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Mismatch Repair System corrects mutations arising from DNA replication that escape from DNA polymerase proofreading activity. This system consists of three main proteins, MutS-L-H, responsible for lesion recognition and repair. MutL is a member of GHKL ATPase family and its ATPase cycle has been proposed to modulate MutL activity during the repair process. Pseudomonas aeruginosa MutL (PaMutL) contains an N-terminal (NTD) ATPase domain connected by a linker to a C-terminal (CTD) dimerization domain that possesses metal ion-dependent endonuclease activity. With the aim to identify characteristics that allow the PaMutL NTD allosteric control of CTD endonuclease activity, we used an in silico and experimental approach to determine the interaction surfaces of P. aeruginosa NTD (PaNTD), and compared it with the well characterized Escherichia coli MutL NTD (EcNTD). Molecular dynamics simulations of PaNTD and EcNTD bound to or free of adenosine nucleotides showed that a significant difference exists between the behavior of the EcNTD and PaNTD dimerization interface, particularly in the ATP lid. Structure based simulations of MutL homologues with endonuclease activity were performed that allowed an insight of the dimerization interface behavior in this family of proteins. Our experimental results show that, unlike EcNTD, PaNTD is dimeric in presence of ADP. Simulations in mixed solvent allowed us to identify the PaNTD putative DNA binding patch and a putative interaction patch located opposite to the dimerization face. Structure based simulations of PaNTD dimer in presence of ADP or ATP suggest that nucleotide binding could differentially modulate PaNTD protein-protein interactions. Far western assays performed in presence of ADP or ATP are in agreement with our in silico analysis. [ABSTRACT FROM AUTHOR]

High-frequency electron paramagnetic resonance (HF-EPR) spectroscopy was employed to examine the oxidation slate and local structure of Ni and Mn ions in Ni,Mn-codoped LiCoO2. The assignment of EPR signals was based on Mg,Mn-codoped LiCoO2 and Ni-doped LiCoO2 used as Mn4+ and low-spin Ni3+ EPFI references. Complementary information on the oxidation state of transition-metal ions was obtained by solid-state 6.7Li NMR spectroscopy. For slightly doped oxides (LiCo1-xNixMnxO2 with x < 0.05), nickel and manganese substitute for cobalt in the CoO2 layers and are stabilized as Ni3+ and Mn4+ ions. The local structure of Mn4+ ions was determined by modeling of the axial zero-field-splitting parameter in the framework of the Newman superposition model, It has been found that the local trigonal distortion around Mn4+ is smaller in compadson with that of the host site. To achieve a local compensation of Mn4+ charge, several detect models are discussed. With an increase in the total dopant content (LiCO1-xNixMnxO2 and 0.05 ⩽ x ⩽5 0.10), a saturation in the Ni3+ amount (up to x < 0.05) is attained, while the Mn4+ content increases gradually. Ni3+ ions are surrounded by Co3+ ions only in the whole concentration range (0< x 5 0.10). The first metal coordination sphere of Mn4+ ions undergoes a transformation with an increase in the total Ni and Mn contents due to a progressive replacement of Co3+ by Mn4+ and Ni2+ ions. For highly doped oxides (LiCo1-xNixMnxO2 wilh x = 0.10), nickel and manganese achieve, with respect to the local charge compensation, their usual oxidation states of 2+ and 4+. [ABSTRACT FROM AUTHOR]

The local coordination of Fe[sup3+] spin probes in trigonal LiAl[suby]Co[sub1-y]O[sub2] was studied using high-frequency electron paramagnetic resonance spectroscopy. This technique allows the determination of Fe[sup3+] ions in respect to axial and rhorhbic zero-field splitting parameters (ZFS). After the progressive replacement of Co by Al, the axial D parameter of Fe[sup3+] increases from +0.0548 to +0.2802 cm[sup-1]. On the same order, the rhombic E parameter decreases. Structural infoiLmation about the Fe[sup3+] site in layered LiAl[suby]Co[sub1-y]O[sub2] oxides was based on modeling of the magnitude of the ZFS parameters by means of the Newman superposition model. It was found that the first metal coordination sphere including Co[sup3+] and AI[sup3+] ions gave rise to differentiation of the Fe[sup3+] dopants in respect to local trigonal and rhombic distortion. The maximum trigonal distortion for the FeO[sub6] octahedron was achieved when Fe[sup3+] spin probes were surrpunded by Al only, while the Co environment yields a rhombic distortion of the FeO[sub6] octahedron. [ABSTRACT FROM AUTHOR]

