity in pharmaceutical innovations. Composite nanogels show capability in gene, drug and diagnostic agents' delivery while providing an ideal platform for theranostic purposes as multifunctional systems. Doxorubicin as an anticancer agent is widely used against numerous cancers. Due to high systemic toxicity of doxorubicin, there is still need for its safe and specific delivery to the site of action. In this regard, so many efforts have been put in by the researchers for preparation of different nanogel formulations of doxorubicin in order to produce more efficient formulations. This review focuses on design, fabrication, advantages and disadvantages of composite nanogel-based doxorubicin formulations.Due to the poor physicochemical properties of drugs and multi-stage biological barriers in vivo, drugs at action site cannot reach up to sufficient concentration to acquire expected therapeutic outcomes, which may conversely lead to side effects to normal tissues and organs. In recent years, nanoscale drug delivery systems (NDDS) have developed rapidly to effectively deliver drugs to target site. In addition to avoiding drug degradation and preserving drug physicochemical properties, NDDS with controllable drug behaviors on tissues, cells, and organelles can be applied to break through multi-stage barriers and manipulate drug metabolism. But poor knowledge of various biological barriers still hinders the development of NDDS. Hence, this review is to dissect the vital influence of biological barriers in pharmaceutical research and introduce strategies for manipulating drug behaviors by crossing multi-stage biological barriers. Moreover, setbacks faced in the development of nanotechnology-based formulations are analyzed, which brings systematic thinking in the field of drug delivery. By addressing these barriers and understanding the principles behind, the current review provides a new insight into the rational design of NDDS and promotes the development of nanomedicine.Tomato (Lycopersicon esculentum Mill) is an important food plant that has been used as a model plant in genetic evolution and molecular biology research. The plant is originated from the tropics; thus, it is sensitive to cold. Its growth and development can be easily affected by cold stress. In this study, cold-regulated gene LeCOR413PM2 was cloned from tomato leaves and then used to generate two types of transgenic tomato plants LeCOR413PM2-overexpressing transgenic plants and RNA-interference-expressing transgenic plants. The functions and expression of LeCOR413PM2 gene in response to cold stress were subsequently assessed. The results showed that LeCOR413PM2 localized in the plasma membrane. Expression of LeCOR413PM2 gene in the leaf of transgenic tomato plant was highest compared to that in other organs (i.e., root, stem, flower and fruit); it was elevated when plants were treated with cold stress. Overexpression of LeCOR413PM2 gene was found to not only reduce damage to cell membrane, accumulation of ROS, and photoinhibition of PSII, but also maintain high activity of antioxidant enzymes and content of osmotic regulators.