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Azole resistance in the pathogenic yeast Candida glabrata is a serious clinical complication and increasing in frequency. The majority of resistant organisms have been found to contain a substitution mutation in the Zn2Cys6 zinc cluster-containing transcription factor Pdr1. These mutations typically lead to this factor driving high, constitutive expression of target genes like the ATP-binding cassette transporter-encoding gene CDR1. Overexpression of Cdr1 is required for the observed elevated fluconazole resistance exhibited by strains containing one of these hyperactive PDR1 alleles. While the identity of hyperactive PDR1 alleles has been extensively documented, the mechanisms underlying how these gain-of-function (GOF) forms of Pdr1 lead to elevated target gene transcription are not well understood. We have used a tandem affinity purification-tagged form of Pdr1 to identify coactivator proteins that biochemically purify with the wild-type and 2 different GOF forms of Pdr1. Three coactivator proteins were found to associate with Pdr1: the SWI/SNF complex Snf2 chromatin remodeling protein and 2 different components of the SAGA complex, Spt7 and Ngg1. We found that deletion mutants lacking either SNF2 or SPT7 exhibited growth defects, even in the absence of fluconazole challenge. To overcome these issues, we employed a conditional degradation system to acutely deplete these coactivators and determined that loss of either coactivator complex, SWI/SNF or SAGA, caused defects in Pdr1-dependent transcription. A double degron strain that could be depleted for both SWI/SNF and SAGA exhibited a profound defect in PDR1 autoregulation, revealing that these complexes work together to ensure high-level Pdr1-dependent gene transcription. [ABSTRACT FROM AUTHOR]

Introduction: Endotoxin or lipopolysaccharide (LPS) is one of the components of the wall of gram-negative bacteria, which, when in contact with blood, stimulates the immune system and causes fever and even serious adverse effects or death. Removing endotoxin is one of the most daunting challenges presented to the purification process in the production of recombinant biological drugs. Bacterial endotoxin (LPS) is resistant to heat and passes through sterilizing filters, and due to its dangerous side effects in living organisms, part of the target protein purification process is always related to removing this disturbing factor from the product. Materials & Methods: The affinity matrix S3E3-S-Sepharose, which was produced by immobilizing the improved variant of antimicrobial peptide S3 called S3E3 peptide on Sepharose chromatography resin, was used to remove endotoxin from active pharmaceutical ingredient (API) of streptokinase, and the performance of this matrix was compared with the performance of a disposable commercial matrix (containing S3 peptide as its ligand) and with ion exchange chromatography resin. Results: The statistical analysis of the results revealed that compared to commercial S3 matrices and ion exchange chromatography, the S3E3-S-Sepharose matrix had higher protein recovery (84.07% compared to 81.50 and 75.31%, respectively) and higher streptokinase biological activity recovery (81.95% vs. 27 76.76 and 61.54%, respectively). Conclusion: As evidenced by the obtained results, the S3E3-S-Sepharose matrix seems to be a suitable candidate for use in the purification processes of Streptokinase API and other recombinant biopharmaceuticals. [ABSTRACT FROM AUTHOR]

Background: Radiolabeled antibody fragments present a promising opportunity as theranostic agents, offering distinct advantages over whole antibodies. In this study, the authors investigate the potential of [177Lu]Lu-DTPA-F(ab′)2-pertuzumab as a theranostic agent for precise targeting of human epidermal growth factor receptor 2 (HER2)-positive cancers. Additionally, the authors aim to quantitatively assess the binding synergism in the presence of cold trastuzumab. Materials and Methods: F(ab′)2-pertuzumab was prepared by pepsin digestion and conjugated with a bifunctional chelator. The immunoconjugate was radiolabeled with 177Lu and characterized by chromatography techniques. Binding parameters (affinity, specificity, and immunoreactivity) and cellular binding enhancement studies were evaluated in HER2-overexpressing and triple-negative cell lines. The in vivo enhancement in tumor uptake of the radiolabeled immunoformulation was assessed in severe combined immunodeficient (SCID) mice bearing tumors, both in the presence and absence of unlabeled trastuzumab. Results: The formulation of [177Lu]Lu-DTPA-F(ab′)2-pertuzumab could be prepared in high yields and with consistent radiochemical purity, ensuring reproducibility. Comprehensive in vitro and in vivo evaluation studies confirmed high specificity and immunoreactivity of the formulation toward HER2 receptors. Binding synergism of radiolabeled pertuzumab fragments in the presence of trastuzumab to HER2 receptors was observed. Conclusions: The radioformulation of [177Lu]Lu-DTPA-F(ab′)2-pertuzumab holds great promise as a targeted approach for addressing HER2-positive cancers. A potentially effective strategy to amplify therapeutic efficacy involves dual epitope targeting by combining radiolabeled pertuzumab with cold trastuzumab. [ABSTRACT FROM AUTHOR]

Elucidation of protein function is one of the central issues in the field of life sciences. To study the function of proteins not in isolation, but in a cell or its lysate, thus, it is necessary to selectively label the target protein in a mixture. Affinity labeling is one of several widely used methods for selective labeling; however, this method has the disadvantage that the labeling reagent is always activated, albeit weakly. Therefore, fine‐tuning of the reactivity and/or reaction conditions is generally required for successful target‐selective labeling. We previously developed a new affinity labeling reagent with N‐sulfanylethylanilide (SEAlide) as a key reactive unit. It was designed based on the following hypotheses. SEAlide is less reactive and does not label in the absence of a target protein. Upon target binding, amino acid side‐chain functional groups on the target surface convert SEAlide into a thioester form via N–S acyl transfer, allowing the target to be labeled. However, no evidence has been obtained so far to directly prove the hypothesis. In this study, we examine whether amino acid side‐chain functional groups can activate SEAlide from the viewpoint of theoretical chemistry. The theoretical studies show that the activation free energy and enthalpy of the acyl transfer of SEAlide are reduced in the presence of methylammonium, which is a model for the protonated side chain of Lys, and acetate, which is a model for the deprotonated side chain of Asp/Glu. It suggests that Lys and Asp/Glu side chains could potentially stabilize the activation transition states to accelerate the thioester formation. Furthermore, the significant decrease in the activation enthalpy indicates that the contribution of entropy to the transition state is large. This result supports the original hypothesis that the SEAlide‐based labeling reagent is efficiently activated by binding to the target protein. [ABSTRACT FROM AUTHOR]

