Directed evolution of artificial repeat proteins as habit modifiers for the morphosynthesis of (111)-terminated gold nanocrystals

Natural biocomposites are shaped by proteins that have evolved to interact with inorganic materials. Protein directed evolution methods which mimic Darwinian evolution have proven highly successful to generate improved enzymes or therapeutic antibodies but have rarely been used to evolve protein–material interactions. Indeed, most reported studies have focused on short peptides and a wide range of oligopeptides with chemical binding affinity for inorganic materials have been uncovered by phage display methods. However, their small size and flexible unfolded structure prevent them from dictating the shape and crystallinity of the growing material. In the present work, a specific set of artificial repeat proteins (αRep), which exhibit highly stable 3D folding with a well-defined hypervariable interacting surface, is selected by directed evolution of a very efficient home-built protein library for their high and selective affinity for the Au(111) surface. The proteins are built from the extendable concatenation of self-compatible repeated motifs idealized from natural HEAT proteins. The high-yield synthesis of Au(111)-faceted nanostructures mediated by these αRep proteins demonstrates their chemical affinity and structural selectivity that endow them with high crystal habit modification performances. Importantly, we further exploit the protein shell spontaneously assembled on the nanocrystal facets to drive protein-mediated colloidal self-assembly and on-surface enzymatic catalysis. Our method constitutes a generic tool for producing nanocrystals with determined faceting, superior biocompatibility and versatile bio-functionalization towards plasmon-based devices and (bio)molecular sensors

Utilization of a deoxynucleoside diphosphate substrate by HIV reverse transcriptase

Background: Deoxynucleoside triphosphates (dNTPs) are the normal substrates for DNA sysnthesis is catalyzed by polymerases such as HIV-1 reverse transcriptase (RT). However, substantial amounts of deoxynucleoside diphosphates (dNDPs) are also present in the cell. Use of dNDPs in HIV-1 DNA sysnthesis could have significant implications for the efficacy of nucleoside RT inhibitors such as AZT which are first line therapeutics fro treatment of HIV infection. Our earlier work on HIV-1 reverse transcriptase (RT) suggested that the interaction between the γ phosphate of the incoming dNTP and RT residue K65 in the active site is not essential for dNTP insertion, implying that this polymerase may be able to insert dNPs in addition to dNTPs. Methodology/Principal Findings: We examined the ability of recombinant wild type (wt) and mutant RTs with substitutions at residue K65 to utilize a dNDP substrate in primer extension reactions. We found that wild type HIV-1 RT indeed catalyzes incorporation of dNDP substrates whereas RT with mutations of residue K645 were unable to catalyze this reaction. Wild type HIV-1 RT also catalyzed the reverse reaction, inorganic phosphate-dependent phosphorolysis. Nucleotide-mediated phosphorolytic removal of chain-terminating 3′-terminal nucleoside inhibitors such as AZT forms the basis of HIV-1 resistance to such drugs, and this removal is enhanced by thymidine analog mutations (TAMs). We found that both wt and TAM-containing RTs were able to catalyze Pi-mediated phosphorolysis of 3′-terminal AZT at physiological levels of Pi with an efficacy similar to that for ATP-dependent AZT-excision. Conclusion: We have identified two new catalytic function of HIV-1 RT, the use of dNDPs as substrates for DNA synthesis, and the use of Pi as substrate for phosphorolytic removal of primer 3′-terminal nucleotides. The ability to insert dNDPs has been documented for only one other DNA polymerase The RB69 DNA polymerase and the reverse reaction employing inorganic phosphate has not been documented for any DNA polymerase. Importantly, our results show that Pi-mediated phosphorolysis can contribute to AZT resistance and indicates that factors that influence HIV resistance to AZT are more complex than previously appreciated. © 2008 Garforth et al

Reversible Keap1 inhibitors are preferential pharmacological tools to modulate cellular mitophagy

