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Abstract Identification of potential human–virus protein–protein interactions (PPIs) contributes to the understanding of the mechanisms of viral infection and to the development of antiviral drugs. Existing computational models often have more hyperparameters that need to be adjusted manually, which limits their computational efficiency and generalization ability. Based on this, this study proposes a kernel Bayesian logistic matrix decomposition model with automatic rank determination, VKBNMF, for the prediction of human–virus PPIs. VKBNMF introduces auxiliary information into the logistic matrix decomposition and sets the prior probabilities of the latent variables to build a Bayesian framework for automatic parameter search. In addition, we construct the variational inference framework of VKBNMF to ensure the solution efficiency. The experimental results show that for the scenarios of paired PPIs, VKBNMF achieves an average AUPR of 0.9101, 0.9316, 0.8727, and 0.9517 on the four benchmark datasets, respectively, and for the scenarios of new human (viral) proteins, VKBNMF still achieves a higher hit rate. The case study also further demonstrated that VKBNMF can be used as an effective tool for the prediction of human–virus PPIs.

Identification of potential human–virus protein–protein interactions (PPIs) contributes to the understanding of the mechanisms of viral infection and to the development of antiviral drugs. Existing computational models often have more hyperparameters that need to be adjusted manually, which limits their computational efficiency and generalization ability. Based on this, this study proposes a kernel Bayesian logistic matrix decomposition model with automatic rank determination, VKBNMF, for the prediction of human–virus PPIs. VKBNMF introduces auxiliary information into the logistic matrix decomposition and sets the prior probabilities of the latent variables to build a Bayesian framework for automatic parameter search. In addition, we construct the variational inference framework of VKBNMF to ensure the solution efficiency. The experimental results show that for the scenarios of paired PPIs, VKBNMF achieves an average AUPR of 0.9101, 0.9316, 0.8727, and 0.9517 on the four benchmark datasets, respectively, and for the scenarios of new human (viral) proteins, VKBNMF still achieves a higher hit rate. The case study also further demonstrated that VKBNMF can be used as an effective tool for the prediction of human–virus PPIs. [ABSTRACT FROM AUTHOR]

Common to all biological systems and living organisms are molecular interactions, which may lead to specific physiological events. Most often, a cascade of events occurs, establishing an equilibrium between possibly competing and/or synergistic processes. Biochemical pathways that sustain life depend on multiple intrinsic and extrinsic factors contributing to aging and/or diseases. This article deals with food antioxidants and human proteins from the circulation, their interaction, their effect on the structure, properties, and function of antioxidant-bound proteins, and the possible impact of complex formation on antioxidants. An overview of studies examining interactions between individual antioxidant compounds and major blood proteins is presented with findings. Investigating antioxidant/protein interactions at the level of the human organism and determining antioxidant distribution between proteins and involvement in the particular physiological role is a very complex and challenging task. However, by knowing the role of a particular protein in certain pathology or aging, and the effect exerted by a particular antioxidant bound to it, it is possible to recommend specific food intake or resistance to it to improve the condition or slow down the process. [ABSTRACT FROM AUTHOR]

Viral infection involves a large number of protein–protein interactions (PPIs) between the virus and the host, and the identification of these PPIs plays an important role in revealing viral infection and pathogenesis. Existing computational models focus on predicting whether human proteins and viral proteins interact, and rarely take into account the types of diseases associated with these interactions. Although there are computational models based on a matrix and tensor decomposition for predicting multi-type biological interaction relationships, these methods cannot effectively model high-order nonlinear relationships of biological entities and are not suitable for integrating multiple features. To this end, we propose a novel computational framework, LTDSSL, to determine human–virus PPIs under different disease types. LTDSSL utilizes logistic functions to model nonlinear associations, sets importance levels to emphasize the importance of observed interactions and utilizes sparse subspace learning of multiple features to improve model performance. Experimental results show that LTDSSL has better predictive performance for both new disease types and new triples than the state-of-the-art methods. In addition, the case study further demonstrates that LTDSSL can effectively predict human-viral PPIs under various disease types. [ABSTRACT FROM AUTHOR]

Background/Aim: Recently, we reported that coiled-coil domain containing 25 (CCDC25) protein is elevated in the sera of patients with cholangiocarcinoma (CCA) and is suggested to be a diagnostic biomarker for CCA. This study aimed to examine whether serum CCDC25 level can be a unique biomarker for CCA. Bioinformatic analyses using Human Protein Atlas (HPA) database and Gene Expression Profiling Interactive Analysis 2 (GEPIA2) indicated that CCDC25 protein and mRNA are expressed not only in CCA but also in other cancers, such as colorectal cancer (CRC), breast cancer (BC), and hepatocellular carcinoma (HCC), all of which are the top 5 cancers highly prevalent in Thailand. Materials and Methods: Using a quantitative dot blot assay, serum CCDC25 levels were measured for 30 healthy controls (HC), 34 CRC, 42 BC, 43 HCC, and 83 CCA. Results: The serum CCDC25 levels of CCA patients (0.193±0.039 ng/µl) were significantly higher than those of CRC (0.019±0.006 ng/µl), BC (0.036±0.015 ng/µl), HCC (0.035±0.016 ng/µl), and higher than those of HC (0.012±0.003 ng/µl). The serum CCDC25 level can discriminate CCA from the HC, CRC, BC, and HCC with a sensitivity of 100, 99, 94, and 94%, respectively, and specificity of 100, 100, 98, and 95%, respectively. Conclusion: CCDC25 is a candidate diagnostic biomarker for CCA. [ABSTRACT FROM AUTHOR]

Background: The coronavirus nonstructural protein 5 (Nsp5) is a cysteine protease required for processing the viral polyprotein and is therefore crucial for viral replication. Nsp5 from several coronaviruses have also been found to cleave host proteins, disrupting molecular pathways involved in innate immunity. Nsp5 from the recently emerged SARS-CoV-2 virus interacts with and can cleave human proteins, which may be relevant to the pathogenesis of COVID-19. Based on the continuing global pandemic, and emerging understanding of coronavirus Nsp5-human protein interactions, we set out to predict what human proteins are cleaved by the coronavirus Nsp5 protease using a bioinformatics approach. Results: Using a previously developed neural network trained on coronavirus Nsp5 cleavage sites (NetCorona), we made predictions of Nsp5 cleavage sites in all human proteins. Structures of human proteins in the Protein Data Bank containing a predicted Nsp5 cleavage site were then examined, generating a list of 92 human proteins with a highly predicted and accessible cleavage site. Of those, 48 are expected to be found in the same cellular compartment as Nsp5. Analysis of this targeted list of proteins revealed molecular pathways susceptible to Nsp5 cleavage and therefore relevant to coronavirus infection, including pathways involved in mRNA processing, cytokine response, cytoskeleton organization, and apoptosis. Conclusions: This study combines predictions of Nsp5 cleavage sites in human proteins with protein structure information and protein network analysis. We predicted cleavage sites in proteins recently shown to be cleaved in vitro by SARS-CoV-2 Nsp5, and we discuss how other potentially cleaved proteins may be relevant to coronavirus mediated immune dysregulation. The data presented here will assist in the design of more targeted experiments, to determine the role of coronavirus Nsp5 cleavage of host proteins, which is relevant to understanding the molecular pathology of coronavirus infection. [ABSTRACT FROM AUTHOR]

Common to all biological systems and living organisms are molecular interactions, which may lead to specific physiological events. Most often, a cascade of events occurs, establishing an equilibrium between possibly competing and/or synergistic processes. Biochemical pathways that sustain life depend on multiple intrinsic and extrinsic factors contributing to aging and/or diseases. This article deals with food antioxidants and human proteins from the circulation, their interaction, their effect on the structure, properties, and function of antioxidant-bound proteins, and the possible impact of complex formation on antioxidants. An overview of studies examining interactions between individual antioxidant compounds and major blood proteins is presented with findings. Investigating antioxidant/protein interactions at the level of the human organism and determining antioxidant distribution between proteins and involvement in the particular physiological role is a very complex and challenging task. However, by knowing the role of a particular protein in certain pathology or aging, and the effect exerted by a particular antioxidant bound to it, it is possible to recommend specific food intake or resistance to it to improve the condition or slow down the process.

