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AIM: Haemangiopericytoma (HPC) represents approximately 3% of all tumours in the head and neck. This tumour is a soft tissue tumour derived from mesenchymal cells with pericytic differentiation. We present the clinicopathological findings of a case. MATERIALS AND METHODS: A 69-year-old man was referred to our Department for a mass located on the right pre-molar maxillary gingiva; this mass caused problems during chewing, but was otherwise asymptomatic. RESULTS: Clinical examination revealed a nodular, pink lesion, 3.5 cm in diameter, which was lined with normal mucosa. The lesion was mobile in relation to the deep and superficial tissues. Microscopic analysis of the neoplasm showed a vascular rich pattern, constituted by vessels covered with flat endothelium and surrounded by abundant spindly cells. On the basis of these histological and immunohistochemical findings, the final diagnosis was HPC. CONCLUSIONS: HPC is an uncommon vascular tumour for which the biological behaviour is difficult to predict. In our patient, no recurrences or distant metastases were present at a 4 years follow-up. [ABSTRACT FROM AUTHOR]

BACKGROUND: Soft tissue myxoma of the oral cavity is rare. Only three cases of myxoma of gingiva have been reported in the literature. We present a case of soft tissue myxoma arising from the left maxillary adherent gingiva in a 42-year-old female patient. METHODS: Histological examination showed spindle-shaped cells in a myxoid stroma. Immunohistochemical stains with S-100 protein were negative, while those with vimentin were positive. RESULTS: Clinical examination revealed a soft tissue mass, with tense elastic consistency on palpation. The overlying mucosa was normal and healthy. A clinical diagnosis of fibroma was given. Histological examination showed spindle-shaped and stellate cells, arranged in a myxoid fibrous stroma, with collagen fibres distributed uniformly. Scattered islands or strands of inactive odontogenic epithelium were present. On the basis of the histological and immunohistochemical findings, the final diagnosis was soft tissue myxoma. CONCLUSIONS: Further studies are necessary to clarify the origin and histogenesis of this lesion. [ABSTRACT FROM AUTHOR]

Introduction: Recently the term Keratocystic Odontogenic Tumor (KCOT) has been recommended for Odontogenic Keratocysts (OKC) to address the neoplastic nature of the lesion compared to radicular and dentigerous cysts. S100 are calcium-binding proteins involved in cell differentiation and inflammation, with a potential role in neoplastic transformation. Aim: The aim of this study was to evaluate whether S100A8 protein expression is different in KCOT compared to radicular cysts (RC) and dentigerous cysts (DC). Methods: A total of 84 consecutive odontogenic cysts, 34 RC, 25 DC, and 25 KCOT, were analyzed in this study. Results: Epithelial cells in KCOT cases were not immunoreactive for S100A8 except focally in cases associated with inflammation, while RC cases showed a variable positivity of all the epithelial layers from the basal to the superficial in 19/34 cases and DC cases showed a weak positivity of the intermediate and superficial layers in 7/25 cases. Conclusion: The lack of S100A8 protein expression seems to be observed more frequently in KCOT compared to RC and DC. This difference might be related to their neoplastic nature and a potential aggressive biological behavior for odontogenic cystic lesions.

Abstract Background Channel proteins like FhuA can be an alternative to artificial chemically synthesized nanopores. To reach such goals, channel proteins must be flexible enough to be modified in their geometry, i.e. length and diameter. As continuation of a previous study in which we addressed the lengthening of the channel, here we report the increasing of the channel diameter by genetic engineering. Results The FhuA Δ1-159 diameter increase has been obtained by doubling the amino acid sequence of the first two N-terminal β-strands, resulting in variant FhuA Δ1-159 Exp. The total number of β-strands increased from 22 to 24 and the channel surface area is expected to increase by ~16%. The secondary structure analysis by circular dichroism (CD) spectroscopy shows a high β-sheet content, suggesting the correct folding of FhuA Δ1-159 Exp. To further prove the FhuA Δ1-159 Exp channel functionality, kinetic measurement using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine) were conducted. The results indicated a 17% faster diffusion kinetic for FhuA Δ1-159 Exp as compared to FhuA Δ1-159, well correlated to the expected channel surface area increase of ~16%. Conclusion In this study using a simple "semi rational" approach the FhuA Δ1-159 diameter was enlarged. By combining the actual results with the previous ones on the FhuA Δ1-159 lengthening a new set of synthetic nanochannels with desired lengths and diameters can be produced, broadening the FhuA Δ1-159 applications. As large scale protein production is possible our approach can give a contribution to nanochannel industrial applications.