We demonstrate that the electron paramagnetic resonance spectrum of Mn2+ impurities in calcite, and therefore also in marble, may be accurately reproduced by a traditional spin Hamiltonian formalism. The success of such a treatment, however, very much depends on the spin Hamiltonian parameters having the correct signs as well as magnitudes. We present data that determine the sign of the axial anisotropy parameter and thereby facilitate future quantum mechanical characterizations of marble electron paramagnetic resonance spectra that supplement provenance determination. [ABSTRACT FROM AUTHOR]

β-Galactosidase is an important biotechnological enzyme used in the dairy industry, pharmacology and in molecular biology. In our laboratory we have overexpressed a recombinant β-galactosidase in Escherichia coli (E. coli). This enzyme differs from its native version (β-Gal WT) in that 6 histidine residues have been added to the carboxyl terminus in the primary sequence (β-Gal His), which allows its purification by immobilized metal affinity chromatography (IMAC). In this work we compared the functionality and structure of both proteins and evaluated their catalytic behavior on the kinetics of lactose hydrolysis. We observed a significant reduction in the enzymatic activity of β-Gal His with respect to β-Gal WT. Although, both enzymes showed a similar catalytic profile as a function of temperature, β-Gal His presented a higher resistance to the thermal inactivation compared to β-Gal WT. At room temperature, β-Gal His showed a fluorescence spectrum compatible with a partially unstructured protein, however, it exhibited a lower tendency to the thermal-induced unfolding with respect to β-Gal WT. The distinctively supramolecular arranges of the proteins would explain the effect of the presence of His-tag on the enzymatic activity and thermal stability. [Display omitted] • His-tag stabilizes the inactive β-galactosidase monomer. • His-tag protein is trapped in a partially folded state. [ABSTRACT FROM AUTHOR]

[Display omitted] • A Novel intein-based self-cleaving affinity tag was designed and implemented. • DnaX-CBD self-cleaving tag allows affinity purification of periplasmic proteins. • DnaX-CBD tag allows purification of recombinant proteins containing disulfide bonds. • DnaX-CBD tag allows purification of recombinant proteins free of extra amino acids. • Simple approach for production of research and therapeutic proteins. We evaluated several intein-based self-cleaving affinity tags for expression and single-step affinity chromatography purification of recombinant proteins produced in Escherichia coli. We used human growth hormone (hGH) as target protein that contains two internal disulfide bridges and an N-terminal phenylalanine. Use of N-terminal thiol-induced Sce VMA1 intein affinity tag resulted in purified hGH deficient in disulfide bonds. Inteins with self-cleavage inducible by pH and/or temperature shift were analyzed. N-terminal Ssp DnaX intein affinity tag resulted in a completely cleaved cytosolic protein, whereas N-terminal Ssp DnaB intein affinity tag resulted in a cytosolic fusion protein incapable of releasing hGH. Periplasmic expression of target protein was analyzed using an N-terminal signal peptide and C-terminal Ssp DnaX pH-inducible self-cleaving affinity tag. The fusion protein was properly expressed in pH 8 buffered culture medium. Fusion of a periplasmic signal peptide to the N-terminus of the POI allowed secretion to the periplasmic region and presence of the natural N-terminal amino acid of the POI following cleavage. Periplasmic expression of hGH fused to this novel C-terminal DnaX intein-based self-cleaving affinity tag made possible expression and purification of hGH protein containing disulfide bonds and free of extra amino acids. [ABSTRACT FROM AUTHOR]