The use of metaproteomics for studying the human gut microbiota can shed light on the taxonomic profile and the functional role of the microbial community. Nevertheless, methods for extracting proteins from stool samples continue to evolve, in the pursuit of optimal protocols for moistening and dispersing the stool sample and for disrupting microbial cells, which are two critical steps for ensuring good protein recovery. Here, we evaluated different stool sample processing (SSP) and microbial cell disruption methods (CDMs). The combination of a longer disintegration period of the stool sample in a tube rotator with sonication increased the overall number of identified peptides and proteins. Proteobacteria, Bacteroidetes, Planctomycetes, and Euryarchaeota identification was favored by mechanical cell disruption with glass beads. In contrast, the relative abundance of Firmicutes, Actinobacteria, and Fusobacteria was improved when sonication was performed before bead beating. Tenericutes and Apicomplexa identification was enhanced by moistening the stool samples during processing and by disrupting cells with medium-sized glass beads combined with or without sonication. Human protein identifications were affected by sonication. To test the reproducibility of these gut metaproteomic analyses, we examined samples from six healthy individuals using a protocol that had shown a good taxonomic diversity and identification of proteins from Proteobacteria and humans. We also detected proteins involved in microbial functions relevant to the host and related mostly to specific taxa, such as B12 biosynthesis and short chain fatty acid (SCFA) production carried out mainly by members in the Prevotella genus and the Firmicutes phylum, respectively. The taxonomic and functional profiles obtained with the different protocols described in this work provides the researcher with valuable information when choosing the most adequate protocol for the study of certain pathologies under suspicion of being related to a specific taxon from the gut microbiota. [ABSTRACT FROM AUTHOR]

The use of metaproteomics for studying the human gut microbiota can shed light on the taxonomic profile and the functional role of the microbial community. Nevertheless, methods for extracting proteins from stool samples continue to evolve, in the pursuit of optimal protocols for moistening and dispersing the stool sample and for disrupting microbial cells, which are two critical steps for ensuring good protein recovery. Here, we evaluated different stool sample processing (SSP) and microbial cell disruption methods (CDMs). The combination of a longer disintegration period of the stool sample in a tube rotator with sonication increased the overall number of identified peptides and proteins. Proteobacteria, Bacteroidetes, Planctomycetes, and Euryarchaeota identification was favored by mechanical cell disruption with glass beads. In contrast, the relative abundance of Firmicutes, Actinobacteria, and Fusobacteria was improved when sonication was performed before bead beating. Tenericutes and Apicomplexa identification was enhanced by moistening the stool samples during processing and by disrupting cells with medium-sized glass beads combined with or without sonication. Human protein identifications were affected by sonication. To test the reproducibility of these gut metaproteomic analyses, we examined samples from six healthy individuals using a protocol that had shown a good taxonomic diversity and identification of proteins from Proteobacteria and humans. We also detected proteins involved in microbial functions relevant to the host and related mostly to specific taxa, such as B12 biosynthesis and short chain fatty acid (SCFA) production carried out mainly by members in the Prevotella genus and the Firmicutes phylum, respectively. The taxonomic and functional profiles obtained with the different protocols described in this work provides the researcher with valuable information when choosing the most adequate protocol for the study of certain pathologies under suspicion of being related to a specific taxon from the gut microbiota.

Aim: To quantitatively and qualitatively compare the host proteomic profile in samples of symptomatic and asymptomatic apical periodontitis (AP) using nano‐liquid chromatography–electron spray tandem mass spectrometry. Methodology: Samples were obtained from 18 patients with radiographically evident AP, divided into symptomatic and asymptomatic groups (nine per group) according to clinical characteristics. After sample collection, protein extraction, purification and quantification of the samples were performed, which were analysed by reverse‐phase liquid chromatography coupled to mass spectrometry. Label‐free quantitative proteomic analysis was performed by Protein Lynx Global Service software. Differences in expression of proteins between the groups were calculated using the Monte Carlo algorithm, considering P < 0.05 for down‐regulated proteins and 1 − P > 0.95 for up‐regulated proteins. Proteins were identified with the embedded ion accounting algorithm in the software and a search of the Homo sapiens UniProt database. Results: A total of 853 individual human proteins were identified. In the quantitative analysis, common proteins to both groups accounted for 143 proteins. Differences in expression between groups resulted in 51 up‐regulated proteins (1 − P > 0.95) in the symptomatic group, including alpha‐1‐antitrypsin, protein S100‐A8, myeloperoxidase, peroxiredoxin and lactotransferrin. This group also had 43 down‐regulated proteins (P < 0.05), comprising immunoglobulin, neutrophil defensin, pyruvate kinase and alpha‐enolase. The qualitative analysis considered only the exclusive proteins of each group. For the symptomatic group, 318 complete proteins and 29 fragments were identified, such as dedicator of cytokinesis protein, intersectin, prostaglandin, phospholipase DDHD2 and superoxide dismutase. For the asymptomatic group, 326 complete proteins and 37 fragments were identified, including azurocidin, C‐reactive protein, collagen alpha, cathepsin, heat shock and laminin. Conclusions: Quantitative differences in the expression of common proteins in cases of symptomatic and asymptomatic AP were found, which were mostly related to host immune response in both groups. Exclusive proteins in the symptomatic group were mainly related to the host response to the presence of viruses in endodontic infections, oxidative stress and proteolytic enzymes. The results provide a basis for a better understanding of cellular and molecular pathways involved in AP, establishing specific proteomic profiles for symptomatic and asymptomatic conditions. [ABSTRACT FROM AUTHOR]

Since 1958, cell culture media supplemented with fetal bovine serum is used, despite the well-known concerns about animal welfare, reproducibility, reliability, relevance, and safety. To obliterate these concerns and increase scientific accuracy, we recently published an open access, publicly available paper on a defined medium composition to make it possible for any lab to prepare this medium. The medium supports routine culturing and cell banking as well as investigations of growth curves, dose response testing of compounds of cells in 2D and 3D, and cell migration; all important aspects for research and toxicology. Here we give a detailed description of how to mix the defined universal cell culture medium in 14 simple steps to support any entity that wishes to make it. We also list different normal and cancer cell lines that have been cultured in the defined medium.•Open source composition of animal product free universal cell culture medium•Protocols for mixing solutions of small xeno free molecules for supplementation•Protocols for mixing solutions of human proteins for supplementation., (© 2024 The Author(s).)

