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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]

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: 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]

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]

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]

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]

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]

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]

Retinal drusen formation is not only a clinical hallmark for the development of age-related macular degeneration (AMD) but also for other disorders, such as Alzheimer's disease and renal diseases. The initiation and growth of drusen is poorly understood. Attention has focused on lipids and minerals, but relatively little is known about the origin of drusen-associated proteins and how they are retained in the space between the basal lamina of the retinal pigment epithelium and the inner collagenous layer space (sub-RPE-BL space). While some authors suggested that drusen proteins are mainly derived from cellular debris from processed photoreceptor outer segments and the RPE, others suggest a choroidal cell or blood origin. Here, we reviewed and supplemented the existing literature on the molecular composition of the retina/choroid complex, to gain a more complete understanding of the sources of proteins in drusen. These "drusenomics" studies showed that a considerable proportion of currently identified drusen proteins is uniquely originating from the blood. A smaller, but still large fraction of drusen proteins comes from both blood and/or RPE. Only a small proportion of drusen proteins is uniquely derived from the photoreceptors or choroid. We next evaluated how drusen components may "meet, greet and stick" to each other and/or to structures like hydroxyapatite spherules to form macroscopic deposits in the sub-RPE-BL space. Finally, we discuss implications of our findings with respect to the previously proposed homology between drusenogenesis in AMD and plaque formation in atherosclerosis. [ABSTRACT FROM AUTHOR]

Highlights • E. dentata and A. nidulans have been identified with robust growth at 37 °C, and strong PAD productivity. • Purified E. dentata and A. nidulans PADs have molecular mass 41 and 48 kDa, respectively. • PADs peptide fingerprints of E. dentata and A. nidulans displayed 18% and 31% similarity to human PAD4. • The orientation of surface trypsinolytic sites on PADs from both fungal isolates were similar. • PADs from both isolates are calcium dependent with participation of serine and cysteine residues on their catalytic sites. Abstract Peptidylarginine deiminases (PADs) are a group of hydrolases, mediating the deimination of peptidylarginine residues into peptidyl-citrulline. Equivocal protein citrullination by PADs of fungal pathogens has a strong relation to the progression of multiple human diseases, however, the biochemical properties of fungal PADs remain ambiguous. Thus, this is the first report exploring the molecular properties of PAD from thermotolerant fungi, to imitate the human temperature. The teleomorph Emericella dentata and anamorph Aspergillus nidulans have been morphologically and molecularly identified, with observed robust growth at 37–40 °C, and strong PAD productivity. The physiological profiles of E. dentata and A. nidulans for PADs production in response to carbon, nitrogen sources, initial medium pH and incubation temperature were relatively identical, emphasizing the taxonomical proximity of these fungal isolates. PADs were purified from E. dentata and A. nidulans with apparent molecular masses 41 and 48 kDa, respectively. The peptide fingerprints of PADs from E. dentata and A. nidulans have been analyzed by MALDI-TOF/MS, displaying a higher sequence similarity to human PAD4 by 18% and 31%, respectively. The conserved peptide sequences of E. dentata and A. nidulans PADs displayed a higher similarity to human PAD than A. fumigatus PADs clade. PADs from both fungal isolates have an optimum pH and pH stability at 7.0–8.0, with putative p I 5.0–5.5, higher structural denaturation at pH 4.0–5.5 and 9.5–12 as revealed from absorbance at λ 280 nm. E. dentata PAD had a higher conformationally thermal stability than A. nidulans PAD as revealed from its lower K r value. From the proteolytic mapping, the orientation of trypsinolytic recognition sites on the PADs surface from both fungal isolates was very similar. PADs from both isolates are calcium dependent, with participation of serine and cysteine residues on their catalytic sites. PADs displayed a higher affinity to deiminate the peptidylarginine residues with a feeble affinity to work as ADI. So, PADs from E. dentata and A. nidulans had a relatively similar conformational and kinetic properties. Further molecular modeling analysis are ongoing to explore the role of PADs in citrullination of human proteins in Aspergillosis, that will open a new avenue for unraveling the vague of protein-protein interaction of human A. nidulans pathogen. [ABSTRACT FROM AUTHOR]

