Termostabilnost topljivih i vezanih peroksidaza iz artičoke te matematički model njihove kinetike inaktivacije

Soluble peroxidases (SP), and ionically (IBP) and covalently (CBP) bound peroxidases were extracted from leaves and edible part of artichoke heads (heart). The peroxidase (POD) forms showed a characteristic electrophoretic pattern; in particular SP and IBP forms showed nearly the same pattern, while CBP was quite different. Several basic and acid POD isoforms were present in SP and CBP forms, whereas in IBP only basic components were found. The thermal stability of different POD forms was tested at different temperatures (70, 80, 90 and 100 °C). The leaf POD forms showed greater heat stability compared to the head ones. The heat sensitivity of three POD forms was different: the bound forms were characterized by a greater heat stability than the soluble form. A series-type mathematical model has been developed to describe the inactivation kinetics of artichoke POD. The activation energies for the three POD forms of artichoke leaves and hearts were estimated by an Arrhenius-type relationship. The second step of the inactivation requires more energy to be carried out in all POD forms, especially in SP and IBP.Iz lišća i jestivog dijela glavice artičoke ekstrahirane su topljive te ionski i kovalentno vezane peroksidaze. Elektroforezom je dobivena karakteristična slika, pri čemu su topljive i ionski vezane peroksidaze imale gotovo isti, a kovalentno vezane sasvim drukčiji raspored. U topljivim i kovalentno vezanim peroksidazama nađeno je nekoliko kiselih i lužnatih izoformi, a u ionski vezanim samo lužnate. Toplinska stabilnost pojedinih peroksidaza ispitana je pri različitim temperaturama (70, 80, 90 i 100 °C). Izoforme iz lišća pokazale su veću termostabilnost od onih iz glavice artičoke. Vezani oblici peroksidaza bili su stabilniji od topljivih. Razvijen je matematički model (series-type mathematical model) za opisivanje kinetike inaktivacije peroksidaza artičoke. Energija aktivacije triju peroksidaza iz lišća i glavice artičoke procijenjena je pomoću Arrheniusova modela, pa je zaključeno da je potrebno utrošiti više energije za provođenje drugog koraka inaktivacije u svim peroksidazama, a osobito u topljivim i ionski vezanim

An analytic model to calculate Voxel S-Values for 177^{177}Lu

Objective: $^{177}$Lu is one of the most employed isotopes in targeted radionuclide therapies and theranostics, and 3D internal dosimetry for such procedures has great importance. Voxel S-Values (VSVs) approach is widely used for this purpose, but VSVs are available for a limited number of voxel dimensions. The aim of this work is to develop an analytic model for the calculation of $^{177}$Lu-VSVs in any cubic voxelized geometry of practical interest. Approach: Monte Carlo (MC) simulations were implemented with the toolkit GAMOS to evaluate VSVs in voxelized geometries of soft tissue from a source of $^{177}$Lu homogeneously distributed in the central voxel. Nine geometric setups, containing 15x15x15 cubic voxels of sides l ranging from 2 mm to 6 mm, in steps of 0.5 mm, were considered. For each l, the VSVs computed as a function of the "normalized radius", Rn = R/l (with R = distance from the center of the source voxel), were fitted with a parametric function. The dependencies of the parameters as a function of l were then fitted with appropriate functions, in order to implement the model for deducing $^{177}$Lu-VSVs for any l within the aforementioned range. Main results: The MC-derived VSVs were satisfactorily compared with literature data for validation, and the VSVs computed with the analytic model agree with the MC ones within 2\% for Rn $\leq$ 2 and within 6\% for Rn > 2. Significance: The proposed model enables the easy and fast calculation, with a simple spreadsheet, of $^{177}$Lu-VSVs in any cubic voxelized geometry of practical interest, avoiding the necessity of implementing ad-hoc MC simulations to estimate VSVs for specific voxel dimensions not available in literature data.Comment: 16 pages, 7 figures, first round review in "Biomedical Physics & Engineering Express

Experimental and Numerical Analysis of Seismic Response of Unreinforced Masonry Cross Vaults

The present paper shows an experimental and numerical analysis to understand the seismic behaviour of unreinforced masonry cross vault. The experimental tests were performed on a 1:5 scale model of a cross vault made of 3D-printed blocks with dry joints. The seismic actions was experimentally simulated as a horizontal force proportional to the vault\u2019s mass by using a quasi-static tilt testing setup. The vault 3D collapse mechanism and its strength expressed in terms of collapse multiplier was investigated, also considering the direction of the seismic action with respect to the vault\u2019s base. The tests results were compared to those obtained from a numerical analysis using a rigid-block model based on 3D limit analysis. The model formulation allows to take into account both associative and non-associative behaviour. A sensitivity analysis on friction angle variation was also investigated to evaluate the accuracy and robustness of the model