Human protein subcellular localization has an important research value in biological processes, also in elucidating protein functions and identifying drug targets. Over the past decade, a number of protein subcellular localization prediction tools have been designed and made freely available online. The purpose of this paper is to summarize the progress of research on the subcellular localization of human proteins in recent years, including commonly used data sets proposed by the predecessors and the performance of all selected prediction tools against the same benchmark data set. We carry out a systematic evaluation of several publicly available subcellular localization prediction methods on various benchmark data sets. Among them, we find that mLASSO-Hum and pLoc-mHum provide a statistically significant improvement in performance, as measured by the value of accuracy, relative to the other methods. Meanwhile, we build a new data set using the latest version of Uniprot database and construct a new GO-based prediction method HumLoc-LBCI in this paper. Then, we test all selected prediction tools on the new data set. Finally, we discuss the possible development directions of human protein subcellular localization. Availability: The codes and data are available from http://www.lbci.cn/syn/. [ABSTRACT FROM AUTHOR]

• A functional segment of BAX protein can be engineered to self-assemble as fluorescent nanoparticles targeted to the tumoral marker CXCR4. • Despite their CXCR4 selectivity, BAX nanoparticles do not show a tumor biodistribution and accumulate in liver and kidney. • The cocktail administration of BAX nanoparticles with equivalent PUMA and BAK constructs results in high tumor accumulation and potent antitumoral effect. • The combined administration of BAX, PUMA and BAK nanoparticles promote potent tumor cell destruction with a profile different from that of any of the single pro-apoptotic factors. A functional 29 amino acid-segment of the helix α5 from the human BAX protein has been engineered for production in recombinant bacteria as self-assembling, GFP-containing fluorescent nanoparticles, which are targeted to the tumoral marker CXCR4. These nanoparticles, of around 34 nm in diameter, show a moderate tumor biodistribution and limited antitumoral effect when systemically administered to mouse models of human CXCR4+ colorectal cancer (at 300 μg dose). However, if such BAX nanoparticles are co-administered in cocktail with equivalent nanoparticulate versions of BAK and PUMA proteins at the same total protein dose (300 μg), protein biodistribution and stability in tumor is largely improved, as determined by fluorescence profiles. This fact leads to a potent and faster destruction of tumor tissues when compared to individual pro-apoptotic factors. The analysis and interpretation of the boosted effect, from both the structural and functional sides, offers clues for the design of more efficient nanomedicines and theragnostic agents in oncology based on precise cocktails of human proteins. Several human pro-apoptotic peptides (namely BAK, BAX and PUMA) have been engineered as self-assembling protein nanoparticles targeted to the tumoral marker CXCR4. The systemic administration of the same final amounts of those materials as single drugs, or as combinations of two or three of them, shows disparate intensities of antitumoral effects in a mouse model of human colorectal cancer, which are boosted in the triple combination on a non-additive basis. The superiority of the combined administration of pro-apoptotic agents, acting at different levels of the apoptotic cascade, opens a plethora of possibilities for the development of effective and selective cancer therapies based on the precise cocktailing of pro-apoptotic nanoparticulate agents. Image, graphical abstract [ABSTRACT FROM AUTHOR]

Immunohistochemistry (IHC) is the accepted standard for spatial analysis of protein expression in tissues. IHC is widely used for cancer diagnostics and in basic research. The development of new antibodies to proteins with unknown expression patterns has created a demand for thorough validation. We have applied resources from the Human Protein Atlas project and the Antibody Portal at National Cancer Institute to generate protein expression data for 12 proteins across 39 cancer cell lines and 37 normal human tissue types. The outcome of IHC on consecutive sections from both cell and tissue microarrays using two independent antibodies for each protein was compared with in situ proximity ligation (isPLA), where binding by both antibodies is required to generate detection signals. Semi-quantitative scores from IHC and isPLA were compared with expression of the corresponding 12 transcripts across all cell lines and tissue types. Our results show a more consistent correlation between mRNA levels and isPLA as compared to IHC. The main benefits of isPLA include increased detection specificity and decreased unspecific staining compared to IHC. We conclude that implementing isPLA as a complement to IHC for analysis of protein expression and in antibody validation pipelines can lead to more accurate localization of proteins in tissue. [ABSTRACT FROM AUTHOR]

The functional and optimal expression of genes is crucial for survival of all living organisms. Numerous experiments and efforts have been performed to reveal the mechanisms required for the functional and optimal expression of human genes. The yeast Saccharomyces cerevisiae has evolved independently of humans for billions of years. Nevertheless, S. cerevisiae has many conserved genes and expression mechanisms that are similar to those in humans. Yeast is the most commonly used model organism for studying the function and expression mechanisms of human genes because it has a relatively simple genome structure, which is easy to manipulate. Many previous studies have focused on understanding the functions and mechanisms of human proteins using orthologous genes and biological systems of yeast. In this review, we mainly introduce two recent studies that replaced human genes and nucleosomes with those of yeast. Here, we suggest that, although yeast is a relatively small eukaryotic cell, its humanization is useful for the direct study of human proteins. In addition, yeast can be used as a model organism in a broader range of studies, including drug screening. [ABSTRACT FROM AUTHOR]

Studies on the structures and functions of individual kinases have been used to understand the biological properties of other kinases that do not yet have experimental structures. The key factor in accurate inference by homology is an accurate sequence alignment. We present a parsimonious, structure-based multiple sequence alignment (MSA) of 497 human protein kinase domains excluding atypical kinases. The alignment is arranged in 17 blocks of conserved regions and unaligned blocks in between that contain insertions of varying lengths present in only a subset of kinases. The aligned blocks contain well-conserved elements of secondary structure and well-known functional motifs, such as the DFG and HRD motifs. From pairwise, all-against-all alignment of 272 human kinase structures, we estimate the accuracy of our MSA to be 97%. The remaining inaccuracy comes from a few structures with shifted elements of secondary structure, and from the boundaries of aligned and unaligned regions, where compromises need to be made to encompass the majority of kinases. A new phylogeny of the protein kinase domains in the human genome based on our alignment indicates that ten kinases previously labeled as "OTHER" can be confidently placed into the CAMK group. These kinases comprise the Aurora kinases, Polo kinases, and calcium/calmodulin-dependent kinase kinases. [ABSTRACT FROM AUTHOR]

Antibodies are widely used for the detection of specific molecules such as peptides, proteins, and chemical compounds. The specificity of an antibody is therefore its most important feature. However, it is very difficult to confirm antibody specificity. Recently, we made a human protein array consisting of 19,712 kinds of recombinant human proteins produced by a wheat cell-free protein production system. Here, we demonstrate a novel protein array technology for antibody validation (CF-PA2Vtech). Full-length human cDNAs were fused to N-terminal FLAG-GST and then synthesized by the wheat cell-free system. To construct a 20 K human protein array, about 10 to 14 kinds of human proteins were mixed and captured in each well by glutathione-conjugated magnetic beads in 12 plates or one plate with 384- or 1536-well format, respectively, using a strong magnetic device. Using this protein array plate, commercially available anti-HA or anti-PD-1 antibody reacted to 13 or three human proteins, respectively. The cross-reactivity of these proteins was also confirmed by immunoblotting. These proteins have a similar epitope, and alanine mutations of these epitope candidates dissolved the reactivity. These results indicated that CF-PA2Vtech is very useful for validation of antibodies against human protein. [ABSTRACT FROM AUTHOR]

This study quantitatively validates the principle that the biological properties associated with a given genotype are determined by the distribution of amino acids. In order to visualize this central law of molecular biology, each protein was represented by a point in 250-dimensional space based on its amino acid distribution. Proteins from the same family are found to cluster together, leading to the principle that the convex hull surrounding protein points from the same family do not intersect with the convex hulls of other protein families. This principle was verified computationally for all available and reliable protein kinases and human proteins. In addition, we generated 2,328,761 figures to show that the convex hulls of different families were disjoint from each other. The classification performs well with high and robust accuracy (95.75% and 97.5%) together with reasonable phylogenetic trees validate our methods further. • We use a new natural vector method to characterize protein sequences. • We propose the convex hull principle on protein families. • The convex hulls formed from the natural vectors of different families are disjoint. • We verify this principle on all reliable protein kinase domains and human proteins. • The classification results and phylogenetic trees perform well. [ABSTRACT FROM AUTHOR]