Protein allostery is ubiquitous phenomena that are important for cellular signalling processes. Despite extensive methodology development, a quantitative model is still needed to accurately measure protein allosteric response upon external perturbation. Here, we introduced the relative entropy concept from information theory as a quantitative metric to develop a method for measurement of the population shift with regard to protein structure during allosteric transition. This method is referred to as relative entropy-based dynamical allosteric network (REDAN) model. Using this method, protein allostery could be evaluated at three mutually dependent structural levels: allosteric residues, allosteric pathways, and allosteric communities. All three levels are carried out using rigorous searching algorithms based on relative entropy. Application of the REDAN model on the second PDZ domain (PDZ2) in the human PTP1E protein provided metric-based insight into its allostery upon peptide binding. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is a common and highly lethal form of cancer. Although the etiologic role of Fusobacterium nucleatum (F. nucleatum) in the development of CRC has been elucidated, the specific tumor molecules involved in the progression of CRC induced by F. nucleatum have not been identified. This study investigated several miRNAs and genes involved in the progression of F. nucleatum-induced CRC by Affymetrix miRNA microarray technology and GeneChip Human Transcriptome Array 2.0. The results suggest that miR-4474 and miR-4717 are up-regulated in CRC tissues in response to F. nucleatum infection, compared with the control group (paracancerous tissues), while other genes associated with signaling pathways in cancer, including CREB-binding protein (CREBBP), STAT1, PRKACB, CAMK2B, JUN, TP53 and EWSR1, were dysregulated. Bioinformatic analysis identified CREBBP as the primary aberrantly expressed gene in F. nucleatum-induced CRC. Consistent with the microarray analysis results, real-time RT-PCR analysis demonstrated that the expression of miR-4474/4717 was upregulated while that of CREBBP mRNA was downregulated in CRC patients infected with F. nucleatum. Additionally, CREBBP was identified as a novel target of miR-4474/4717. The results of this study suggest that miR-4474 and miR-4717 are involved in the progression of F. nucleatum-induced CRC by posttranscriptionally regulating the target gene CREBBP. [ABSTRACT FROM AUTHOR]

Highlights • CYFRA 21-1 and HE4 may be useful as biomarkers of recurrence in ADC. • Increased serum CYFRA 21-1 or HE4 alone indicated ADC recurrence. • CYFRA 21-1 and HE4 combined increased test specificity and recurrence detection. Abstract Objectives The biomarkers cytokeratin 19 fragment (CYFRA 21-1) and human epididymis protein 4 (HE4) are useful in the diagnosis, prognosis, and monitoring of non-small cell lung cancer (NSCLC), but their combination has not been investigated yet. The objective of this analysis was to evaluate the ability of CYFRA 21-1 and HE4 to predict recurrence as part of follow-up monitoring in patients with adenocarcinoma (ADC) of the lung. Materials and methods Serum samples were collected from patients with stage I-IIIA ADC preoperatively and during follow-up at 3, 6, 12, 18, and 24 months and then every 6–12 months up to 5 years post-R0 resection. Samples were analyzed for CYFRA 21-1 and HE4 via electrochemiluminescence immunoassay. All cases of disease recurrence were verified by imaging. The diagnostic performance of CYFRA 21-1, HE4, and their combination to predict recurrence was assessed by Receiver Operating Characteristic (ROC) and corresponding area under the curve (AUC). Results 115 patients with ADC were included (N = 612 biomarker measurements); median age was 63 years; most had stage I–II disease (n = 97; 84.3%). All patients underwent surgical resection; 44 patients (38%) also received adjuvant chemotherapy and 16 (14%) received radiation therapy. At the median timepoint for the last blood sample collection (37 months), 31 patients (27%) had experienced recurrence. Both CYFRA 21-1 and HE4 were able to detect recurrence (AUC and 95% confidence interval [CI]): 75.9% (66.0–85.8%) and 75.4% (65.9–84.8%), respectively, but this increased with the combination (78.8% [69.0–88.6%]). At a sensitivity of 80%, the respective specificities (95% CI) for CYFRA 21-1, HE4, and the combination were 57.1% (53.0–61.2%), 57.1% (53.0–61.2%), and 69.7% (65.8–73.4%). Conclusion Serial measurements of serum CYFRA 21-1 and HE4 levels could provide a valuable method for follow-up monitoring of patients with ADC to detect recurrence. [ABSTRACT FROM AUTHOR]