The first person-to-person transmission of the 2019 novel coronavirus in Italy on 21 February 2020 led to an infection chain that represents one of the largest known COVID-19 outbreaks outside Asia. In northern Italy in particular, we rapidly experienced a critical care crisis due to a shortage of intensive care beds, as we expected according to data reported in China. Based on our experience of managing this surge, we produced this review to support other healthcare services in preparedness and training of hospitals during the current coronavirus outbreak. We had a dedicated task force that identified a response plan, which included: (1) establishment of dedicated, cohorted intensive care units for COVID-19-positive patients; (2) design of appropriate procedures for pre-triage, diagnosis and isolation of suspected and confirmed cases; and (3) training of all staff to work in the dedicated intensive care unit, in personal protective equipment usage and patient management. Hospital multidisciplinary and departmental collaboration was needed to work on all principles of surge capacity, including: space definition; supplies provision; staff recruitment; and ad hoc training. Dedicated protocols were applied where full isolation of spaces, staff and patients was implemented. Opening the unit and the whole hospital emergency process required the multidisciplinary, multi-level involvement of healthcare providers and hospital managers all working towards a common goal: patient care and hospital safety. Hospitals should be prepared to face severe disruptions to their routine and it is very likely that protocols and procedures might require re-discussion and updating on a daily basis. [ABSTRACT FROM AUTHOR]

The aim of this work was to identify risk genes related to the development and progression of squamous cell carcinoma head and neck (SCCHN) and do a meta-analysis of available estimates. Eligible gene/polymorphism studies were identified by electronic searches. Individual participant data of 8540 patients with HNC and 9844 controls from 19 genetic studies were analyzed, yielding adjusted (tobacco, gender, age and alcohol) odds ratios (OR) and 95% confidence intervals (CIs) comparing cases with controls. A meta-analysis was done on the studies that applied fixed and random models. People have an increase of polymorphism expression related to inflammation (NFKB1-294-ATTG, TNF[alpha]308-A2A2/A2A1, and TNF[beta]252- B2B2/B2B1) or carcinogenic metabolism (GSTM1 null, and CYP1A1 m1/m1), representative of malignancy development. Furthermore, the increased expression of genes associated with the stabilization and repair of the cellular (OGG1-Asp267Asn, Ser279Gly Ile253Phe, 1578A>T, 1582C>T Ala399Glu (1542C>A) 1582insG 1543_1544delCT), and genes associated with the regulation of proliferation, apoptosis or tumor survival (miRNA499-CT/CC, CRYABC802G-CG/GG) are considered as risk factors. In this scheme, only the polymorphisms of ADH7A92G-GG and DEC1606-T/C genes are protective against malignancy transformation. The TP53, GSTM1 and CYPA1genes have been evaluated in more than one study and analyzed for homogeneity in each genotype. The meta-analysis showed no significant association between different allelic variants of Arg72Pro rs1042522 and SCCHN risk. In a model of tumorigenesis, an increased risk of SCCHN is associated with DNA repair and DNA stabilization genes. In addition, the polymorphisms involved in inflammation and carcinogenic metabolism processes represent an increased risk of SCCHN. [ABSTRACT FROM AUTHOR]

The hallmark of the mismatch repair system in bacterial and eukaryotic organisms devoid of MutH is the presence of a MutL homologue with endonuclease activity. The aim of this study was to analyse whether different DNA structures affect Pseudomonas aeruginosa MutL (PaMutL) endonuclease activity and to determine if a specific nucleotide sequence is required for this activity. Our results showed that PaMutL was able to nick covalently closed circular plasmids but not linear DNA at high ionic strengths, while the activity on linear DNA was only found below 60 mM salt. In addition, single strand DNA, ss/ds DNA boundaries and negatively supercoiling degree were not required for PaMutL nicking activity. Finally, the analysis of the incision sites revealed that PaMutL, as well as Bacillus thuringiensis MutL homologue, did not show DNA sequence specificity. [ABSTRACT FROM AUTHOR]

Abstract: Human and Saccharomyces cerevisiae MutLα, and some bacterial MutL proteins, possess a metal ion-dependent endonuclease activity which is important for the in vivo function of these proteins. Conserved amino acids of the C-terminal region of human PMS2, S. cerevisiae PMS1 and of some bacterial MutL proteins have been implicated in the metal-binding/endonuclease activity. However, the contribution of individual amino acids to these activities has not yet been fully elucidated. In this work we show that Pseudomonas aeruginosa MutL protein possess an in vitro metal ion-dependent endonuclease activity. In agreement with previous published results, we observed that mutation of the aspartic acid, the first histidine or the first glutamic acid of the conserved C-terminal DMHAAHERITYE region results in nonfunctional in vivo proteins. We also determined that the arginine residue is essential for the in vivo function of this protein. However, we unexpectedly observed that although the first glutamic acid mutant derivative is not functional in vivo, its in vitro endonuclease activity is even higher than that of the wild-type protein. [Copyright &y& Elsevier]