A cell contains numerous protein molecules. One of the fundamental goals in molecular cell biology is to determine their subcellular locations since this information is extremely important to both basic research and drug development. In this paper, we report a novel and very powerful predictor called "pLoc_bal-mHum" for predicting the subcellular localization of human proteins based on their sequence information alone. Cross-validation tests on exactly the same experiment-confirmed dataset have indicated that the new predictor is remarkably superior to the existing state-of-the-art predictor in identifying the subcellular localization of human proteins. To maximize the convenience for the majority of experimental scientists, a user-friendly web-server for the new predictor has been established at http://www.jci-bioinfo.cn/pLoc%5fbal-mHum/ , by which users can easily get their desired results without the need to go through the detailed mathematics. • A very powerful method has been developed for predicting the subcellular localization of human proteins with both single location site and multiple location sites • A user-friendly web-server called "pLoc_bal-mHum" has been established for the new method by which user can easily get their desired results [ABSTRACT FROM AUTHOR]

Protein–protein interactions (PPIs) have been widely used to understand different biological processes and cellular functions associated with several diseases like cancer. Although some cancer‐related protein interaction databases are available, lack of experimental data and conflicting PPI data among different available databases have slowed down the cancer research. Therefore, in this study, the authors have focused on various proteins that are directly related to different types of cancer disease. They have prepared a PPI database between cancer‐associated proteins with the rest of the human proteins. They have also incorporated the annotation type and direction of each interaction. Subsequently, a biclustering‐based association rule mining algorithm is applied to predict new interactions with type and direction. This study shows the prediction power of association rule mining algorithm over the traditional classifier model without choosing a negative data set. The time complexity of the biclustering‐based association rule mining is also analysed and compared to traditional association rule mining. The authors are able to discover 38 new PPIs which are not present in the cancer database. The biological relevance of these newly predicted interactions is analysed by published literature. Recognition of such interactions may accelerate a way of developing new drugs to prevent different cancer‐related diseases. [ABSTRACT FROM AUTHOR]

Numerous studies have demonstrated the advantages of plant cell suspension culture systems in producing bioactive recombinant human growth factors. This study investigated the biological activity of recombinant basic human fibroblast growth factor (rhFGF2) protein produced by a plant culture system to enhance new bone formation in a bone defect mouse model. The human FGF2 cDNA gene was cloned into a plant expression vector driven by the rice α-amylase 3D promoter. The vector was introduced into rice calli (Oryza sativa L. cv. Dongjin), and the clone with the highest expression of rhFGF2 was selected. Maximum accumulation of rhFGF2 protein (approximately 28 mg/l) was reached at 13 day post-incubation. Male C57BL/6 mice underwent calvarial defect surgery and the defects were loaded with absorbable collagen sponge (ACS) only (ACS group) or ACS impregnated with 5 μg of plant-derived rhFGF2 (p-FGF2) protein or E. coli-derived rhFGF2 (e-FGF2) protein. Similar to the effects of e-FGF2, local delivery with p-FGF2 enhanced bone healing in the damaged region to higher levels than the ACS group. Exogenous addition of p-FGF2 or e-FGF2 exhibited similar effects on proliferation, mineralization, and osteogenic marker expression in MC3T3-E1 cells. Together, the current findings support the usefulness of this plant-based expression system for the production of biologically active rhFGF2. [ABSTRACT FROM AUTHOR]

Bisphenol A (BPA) negatively affects steroid production in human luteinized granulosa cells (GC). This study was designed to address two important questions: (1) whether BPA exerts the same disruptive effect in human cumulus granulosa cells (hCGC) and (2) to reveal the molecular mechanism underlying the BPA's action on steroidogenesis. We used cultured hCGC since these cells exert the properties of GC from early antral follicles. Results showed that BPA at 100 μM decreased estradiol level and CYP19A1 mRNA, but increased progesterone production, steroidogenic acute regulatory protein (STAR) and peroxisome proliferator-activated receptor gamma (PPARγ) mRNA expression after 48 h. Shorter (6 h) exposure to BPA elevated PPAR γ mRNA level in hCGC. Addition of ERK1/2 (U0126), EGFR (AG1478) and PPARγ (GW9662) inhibitors prevented the BPA-induced STAR and PPAR γ mRNA expression. Western blot analysis showed that BPA induced a rapid EGFR and ERK1/2 activation. The BPA-induced EGFR phosphorylation was prevented by addition of the PPARγ inhibitor, whereas the BPA-induced ERK1/2 activation was prevented by addition of the EGFR or PPARγ inhibitor. These data show that BPA increases the progesterone and decreases the estradiol biosynthetic pathway in hCGC. Augmentation of the progesterone biosynthetic pathway is mediated through the PPARγ-dependent activation of EGFR and ERK1/2, leading to increased expression of STAR mRNA. • BPA increases STAR mRNA expression in human cumulus granulosa cells. • BPA increases progesterone production in human cumulus granulosa cells. • BPA increases STAR mRNA through the PPARγ dependent activation of EGFR and ERK1/2. [ABSTRACT FROM AUTHOR]

• Detection of free extracellular heme is challenging issue. • Cysteine can be used as an absorbance spectroscopy probe for distinguishing protein-bound from free heme. • Use of cysteine allow detection of heme bound to proteins with low affinity. • The strategy might be used for assessment of heme-binding capacity of plasma or serum of patients with hemolytic diseases. Heme serves as a prosthetic group of numerous proteins involved in the oxidative metabolism. As result of various pathological conditions associated with hemolysis or tissue damage, large quantities of hemoproteins and heme can be released extracellularly. Extracellular heme has pronounced pathogenic effects in hemolytic diseases, mediated by its pro-oxidative and pro-inflammatory activities. The pathogenic potential of heme is mostly expressed when the molecule is in protein unbound form. The pathological relevance of free heme deems it necessary to develop reliable approaches for its assessment. Here we developed a technique based on UV–vis absorbance spectroscopy, where cysteine was used as a spectroscopy probe to distinguish between heme-bound to plasma proteins or hemoglobin from free heme. This technique allowed estimation of the heme-binding capacity of human serum, of particular heme scavenging proteins (albumin, hemopexin) or of immunoglobulins. The main advantage of the proposed approach is that it can distinguish free heme from heme associated with proteins with a wide range of affinities. The described strategy can be used for evaluation of heme-binding capacity of human plasma or serum following intravascular hemolysis or for estimation of stoichiometry of interaction of heme with a given protein. [ABSTRACT FROM AUTHOR]

Bence-Jones protein is a biomarker in urine for multiple myeloma. Traditional methods for urine Bence-Jones protein detection are either less-sensitive or laborious. Herein, we describe a new method for the detection of urine Bence-Jones protein using nanoporous materials and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Macroporous ordered silica foams (MOSF) were used to enrich proteins in urine, and then the materials-proteins composites were analyzed by MALDI-TOF MS. Based on the presence of specific mass spectrometric signals, Bence-Jones protein can be detected for the diagnosis of multiple myeloma. Twenty-one clinical positive and twenty-seven clinical negative urine samples were analyzed by the method. High sensitivity (95.24%, 20/21) and specificity (100%, 27/27) for the diagnosis of multiple myeloma were achieved. Compared to other methods for multiple myeloma diagnosis, e.g. immunofixation electrophoresis and immunonephelometry, our approach is more rapid, economical and convenient, which can be a new choice for the clinical diagnosis of Bence-Jones protein related diseases. fx1 • Macroporous ordered silica foams (MOSF) for Bence-Jones protein enrichment. • MOSF-protein composites detected by MALDI-TOF MS. • High sensitivity (95.24%, 20/21) and specificity (100%, 27/27) for clinical multiple myeloma diagnosis. [ABSTRACT FROM AUTHOR]