Mitophagy orchestrates the autophagic degradation of dysfunctional mitochondria preventing their pathological accumulation and contributing to cellular homeostasis. We previously identified a novel chemical tool (hereafter referred to as PMI), which drives mitochondria into autophagy without collapsing their membrane potential (ΔΨm). PMI is an inhibitor of the protein-protein interaction (PPI) between the transcription factor Nrf2 and its negative regulator, Keap1 and is able to up-regulate the expression of autophagy-associated proteins, including p62/SQSTM1. Here we show that PMI promotes mitochondrial respiration, leading to a superoxide-dependent activation of mitophagy. Structurally distinct Keap1-Nrf2 PPI inhibitors promote mitochondrial turnover, while covalent Keap1 modifiers, including sulforaphane (SFN) and dimethyl fumarate (DMF), are unable to induce a similar response. Additionally, we demonstrate that SFN reverses the effects of PMI in co-treated cells by reducing the accumulation of p62 in mitochondria and subsequently limiting their autophagic degradation. This study highlights the unique features of Keap1-Nrf2 PPI inhibitors as inducers of mitophagy and their potential as pharmacological agents for the treatment of pathological conditions characterized by impaired mitochondrial quality control

In vitro effects of zinc on the cytokine production from peripheral blood mononuclear cells in patients with zinc allergy.

Metals, such as nickel, cobalt, chromium and zinc, are ubiquitous in the environment. Systemic reactions, including hand dermatitis and generalized eczematous reactions, can be caused by the dietary ingestion of metals. In this study, we aimed to determine whether the cytokine production from peripheral blood mononuclear cells (PBMCs) obtained from zinc allergy patients can be used as a sensitive marker to investigate zinc-allergic contact dermatitis. The diagnosis of sensitivity to metal was made based on the results of a metal patch test. The PBMCs were stimulated with various concentrations (5-100 μM) of zinc sulfate (ZnSO4) for 24 h. The culture supernatants were collected and analyzed using ELISA for measurement of the cytokine production. The levels of IFN-γ, TNF-α, IL-1β, IL-5, IL-13 and MIF were significantly higher in the zinc-allergic patients (n = 5) than in the healthy controls (n = 5) at 100 μM of ZnSO4 stimulation. Although, patch testing is considered as standard test to diagnose metal allergy but false-positive and -negative reactions may limit its use in conditions of existing dermatitis. Therefore, this study suggest that in support of patch testing the determination of cytokine production using PBMCs cultures would be helpful for making an early diagnosis of such conditions

Molecular Analysis of Precursor Lesions in Familial Pancreatic Cancer

PMCID: PMC3553106This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Nrt1 and Tna1-Independent Export of NAD+ Precursor Vitamins Promotes NAD+ Homeostasis and Allows Engineering of Vitamin Production

NAD+ is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD+ consuming enzymes. NAD+ biosynthesis is required for two different regimens that extend lifespan in yeast. NAD+ is synthesized from tryptophan and the three vitamin precursors of NAD+: nicotinic acid, nicotinamide and nicotinamide riboside. Supplementation of yeast cells with NAD+ precursors increases intracellular NAD+ levels and extends replicative lifespan. Here we show that both nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the nicotinamide riboside transporter, Nrt1, leads to increased export of nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD+ metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD+ metabolism by balancing import and export of NAD+ precursor vitamins