Abstract Background Channel proteins like the engineered FhuA Δ1-159 often cannot insert into thick polymeric membranes due to a mismatch between the hydrophobic surface of the protein and the hydrophobic surface of the polymer membrane. To address this problem usually specific block copolymers are synthesized to facilitate protein insertion. Within this study in a reverse approach we match the protein to the polymer instead of matching the polymer to the protein. Results To increase the FhuA Δ1-159 hydrophobic surface by 1 nm, the last 5 amino acids of each of the 22 β-sheets, prior to the more regular periplasmatic β-turns, were doubled leading to an extended FhuA Δ1-159 (FhuA Δ1-159 Ext). The secondary structure prediction and CD spectroscopy indicate the β-barrel folding of FhuA Δ1-159 Ext. The FhuA Δ1-159 Ext insertion and functionality within a nanocontainer polymeric membrane based on the triblock copolymer PIB1000-PEG6000-PIB1000 (PIB = polyisobutylene, PEG = polyethyleneglycol) has been proven by kinetic analysis using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine). Identical experiments with the unmodified FhuA Δ1-159 report no kinetics and presumably no insertion into the PIB1000-PEG6000-PIB1000 membrane. Furthermore labeling of the Lys-NH2 groups present in the FhuA Δ1-159 Ext channel, leads to controllability of in/out flux of substrates and products from the nanocontainer. Conclusion Using a simple "semi rational" approach the protein's hydrophobic transmembrane region was increased by 1 nm, leading to a predicted lower hydrophobic mismatch between the protein and polymer membrane, minimizing the insertion energy penalty. The strategy of adding amino acids to the FhuA Δ1-159 Ext hydrophobic part can be further expanded to increase the protein's hydrophobicity, promoting the efficient embedding into thicker/more hydrophobic block copolymer membranes.

Abstract Background Recently we reported a nanocontainer based reduction triggered release system through an engineered transmembrane channel (FhuA Δ1-160; Onaca et al., 2008). Compound fluxes within the FhuA Δ1-160 channel protein are controlled sterically through labeled lysine residues (label: 3-(2-pyridyldithio)propionic-acid-N-hydroxysuccinimide-ester). Quantifying the sterical contribution of each labeled lysine would open up an opportunity for designing compound specific drug release systems. Results In total, 12 FhuA Δ1-160 variants were generated to gain insights on sterically controlled compound fluxes: Subset A) six FhuA Δ1-160 variants in which one of the six lysines in the interior of FhuA Δ1-160 was substituted to alanine and Subset B) six FhuA Δ1-160 variants in which only one lysine inside the barrel was not changed to alanine. Translocation efficiencies were quantified with the colorimetric TMB (3,3',5,5'-tetramethylbenzidine) detection system employing horseradish peroxidase (HRP). Investigation of the six subset A variants identified position K556A as sterically important. The K556A substitution increases TMB diffusion from 15 to 97 [nM]/s and reaches nearly the TMB diffusion value of the unlabeled FhuA Δ1-160 (102 [nM]/s). The prominent role of position K556 is confirmed by the corresponding subset B variant which contains only the K556 lysine in the interior of the barrel. Pyridyl labeling of K556 reduces TMB translocation to 16 [nM]/s reaching nearly background levels in liposomes (13 [nM]/s). A first B-factor analysis based on MD simulations confirmed that position K556 is the least fluctuating lysine among the six in the channel interior of FhuA Δ1-160 and therefore well suited for controlling compound fluxes through steric hindrance. Conclusions A FhuA Δ1-160 based reduction triggered release system has been shown to control the compound flux by the presence of only one inner channel sterical hindrance based on 3-(2-pyridyldithio)propionic-acid labeling (amino acid position K556). As a consequence, the release kinetic can be modulated by introducing an opportune number of hindrances. The FhuA Δ1-160 channel embedded in liposomes can be advanced to a universal and compound independent release system which allows a size selective compound release through rationally re-engineered channels.