Okadaic acid (OA) is a lipophilic phycotoxin that accumulates in the hepatopancreas and fatty tissue of shellfish. Consumption of highly OA-contaminated seafood leads to diarrhetic shellfish poisoning which provokes severe gastrointestinal symptoms associated with a disruption of the intestinal epithelium. Since the molecular mechanisms leading to intestinal barrier disruption are not fully elucidated, we investigated the influence of OA on intestinal tight junction proteins (TJPs) in differentiated Caco-2 cells. We found a concentration- and time-dependent deregulation of genes encoding for intestinal TJPs of the claudin family, occludin, as well as zonula occludens (ZO) 1 and 2. Immunofluorescence staining showed concentration-dependent effects on the structural organization of TJPs already after treatment with a subtoxic but human-relevant concentration of OA. In addition, changes in the structural organization of cytoskeletal F-actin as well as its associated protein ZO-1 were observed. In summary, we demonstrated effects of OA on TJPs in intestinal Caco-2 cells. TJP expressions were affected after treatment with food-relevant OA concentrations. These results might explain the high potential of OA to disrupt the intestinal barrier in vivo as its first target. Thereby the present data contribute to a better understanding of the OA-dependent induction of molecular effects within the intestinal epithelium. • Exposure of differentiated Caco-2 cells to okadaic acid (OA) induces cytotoxicity. • Intestinal TJPs were affected by OA concentration- and time-dependently. • OA regulates TJPs on gene and protein expression levels. • OA affects TJPs expression after treatment with food-relevant concentrations. • OA destroys the TJP networks resulting in loss of intestinal epithelial function. [ABSTRACT FROM AUTHOR]

Early stage diagnosis of breast cancer by monitoring the proteins in human biological fluids is the best method and the first section toward efficient therapy, delaying metastasis and hindrance mortality. In this study, graphite ink was synthesized and combined with CA 15-3 antibody in order to develop a novel immunosensor for detection of CA 15-3, breast cancer biomarker. Conductivity of bioink was examined by designing various conductive patterns on paper using a rollerball pen. Electrochemical behavior of engineered immunosensor was evaluated through employing sensitive diagnostic technique, DPV (differential pulse voltammetry). Under optimal conditions, the linear concentration range and low limit of quantification (LLOQ) of designed immunosensor was 15–250 U/mL and 15 U/mL, respectively. The process was successfully applied to assay of breast cancer biomarker (CA15-3) in unprocessed human plasma specimens. Unlabelled Image [ABSTRACT FROM AUTHOR]

Aim: Bronchial epithelium acts as a defensive barrier against inhaled pollutants and microorganisms. This barrier is often compromised in inflammatory airway diseases that are characterized by excessive oxidative stress responses, leading to bronchial epithelial shedding, barrier failure, and increased bronchial epithelium permeability. Among proteins expressed in the junctional barrier and participating to the regulation of the response to oxidative and to environmental stresses is the cellular prion protein (PrPC). However, the role of PrPC is still unknown in the bronchial epithelium. Herein, we investigated the cellular mechanisms by which PrPC protein participates into the junctional complexes formation, regulation, and oxidative protection in human bronchial epithelium. Results: Both PrPC messenger RNA and mature protein were expressed in human epithelial bronchial cells. PrPC was localized in the apical domain and became lateral, at high degree of cell polarization, where it colocalized and interacted with adherens (E-cadherin/γ-catenin) and desmosomal (desmoglein/desmoplakin) junctional proteins. No interaction was detected with tight junction proteins. Disruption of such interactions induced the loss of the epithelial barrier. Moreover, we demonstrated that PrPC protection against copper-associated oxidative stress was involved in multiple processes, including the stability of adherens and desmosomal junctional proteins. Innovation: PrPC is a pivotal protein in the protection against oxidative stress that is associated with the degradation of adherens and desmosomal junctional proteins. Conclusion: Altogether, these results demonstrate that the loss of the integrity of the epithelial barrier by oxidative stress is attenuated by the activation of PrPC expression, where deregulation might be associated with respiratory diseases. [ABSTRACT FROM AUTHOR]

HRSV is a principle cause of infant hospitalization, childhood wheezing and a common pathogen in the elderly. Limited information exists regarding HRSV genotypes in Iran. In order to better understand HRSV strain diversity, we performed an in-depth evaluation of the genetic variability of the HRSV F protein detected in children under two years of age that, presented with acute respiratory symptoms during 2015–2016 in Tehran. A total of 180 nasopharyngeal swabs were evaluated. The HRSV positive samples were genotyped for G and F gene sequences using RT-PCR and sequencing methods. Phylogenetic analysis was performed using the neighbor-joining and maximum likelihood methods. Genetic and antigenic characteristics of the F gene, nucleotide and amino acids in significant positions and immune system binding regions, as well as the p-distance, positive/negative selection site, linear epitopes and glycosylation sites were investigated in all selected sequences. Among the 83 HRSV positive samples, the Fifty-five cases were successfully sequenced. All of them were classified as subgroup A and belonged to the ON-1 genotype, which possessed 72-nt duplication in the G gene. This study is the first report on the emergence of ON-1 in Iran. ON-1 Iranian sequences clustered in three lineages according to virus fusion (F) gene variations. F gene sequence analysis showed that all genetic changes in the isolates from Iran were base substitutions and no deletion/insertions were identified. The low dN/dS ratio and lack of positively selected sites showed that the fusion genes found in the strains from Iran are not under host selective pressure. Continuing and long-term molecular epidemiological surveys for early detection of circulating and newly emerging genotypes are necessary to gain a better understanding of their epidemic potential. • We performed an in-depth evaluation of the genetic variability of the HRSV F protein, during 2015–2016 in Tehran. • Based on our findings, a striking shift has been occurred from GA2 to ON1 in the circulation pattern of HRSV in Iran. • This study is the first report regarding the emergence of ON-1 in Iran. [ABSTRACT FROM AUTHOR]

Objective: Natural products are well known as the source of drugs in the treatment of allergic inflammation. Chrysophanol, an anthraquinone from the AST2017-01 extract, showed a beneficial anti-inflammatory effect on activated human mast cells in our previous study. However, a regulatory effect of AST2017-01 and chrysophanol on mast cell proliferation induced by thymic stromal lymphopoietin (TSLP) remains unclear. The present study determined the anti-proliferative effect and the fundamental mechanism of AST2017-01 and chrysophanol in mast cells. Methods: We evaluated an anti-proliferative effect of AST2017-01 and chrysophanol in TSLP-stimulated human mast cell line, HMC-1. Results: Without cytotoxicity, AST2017-01 and chrysophanol decreased mast cells growth and Ki67 mRNA expression increased by TSLP. AST2017-01 and chrysophanol enhanced expressions of p53 and Bax, whereas inhibited expression of Bcl-2. AST2017-01 and chrysophanol restored caspase-3 activity which was decreased by TSLP. AST2017-01 and chrysophanol suppressed expressions of murine double minute-2 protein and phosphorylated-signal transducer and activator of transcription six which are associated with the regulation of p53 protein. AST2017-01 and chrysophanol decreased levels of interleukin (IL)-13, IL-6, and tumor necrosis factor-α. Moreover, AST2017-01 and chrysophanol reduced mRNA expressions of TSLP receptor and IL-7 receptor α. Conclusions: Therefore, this study proposes that AST2017-01 and chrysophanol may be promising candidates for the development of potent anti-inflammatory or health functional foods. [ABSTRACT FROM AUTHOR]