Most of the breast cancers are estrogen receptor-positive recurring with a steady rate of up to 20 years dysregulating the normal cell cycle. Dinaciclib is still in clinical trials and considered as a research drug against such cancers targeting CDK2. The major goal of this study was to identify the potential inhibitors of CDK-2 present in Moringa oleifera for treating hormonal receptor positive breast cancers. For this purpose, in silico techniques; molecular docking, MM-GBSA and molecular dynamics simulations were employed to screen Moringa oleifera compounds and their anticancer potential was determined against CDK-2 protein targets. Among 36 compounds of Moringa oleifera reported in literature, chlorogenic acid (1), quercetin (2), ellagic acid (3), niazirin (4), and kaempferol (5) showed good affinity with the target. The interaction of the compounds was visualized using PYMOL software. The profiles of absorption, distribution, metabolism, excretion (ADME) and toxicity were determined using SWISS and ProTox II webservers. The MTT assay was performed in-vitro using MCF-7 cancer cell lines to validate the anticancer potential of Moringa oleifera leaf extract. MTT assay results revealed no significant change in proliferation of Mcf-7 cells following 24 h treatment with fraction A (petroleum ether). However, significant antiproliferative effect was observed at 200 µg/mL dose of fraction B (ethyl acetate) and cell viability was reduced to 40%. In conclusion, the data suggested that all the compounds with highest negative docking score than the reference could be the potential candidates for cyclin dependent kinase-2 (CDK-2) inhibition while ellagic acid, chlorogenic acid and quercetin being the most stable and potent inhibitors to treat estrogen receptor positive breast cancer targeting CDK-2. Moreover, the data suggested that further investigation is required to determine the optimum dose for significant antiproliferative effects using in-vivo models to validate our findings of in-silico analysis. [ABSTRACT FROM AUTHOR]

Simple Summary: The development of malignant pleural mesothelioma, a rare and often aggressive cancer associated with asbestos exposure, can take decades to develop. The existing methods for diagnosis are insufficient, hence, better detection methods are required. Given that pleural effusions are close to the tumor and reasonably accessible, it is believed that pleural effusion contains biomarkers that can provide insight into the disease. As part of our explorative analysis of pleural effusion, we applied a novel mass spectrometry-based method, and as a result, we have identified several proteins with diagnostic potential as markers for malignant pleural mesothelioma. The research into the potential use of pleural effusion biomarkers as a viable future diagnostic tool for malignant pleural mesothelioma will be advanced with the addition of the knowledge gained from our study. Malignant pleural mesothelioma (MPM) is an asbestos-associated, highly aggressive cancer characterized by late-stage diagnosis and poor prognosis. Gold standards for diagnosis are pleural biopsy and cytology of pleural effusion (PE), both of which are limited by low sensitivity and markedly inter-observer variations. Therefore, the assessment of PE biomarkers is considered a viable and objective diagnostic tool for MPM diagnosis. We applied a novel affinity-enrichment mass spectrometry-based proteomics method for explorative analysis of pleural effusions from a prospective cohort of 84 patients referred for thoracoscopy due to clinical suspicion of MPM. Protein biomarkers with a high capability to discriminate MPM from non-MPM patients were identified, and a Random Forest algorithm was applied for building classification models. Immunohistology of pleural biopsies confirmed MPM in 40 patients and ruled out MPM in 44 patients. Proteomic analysis of pleural effusions identified panels of proteins with excellent diagnostic properties (90–100% sensitivities, 89–98% specificities, and AUC 0.97–0.99) depending on the specific protein combination. Diagnostic proteins associated with cancer growth included galactin-3 binding protein, testican-2, haptoglobin, Beta ig-h3, and protein AMBP. Moreover, we also confirmed previously reported diagnostic accuracies of the MPM markers fibulin-3 and mesothelin measured by two complementary mass spectrometry-based methods. In conclusion, a novel affinity-enrichment mass spectrometry-based proteomics identified panels of proteins in pleural effusion with extraordinary diagnostic accuracies, which are described here for the first time as biomarkers for MPM. [ABSTRACT FROM AUTHOR]

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The Qinglongyi-Buguzhi herbal pair (QB) is one of commonly used herbal combinations for treating vitiligo in traditional Chinese medicine, consisting of the exocarp of the immature fruit of Juglans regia L. or Juglans mandshurica Maxim., and dried, mature fruit of Psoralea corylifolia L. However, the active components and potential mechanisms of QB in the treatment of vitiligo are still unclear. The purpose of this study is to clarify the effects and mechanisms of QB on vitiligo treatment through integration of network pharmacology and empirical examinations. The active components and targets of QB as well as the targets linked to vitiligo were obtained from network databases. Visualization networks were constructed with Cytoscape 3.9.1. GO and KEGG enrichment analysis were conducted to investigate the possible mechanism. Molecular docking was employed to evaluate the binding affinities between the primary active ingredients of QB and essential targets of the PI3K/Akt/Nrf2 pathway. In vivo and in vitro experiments were carried out to confirm the results of network pharmacology. We evaluated 44 active compounds and 602 genes from QB, and 107 of these genes linked to vitiligo. GO analysis suggested QB might lessen vitiligo by regulating oxidative stress. KEGG pathway analysis indicated the PI3K/Akt pathway may be crucial for treating vitiligo. Molecular docking results demonstrated the key active ingredients of QB had good binding activity with the major targets in the PI3K/Akt/Nrf2 pathway. In vivo, QB significantly ameliorated vitiligo model mouse's skin pathologies by reducing ROS, elevating CAT and SOD levels. Western blot showed that QB increased the phosphorylation of PI3K and Akt and the expressions of Nrf2 and HO-1 in the skin. In vitro, QB reversed H 2 O 2 -induced oxidative injury of melanocytes, enhanced cell survival rate, reduced ROS level, upregulated SOD and CAT activities, and raised the content of melanin. Moreover, QB upregulated the expression levels of Akt, Nrf2, HO-1 mRNA, Akt phosphorylation, HO-1, and nuclear Nrf2 proteins, and also encouraged the nuclear translocation of Nrf2. However, LY294002 treatment significantly reversed the regulatory effect of QB on oxidative damage of melanocytes. This study revealed that the therapeutic effect of QB on vitiligo was achieved through multiple components, targets and pathways. Experimental investigation demonstrated that QB could improve vitiligo via reducing oxidative stress, which was probably accomplished by activating the PI3K/Akt/Nrf2 signaling pathway. [Display omitted] • QB is safe and effective in the treatment of vitiligo. • The mechanisms of QB in the treatment of vitiligo are explored by the combination of network pharmacology and in vivo and in vitro experiments. • QB may play a therapeutic role in vitiligo by activating PI3K/Akt/Nrf2 signaling pathway to reduce oxidative stress. [ABSTRACT FROM AUTHOR]