By means of quantum chemistry (PBE0/def2-TZVPP; DLPNO-CCSD(T)/cc-pVTZ) and small, but reliable models of Polyhedral Oligomeric Silsesquioxanes (POSS), an array of astrochemically-relevant catalysis products, related to prebiotic and origin of life chemistry, has been theoretically explored. In this work, the heterogeneous phase hydrocyanation reaction of an unsaturated CC bond (propene) catalyzed by a Ni center complexed to a silica surface is analyzed. Of the two possible regioisomers, the branched iso-propyl-cyanide is thermodynamically and kinetically preferred over the linear n -propyl-cyanide ( T = 200 K). The formation of nitriles based on a regioselective process has profound implications on prebiotic and origin of life chemistry, as well as deep connections to terrestrial surface chemistry and geochemistry.

Purpose of Review: In response to the COVID-19 pandemic, there has been a remarkably accelerated development of vaccines worldwide. However, an effective distribution system is crucial for vaccination at a national level. Ecuador was one of the first Latin American countries to be most severely affected by the pandemic. It has been struggling to expand its vaccination drive and requires a strategy that provides an achievable vaccination rate and maintains its primary care services. This study aims to provide an efficient vaccination model to achieve herd immunity by utilizing the country's existing infrastructure (the centralized electoral system) for mass vaccination., Recent Findings: The national electoral data from 2017 and 2021 were used to create estimates for the proposed vaccination model. Two model variations, total personnel, needed, and the number of days needed to vaccinate 50%, 75%, and 100% of the population were considered. The numbers of vaccines needed, and vaccination sites were estimated based on the current number of registered voters and polling stations. The results from the proposed model show that 17,892,353 people can be vaccinated, at 40,093 polling stations, by 90,209 personnel if one vaccinator was available per polling station., Summary: Based on this model, even a conservative estimate shows that 12.56 days are needed to achieve herd immunity, and 16.74 days are needed to vaccinate the entire population of Ecuador. Additionally, we propose that this vaccination model can be used as a blueprint for any country to address similar catastrophes in the future., Supplementary Information: The online version contains supplementary material available at 10.1007/s40475-022-00251-y., Competing Interests: Conflict of InterestThe authors declare no competing interests., (© The Author(s), under exclusive licence to Springer Nature Switzerland AG 2022.)

Heterogeneous phase astrochemistry plays an important role in the synthesis of complex organic matter (COM) as found on comets and rocky body surfaces like asteroids, planetoids, moons and planets. The proposed catalytic model is based on two assumptions: (a) siliceous rocks in both crystalline or amorphous states show surface-exposed defective centers such as siloxyl (Si-O•) radicals; (b) the second phase is represented by gas phase CO molecules, an abundant C 1 building block found in space. By means of quantum chemistry; (DFT, PW6B95/def2-TZVPP); the surface of a siliceous rock in presence of CO is modeled by a simple POSS (polyhedral silsesquioxane) where a siloxyl (Si-O•) radical is present. Four CO molecules have been consecutively added to the Si-O• radical and to the nascent polymeric CO (pCO) chain. The first CO insertion shows no activation free energy with ΔG 200 K = -21.7 kcal/mol forming the SiO-CO• radical. The second and third CO insertions show Δ G 200 K ‡ ≤ 10.5 kcal/mol. Ring closure of the SiO-CO-CO• (oxalic anhydride) moiety as well as of the SiO-CO-CO-CO• system (di-cheto form of oxetane) are thermodynamically disfavored. The last CO insertion shows no free energy of activation resulting in the stable five member pCO ring, precursor to 1,4-epoxy-1,2,3-butanone. Hydrogenation reactions of the pCO have been considered on the SiO oxygen or on the carbons and oxygens of the pCO chains. The formation of the reactive aldehyde SiO-CHO on the siliceous surface is possible. In principle, the complete hydrogenation of the (CO) 1-4 series results in the formation of methanol and polyols. Furthermore, all the SiO-pCO intermediates and the lactone 1,4-epoxy-1,2,3-butanone product in its radical form can be important building blocks in further polymerization reactions and/or open ring reactions with H (aldehydes, polyols) or CN (chetonitriles), resulting in highly reactive multi-functional compounds contributing to COM synthesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fioroni and DeYonker.)