Degradation of Aflatoxin B1 by a Sustainable Enzymatic Extract from Spent Mushroom Substrate of Pleurotus eryngii

Ligninolytic enzymes from white-rot fungi, such as laccase (Lac) and Mn-peroxidase (MnP), are able to degrade aflatoxin B1 (AFB1), the most harmful among the known mycotoxins. The high cost of purification of these enzymes has limited their implementation into practical technologies. Every year, tons of spent mushroom substrate (SMS) are produced as a by-product of edible mushroom cultivation, such as Pleurotus spp., and disposed at a cost for farmers. SMS may still bea source of ligninolytic enzymes useful for AFB1 degradation. The in vitro AFB1-degradative activity of an SMS crude extract (SMSE) was investigated. Results show that: (1) in SMSE, high Lac activity (4 U g−1 dry matter) and low MnP activity (0.4 U g−1 dry matter) were present; (2) after 1 d of incubation at 25 °C, the SMSE was able to degrade more than 50% of AFB1, whereas after 3 and 7 d of incubation, the percentage of degradation reached the values of 75% and 90%, respectively; (3) with increasing pH values, the degradation percentage increased, reaching 90% after 3 d at pH 8. Based on these results, SMS proved to be a suitable source of AFB1 degrading enzymes and the use of SMSE to detoxify AFB1 contaminated commodities appears conceivable

Biokemijska svojstva topljivih i vezanih peroksidaza ekstrahiranih iz glavica i lišća artičoke

Soluble (SP), ionically bound (IBP) and covalently bound (CBP) peroxidases (POD) from artichoke leaves and heads have been characterized for the main biochemical parameters. The three PODs, in both leaves and heads, showed the major apparent catalytic efficiency (vmax,app/Km,app) towards ferulic acid, even though, in some cases, they showed higher affinity (Km,app) for other substrates. In leaves, SP and IBP showed higher Km,app for ferulic and chlorogenic acids, and CBP for ferulic and caffeic acids. In heads, SP showed higher Km,app for chlorogenic acid, IBP for caffeic and ferulic acids, and CBP for ferulic acid. It was shown that pH optimum for PODs ranged between 5.0 and 6.0 in leaves. In heads, pH optimum for SP and IBP was 5.5, while CBP presented a very low activity in a wide pH range. All PODs showed high thermal stability but different ability to regenerate: the bound forms were more able to regenerate than the soluble one. The results obtained show that (i) CBP from heads is able to work under very different cellular conditions, (ii) all PODs, in both tissues, have a high apparent catalytic efficiency for ferulic acid, which could explain the effective involvement of POD in lignin biosynthesis, (iii) in heads, high Km,app of SP for chlorogenic acid, particularly abundant in artichoke, could justify the possible involvement of PODs in browning mechanism, and (iv) in heat-processed artichoke, the ability of PODs to regenerate could contribute to oxidation and loss of product quality.Okarakterizirana su mnoga biokemijska svojstva topljivih, ionski i kovalentno vezanih peroksidaza ekstrahiranih iz lišća i glavica artičoke. Sva tri oblika peroksidaza, iz lišća i glavica, pokazala su veliku prividnu katalitičku djelotvornost (vmax,app/Km,app) prema feruličnoj kiselini, iako su u nekoliko slučajeva imala veći afinitet (Km,app) prema drugim supstratima. Topljive i ionski vezane peroksidaze iz lišća artičoke imale su veći afinitet za feruličnu i klorogenu kiselinu, a kovalentno vezane za feruličnu i kafeinsku kiselinu. Topljive peroksidaze iz glavica artičoke pokazale su veći afinitet prema klorogenoj kiselini, ionski vezane prema kafeinskoj i feruličnoj, a kovalentno vezane peroksidaze samo prema feruličnoj kiselini. Optimalna pH-vrijednost za aktivnost peroksidaza iz lišća bila je od 5 do 6, a za aktivnost topljivih i ionski vezanih peroksidaza iz glavica 5,5. Kovalentno vezane peroksidaze ekstrahirane iz glavica artičoke imale su malu aktivnost pri svim pH-vrijednostima. Sve su peroksidaze imale veliku termostabilnost, ali različitu sposobnost regeneracije: vezani su se oblici bolje regenerirali od topljivih oblika. Iz dobivenih rezultata izvedeni su sljedeći zaključci: (i) kovalentno vezane peroksidaze iz glavica djeluju u različitim staničnim uvjetima, (ii) sve ekstrahirane peroksidaze imaju veliku prividnu katalitičku djelotvornost prema feruličnoj kiselini, što objašnjava ulogu peroksidaza u biosintezi lignina, (iii) veliki afinitet topljive peroksidaze iz glavica prema klorogenoj kiselini, osobito zastupljenoj u artičokama, mogao bi objasniti ulogu peroksidaza u posmeđivanju glavica i (iv) sposobnost regeneracije peroksidaza uzrokuje oksidaciju i gubitak kakvoće artičoke tijekom kuhanja