Recent advances in the field of translational chemical biology use diverse "proximity-inducing" synthetic modalities to elicit new modes of "event driven" pharmacology. These include mechanisms of targeted protein degradation and immune clearance of pathogenic cells. Heterobifunctional "chimeric" compounds like Proteolysis TArgeting Chimeras (PROTACs) and Antibody Recruiting Molecules (ARMs) leverage these mechanisms, respectively. Both systems function through the formation of reversible "ternary" or higher-order biomolecular complexes. Critical to function are key parameters, such as bifunctional molecule affinity for endogenous proteins, target residence time, and turnover. To probe the mechanism and enhance function, covalent chemical approaches have been developed to kinetically stabilize ternary complexes. These include electrophilic PROTACs and Covalent Immune Recruiters (CIRs), the latter designed to uniquely enforce cell-cell induced proximity. Inducing cell-cell proximity is associated with key challenges arising from a combination of steric and/or mechanical based destabilizing forces on the ternary complex. These factors can attenuate the formation of ternary complexes driven by high affinity bifunctional/proximity inducing molecules. This Account describes initial efforts in our lab to address these challenges using the CIR strategy in antibody recruitment or receptor engineered T cell model systems of cell-cell induced proximity. ARMs form ternary complexes with serum antibodies and surface protein antigens on tumor cells that subsequently engage immune cells via Fc receptors. Binding and clustering of Fc receptors trigger immune cell killing of the tumor cell. We applied the CIR strategy to convert ARMs to covalent chimeras, which "irreversibly" recruit serum antibodies to tumor cells. These covalent chimeras leverage electrophile preorganization and kinetic effective molarity to achieve fast and selective covalent engagement of the target ternary complex protein, e.g., serum antibody. Importantly, covalent engagement can proceed via diverse binding site amino acids beyond cysteine. Covalent chimeras demonstrated striking functional enhancements compared to noncovalent ARM analogs in functional immune assays. We revealed this enhancement was in fact due to the increased kinetic stability and not concentration, of ternary complexes. This finding was recapitulated using analogous CIR modalities that integrate peptidic or carbohydrate binding ligands with Sulfur(VI) Fluoride Exchange (SuFEx) electrophiles to induce cell-cell proximity. Mechanistic studies in a distinct model system that uses T cells engineered with receptors that recognize covalent chimeras or ARMs, revealed covalent receptor engagement uniquely enforces downstream activation signaling. Finally, this Account discusses potential challenges and future directions for adapting and optimizing covalent chimeric/bifunctional molecules for diverse applications in cell-cell induced proximity.

The Potentilla genus has long been used traditionally as food and a folklore medicine. In the present study, aerial parts of two Potentilla species, Potentilla fulgens and Potentilla atrosanguinea , of western Himalayan origin, were studied for their anti-breast cancer activity. Ethyl acetate (PAA-EA, PFA-EA), methanolic (PAA-ME, PFA-ME) and hydro-methanolic extract (PAA-HM, PFA-HM) of the plants were tested for their antiproliferative activities against MCF-7 and T-47D breast cancer cell lines. The extracts showed good antiproliferative activity against ER- α dominant breast cancer cell line T-47D, having IC 50 values 6.19 ± 0.01 to 33.23 ± 0.04 μg/ml. Eight compounds were isolated, characterized, and quantified from ethyl acetate and methanolic extracts by column chromatography, 1D, 2D-NMR, HRMS and TLC densitometric analysis. Two compounds (4 and 6) have shown better antiproliferative activity than standard bazedoxifene and were further evaluated for their ER- α binding affinity via—fluorescence polarization-based competitive binding assay. The antiestrogenic properties of both compounds were assessed using western blotting. Compounds 4 and 6 were found to have significant affinity for the ER- α and managed to decrease its expression by 38 and 54% respectively. Compounds 4 and 6 also had good stability and reactivity as measured by minimal fluctuations in molecular dynamic simulation analysis, a good dock score in molecular docking, and a respectable HOMO-LUMO energy gap in DFT calculations. Compounds 4 and 6 have shown reliable results and can be used in the development of natural product-based anti-breast cancer agents. [Display omitted] • Natural products are source of leads in cancer drug discovery. • P. atrosanguinea and P. fulgens are traditionally being used as food in India were studied for their anti-breast cancer potential. • Eight compounds were isolated and evaluated for their antiproliferative activity alongwith quantification by HPTLC. • Compounds 4 and 6 were found to possess significant antiproliferative activity, ER-α binding affinity, and anti-estrogenic activity. • These compounds have shown dynamic stability In silico via. DFT and MD simulation. [ABSTRACT FROM AUTHOR]