The formation of phosphorous-containing polycyclic aromatic hydrocarbons (PAPHs) in astrophysical contexts is proposed and analyzed by means of computational methods [B3LYP-D3BJ/ma-def2-TZVPP, MP2-F12, CCSD-F12b and CCSD(T)-F12b levels of theory]. A "bottom-up" approach based on a radical-neutral reaction scheme between acetylene (C 2 H 2 ) and the CP radical was used investigating: (a) the synthesis of the first PAPH (C 5 H 5 P) "phosphinine"; (b) PAPH growth by addition of C 2 H 2 to the C 5 H 4 P radical; (c) PAPH synthesis by addition reactions of one CP radical and nC 2 H 2 to a neutral PAH. Results show: (I) the formation of the phosphinine radical has a strong thermodynamic tendency (-133.3 kcal mol -1 ) and kinetic barriers ≤5.4 kcal mol -1 ; (II) PAPH growth by nC 2 H 2 addition on the radical phosphinine easily and exothermically produces radicals (1a- or 1-phospha-naphtalenes with kinetic barriers ≤7.1 kcal mol -1 and reaction free energies ≤-102.5 kcal mol -1 ); (III) the addition of a single CP + nC 2 H 2 to a neutral benzene generates a complex chemistry where the main product is 2-phospha-naphtalene; (IV) because of the CP radical character, its barrierless addition to a PAH produces a resonant stabilized PAPH, becoming excellent candidates for addition reactions with neutral or radical hydrocarbons and PAHs; (V) the same energy trend between all four levels of theory continues a well-calibrated computational protocol to analyze complex organic reactions with astrochemical interest using electronic structure theory.

The formation of the chiral molecule propylene oxide (CH 3 CHCH 2 O) recently detected in the interstellar medium (ISM) is proposed to take place on an amorphous silicate grain surface where peroxo defects are present. A computational analysis conducted at the DFT and MP2-F12 levels of theory on a neat amorphous silica model supports such a hypothesis resulting in (a) strong thermodynamic driving forces and low activation energies allowing the synthesis of CH 3 CHCH 2 O at low temperatures, (b) chemical defects on silica surfaces promoting heterogeneous catalysis of the increasing molecular complexity found in interstellar and circumstellar medium, and (c) chemical defects that have implications on understanding how processing phases modify the nature of the reactive groups on a silica surface affecting the surface catalytic activity.

Cosmic siliceous dust grains are involved in the synthesis of H 2 in the inter-stellar medium. In this work, the dust grain siliceous surface is represented by a hydrogen Fe-metalla-silsesquioxane model of general formula: [Fe(H 7 Si 7 O 12-n )(OH) n ] + (n=0,1,2) where Fe + behaves like a single-site heterogeneous catalyst grafted on a siliceous surface synthesizing H 2 from H. A computational analysis is performed using two levels of theory (B3LYP-D3BJ and MP2-F12) to quantify the thermodynamic driving force of the reaction: [Fe-T7H 7 ] + +4H→[Fe-T7H 7 (OH) 2 ] + +H 2 . The general outcomes are: 1) H 2 synthesis is thermodynamically strongly favored; 2) Fe-H / Fe-H 2 barrier-less formation potential; 3) chemisorbed H-Fe leads to facile H 2 synthesis at 20≤T≤100 K; 4) relative spin energetics and thermodynamic quantities between the B3LYP-D3BJ and MP2-F12 levels of theory are in qualitative agreement. The metalla-silsesquioxane model shows how Fe + fixed on a siliceous surface can potentially catalyze H 2 formation in space., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The aim of this study was to evaluate whether or not the expression of cGMP- phosphodiesterases (cGMP-PDE) varies in different thyroid pathologies and to elucidate the relationship between the expression of cGMP-PDE, cGMP, and autophagy. Fifty-four thyroid biopsy samples, excised to perform the biopsy, were split into two parts and randomly assigned: one part was microscopically examined and histological classified, and the other was frozen and analysed in order to evaluate the cGMP-PDE activity. Intracellular cGMP was also measured. A strong expression of intracellular cGMP and cGMP-PDE activity was observed in carcinoma in respect to controls and benign pathologies. The level of cGMP-PDE in papillary carcinoma without lymph node involvement (N-) was approximately four-fold higher compared to those with lymph node invasion (N±). On the contrary, the cGMP was one and a half times higher in N± than N-. Our results are promising, although further epigenetical studies are needed to confirm this association. A correlation between the cGMP-degrading activity and the severity of thyroid pathology has been shown. The decrease of cGMP-PDE and the increase of cGMP in N± papillar carcinoma could be an autophagic stimulus, a defence mechanism of the body, against the cancer that is expanding and invading other tissues and organs.