Cryopreservation can slow down the metabolism and decrease the risk of bacterial contamination. But, chilled platelets (PLTs) show a reduced period in circulation due to the rapid clearance by hepatic cells or spleen macrophages after transfusion. The deleterious changes that PLTs undergo are mainly considered the result of PLT protein variation. However, the basis for proteomic variation of stored PLTs remains poorly understood. Besides count, activation markers (CD62P and Annexin V), and aggregation, we used quantitative mass spectrometry to create the first comprehensive and quantitative human PLT proteome of samples stored at different temperatures (22°C, 10°C and −80°C). We found different conditions caused different platelet storage lesion (PSL). PLT count was decreased no matter at what temperature stored. PLTs viability at low temperature dropped by 21.78% and 11.21%, respectively, as compared 10.26% at room temperature, there were no significant differences between the storage methods. Membrane expression of CD62P gradually increased in all groups especially stored at 22°C up to 40% and 10°C up to 30%. However, exposure of PS on the PLT membrane was below 1% in every group. The PLT proteome showed there were 575 and 454 potential proteins identified by general iTRAQ analysis and phosphorylation iTRAQ a nalysis, respectively, among them, 33 common differentially expressed proteins caused by storage time and 44 caused by storage temperature Especially, membrane-bound proteins (such as FERMT3, STX4, MYL9 and TAGLN2) played key roles in PLT storage lesion. The pathways "Endocytosis", "Fc gamma R-mediated phagocytosis" and "Regulation of actin cytoskeleton" were affected predominantly by storage time. And the pathways "SNARE interactions in vesicular transport" and "Vasopressin-regulated water reabsorption" were affected by cold storage in our study. Proteomic results can help us to understand PLT biochemistry and physiology and thus unravel the mechanisms of PSL in time and space for more successful PLT transfusion therapy. [ABSTRACT FROM AUTHOR]

Tudor domain-containing (TDRD) proteins, as a family of evolutionarily conserved proteins, have been studied extensively in recent years in terms of their biological and biochemical functions. A major function of the TDRD proteins is to recognize the N-terminal arginine-rich motifs of the P-element-induced wimpy testis (PIWI) proteins via their conserved extended Tudor (eTudor or eTud) domains, which is essential in piRNA biogenesis and germ cell development. In this review, we summarize recent progress in the study of the TDRD proteins, and discuss the molecular mechanisms for the different binding selectivity of these eTudor domains to PIWI proteins based on the available binding and structural data. Understanding the binding differences of these TDRDs to PIWI proteins will help us better understand their functional differences and aid us in developing the target-specific therapeutics, because overexpression or mutations of the human TDRD proteins have been demonstrated to associate with various diseases. [ABSTRACT FROM AUTHOR]

Abstract WIPI proteins are mammalian PROPPIN family members that bind to phosphoinositides and play prominent roles in autophagosome biogenesis. Two phosphoinositide-binding sites were previously described in yeast PROPPIN Hsv2 but remain to be determined in mammalian WIPI proteins. Here, we characterized four human WIPI proteins (WIPI1–4) and solved the structure of WIPI3. WIPI proteins can bind to PI(3)P and PI(3,5)P 2 and adopt a conventional seven-bladed β-propeller fold. The structure of WIPI3 revealed that WIPI proteins also contain two sites embedded in blades 5 and 6 for recognizing phosphoinositides, resembling that in Hsv2. Structural comparison further demonstrated that the two conserved phosphoinositide-binding sites in PROPPIN proteins are not identical but intrinsically tend to recognize different types of phosphoinositides. This work provides the structural evidence to support the conservation of the two phosphoinositide-binding sites in WIPI proteins and also uncovers the potential phosphoinositide-binding selectivity for each site. Graphical Abstract Unlabelled Image Highlights • The structure of WIPI3 was determined at 1.8-Å resolution. • WIPI proteins contain two phosphoinositide-binding sites in blades 5 and 6. • The peripheries of two phosphoinositide-binding sites are distinct. • Two phosphoinositide-binding sites tend to recognize different phosphoinositides. [ABSTRACT FROM AUTHOR]