Kaempferol (KP), as a natural anti-inflammatory compound, has been reported to have curative effects on alleviating senile osteoporosis (SOP), which is an inflammation-related musculoskeletal disease, but the molecular mechanisms remain unclear due to scanty relevant studies. We predicted the targets of KP and SOP, and the common targets of them were subsequently used to carry out PPI analysis. Moreover, we adopted GO and KEGG enrichment analysis and molecular docking to explore potential mechanisms of KP against SOP. There were totally 152 KP-related targets and 978 SOP-related targets, and their overlapped targets comprised 68 intersection targets. GO enrichment analysis showed 1529 biological processes (p < 0.05), which involved regulation of inflammatory response, oxidative stress, regulation of bone resorption and remodeling, osteoblast and osteoclast differentiation, etc. Moreover, KEGG analysis revealed 146 items including 44 signaling pathways (p < 0.05), which were closely linked to TNF, IL-17, NF-kappa B, PI3K-Akt, MAPK, estrogen, p53, prolactin, VEGF, and HIF-1 signaling pathways. By means of molecular docking, we found that kaempferol is bound with the key targets' active pockets through some connections such as hydrogen bond, pi-alkyl, pi-sigma, pi-pi Stacked, pi-pi T-shaped, and van der Waals, illustrating that kaempferol has close combination with the key targets. Collectively, various targets and pathways involve in the process of kaempferol treatment against SOP through regulating inflammatory response, oxidative stress, bone homeostasis, etc. Moreover, our study first reported that kaempferol may regulate core targets' expression with involvement of inflammatory response, oxidative stress, and bone homeostasis, thus treating SOP. [ABSTRACT FROM AUTHOR]

Here we explored the reactivity of a set of multivalent electrophiles cofunctionalized with a carbohydrate ligand on gold nanoparticles to achieve efficient affinity labeling for target protein analysis. Evaluation of the reactivity and selectivity of the electrophiles against three different cognate binding proteins identified arylsulfonyl fluoride as the most efficient protein‐reactive group in this study. We demonstrated that multivalent arylsulfonyl fluoride probe 4 at 50 nm concentration achieved selective affinity labeling and enrichment of a model protein PNA in cell lysate, which was more effective than photoaffinity probe 1 with arylazide group. Labeling site analysis by LC–MS/MS revealed that the nanoparticle‐immobilized arylsulfonyl fluoride group can target multiple amino acid residues around the ligand binding site of the target proteins. Our study highlights the utility of arylsulfonyl fluoride as a highly effective multivalent affinity label suitable for covalently capturing unknown target proteins. [ABSTRACT FROM AUTHOR]

Weakly Supervised Semantic Segmentation (WSSS) techniques explore individual regularization strategies to refine Class Activation Maps (CAMs). In this work, we first analyze complementary WSSS techniques in the literature, their segmentation properties, and the conditions in which they are most effective. Based on these findings, we devise two new techniques: P-NOC and C 2 AM-H. In the first, we promote the conjoint training of two adversarial CAM generating networks: the generator, which progressively learns to erase regions containing class-specific features, and a discriminator, which is refined to gradually shift its attention to new class discriminant features. In the latter, we employ the high quality pseudo-segmentation priors produced by P-NOC to guide the learning to saliency information in a weakly supervised fashion. Finally, we employ both pseudo-segmentation priors and pseudo-saliency proposals in the random walk procedure, resulting in higher quality pseudo-semantic segmentation masks, and competitive results with the state of the art. • Complementary techniques improve segmentation effectiveness in WSSS tasks. • GAN-style training can improve Class-Specific Adversarial Erasing (CSE) methods. • Foreground hints devised from CAMs regularize contrastive WSSS methods. • Adding saliency information to affinity labels decreases uncertainty. [ABSTRACT FROM AUTHOR]

Introduction: According to the statistics, vascular injury occurs during the onset of diabetic changes after the production of several byproducts. Many authorities have focused to find an alternative therapy for diabetic patients. In this study, we investigated the therapeutic effects of natural polyphenol like resveratrol on human endothelial cells exposed to malondialdehyde for 48 hours. Methods: Human Umbilical Vein Endothelial Cells were randomly classified into four groups;control, malondialdehyde (2.5 mM), resveratrol (100 μM), and cells received the combined regime for 48 hours. Cell viability was determined by 3-(4, 5-dimethyl thiazol-2-yl) 2, 5-diphenyl-tetrazoliumbromide (MTT) assay. Griess reaction was performed to measure the content of Nitric oxide (NO). Apoptosis was studied by using real-time polymerase chain reaction (RT-PCR) and western blotting assays. Levels of receptor tyrosine kinases like VEGFR-1, -2, Tie-1, and -2 were analyzed by enzyme-linked immunosorbent assay(ELISA). The affinity of resveratrol and malondialdehyde to serum albumin was measured by Surface Plasmon Resonance Assay. Any changes in chromatin remodeling were detected by PCR array analysis. Results: Resveratrol reduced cytotoxicity and NO content inside cells induced by malondialdehyde(MDA) (P < 0.05). Endothelial cell apoptosis was decreased by the reduction of pro-apoptotic factor Bax and increase of Bcl-2 following the incubation with resveratrol (P < 0.05). MDA-induced receptor tyrosine kinases increase was inhibited by resveratrol and reached near-to-normal levels (P < 0.05). Surface Plasmon Resonance revealed a higher affinity of resveratrol to albumin compared to the malondialdehyde-albumin complex. Polymerase chain reaction (PCR) array revealed the potency of resveratrol in chromatin remodeling following the treatment with malondialdehyde (P < 0.05). Conclusion: Based on our findings, resveratrol has the potential to decrease diabetic vascular injury induced by lipid byproducts such as MDA. [ABSTRACT FROM AUTHOR]