A weak group inverse for rectangular matrices

[EN] In this paper, we extend the notion of weak group inverse to rectangular matrices (called WweightedWGinverse) by using the weighted core EP inverse recently investigated. This new generalized inverse also generalizes the well-known weighted group inverse given by Cline and Greville. In addition, we give several representations of the W-weighted WG inverse, and derive some characterizations and properties.First author was partially supported by UNRC (Grant PPI 18/C472) and CONICET (Grant PIP 112-201501-00433CO). Third author was partially supported by Ministerio de Economia, Industria y Competitividad of Spain (Grants DGI MTM2013-43678-P and Red de Excelencia MTM2017-90682-REDT).Ferreyra, DE.; Orquera, V.; Thome, N. (2019). A weak group inverse for rectangular matrices. Revista de la Real Academia de Ciencias Exactas Físicas y Naturales Serie A Matemáticas. 113(4):3727-3740. https://doi.org/10.1007/s13398-019-00674-9S372737401134Ben-Israel, A., Greville, T.N.E.: Generalized Inverses: Theory and Applications, 2nd edn. Springer, New York (2003)Baksalary, O.M., Trenkler, G.: Core inverse of matrices. Linear Multilinear Algebra 58, 681–697 (2010)Baksalary, O.M., Trenkler, G.: On a generalized core inverse. Appl. Math. Comput. 236, 450–457 (2014)Bajodah, A.H.: Servo-constraint generalized inverse dynamics for robot manipulator control design. Int. J. Robot. Autom. 25, (2010). https://doi.org/10.2316/Journal.206.2016.1.206-3291Campbell, S.L., Meyer Jr., C.D.: Generalized Inverses of Linear transformations. SIAM, Philadelphia (2009)Cline, R.E., Greville, T.N.E.: A Drazin inverse for rectangular matrices. Linear Algebra Appl. 29, 53–62 (1980)Dajić, A., Koliha, J.J.: The weighted g-Drazin inverse for operators. J. Aust. Math. Soc. 2, 163–181 (2007)Doty, K.L., Melchiorri, C., Bonivento, C.: A theory of generalized inverses applied to robotics. Int. J. Rob. Res. 12, 1–19 (1993)Drazin, M.P.: Pseudo-inverses in associate rings and semirings. Am. Math. Mon. 65, 506–514 (1958)Ferreyra, D.E., Levis, F.E., Thome, N.: Revisiting of the core EP inverse and its extension to rectangular matrices. Quaest. Math. 41, 265–281 (2018)Ferreyra, D.E., Levis, F.E., Thome, N.: Maximal classes of matrices determining generalized inverses. Appl. Math. Comput. 333, 42–52 (2018)Gigola, S., Lebtahi, L., Thome, N.: The inverse eigenvalue problem for a Hermitian reflexive matrix and the optimization problem. J. Comput. Appl. Math. 291, 449–457 (2016)Hartwig, R.E.: The weighted $$*$$ ∗ -core-nilpotent decomposition. Linear Algebra Appl. 211, 101–111 (1994)Kirkland, S.J., Neumann, M.: Group inverses of M-matrices and their applications. Chapman and Hall/CRC, London (2013)Malik, S., Thome, N.: On a new generalized inverse for matrices of an arbitrary index. Appl. Math. Comput. 226, 575–580 (2014)Male $${{\check{\rm s}}}$$ s ˇ ević, B., Obradović, R., Banjac, B., Jovović, I., Makragić, M.: Application of polynomial texture mapping in process of digitalization of cultural heritage. arXiv:1312.6935 (2013). Accessed 14 June 2018Manjunatha Prasad, K., Mohana, K.S.: Core EP inverse. Linear Multilinear Algebra 62, 792–802 (2014)Mehdipour, M., Salemi, A.: On a new generalized inverse of matrices. Linear Multilinear Algebra 66, 1046–1053 (2018)Meng, L.S.: The DMP inverse for rectangular matrices. Filomat 31, 6015–6019 (2017)Mosić, D.: The CMP inverse for rectangular matrices. Aequaetiones Math. 92, 649–659 (2018)Penrose, R.: A generalized inverse for matrices. Proc. Cambrid. Philos. Soc. 51, 406–413 (1955)Soleimani, F., Stanimirović, P.S., Soleymani, F.: Some matrix iterations for computing generalized inverses and balancing chemical equations. Algorithms 8, 982–998 (2015)Xiao, G.Z., Shen, B.Z., Wu, C.K., Wong, C.S.: Some spectral techniques in coding theory. Discrete Math. 87, 181–186 (1991)Wang, H.: Core-EP decomposition and its applications. Linear Algebra Appl. 508, 289–300 (2016)Wang, H., Chen, J.: Weak group inverse. Open Math. 16, 1218–1232 (2018)Wei, Y.: A characterization for the $$W$$ W -weighted Drazin inverse and a Crammer rule for the $$W$$ W -weighted Drazin inverse solution. Appl. Math. Comput. 125, 303–310 (2002