Microrchidia 3 (MORC3) is a human protein linked to autoimmune disorders, Down syndrome, and cancer. It is a member of a newly identified family of human ATPases with an uncharacterized mechanism of action. Here, we elucidate the molecular basis for inhibition and activation of MORC3. The crystal structure of the MORC3 region encompassing the ATPase and CW domains in complex with a nonhydrolyzable ATP analog demonstrates that the two domains are directly coupled. The extensive ATPase:CW interface stabilizes the protein fold but inhibits the catalytic activity of MORC3. Enzymatic, NMR, mutational, and biochemical analyses show that in the autoinhibited, off state, the CW domain sterically impedes binding of the ATPase domain to DNA, which in turn is required for the catalytic activity. MORC3 autoinhibition is released by disrupting the intramolecular ATPase:CW coupling through the competitive interaction of CW with histone H3 tail or by mutating the interfacial residues. Binding of CW to H3 leads to a marked rearrangement in the ATPase-CW cassette, which frees the DNA-binding site in active MORC3 (on state). We show that ATP-induced dimerization of the ATPase domain is strictly required for the catalytic activity and that the dimeric form of ATPase-CW might cooperatively bind to dsDNA. Together, our findings uncovered a mechanism underlying the fine-tuned regulation of the catalytic domain of MORC3 by the epigenetic reader, CW. [ABSTRACT FROM AUTHOR]

Abstract Background We have recently shown that human epididymis protein 4 (HE4) levels correlate with the severity of cystic fibrosis (CF) lung disease. However, there are no data on how HE4 levels alter in patients receiving CFTR modulating therapy. Methods In this retrospective clinical study, 3 independent CF patient cohorts (US-American: 29, Australian: 12 and Irish: 19 cases) were enrolled carrying at least one Class III CFTR CF-causing mutation (p.Gly551Asp) and being treated with CFTR potentiator ivacaftor. Plasma HE4 was measured by immunoassay before treatment (baseline) and 1–6 months after commencement of ivacaftor , and were correlated with FEV 1 (% predicted), sweat chloride, C-reactive protein (CRP) and body mass index (BMI). Results After 1 month of therapy, HE4 levels were significantly lower than at baseline and remained decreased up to 6 months. A significant inverse correlation between absolute and delta values of HE4 and FEV 1 (r = −0.5376; P <.001 and r = −0.3285; P <.001), was retrospectively observed in pooled groups, including an independent association of HE4 with FEV 1 by multiple regression analysis (β = −0.57, P =.019). Substantial area under the receiver operating characteristic curve (ROC-AUC) value was determined for HE4 when 7% mean change of FEV 1 (0.722 [95% CI 0.581–0.863]; P =.029) were used as classifier, especially in the first 2 months of treatment (0.806 [95% CI 0.665–0.947]; P <.001). Conclusions This study shows that plasma HE4 levels inversely correlate with lung function improvement in CF patients receiving ivacaftor. Overall, this potential biomarker may be of value for routine clinical and laboratory follow-up of CFTR modulating therapy. [ABSTRACT FROM AUTHOR]