Use of a codebook to categorize meaning units is a well-known research strategy in qualitative inquiry. However, methodology for the creation of a codebook in practice is not standardized, and specific guidance for codebook ideation is sometimes unclear, especially to novice qualitative researchers. This article describes the procedure that was utilized to create a codebook, which adapted an affinity diagram methodology (Scupin, 1997), an approach used in user-centered design. For this research, affinity diagramming was applied to a method outlined by Kurasaki (2000) for codebook ideation. Annotations of a subset of military veterans’ transcripts were utilized in congruence with affinity diagramming to create a codebook to categorize the phenomenon of reintegration into the civilian community after service (Haskins Lisle, 2017). This method could be useful in exploratory research that utilizes a codebook generated in vivo from annotations, especially for novice researchers who are overwhelmed by the codebook creation phase. [ABSTRACT FROM AUTHOR]

Structural proteomics refers to large‐scale mapping of protein structures in order to understand the relationship between protein sequence, structure, and function. Chemical labeling, in combination with mass‐spectrometry (MS) analysis, have emerged as powerful tools to enable a broad range of biological applications in structural proteomics. The key to success is a biocompatible reagent that modifies a protein without affecting its high‐order structure. Fluorine, well‐known to exert profound effects on the physical and chemical properties of reagents, should have an impact on structural proteomics. In this Minireview, we describe several fluorine‐containing reagents that can be applied in structural proteomics. We organize their applications around four MS‐based techniques: a) affinity labeling, b) activity‐based protein profiling (ABPP), c) protein footprinting, and d) protein cross‐linking. Our aim is to provide an overview of the research, development, and application of fluorine‐containing reagents in protein structural studies. [ABSTRACT FROM AUTHOR]

Very accurate electron affinity (EA) calculations of 6Li and 7Li (and ∞Li) have been performed using explicitly correlated Gaussian functions and a variational approach that explicitly includes the nuclear motion in the calculations (i.e., the approach that does not assume the Born–Oppenheimer approximation). The leading relativistic and quantum electrodynamics corrections to the electron affinities were also calculated. The results are the most accurate theoretical values obtained for the studied systems to date. Our best estimates of the 7Li and 6Li EAs are 4984.9842(30) and 4984.9015(30) cm-1, respectively, and of the 7Li/6Li EA isotope shift is 0.0827 cm-1. [ABSTRACT FROM AUTHOR]

In this data article, we described the synthetic routes to four biotinylated probes (2, 3, 4, and 5) of artemisinin and the associated experimental procedures. We also provided the physical data for the synthesized compounds. These synthesized biotinylated probes of artemisinin are useful molecular tools for the affinity-labeling study of target receptor proteins of artemisinin in tropical pathogens such as Trypanosoma, Leishmania, and Schistosoma. The data provided herein are related to “Biotinylated probes of artemisinin with labeling affinity toward Trypanosoma brucei brucei target proteins”, by Konziase (Anal. Biochem. (2015)).

Abstract As cardiovascular diseases (CVD) are responsible for millions of deaths annually, there is a need for rapid and sensitive diagnosis of CVD at earlier stages. Aptamers generated by systematic evolution of ligands by exponential enrichment (SELEX) processes have been shown to be superior to conventional antibody-based cardiac biomarker detection. However, SELEX is a complicated, lengthy procedure requiring multiple rounds of extraction/amplification and well-trained personnel. To circumvent such issue, we designed an automated, miniaturized SELEX platform for the screening of aptamers towards three protein biomarkers associated with CVDs: N-terminal pro-peptide of B-type natriuretic peptide, human cardiac troponin I, and fibrinogen. The developed microfluidic platform was equipped with microfluidic devices capable of sample transport and mixing along with an on-chip nucleic acid amplification module such that the entire screening process (5 rounds of selection in 8 h.) could be performed consecutively on a single chip while consuming only 35 µL of reagents in each cycle. This system may therefore serve as a promising, sensitive, cost-effective platform for the selection of aptamers specific for CVD biomarkers. Highlights • An automated, miniaturized microfluidic platform for screening of aptamers towards three protein biomarkers. • Biomarkers related to cardiovasculart disease namely N-terminal propeptide of B-type natriuretic peptide, human cardiac troponin I, and fibrinogen were used. • The entire screening process (5 rounds of selection in 8 h) could be performed continuously on a single chip while consuming only 35 µL of reagents. [ABSTRACT FROM AUTHOR]

Fc γ receptors (FcγRs) bind circulating IgG (IgG1) at the surface of leukocytes. Antibodies clustered at the surface of a targeted particle trigger a protective immune response through activating FcγRs. Three recent reports indicate that the composition of the asparagine-linked carbohydrate chains (N-glycans) of FcγRIIIa/CD16a impacted IgG1-binding affinity. Here we determined how N-glycan composition affected the affinity of the “low-affinity" FcγRs for six homogeneous IgG1 Fc N-glycoforms (G0, G0F, G2, G2F, A2G2, and A2G2F). Surprisingly, CD16a with oligomannose N-glycans bound to IgG1 Fc (A2G2) with a KD = 1.0 ± 0.1 nM. This affinity represents a 51-fold increase over the affinity measured for CD16a with complextypeN- glycans (51±8nM) and is comparable with the affinity of FcγRI/CD64, the sole “high-affinity" FcγR. CD16a N-glycan composition accounted for increases in binding affinity for the other IgG1 Fc glycoforms tested (10 -50-fold). This remarkable sensitivity could only be eliminated by preventing glycosylation at Asn162 with an Asn-to-Gln mutation; mutations at the four other N-glycosylation sites preserved tighter binding in the Man5 glycoform. None of the other low-affinity FcγRs showed more than a 3.1-fold increase upon modifying the receptor N-glycan composition, including CD16b, which differs from CD16a by only four amino acid residues. This result indicates that CD16a is unique among the low-affinity FcγRs, and modifying only the glycan composition of both the IgG1 Fc ligand and receptor provides a 400-fold range in affinities. [ABSTRACT FROM AUTHOR]