Transition metals (TMs) are proposed to play a role in astrophysical environments in both gas and solid state astrochemistry by co-determining the homogeneous/heterogeneous chemistry represented by the gas-gas and gas-dust grain interactions. Their chemistry is a function of temperature, radiation field and chemical composition/coordination sphere and as a consequence, dependent on the astrophysical object in which TMs are localized. Here five main categories of TM compounds are proposed and classified as: (a) pure bulk and clusters; (b) TM naked ions; (c) TM oxides/minerals or inorganic compounds; (d) TM-L (L = ligand) with L = (σ and/or π)-donor/acceptor species like H/H2, N/N2, CO, and H2O and (e) TM-organoligands such as Cp, PAH, and R1=˙=˙=R2. Each of the classes is correlated to their possible localization within astrophysical objects. Because of this variety coupled with their ability to modulate reactivity and regio/enantioselectivity by ligand sphere composition, TM compounds can introduce a fine organic synthesis in astrochemistry. For the selection of small TM parental compounds to be analyzed as first examples, by constraining the TMs and the second element/molecule on the basis of their cosmic abundance and mutual reactivity, Fe atoms coupled with N and CO are studied by developing the chemistry of [FeN](+), [FeNH](+) and [(CO)2FeN](+). These molecules, due to their ability to perform C-C and C-H bond activation, are able to open the pathway toward nitrogenation/amination and carbonylation of organic substrates. By considering the simplest organic substrate CH4, the parental reaction schemes (gas phase, T = 30 K): (I) [FeN](+) + CH4 + H → [Fe](+) + H3C-NH2; (II) [FeNH](+) + CH4 → [Fe](+) + H3C-NH2 and (III) [(CO)2FeN](+) + H → [FeCO](+) + HNCO are analyzed by theoretical methods (B2PLYP double hybrid functional/TZVPPP basis set). All reactions are thermodynamically favored and first step transition states can follow a minimal energy path by spin crossing, while H extraction in reaction II shows very high activation energies. The need to overcome high activation energy barriers underlines the importance of molecular activation by radiation and particle collision. TM chemistry is expected to contribute to the known synthesis of organic compounds in space leading towards a new direction in the astrochemistry field whose qualitative (type of compounds) and quantitative contributions must be unraveled.

An EGFP construct interacting with the PIB1000-PEG6000-PIB1000 vesicles surface reported a ~2-fold fluorescence emission enhancement. Because of the constructs nature with the amphiphilic peptide inserted into the PIB core, EGFP is expected to experience a "pure" PEG environment. To unravel this phenomenon PEG/water solutions at different molecular weights and concentrations were used. Already at ~1:10 protein/PEG molar ratio the increase in fluorescence emission is observed reaching a plateau correlating with the PEG molecular weight. Parallel experiments in presence of glycerol aqueous solutions did show a slight fluorescence enhancement however starting at much higher concentrations. Molecular dynamics simulations of EGFP in neat water, glycerol, and PEG aqueous solutions were performed showing that PEG molecules tend to "wrap" the protein creating a microenvironment where the local PEG concentration is higher compared to its bulk concentration. Because the fluorescent emission can be perturbed by the refractive index surrounding the protein, the clustering of PEG molecules induces an enhanced fluorescence emission already at extremely low concentrations. These findings can be important when related to the use of EGFP as reported in molecular biology experiments.