Epigenetic readers are specific proteins which recognize histone marks and represents the underlying mechanism for chromatin regulation. Histone H3 lysine methylation is a potential epigenetic code for the chromatin organization and transcriptional control. Recognition of histone methylation is achieved by evolutionary conserved reader modules known as chromodomain, identified in several proteins, and is involved in transcriptional silencing and chromatin remodelling. Genetic perturbations within the structurally conserved chromodomain could potentially mistarget the reader protein and impair their regulatory pathways, ultimately leading to cellular chaos by setting the stage for tumor development and progression. Here, we report the structural conservations associated with diverse functions, prognostic significance and functional consequences of mutations within chromodomain of human proteins in distinct cancers. We have extensively analysed chromodomain containing human proteins in terms of their structural-functional ability to act as a molecular switch in the recognition of methyl-lysine recognition. We further investigated the combinatorial potential, target promiscuity and binding specificity associated with their underlying mechanisms. Indeed, the molecular mechanism of epigenetic silencing significantly underlies a newer cancer therapy approach. We hope that a critical understanding of chromodomains will pave the way for novel paths of research providing newer insights into the designing of effective anti-cancer therapies. [ABSTRACT FROM AUTHOR]

The early control and prevention of cancer contributes effectively interventions and cancer therapies. Secretory protein, one of the richest biomarkers, is proved important as molecular signposts of the physiological state of a cell. In this work, we aim to propose a proteomic high-throughput technology platform to facilitate detection of early cancer by means of biomarkers that secreted into the bloodstream. We compile a new benchmark dataset of human secretory proteins in plasma. A series of sequence-derived features, which have been proved involved in the structure and function of the secretory proteins, are collected to mathematically encode these proteins. Considering the influence of potential irrelevant or redundant features, we introduce discrete firefly optimization algorithm to perform feature selection. We evaluate and compare the proposed method SCRIP (Secretory proteins in plasma) with state-of-the-art approaches on benchmark datasets and independent testing datasets. SCRIP achieves the average AUC values of 0.876 and 0.844 in five-fold the cross-validation and independent test, respectively. Besides that, we also test SCRIP on proteins in four types of cancer tissues and successfully detect 66∼77% potential cancer biomarkers. [ABSTRACT FROM AUTHOR]

In this study it is aimed to construct an immunosensor system for detection of Sex-determining region Y-box 2 (SOX2) antigen by using Indium tin oxide- polyethylene terephthalate (ITO-PET) as working electrode. Firstly, ITO-PET electrode surfaces were hydroxylated by using NH 4 OH/H 2 O 2 /H 2 O and were incubated with 11-cyanoundecyltrimethoxysilane (11-CUTMS), respectively. After silanization process, anti- SOX2 was immobilized on modified ITO-PET electrodes. All immobilization processes were examined with Electrochemical impedance spectroscopy (EIS) and Cyclic voltammetry (CV). The basic parameters such as 11-CUTMS and anti-SOX2 concentrations, anti-SOX2 and SOX2 incubation period were optimized. The immunosensor prepared under optimal conditions gave a response for a wide concentration range of SOX2 protein (0.02 pg mL−1–2 pg mL−1) and the limit of detection was determined as low as 0.013 pg mL−1. And also, the immunosensor had good repeatability, reproducibility, reusability and long shelf life. Scanning Electron Microscope (SEM) method was utilized to observe the morphologies of the electrode surfaces belonging to all steps. Lastly, seven different real human serum were analyzed with the constructed immunosensor and Enzyme-Linked ImmunoSorbent Assay (ELISA). The results found with these methods were analogised with each other. Unlabelled Image • The biosensor was designed by using disposable and very low-cost ITO-PET working electrode. • The new biosensor showed high analytical performance with a linear range 0.02 pg mL-1 -2 pg mL-1. • 11-cyanoundecyltrimethoxysilane, a novel silanization agent, was used to modify ITO-PET electrode surface. • The proposed biosensor was applied to the real human serum and compared with the results obtained from ELISA test. • Kruskal Wallis test was used to interpret the analysis results obtained with the biosensor and ELISA test for human serum. [ABSTRACT FROM AUTHOR]

Indole- and azaindole-based glyoxylyl amide derivatives have been described as HIV-1 attachment inhibitors (AIs) that act by blocking the interaction between the viral gp120 coat protein and the human host cell CD4 receptor. As part of an effort to more deeply understand the role of the indole/azaindole heterocycle in the expression of antiviral activity, a survey of potential replacements was conducted using parallel synthesis methodology. The design and optimization was guided by a simple 2-dimensional overlay based on an overall planar topography between the indole/azaindole and C-7 substituents that had been deduced from structure-activity studies leading to the discovery of temsavir (3). 2-Substituted naphthalene- and quinoline-derived chemotypes emerged as the most interesting prototypes, with C-5 and C-6 substituents enhancing antiviral potency. Despite the fact that neither of these chemotypes incorporated a H-bond donor that has been shown to engage the side chain carboxylate of Asp 113 in gp120, the antiviral potency of several analogues met or exceeded that of 3 , demonstrating that engaging Asp 113 is not a prerequisite for potent antiviral activity. [ABSTRACT FROM AUTHOR]

Objective: The heterodimeric IL-12 family member cytokines including, IL-12, IL-23, IL-27, and IL-35 and have multiple roles in regulating innate and adaptive immunity with crucial functions in inflammatory disorders such as psoriasis. Chain pairing promiscuity is a feature of the IL-12 family. Recently, based on murine data, a new family member, IL-39, was proposed, consisting of IL23p19 (shared with IL-23) and EBI3 (shared with IL-27 and IL-35). IL-39 has subsequently been implicated in experimental murine lupus. Given the success of IL-23p19 therapeutic targeting in diseases including psoriasis, it is of great interest to confirm the presence of IL-39 in man. Human IL-39 is yet to be either detected or expressed, which has halted research in this area. Methods: Using a disulphide-linked human chimera protein composing of IL-23p19 and EBI3 human chains, we stimulated human leukocytes, and analysed cytokine secretion and STAT3 phosphorylation. Results and Conclusion: We report that this cytokine shows no activity in human cells. IL-39 chimera protein failed to induce either IL-6, IL-8, TNF, or IL-17A from leukocytes or STAT3 phosphorylation and thus, remains a 'theoretical cytokine' in humans. [ABSTRACT FROM AUTHOR]

Abstract Next-generation sequencing (NGS) technology is highly expected to help researchers disclose the complexity of alternative splicing and understand its association with carcinogenesis. Alternative splicing alterations are firmly associated with multiple malignancies, in terms of functional roles in malignant transformation, motility, and/or metastasis of cancer cells. One perfect example illustrating the connection between alternative splicing and cancer is the human protein arginine methyltransferase 1 (PRMT1) gene, previously cloned from members of our research group and involved in a variety of processes including transcription, DNA repair, and signal transduction. Two splice variants of PRMT1 (variants v.1 and v.2) are downregulated in breast cancer. In addition, PRMT1 v.2 promotes the survival and invasiveness of breast cancer cells, while it could serve as a biomarker of unfavorable prognosis in colon cancer patients. The aim of this study was the molecular cloning of novel alternative splice variants of PRMT1 with the use of 3′ RACE coupled with NGS technology. Extensive bioinformatics and computational analysis revealed a significant number of 19 novel alternative splicing events between annotated exons of PRMT1 as well as one novel exon, resulting in the discovery of multiple PRMT1 transcripts. In order to validate the full sequence of the novel transcripts, RT-PCR was carried out with the use of variant-specific primers. As a result, 58 novel PRMT1 transcripts were identified, 34 of which are mRNAs encoding new protein isoforms, whereas the rest 24 transcripts are candidates for nonsense-mediated mRNA decay (NMD). Highlights • We used NGS technology to identify novel splice variants of the PRMT1 gene. • 58 novel and 5′-partial splice variants of the PRMT1 gene were identified. • 34 novel splice variants of PRMT1 are predicted to encode new protein isoforms. • The rest 24 transcripts are probably nonsense-mediated mRNA decay (NMD) candidates. [ABSTRACT FROM AUTHOR]