Genetically site-specific introduction of noncanonical amino acids (ncAAs) for protein conjugation generally requires incorporation through exogenous feeding of chemically synthesized ncAAs. We developed a p-amino-phenylalanine (pAF)-based relay system that enables site-specific functionalization of proteins without chemical synthesis of the building blocks. pAF was biosynthesized under optimized conditions, followed by site-specific incorporation into a specific protein residue. The resulting protein was ready for functionalization using an oxidative conjugation reaction. We demonstrated the use of this relay system by preparing a fluorophore-labeled anti-HER2 single-chain variable fragment antibody for fluorescent imaging. [ABSTRACT FROM AUTHOR]

Chemoselective and affinity-based probes of steviol were applied to the discovery of glycosyltransferase activity and corresponding UDP-glycosyltransferase in Arabidopsis thaliana, which illustrates a simple strategy for rapidly harnessing minable novel biological parts from plants for synthetic biology. [ABSTRACT FROM AUTHOR]

Background. The diagnosis of von Willebrand disease is complex due to the heterogeneity of the disease. About eighty percent of von Willebrand disease patients are diagnosed with a quantitative defect of von Willebrand factor (VWF) where fifty percent is due to an increased clearance of von Willebrand factor. These patients do not respond well to the treatment of choice, Desmopressin (DDAVP) due to decreased efficacy. The ratio between the VWF propeptide and the mature VWF antigen is used to diagnose these patients. Commercial VWF propeptide assays are too expensive for use in developing countries. In this study, we developed a cost-effective ELISA assay. Methods. We first displayed VWF propeptide on yeast. Antibody fragments were selected against the displayed VWF propeptide by using phage display technology. The antibodies were used to develop a cost-effective VWF propeptide assay and compared to a commercial VWF propeptide assay. Results. Two of these antibody fragments bound specific to the VWF propeptide and not to the yeast used for the expression of the propeptides. These purified antibody fragments were able to detect VWF propeptide in normal plasma. Conclusion. Our assay performed well when compared to a commercial kit. It also showed a higher binding affinity for VWF propeptide in plasma at especially lower plasma concentrations. [ABSTRACT FROM AUTHOR]

Background: Several circumstances require the accurate measurement of red blood cell (RBC) survival and clearance, such as determination of posttransfusion recovery of stored RBCs to investigate the effect of new additive solutions. To this end, biotin as a marker of RBCs to track donor RBCs in the blood of the recipient has been used in many studies. However, so far only experimental, nonvalidated, biotin-labeled red cell concentrates (RCCs) are transfused. The goal of this study was to produce a standardized biotin-labeled RCC product in a fast, simple, and sterile manner that can be used for clinical research and for the evaluation of new blood products according to Good Practice Guidelines (GPG) for blood establishments.Study Design and Methods: RCC fractions were labeled with two different concentrations of biotinylation reagent in a closed system, to prevent bacterial contamination of the end product. Using flow cytometry, the reproducibility and robustness of the biotin labeling was assessed, as well as the stability of the biotin label on the (un-)irradiated RCC fraction. Additionally, parameters such as phosphatidylserine (PS) exposure, sodium (Na), potassium (K), free hemoglobin, adenosine triphosphate (ATP), pH, and morphology were determined prior to and after biotin labeling to rule out detrimental effects of the labeling procedure on the RCC.Results: Our data show that RCCs can be labeled under sterile conditions in a closed system with two different biotinylation reagent concentrations, without affecting the biological activity.Conclusion: An easy, rapid (<2 hr), and robust method was developed to manufacture biotin-labeled RCCs for clinical research compliant to GPG. [ABSTRACT FROM AUTHOR]

Few studies have investigated the extractable organic nitrogen (EON) formation mechanisms, and the sources of EON have long been debated. Using 15 N labeling, we performed a 120-day laboratory incubation experiment to explore the dynamic contributions of different types of added N (ammonium-N, ryegrass-N and their combination) to soil EON and the role that microorganisms play in N transformation into EON. We show that the 15 N abundances and recoveries in soil EON pool were relatively low during the incubation, except the first hours after ryegrass addition in 15 N-ryegrass addition treatments. In general, most of the EON during the incubation was soil derived, and both ammonium-N (80 mg kg −1 ) and ryegrass-N (160 mg kg −1 ) additions made minor contributions (3–4% and 8–13% during day 1–120) to the soil EON pool. Moreover, along with the decline in 15 N recoveries in microbial biomass nitrogen (MBN) pool, the lost MB 15 N did not enter into the EO 15 N pool. Our study demonstrates 1) that EON is a stable N pool in agricultural soil and is less affected by exogenous N addition and 2) that microbial N uptake and release processes contribute little to the soil EON pool. [ABSTRACT FROM AUTHOR]

Endocrine disrupting effect has become a central point of concern, and various biological mechanisms involve in the disruption of endocrine system. Recently, we have explored the mechanism of disrupting hormonal transport protein, through the binding affinity of sex hormone-binding globulin in different fish species. This study, serving as a companion article, focused on the mechanism of activating/inhibiting hormone receptor, by investigating the binding interaction of chemicals with the estrogen receptor (ER) of different fish species. We collected the relative binding affinity ( RBA ) of chemicals with 17β-estradiol binding to the ER of eight fish species. With this parameter as the endpoints, quantitative structure-activity relationship (QSAR) models were established using DRAGON descriptors. Statistical results indicated that the developed models had satisfactory goodness of fit, robustness and predictive ability. The Euclidean distance and Williams plot verified that these models had wide application domains, which covered a large number of structurally diverse chemicals. Based on the screened descriptors, we proposed an appropriate mechanism interpretation for the binding potency. Additionally, even though the same chemical had different affinities for ER from different fish species, the affinity of ER exhibited a high correlation for fish species within the same Order (i.e., Salmoniformes, Cypriniformes, Perciformes), which consistent with that in our previous study. Hence, when performing the endocrine disrupting effect assessment, the species diversity should be taken into account, but maybe the fish species in the same Order can be grouped together. [ABSTRACT FROM AUTHOR]