Abstract Recombinant antibodies have emerged over the last few decades as the fastest growing class of therapeutic proteins for autoimmune diseases. Post-translation modifications of antibodies produced by human cell lines are highly consistent with those existing in natural human proteins and this is a major advantage of utilizing these cell lines. Cinorra is a biosimilar form of the antibody Adalimumab, which is an antagonist of TNF-α used for the treatment of autoimmune diseases. Adalimumab and Cinorra were produced by stable expression from CHO cells. The aim of this study was to select HEK cells as a host for producing Adalimumab to reveal whether the antibody produced by this human-derived cell line has similar characterization to Cinorra. Adalimumab was transiently produced in HEK-293T cells, characterized and analyzed for its properties. Circular dichroism spectroscopy confirmed a strong structural similarity of the expressed antibody with Cinorra. Likewise its binding activity and kinetic affinity to TNF-α (EC 50 = 416.5 ng/ml, KD = 3.89 E-10 M,) were highly similar to that of Cinorra (EC 50 = 421.2 ng/ml and KD = 3.34 E-10 M,). Additionally there was near identical neutralization of TNF-α-mediated cellular cytotoxicity (IC 50 of the expressed = 4.93 nM; IC 50 of Cinorra = 4.5 nM). Results indicate that Adalimumab produced by HEK-293T cells possesses a similarly efficient function and biological activity to Cinorra. Consequently, human-derived host cells with human post-translational modifications might potentially provide a basis for the development of Adalimumab with pharmaceutical properties for research and therapeutic use. Highlights • Cinorra is a new biosimillar form of the antibody Adalimumab developed using CHO cell lines. • Adalimumab produced by HEK-293T cells has a similar structure to Cinorra. • Adalimumab produced by HEK-293T cells has a similar binding activity and affinity to Cinorra. • Adalimumab produced by HEK-293T cells has analogous biological activity to Cinorra. • Human-derived cells with human PTMs are a preferred option for developing therapeutic antibodies. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is a chronic T cell-mediated autoimmune disease. Serum autoantibodies against cyclic citrullinated peptides (anti-CCP) are significant markers for diagnosis and prognosis of this disease. Induction of immune tolerance as therapeutic approach for RA constitutes a current research focal point. In this sense, we carried out a phase I clinical trial in RA patients with a new therapeutic candidate (called CIGB-814); which induced mechanisms associated with restoration of peripheral tolerance in preclinical studies. CIGB 814 is an altered peptide ligand (APL), derived from a CD4+ T cell epitope of human heat-shock protein 60 (HSP60), an autoantigen involved in the pathogenesis of RA. Twenty patients with moderate disease activity were included in this open label trial. Sequential dose-escalation of 1, 2.5 and 5 mg of CIGB-814 was studied. Consecutive groups of six, five, and nine patients received a subcutaneous dose weekly of the peptide during the first month and one dose monthly during the next 5 months. The peptide was well tolerated and reduced disease activity. Here, we reported the quantification of anti-CCP antibodies during the treatment with this APL and in the follow-up stage. Anti-CCP antibodies were quantified in the plasma from patients by a commercial enzyme immunoassay at baseline (T0) and at weeks 28 and 48. Results showed that CIGB-814 induced a significant reduction of anti-CCP antibodies. In addition, this decrease correlated with clinical improvement in patients assessed by Disease Activity Score in 28 joints (DAS28) criteria. These findings reinforce the therapeutic potential of CIGB-814. [ABSTRACT FROM AUTHOR]

Abstract Human Cripto-1 (Cripto-1), the founding member of the EGF-CFC superfamily, is a key regulator of many processes during embryonic development and oncogenesis. Cripto-1 is barely present or even absent in normal adult tissues while it is aberrantly re-expressed in various tumors. Blockade of the CFC domain-mediated Cripto-1 functions is acknowledged as a promising therapeutic intervention point to inhibit the tumorigenic activity of the protein. In this work, we report the generation and characterization of murine monoclonal antibodies raised against the synthetic folded CFC [112−150] domain of the human protein. Through subtractive ELISA assays clones were screened for the ability to specifically recognize "hot spot" residues on the CFC domain, which are crucial for the interaction with Activin Type I receptor (ALK4) and GRP78. On selected antibodies, SPR and epitope mapping studies have confirmed their specificity and have revealed that recognition occurs only on a conformational epitope. Furthermore, FACS analyses have confirmed the ability of 1B4 antibody to recognize the membrane-anchored and soluble native Cripto-1 protein in a panel of human cancer cells. Finally, we have evaluated its functional effects through in vitro cellular signaling assays and cell cycle analysis. These findings suggest that the selected anti-CFC mAbs have the potential to neutralize the protein oncogenic activity and may be used as theranostic molecules suitable as tumor homing agents for Cripto-1-overexpressing cancer cells and tissues and to overcome drug-resistance in routine cancer therapies. Highlights • New monoclonal antibodies that target Cripto-1 have been developed using the synthetic CFC domain. • Residues involved in antibody recognition have been identified. • The new mAbs bind the folded protein, block ALK4 receptor binding and are useful for protein detection in intact cells. • The new mAbs prevent ERK and c-Src activation in the Cripto-1-overexpressing C8161 melanoma cell line. [ABSTRACT FROM AUTHOR]