Engineered channel proteins are promising nano-components with applications in nanodelivery and nanoreactors technology. Because few of the engineered channel proteins have been crystallized, solution studies based on Neutron Scattering, Circular Dichroism and NMR play a major role. Consequently, the understanding of membrane proteins dynamics in water/detergent solutions or when embedded in a lipid membrane, can clarify how the environment affects protein behavior. In this study, molecular dynamics simulations of the FhuA Escherichia coli outer membrane channel protein and its engineered FhuA Δ1-159 variant have been performed in two different environments: a DNPC (1,2-dinervonyl-sn-glycero-3-phosphocholine) lipid bilayer and a water/OES (N-octyl-2-hydroxyethyl sulfoxide) detergent solution. Furthermore the FhuA Δ1-159 variant has been simulated in the open and closed states, the last induced by the presence of six 3-(2-pyridyldithio)-propionic-acid in the channel inner core. Differences in protein structural and dynamical behavior between the two environments have been found. Considering the FhuA protein characterized by an elliptical-cylindrical symmetry: (a) neither variations on the secondary structure nor axial deformation have been observed in any of the systems; (b) the ellipticity of the channel section (open state) and its fluctuations are enhanced in presence of water/OES, while diminished or suppressed in the DNPC bilayer; (c) the insertion of hydrophobic pyridyl groups into the FhuA Δ1-159 channel (closed state) induces a higher ellipticity in water/OES solution, while shifting to a circular section in the DNPC membrane; (d) the cork domain represented by the first 159 amino acids does not play a major role for protein stability., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Polymer based nanocompartments have potential applications in synthetic biology, medicine (drug release) and industrial biotechnology (chiral nanoreactors, multistep syntheses, selective product recovery). A step towards the aforementioned goals is the polymer membrane functionalization through covalent bonding of chemical anchors or insertion of proteins/peptides, to obtain specific properties like recognition, catalytic activity and facilitated diffusion, mimicking the complexity of a biological membrane. The use of genetic engineering techniques widens the possible applications of peptides and proteins specifically designed for polymer membrane interactions. A fusion protein (CecEGFP) based on the antibacterial peptide Cecropin A and the EGFP (Enhanced Green Fluorescent Protein) was designed, expressed and biophysically characterized. CecEGFP interaction with the tri-block copolymer PIB-PEG-PIB (PIB=polyisobutylene, PEG=polyethylene glycol) based polymersome membrane was analyzed by circular dichroism as well as EGFP and Trp fluorescence measurements. Results proved that Cecropin A is usable as a "membrane surface anchor" for water soluble proteins, as it inserts into the polymer membrane. The aim and novelty of this study is within the design of fusion proteins specifically developed for polymer membrane interactions. The use of amphiphilic Cecropin A "anchoring" water soluble proteins to the polymersome surface, avoids chemical coupling between polymers and proteins., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The objective of the present study was to evaluate vascular endothelial growth factor (VEGF) expression in different types of odontogenic cysts. A total of 25 parakeratotic odontogenic keratocysts (POKCs), 16 orthokeratotic odontogenic keratocysts (OOKCs), and 28 follicular cysts (FCs) were evaluated semiquantitatively for immunohistochemical analysis of VEGF in epithelial cells, endothelial cells of blood vessels, inflammatory cells and focally stromal cells. A significant different expression of VEGF in all cell components was found in keratocysts compared to FCs. The POKCs (80%) and OOKCs (68%) showed more than 50% VEGF positive epithelial cells, whereas the majority of FCs (71%) were either negative in the epithelium or showed less than 10% positive cells. Similarly, the POKCs (88%) and OOKCs (68%) showed more than 50% positive endothelial cells, whereas the FCs (75%) were either negative or showed less than 10% VEGF positive endothelial cells. The highest percentage of cases with score 2 positivity in the stromal cells was observed in POKCs (68%); OOKCs showed a score 2 positivity in 44%, score 1 in 31% and score 0 in 25%, whereas 68% of FCs showed a score 0, 25% a score 1 and only 7% of cases showed a score 2. No statistically significant differences were observed between POKCs and OOKCs in VEGF expression in the epithelial and endothelial cells, whereas the positivity score in stromal cells was significantly higher in POKCs compared to OOKCs. The present results can support the hypothesis that angiogenesis is an active mechanism in the invasive growth of the OKC.