Venomous marine cone snails produce peptide toxins (conotoxins) that bind ion channels and receptors with high specificity and therefore are important pharmacological tools. Conotoxins contain conserved cysteine residues that form disulfide bonds that stabilize their structures. To gain structural insight into the large, yet poorly characterized conotoxin H-superfamily, we used NMR and CD spectroscopy along with MS-based analyses to investigate H-Vc7.2 from Conus victoriae, a peptide with a VI/VII cysteine framework. This framework has CysI-CysIV/CysII-CysV/CysIII-CysVI connectivities, which have invariably been associated with the inhibitor cystine knot (ICK) fold. However, the solution structure of recombinantly expressed and purified H-Vc7.2 revealed that although it displays the expected cysteine connectivities, H-Vc7.2 adopts a different fold consisting of two stacked β-hairpins with opposing β-strands connected by two parallel disulfide bonds, a structure homologous to the N-terminal region of the human granulin protein. Using structural comparisons, we subsequently identified several toxins and nontoxin proteins with this "mini-granulin" fold. These findings raise fundamental questions concerning sequence-structure relationships within peptides and proteins and the key determinants that specify a given fold. [ABSTRACT FROM AUTHOR]

Abstract Autophagy, a lysosomal self-degradative process of cellular components, is essential for cellular homeostasis to response cellular stress and is tightly controlled by autophagy-related genes (ATGs). Autophagy-related gene 6 (ATG6, also known as Beclin-1 in human) is an essential factor regulating autophagy and apoptosis. RNA binding proteins (RBPs) regulate gene expression at the post-transcriptional level and their differential expression is linked to the pathogenesis of several human diseases. Here, we demonstrate the role of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) as a novel factor regulating ATG6 expression. hnRNPA1 associates with the 3′ untranslated region (3′UTR) of ATG6 mRNA and promotes its expression without significant changes at the mRNA level. Knockdown of hnRNPA1 decreases ATG6 expression, which is enhanced by the overexpression of hnRNPA1. Also, we show augmented expression of both hnRNPA1 and ATG6 in the colorectal cancer (CRC) tissues obtained from patients and demonstrate a positive correlation of their expression in CRC tissues. Our results suggest the potential role of hnRNPA1-mediated ATG6 regulation in the pathogenesis of CRC. Highlights • hnRNPA1 binds to the 3′UTR of ATG6 mRNA. • hnRNPA1 positively regulates ATG6 expression. • hnRNPA1 and ATG6 is augmented in the cancerous tissues obtained from CRC patients. • hnRNPA1 level positively correlates to ATG6 level in CRC tissues. [ABSTRACT FROM AUTHOR]

Abstract CQDs have emerged with outstanding properties as a star member of carbon nanomaterial family and in order to reveal its wide-range of application in biological microenvironment the interactions between human hemoglobin (HHb) and CQD and also with ethylenediamine-functionalized CQD (NCQD) are assessed using several techniques. Firstly, UV–vis absorption spectra of HHb reveal hyperchromic effect in the region of absorbance of tryptophan and tyrosine residues and also hypochromicity of Soret band in presence of CQD and NCQD. Interestingly, steady-state fluorescence spectroscopy reveal distinct fluorescence enhancement of HHb with significant red shift thereby indicating exposures of tryptophan and tyrosine residues to a more hydrophilic environment. However synchronous fluorescence spectra reveal that the microenvironment of tryptophan and tyrosine residues is altered in opposite manner, i.e. exposure of tryptophan residues to a more hydrophilic environment and the tyrosine residues to a more hydrophobic environment. Moreover the fluorescence enhancement is observed to be accompanied by increase in average fluorescence-lifetime and decrease in steady-state anisotropy thus signifying a decrease in restriction of rotational motion. Furthermore tryptophan residues within HHb appear to interact more with CQD compared to NCQD. Thermodynamic parameters as revealed by Isothermal Titration Calorimetry (ITC) demonstrate that electrostatic, hydrogen bonding and hydrophobic interactions are the predominant modes of interactions in presence of CQD. Whereas hydrophobic and hydrogen bonding interactions are the major interacting forces in presence of NCQD with five-site sequential binding as best-fit model in both the cases. Such interactions also appear to be associated with an increase in aggregation of HHb as evident from the measurements by atomic force microscopy (AFM) and dynamic light scattering (DLS) study. Although FT-IR spectra display alteration of amide I band, but the overall secondary structure of HHb seems to be nearly retained even in presence of CQDs, as evident in the CD spectra. These observations thus highlight the potential biomedical application of CQDs in biological microenvironment of human especially as drug-delivery system. Also bimolecular interaction of HHb as a model protein with other nanoparticles at the nano bio-interface has been outlined. Graphical abstract It is apparent that the fluorescence enhancement is accompanied by an increase in average lifetime. Since the lifetime determines the time available for the fluorophore to interact with or diffuse in its environment, thus the tryptophan and tyrosine residues within HHb are probably getting more exposed to the hydrophilic environment in presence of CQD as well as NCQD. Also there are possibilities that the CQD and NCQD can induce motional freedom within HHb as a result of which the tryptophan and tyrosine residues get exposed to the polar environment and such phenomenon in turn may bring about an increase in average lifetime. Unlabelled Image Highlights • Fluorescence enhancement of hemoglobin is viewed with CQD and NCQD • Tyrosine residues getting exposed to hydrophobic environment (blue shift) with CQDs • Tryptophan residues getting exposed to hydrophilic environment (red shift) with CQDs. • ITC measurements reveal the mechanisms of the nature of interactions. • The present results indicate potential applications of CQDs in drug-delivery systems [ABSTRACT FROM AUTHOR]

Multisubunit tethering complexes (MTCs) are multitasking hubs that form a link between membrane fusion, organelle motility and signaling. CORVET, CHEVI and HOPS are MTCs of the endo-lysosomal system. They regulate the major membrane flows required for endocytosis, lysosome biogenesis, autophagy and phagocytosis. In addition, individual subunits control complex-independent transport of specific cargoes and exert functions beyond tethering, such as attachment to microtubules and SNARE activation. Mutations in CHEVI subunits lead to arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome, while defects in CORVET and, particularly, HOPS are associated with neurodegeneration, pigmentation disorders, liver malfunction and various forms of cancer. Diseases and phenotypes, however, vary per affected subunit and a concise overview of MTC protein function and associated human pathologies is currently lacking. Here, we provide an integrated overview on the cellular functions and pathological defects associated with CORVET, CHEVI or HOPS proteins, both with regard to their complexes and as individual subunits. The combination of these data provides novel insights into how mutations in endo-lysosomal proteins lead to human pathologies. [ABSTRACT FROM AUTHOR]