Expression analysis of E-cadherin, Slug and GSK3β in invasive ductal carcinoma of breast

<p>Abstract</p> <p>Background</p> <p>Cancer progression is linked to a partially dedifferentiated epithelial cell phenotype. The signaling pathways Wnt, Hedgehog, TGF-β and Notch have been implicated in experimental and developmental epithelial mesenchymal transition (EMT). Recent findings from our laboratory confirm that active Wnt/β-catenin signaling is critically involved in invasive ductal carcinomas (IDCs) of breast.</p> <p>Methods</p> <p>In the current study, we analyzed the expression patterns and relationships between the key Wnt/β-catenin signaling components- E-cadherin, Slug and GSK3β in IDCs of breast.</p> <p>Results</p> <p>Of the 98 IDCs analyzed, 53 (54%) showed loss/or reduced membranous staining of E-cadherin in tumor cells. Nuclear accumulation of Slug was observed in 33 (34%) IDCs examined. Loss or reduced level of cytoplasmic GSK3β expression was observed in 52/98 (53%) cases; while 34/98 (35%) tumors showed nuclear accumulation of GSK3β. Statistical analysis revealed associations of nuclear Slug expression with loss of membranous E-cadherin (p = 0.001); nuclear β-catenin (p = 0.001), and cytoplasmic β-catenin (p = 0.005), suggesting Slug mediated E-cadherin suppression via the activation of Wnt/β-catenin signaling pathway in IDCs. Our study also demonstrated significant correlation between GSK3β nuclear localization and tumor grade (p = 0.02), suggesting its association with tumor progression.</p> <p>Conclusion</p> <p>The present study for the first time provided the clinical evidence in support of Wnt/β-catenin signaling upregulation in IDCs and key components of this pathway - E-cadherin, Slug and GSK3β with β-catenin in implementing EMT in these cells.</p

Suspected retinopathies in Norwegian optometric practice with emphasis on patients with diabetes: a cross-sectional study

<p>Abstract</p> <p>Background</p> <p>The scope of optometry differs worldwide. In Norway the vast majority of optometrists perform ophthalmoscopy as part of their routine examinations. The aim of this study was to describe the frequency of <it>suspected </it>retinopathies in patients seen for routine optometric examination and to determine how optometrists deal with these patients.</p> <p>Methods</p> <p>212 optometrists participated in a questionnaire survey and a practice registration during November 2004 – May 2005. In the practice registration, details for 20 consecutive patient encounters were recorded. Data were analysed by chi-square tests and multiple logistic regression.</p> <p>Results</p> <p>All optometrist stated that ocular history taking was an integrated part of their routine examination, while general health and diabetes history were routinely addressed by 59% and 42% of the optometrists, respectively. During the practice registration 4,052 patient encounters were recorded. Ophthalmoscopy was performed in 88% of the patients, of which 2% were dilated fundus examinations. Retinopathy was <it>suspected </it>in 106 patients, of whom 31 did not report a previous history of ocular or systemic disease. Old age (75+), hypertension and diabetes strongly predicted retinopathy with odds ratio (95% CI) of 6.4 (4.2 to 9.9), 3.8 (2.4 to 6.0) and 2.5 (1.4 to 4.7), respectively. Diabetic retinopathy was seen in 10% of diabetic patients and <it>suspected </it>in 0.2% of patients with no established history of diabetes. Retinopathy was not confirmed in 9 out 18 patients with a history of diabetic retinopathy; seven of these had undergone laser treatment. Out of the 106 patients with findings of retinopathy, 28 were referred to an ophthalmologist or a general practitioner (GP), written reports were sent to a GP in 16 cases, ten patients were urged to contact their GP for further follow up, while 52 were considered in need of routine optometric follow up only.</p> <p>Conclusion</p> <p>Optometric practice provides a low threshold setting for detecting cases of ocular disease and retinal manifestations of systemic disease in the population. At present diagnosis of retinopathy in Norwegian optometric practice is unreliable. There are potentials for improving the optometrists' routine examination, their patient management patterns and collaboration routines with medical doctors.</p