Oligohistidine affinity tags (His-tags) are commonly fused to proteins to aid in their purification via metal affinity chromatography. These His-tags are generally assumed to have minimal impact on the properties of the fusion protein, as they have no propensity to form ordered elements, and are small enough not to significantly affect the solubility or size. Here we report structures of two variants of truncated green fluorescent protein (GFP), i.e., split GFP with a β-strand removed, that were found to behave differently in the presence of light. In these structures, the N-terminal His-tag and several neighboring residues play a highly unusual structural and functional role in stabilizing the truncated GFP by substituting as a surrogate β-strand in the groove vacated by the native strand. This finding provides an explanation for the seemingly very different peptide binding and photodissociation properties of split proteins involving β-strands 10 and 11. We show that these truncated GFPs can bind other non-native sequences, and this promiscuity invites the possibility for rational design of sequences optimized for strand binding and photodissociation, both useful for optogenetic applications. [ABSTRACT FROM AUTHOR]

Life scientists often desire to display the signal from two different molecular probes as a single colour image, so as to convey information about the probes' relative concentrations as well as their spatial corelationship. Traditionally, such colour images are created through a merge display, where each greyscale signal is assigned to different channels of an RGB colour image. However, human perception of colour and greyscale intensity is not equivalent. Thus, a merged image display conveys to the typical viewer only a subset of the absolute and relative intensity information present in and between two greyscale images. The Commission Internationale de l'Eclairage L*a*b* colour space (CIELAB) has been designed to specify colours according to the perceptually defined quantities of hue (perceived colour) and luminosity (perceived brightness). Here, we use the CIELAB colour space to encode two dimensions of information about two greyscale images within these two perceptual dimensions of a single colour image. We term our method a Perceptually Uniform Projection display and show using biological image examples how these displays convey more information about two greyscale signals than comparable RGB colour space-based techniques. [ABSTRACT FROM AUTHOR]

Combination of biophysical and structural techniques allowed characterizing and uncovering the mechanisms underlying increased binding affinity of lactosamine derivatives for galectin 3. In particular, complementing information gathered from X-ray crystallography, native mass spectrometry and isothermal microcalorimetry showed favorable enthalpic contribution of cation-π interaction between lactosamine aryl substitutions and arginine residues from the carbohydrate recognition domain, which resulted in two log increase in compound binding affinity. This incrementing strategy allowed individual contribution of galectin inhibitor moieties to be dissected. Altogether, our results suggest that core and substituents of these saccharide-based inhibitors can be optimized separately, providing valuable tools to study the role of galectins in diseases. [ABSTRACT FROM AUTHOR]

Protein-protein interactions (PPIs) trigger a wide range of biological signaling pathways that are crucial for biomedical research and drug discovery. Various techniques have been used to study specific proteins, including affinity chromatography, activity-based probes, affinity-based probes and photo-affinity labeling (PAL). PAL has become one of the most powerful strategies to study PPIs. Traditional photocrosslinkers are used in PAL, including benzophenone, aryl azide and diazirine. Upon photoirradiation, these photocrosslinkers (Pls) generate highly reactive species that react with adjacent molecules, resulting in a direct covalent modification. This review introduces recent examples of chemical proteomics study using PAL for PPIs. [ABSTRACT FROM AUTHOR]

Background: Ovarian cancer is a serious threat for women health and the early diagnosis of this cancer might improves the survival rate of patients. The use of the targeted radiopharmaceuticals could be a non-invasive and logical method for tumor imaging. The aim of this study was to radiolabel GE11 peptide as a new specific probe for imaging of ovarian tumor. Methods: HYNIC-SSS-GE11 peptide was labeled with 99mTc using tricine as a coligand. The 99mTc-tricine-HYNIC-SSS-GE11 peptide was evaluated for specific cellular binding in three cell lines with different levels of EGFR expression. Tumor targeting was assessed in SKOV3 tumor bearing mice. Results: By using tricine as a coligand, labeling yield was more than 98% and the stability of the radiolabelled peptide in human serum up to 4 h was 96%. The in vitro cell uptake test showed that this radiolabeled peptide had a good affinity to SKOV3 cells with dissociation constant of 73 nM. The in vivo results showed a tumor/muscle ratio of 3.2 at 4 h following injection of 99mTc-tricine-HYNIC-SSS-GE11 peptide. Conclusions: Results of this study showed that 99mTc-tricine-HYNIC-SSS-GE11 peptide could be a promising tool for diagnosis and staging of ovarian cancer. [ABSTRACT FROM AUTHOR]

Receptor-targeting radiolabeled molecular probes with high affinity and specificity are useful in studying and monitoring biological processes and responses. Dual- or multiple-targeting probes, using radiolabeled metal chelates conjugated to peptides, have potential advantages over single-targeting probes as they can recognize multiple targets leading to better sensitivity for imaging and radiotherapy when target heterogeneity is present. Two natural hormone peptide receptors, gastrin-releasing peptide (GRP) and Y1, are specifically interesting as their expression is upregulated in most breast and prostate cancers. One of our goals has been to develop a dual-target probe that can bind both GRP and Y1 receptors. Consequently, a heterobivalent dual-target probe, t-BBN/BVD15-DO3A (where a GRP targeting ligand J-G-Abz4-QWAVGHLM-NH2 and Y1 targeting ligand INP-K [ɛ-J-(α-DO3A-ɛ-DGa)-K] YRLRY-NH2 were coupled), that recognizes both GRP and Y1 receptors was synthesized, purified, and characterized in the past. Competitive displacement cell binding assay studies with the probe demonstrated strong affinity (IC50 values given in parentheses) for GRP receptors in T-47D cells (18 ± 0.7 nM) and for Y1 receptors in MCF7 cells (80 ± 11 nM). As a further evaluation of the heterobivalent dual-target probe t-BBN/BVD15-DO3A, the objective of this study was to determine its mouse and human serum stability at 37°C. The in vitro metabolic degradation of the dual-target probe in mouse and human serum was studied by using a 153Gd-labeled t-BBN/BVD15-DO3A and a high-performance liquid chromatography/radioisotope detector analytical method. The half-life (t1/2) of degradation of the dual-target probe in mouse serum was calculated as 7 hours and only ∼20% degradation was seen after 6 hours incubation in human serum. The slow in vitro metabolic degradation of the dual-target probe can be compared with the degradation t1/2 of the corresponding monomeric probes, BVD15-DO3A and AMBA: 15, and ∼40 minutes for BVD15-DO3A and 3.1 and 38.8 hours for AMBA in mouse and human serum, respectively. A possible pathway for in vitro metabolic degradation of the t-BBN/BVD15-DO3A in mouse serum is proposed based on the chromatographic retention times of the intact probe and its degradants. [ABSTRACT FROM AUTHOR]