Background: Channel proteins like FhuA can be an alternative to artificial chemically synthesized nanopores. To reach such goals, channel proteins must be flexible enough to be modified in their geometry, i.e. length and diameter. As continuation of a previous study in which we addressed the lengthening of the channel, here we report the increasing of the channel diameter by genetic engineering., Results: The FhuA Δ1-159 diameter increase has been obtained by doubling the amino acid sequence of the first two N-terminal β-strands, resulting in variant FhuA Δ1-159 Exp. The total number of β-strands increased from 22 to 24 and the channel surface area is expected to increase by ~16%. The secondary structure analysis by circular dichroism (CD) spectroscopy shows a high β-sheet content, suggesting the correct folding of FhuA Δ1-159 Exp. To further prove the FhuA Δ1-159 Exp channel functionality, kinetic measurement using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine) were conducted. The results indicated a 17% faster diffusion kinetic for FhuA Δ1-159 Exp as compared to FhuA Δ1-159, well correlated to the expected channel surface area increase of ~16%., Conclusion: In this study using a simple "semi rational" approach the FhuA Δ1-159 diameter was enlarged. By combining the actual results with the previous ones on the FhuA Δ1-159 lengthening a new set of synthetic nanochannels with desired lengths and diameters can be produced, broadening the FhuA Δ1-159 applications. As large scale protein production is possible our approach can give a contribution to nanochannel industrial applications.

The effect on the structural and thermodynamic properties in water/n-heptane interfaces on addition of NaCl, MgCl(2), and ZnCl(2) has been examined through five independent 100-ns molecular dynamics simulations. Results indicate that the interfacial thickness within the framework of the capillary-wave model decreases on addition of electrolytes in the order Na(+) < Mg(2+) < Zn(2+), whereas the interfacial tension increases in the same order. Ionic density profiles and self-diffusion coefficients are strongly influenced by the strength of the first hydration shell, which varies in the order Na(+) < Mg(2+) < Zn(2+). On the other hand, the Cl(-) behavior, that is, diffusion and solvation sphere, is influenced by its counterion. Accordingly, cations are strongly expelled from the interface, which is especially remarkable for the small divalent cations. This fact alters the water geometry near the interface and in a lesser extent n-heptane order and number of hydrogen bonds per water molecule close to the interface., (Copyright © 2011 Wiley Periodicals, Inc.)

Cel5A is a highly active endoglucanase from Thermoanaerobacter tengcongensis MB4, displaying an optimal temperature range between 75 and 80°C. After three rounds of error-prone PCR and screening of 4700 mutants, five variants of Cel5A with improved activities were identified by Congo Red based screening method. Compared with the wild type, the best variants 3F6 and C3-13 display 135±6% and 193±8% of the wild type specific activity for the substrate carboxymethyl cellulose (CMC), besides improvements in the relative expression level in Escherichia coli system. Remarkable are especially the improvements in activities at reduced temperatures (50% of maximum activity at 50°C and about 45°C respectively, while 65°C for the wild type). Molecular Dynamics simulations performed on the 3F6 and C3-13 variants show a decreased number of intra-Cel5A hydrogen bonds compared to the wild type, implying a more flexible protein skeleton which correlates well to the higher catalytic activity at lower temperatures. To investigate functions of each individual amino acid position site-directed (saturation) mutagenesis were generated and screened. Amino acid positions Val249 and Ile321 were found to be crucial for improving activity and residue Ile13 (encoded by rare codon AUA) yields an improved expression level in E. coli., (Copyright © 2011 Elsevier B.V. All rights reserved.)