Circulating hematopoietic stem cells and putative intestinal stem cells in coeliac disease

Background: The intestinal stem cells (ISC) modulation and the role of circulating hematopoietic stem cells (HSC) in coeliac disease (CD) are poorly understood. Our aim was to investigate the longitudinal modifications in peripheral blood HSC traffic and putative ISC density induced by gluten-free diet (GFD) in CD. Methods: Thirty-one CD patients and 7 controls were enrolled. Circulating CD133+ and CD34+ HSC were measured by flow cytometry, at enrolment and after 7 days and 1, 3, 6, 12, and 24 months of GFD. Endoscopy was performed at diagnosis and repeated at 6, 12, and 24 months following GFD. We used the Marsh-Oberhuber score to evaluate the histological severity of duodenal damage; immunohistochemistry was employed to measure the intraepithelial lymphoid infiltrate (IEL, CD3+ lymphoid cells) and the putative ISC compartment (CD133+ and Lgr5+ epithelial cells). Results: At enrolment, circulating HSCs were significantly increased in CD patients and they further augmented during the first week of GFD, but progressively decreased afterwards. CD patients presented with villous atrophy, abundant IEL and rare ISC residing at the crypt base. Upon GFD, IEL progressively decreased, while ISC density increased, peaking at 12 months. After 24 months of GFD, all patients were asymptomatic and their duodenal mucosa was macroscopically and histologically normal. Conclusions: In active CD patients, the ISC niche is depleted and there is an increased traffic of circulating HSC versus non-coeliac subjects. GFD induces a precocious mobilization of circulating HSC, which is followed by the expansion of the local ISC compartment, leading to mucosal healing and clinical remission

Carriers of ADAMTS13 Rare Variants Are at High Risk of Life-Threatening COVID-19

Thrombosis of small and large vessels is reported as a key player in COVID-19 severity. However, host genetic determinants of this susceptibility are still unclear. Congenital Thrombotic Thrombocytopenic Purpura is a severe autosomal recessive disorder characterized by uncleaved ultra-large vWF and thrombotic microangiopathy, frequently triggered by infections. Carriers are reported to be asymptomatic. Exome analysis of about 3000 SARS-CoV-2 infected subjects of different severities, belonging to the GEN-COVID cohort, revealed the specific role of vWF cleaving enzyme ADAMTS13 (A disintegrin-like and metalloprotease with thrombospondin type 1 motif, 13). We report here that ultra-rare variants in a heterozygous state lead to a rare form of COVID-19 characterized by hyper-inflammation signs, which segregates in families as an autosomal dominant disorder conditioned by SARS-CoV-2 infection, sex, and age. This has clinical relevance due to the availability of drugs such as Caplacizumab, which inhibits vWF-platelet interaction, and Crizanlizumab, which, by inhibiting P-selectin binding to its ligands, prevents leukocyte recruitment and platelet aggregation at the site of vascular damage

Gain- and Loss-of-Function CFTR Alleles Are Associated with COVID-19 Clinical Outcomes

Carriers of single pathogenic variants of the CFTR (cystic fibrosis transmembrane conductance regulator) gene have a higher risk of severe COVID-19 and 14-day death. The machine learning post-Mendelian model pinpointed CFTR as a bidirectional modulator of COVID-19 outcomes. Here, we demonstrate that the rare complex allele [G576V;R668C] is associated with a milder disease via a gain-of-function mechanism. Conversely, CFTR ultra-rare alleles with reduced function are associated with disease severity either alone (dominant disorder) or with another hypomorphic allele in the second chromosome (recessive disorder) with a global residual CFTR activity between 50 to 91%. Furthermore, we characterized novel CFTR complex alleles, including [A238V;F508del], [R74W;D1270N;V201M], [I1027T;F508del], [I506V;D1168G], and simple alleles, including R347C, F1052V, Y625N, I328V, K68E, A309D, A252T, G542*, V562I, R1066H, I506V, I807M, which lead to a reduced CFTR function and thus, to more severe COVID-19. In conclusion, CFTR genetic analysis is an important tool in identifying patients at risk of severe COVID-19