The nutritional regulation of protein and amino acid balance in human skeletal muscle carried out by the authors with Mike Rennie is reviewed in the context of a simple physiological model for the regulation of the maintenance and growth of skeletal muscle, the "Bag Theory". Beginning in London in the late 1970s the work has involved the use of stable isotopes to probe muscle protein synthesis and breakdown with two basic experimental models, primed‐dose continuous tracer infusions combined with muscle biopsies and arterio‐venous (A‐V) studies across a limb, most often the leg, allowing both protein synthesis and breakdown as well as net balance to be measured. In this way, over a 30 year period, the way in which amino acids and insulin mediate the anabolic effect of a meal has been elaborated in great detail confirming the original concepts of bag filling within the muscle endomysial "bag", which is limited by the "bag" size unless bag enlargement occurs requiring new collagen synthesis. Finally we briefly review some new developments involving 2H2O labelling of muscle proteins. Skeletal muscle intracellular protein is contained within an inelastic connective‐tissue framework, in effect collagen "bags", which define the myofibre volume, and the maximum protein content. A "bag full" signal to the two processes of protein synthesis (MPS), and protein breakdown (MPB) controls maximum protein content.In the fasted state protein is lost through a reduction in MPS below the rate of MPB, but food intake reverses this response by an insulin‐mediated inhibition of MPB and an essential amino acid, (EAA), mediated stimulation of MPS. This restores muscle protein to a "muscle full" level at which point the "bag full" signal switches off the anabolic response usually after 1.5‐2h. In a healthy adult the EAA stimulation of MPS shows a dose response from 2 to 20g of EAAs with the insulin inhibition of MPB maximal at low insulin levels (5 mU). The elderly exhibit anabolic blunting with reduced stimulation of MPS and reduced inhibition of MPB by feeding. Resistance exercise induces increased collagen synthesis and connective tissue remodellingallowing "bag" enlargement and increased muscle mass which is observed as an extended "bag full" point to 3‐4h after feeding. [ABSTRACT FROM AUTHOR]

Highlights • Salivary proteins participate in caries counteraction through several mechanisms. • Salivary proteins and peptides may be potential biomarkers for dental caries. • Bioactive peptides may augment anti-caries properties of native salivary proteins. • Functions of peptide analogues as anti-microbial and remineralization are described. Abstract Saliva contains a large number of proteins that play various crucial roles to maintain the oral health and tooth integrity. This oral fluid is proposed to be one of the most important host factors, serving as a special medium for monitoring aspects of microorganisms, diet and host susceptibility involved in the caries process. Extensive salivary proteomic and peptidomic studies have resulted in considerable advances in the field of biomarkers discovery for dental caries. These salivary biomarkers may be exploited for the prediction, diagnosis, prognosis and treatment of dental caries, many of which could also provide the potential templates for bioactive peptides used for the biomimetic management of dental caries, rather than repairing caries lesions with artificial materials. A comprehensive understanding of the biological function of salivary proteins as well as their derived biomimetic peptides with promising potential against dental caries has been long awaited. This review overviewed a collection of current literature and addressed the majority of different functions of salivary proteins and peptides with their potential as functional biomarkers for caries risk assessment and clinical prospects for the anti-caries application. [ABSTRACT FROM AUTHOR]