Abstract Circulating insulin-like growth factor-binding proteins (IGFBPs) continue to gain attention as biomarkers of drug activities on insulin like growth factor (IGF)/IGF receptor signaling pathways. A multiplexed LC–MS/MS method was validated for the absolute quantitation of IGFBPs in human serum. The method was used to measure screening concentrations of IGFBPs in spinal and bulbar muscular atrophy (SBMA) patients in a phase 2 clinical trial. Concentrations of IGFBP 1, 2, 3, and 5 were simultaneously determined based on representative signature peptides derived from an optimized trypsin digestion procedure. Signature peptide levels were absolutely quantitated using a sensitive/specific targeted LC–MS/MS method. Corresponding mass-shifted, stable isotope-labeled peptides were employed as internal standards. A true blank matrix for the quantitation of IGFBPs was not available since they are endogenous proteins in human serum. In this method, calibration standards/curves were prepared using authentic synthetic peptides spiked into a surrogate matrix. The surrogate matrix was generated from human serum treated in the same way as the study samples, but using iodoacetic acid instead of iodoacetamide as the alkylation reagent. This surrogate matrix approach allowed for the direct and sensitive/specific quantification of IGFBP 1, 2, 3, and 5 due to the lack of any endogenous background. Equivalent matrix effect and recovery of analytes was achieved for the authentic and surrogate matrices. The fully validated LC–MS/MS assay will allow further evaluation of the utility of IGFBP biomarkers in clinical trials. Graphical abstract Unlabelled Image Highlights • GLP validated multiplex LC–MS/MS assay for efficacy protein biomarker quantitation. • Simultaneous monitoring of four IGFBPs (IGFBP1, 2, 3, and 5) in human serum • Surrogate matrix approach for sensitive/specific measurement of endogenous proteins • Measure concentrations of serum IGFBPs in subjects in a phase 2 clinical trial • Increase quality and throughput of protein biomarker analyses in clinical trials [ABSTRACT FROM AUTHOR]

Highlights • Diallyl maleate is introduced for the first time to prepare a polymer-based monolith. • The monolith presents a macroporous structure with a reversed-phase mechanism. • The interference of the three most abundant proteins in human plasma is avoided. • There is good correlation between the retention time and protein hydrophobicity. • Identification by LC/MS shows the method's importance in plasma proteomic research. Abstract A monolithic diallyl maleate-based column was developed and used to separate intact proteins from complex bio-samples with high-performance liquid chromatography. The resulting monolith exhibited a relatively uniform porous structure with macro-pores. A sample of standard proteins mixture was used to explore the reversed-phase mechanism for fast separation on the homemade monolithic column. The observed retention time increased with the values of the diameters, relative hydrophobicities and molecular weights of the standard proteins, which mainly depends on the relative hydrophobicity of the proteins. It is worth mentioning that the mask of the three most abundant proteins in human plasma can be avoided, thus providing an opportunity for the middle- and low-abundance proteins to be detected. According to this exploration, the fast separation of human plasma proteins on the diallyl maleate-based monolithic column was achieved in 15 min. The chromatographic fractions were identified by liquid chromatography-tandem mass spectrometry, and the results indicate that the present method is an outstanding method for the fast and efficient fractionation of human plasma that will be significant sense for plasma proteomics research, especially for exploring new disease marker and drug target. [ABSTRACT FROM AUTHOR]

Aims: Peptide receptor radionuclide therapy (PRRT) is in clinical use today to treat metastatic neuroendocrine tumors. Infused, radiolabeled, somatostatin analog peptides target tumors that are killed by irradiation damage. The peptides, however, are also retained in kidneys due to glomerular filtration, and the administered doses must be limited to avoid kidney damage. The human radical scavenger and antioxidant, α1-microglobulin (A1M), has previously been shown to protect bystander tissue against irradiation damage and has pharmacokinetic and biodistribution properties similar to somatostatin analogs. In this study, we have investigated if A1M can be used as a renal protective agent in PRRT. Results: We describe nephroprotective effects of human recombinant A1M on the short- and long-term renal damage observed following lutetium 177 (177Lu)-DOTATATE (150 MBq) exposure in BALB/c mice. After 1, 4, and 8 days (short term), 177Lu-DOTATATE injections resulted in increased formation of DNA double-strand breaks in the renal cortex, upregulated expression of apoptosis and stress response-related genes, and proteinuria (albumin in urine), all of which were significantly suppressed by coadministration of A1M (7 mg/kg). After 6, 12, and 24 weeks (long term), 177Lu-DOTATATE injections resulted in increased animal death, kidney lesions, glomerular loss, upregulation of stress genes, proteinuria, and plasma markers of reduced kidney function, all of which were suppressed by coadministration of A1M. Innovation and Conclusion: This study demonstrates that A1M effectively inhibits radiation-induced renal damage. The findings suggest that A1M may be used as a radioprotector during clinical PRRT, potentially facilitating improved tumor control and enabling more patients to receive treatment. [ABSTRACT FROM AUTHOR]

Membrane tethering is a highly regulated event occurring during the initial physical contact between membrane-bounded transport carriers and their target subcellular membrane compartments, thereby ensuring the spatiotemporal specificity of intracellular membrane trafficking. Although Rab-family small GTPases and specific Rab-interacting effectors, such as coiled-coil tethering proteins and multisubunit tethering complexes, are known to be involved in membrane tethering, how these protein components directly act upon the tethering event remains enigmatic. Here, using a chemically defined reconstitution system, we investigated the molecular basis of membrane tethering by comprehensively and quantitatively evaluating the intrinsic capacities of 10 representative human Rab-family proteins (Rab1a, -3a, -4a, -5a, -6a, -7a, -9a, -11a, -27a, and -33b) to physically tether two distinct membranes via homotypic and heterotypic Rab-Rab assembly. All of the Rabs tested, except Rab27a, specifically caused homotypic membrane tethering at physiologically relevant Rab densities on membrane surfaces (e.g., Rab-to-lipid molar ratios of 1:100-1:3000). Notably, endosomal Rab5a retained its intrinsic potency to drive efficient homotypic tethering even at concentrations below the Rab-to-lipid ratio of 1:3000. Comprehensive reconstitution experiments further uncovered that heterotypic combinations of human Rab-family isoforms, including Rab1a/6a, Rab1a/9a, and Rab1a/33b, can directly and selectively mediate membrane tethering. Rab1a and Rab9a in particular synergistically triggered very rapid and efficient membrane tethering reactions through their heterotypic trans-assembly on two opposing membranes. In conclusion, our findings establish that, in the physiological context, homotypic and heterotypic trans-assemblies of Rab-family small GTPases can provide the essential molecular machinery necessary to drive membrane tethering in eukaryotic endomembrane systems. [ABSTRACT FROM AUTHOR]

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) hold great promise for regenerative medicine and in vitro screening. Despite displaying key cardiomyocyte phenotypic characteristics, they more closely resemble fetal/neonatal cardiomyocytes, and further characterization is necessary. By combining the use of tandem mass tags to label cell lysates, followed by multiplexing, we have determined the effects of short-term (30 day) in vitro culture on hiPSC-CM protein expression. We found that hiPSC-CM exhibit temporal changes in global protein expression; alterations in protein expression were pronounced during the first 2 weeks following thaw and dominated by reductions in proteins associated with protein synthesis and ubiquitination. Between 2 and 4 weeks, proceeding thaw alterations in protein expression were dominated by metabolic pathways, indicating a potential temporal metabolic shift from glycolysis toward oxidative phosphorylation. Time-dependent changes in proteins associated with cardiomyocyte contraction, excitation–contraction coupling, and metabolism were detected. While some were associated with expected functional outcomes in terms of morphology or electrophysiology, others such as metabolism did not produce the anticipated maturation of hiPSC-CM. In several cases, a predicted outcome was not clear because of the concerted changes in both stimulatory and inhibitory pathways. Nevertheless, clear development of hiPSC-CM over this time period was evident. [ABSTRACT FROM AUTHOR]