An ESIPT-based fluorescent probe (Probe 1 ) with a large Stokes shift (202 nm) for the sensing of biothiols has been developed based on cyanobiphenyl benzothiazole derivative exhibiting larger Stokes shift than 2-(benzothiazol-2-yl)phenol. This probe with 2,4-dinitrobenzenesulfonate (DNBS) as a highly thiol-selective group was constructed based on the combination of excited state intramolecular proton transfer (ESIPT) and photoinduced electron transfer (PET) mechanisms. Upon the treatment with biothiols, this probe produced a remarkable fluorescence enhancement at 482 nm. The detect limits for Cys, GSH and Hcy was calculated to be as low as 2.0 × 10 −8 M, 1.7 × 10 −7 M and 1.2 × 10 −7 M, respectively (based on S/N = 3). Importantly, application of this probe was successfully demonstrated by imaging thiols in living NCI-H226 cells. [ABSTRACT FROM AUTHOR]

ABSTRACT We previously showed that an antagonist-based peptide ligand, H-Cys(Npys)-Arg-Tyr-Tyr-Arg- Ile-Lys-NH2, captures the free thiol groups in the ligand-binding site of the nociceptin receptor ORL1. However, the exact receptor sites of this thiol-disulfide exchange reaction have not been uncovered, although such identification would help to clarify the ligand recognition site. Since the Cys→Ala substitution prevents the reaction, we performed the so-called Ala scanning for all the Cys residues in the transmembrane (TM) domains of the ORL1 receptor. Seven different mutant receptors were soundly expressed in the COS-7 cells and examined for their specific affinity labeling by a competitive binding assay using nociceptin and [3H]nociceptin. The results of in vitro Ala scanning analyses revealed that the labeled residues were Cys59 in TM1, Cys215 and Cys231 in TM5, and Cys310 in TM7. The present study has provided a novel method of Cys(Npys)-affinity labeling for identification of the ligand-binding sites in the ORL1 receptor. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 460-469, 2016. [ABSTRACT FROM AUTHOR]

The fluorescence detection of nitroreductase (NTR) and evaluation of the hypoxia status of tumor cells are vital, not only for clinical diagnoses and therapy, but also for biomedical research. Herein, we report the synthesis and application of a new fluorometric 'turn-on' probe for the detection of NTR ( TPE−NO2) that takes advantage of the aggregation-induced emission of tetraphenylethylene. TPE−NO2 can detect NTR at concentrations as low as 5 ng mL−1 in aqueous solution. The detection mechanism relied on the aggregation and deaggregation of tetraphenylethylene molecules. Moreover, this fluorescent probe can be used to monitor the hypoxia status of tumor cells through the detection of endogenous NTR. [ABSTRACT FROM AUTHOR]

High specificity has been an important feature in affinity labeling for target profiling. Especially, to label targets via rapidly progressing reactions with consumption of ligand (probe), high specificity of reaction with common functional groups of target protein should be achieved without reactions with similar groups of non-target proteins. Herein, we demonstrate the kinetic controlled affinity labeling of acyl CoA synthetase using a fatty acid analogue containing a phenylthioester linkage. High specificity was attained by accelerating the labeling rate in the binding pocket. This approach could be useful for profiling a series of target enzymes and transporters in signal transduction pathways. [ABSTRACT FROM AUTHOR]

The combined action of reactive metabolites of benzo[a]pyrene (B[a]P) and oxidative stress can lead to cluster-type DNA damage that includes both a bulky lesion and an apurinic/apyrimidinic (AP) site, which are repaired by the nucleotide and base excision repair mechanisms - NER and BER, respectively. Interaction of NER protein XPC-RAD23B providing primary damage recognition with DNA duplexes containing a B[a]P-derived residue linked to the exocyclic amino group of a guanine (BPDE-N-dG) in the central position of one strand and AP site in different positions of the other strand was analyzed. It was found that XPC-RAD23B crosslinks to DNA containing (+)- trans-BPDE-N-dG more effectively than to DNA containing cis-isomer, independently of the AP site position in the opposite strand; protein affinity to DNA containing one of the BPDE-N-dG isomers depends on the AP site position in the opposite strand. The influence of XPC-RAD23B on hydrolysis of an AP site clustered with BPDE-N-dG catalyzed by the apurinic/apyrimidinic endonuclease 1 (APE1) was examined. XPC-RAD23B was shown to stimulate the endonuclease and inhibit the 3′-5′ exonuclease activity of APE1. These data demonstrate the possibility of cooperation of two proteins belonging to different DNA repair systems in the repair of cluster-type DNA damage. [ABSTRACT FROM AUTHOR]

Message passing algorithms (MPAs) have been traditionally used as an inference method in probabilistic graphical models. Some MPA variants have recently been introduced in the field of estimation of distribution algorithms (EDAs) as a way to improve the efficiency of these algorithms. Multiple developments on MPAs point to an increasing potential of these methods for their application as part of hybrid EDAs. In this paper we review recent work on EDAs that apply MPAs and propose ways to further extend the useful synergies between MPAs and EDAs. Furthermore, we analyze some of the implications that MPA developments can have in their future application to EDAs and other evolutionary algorithms. [ABSTRACT FROM AUTHOR]