Kinetic study of enzymatic urea hydrolysis in the pH range 4-9

The enzymatic hydrolysis of urea by jack bean urease was investigated at 25degreesC over the pH range 4-9. Reaction rate data were found to be well described by a modified Michaelis-Menten equation with a pH-dependent rate coefficient and a product inhibition term. The influence of pH on activity was interpreted in terms of perturbation of the enzyme distribution among three differently protonated forms. Kinetic analysis yielded a Michaelis constant of 3.21 mmol l(-1) and indicated that the inhibition mechanism was of the fully non-competitive type, with K-P = 12.2 mmol l(-1). The estimated activation energy was 35.3 kJ mol(-1). The resulting kinetic expression was tested by comparing model predictions with the experimental behaviour observed in unbuffered media and over a long-teen period

Bose Einstein condensation on inhomogeneous amenable graphs

We investigate the Bose-Einstein Condensation on nonhomogeneous amenable networks for the model describing arrays of Josephson junctions. The resulting topological model, whose Hamiltonian is the pure hopping one given by the opposite of the adjacency operator, has also a mathematical interest in itself. We show that for the nonhomogeneous networks like the comb graphs, particles condensate in momentum and configuration space as well. In this case different properties of the network, of geometric and probabilistic nature, such as the volume growth, the shape of the ground state, and the transience, all play a role in the condensation phenomena. The situation is quite different for homogeneous networks where just one of these parameters, e.g. the volume growth, is enough to determine the appearance of the condensation.Comment: 43 pages, 12 figures, final versio

Altered vitamin B12 metabolism in the central nervous system is associated with the modification of ribosomal gene expression: new insights from comparative RNA dataset analysis

Recent studies have confirmed the direct role of vitamin B12 (VitB12) in the central nervous system (CNS) homeostasis; nevertheless, the detailed mechanisms are poorly understood. By analyzing RNA-Seq and microarray datasets obtained from databanks, this study aims to identify possible basic mechanisms, related to the brain, involved in altering the gene expression under VitB12 deficiency mimicking conditions. The database inquiry returned datasets generated from distinctly heterogeneous experimental sets and considering the quality and relevance requirements, two datasets from mouse and one from rat models were selected. The analyses of individual datasets highlighted a change in ribosomal gene expression in VitB12 deficiency mimicking conditions within each system. Specifically, a divergent regulation was observed depending on the animal model: mice showed a down regulation of the ribosomal gene expression, while rats an upregulation. Interestingly, E2f1 was significantly upregulated under VitB12 deficiency mimicking conditions in the animal models, with a greater upregulation in rats. The rat model also revealed putative E2F1 Transcription Factor Binding Sites (TFBSs) in the promoter of the differently regulated genes involved in ribosomal gene expression. This suggested the possibility that E2F1, being greater expressed in rats, could activate the ribosomal genes having E2F1 TFBSs, thus giving a plausible explication to the divergent regulation observed in animal models. Despite the great diversity of the experimental sets used to generate the datasets considered, a common alteration of the ribosomes exists, thereby indicating a possible basic and conserved response to VitB12 deficiency. Moreover, these findings could provide new insights on E2F1 and its association with CNS homeostasis and VitB12 deficiency

Modelling diffusivity in porous polymeric membranes with an intermediate layer containing microbial cells

Three-layer systems (membrane – composite layer (cells + polymer) – membrane) are important in different biochemical applications. Models of latex layered-membranes were evaluated and compared with experimental data in order to predict the diffusivity of substrates in the composite layer containing living E.coli microbial cells. Diffusivity predictions are dependent on the presence or the absence of a ‘skin’ layer, on the degree of polymer particle coalescence and on the thickness of each layer. Simulations with layered models were made to identify the dominant mechanisms in the three-layer system. It was found that the layered system is sensitive to the latex coatings porosity when the composite layer occupies less than 50% of the total membrane system thickness. Whenever the control of polymer particle coalescence and of the layers (coating/composite layer) thickness may be achieved, multi-layer systems presenting a wide range of relative diffusion conductivities may be built for different types of living cells and for a wide variety of practical applications. The diffusivity of the latex layer is proportional to the square of latex porosity.Fundação para a Ciência e a Tecnologia (FCT); FEDER

HNRNPM guides an alternative splicing program in response to inhibition of the PI3K/AKT/mTOR pathway in Ewing sarcoma cells

Ewing sarcomas (ES) are biologically aggressive tumors of bone and soft tissues for which no cure is currently available. Most ES patients do not respond to chemotherapeutic treatments or acquire resistance. Since the PI3K/AKT/mTOR axis is often deregulated in ES, its inhibition offers therapeutic perspective for these aggressive tumors. Herein, by using splicing sensitive arrays, we have uncovered an extensive splicing program activated upon inhibition of the PI3K/AKT/mTOR signaling pathway by BEZ235. Bioinformatics analyses identified hnRNPM as a key factor in this response. HnRNPMmotifswere significantly enriched in introns flanking the regulated exons and proximity of binding represented a key determinant for hnRNPM-dependent splicing regulation. Knockdown of hnRNPM expression abolished a subset of BEZ235-induced splicing changes that contained hnRNPM binding sites, enhanced BEZ235 cytotoxicity and limited the clonogenicity of ES cells. Importantly, hnRNPM up-regulation correlates with poor outcome in sarcoma patients. These findings uncover an hnRNPM-dependent alternative splicing program set in motion by inhibition of the mTOR/AKT/PI3K pathway in ES cells that limits therapeutic efficacy of pharmacologic inhibitors, suggesting that combined inhibition of the PI3K/AKT/mTOR pathway and hnRNPM activity may represent a novel approach for ES treatment

Genotoxic stress inhibits ewing sarcoma cell growth by modulating alternative pre-mRNA processing of the RNA helicase DHX9

Alternative splicing plays a key role in the DNA damage response and in cancer. Ewing Sarcomas (ES) are aggressive tumors caused by different chromosomal translocations that yield in-frame fusion proteins driving transformation. RNA profiling reveals genes differentially regulated by UV light irradiation in two ES cell lines exhibiting different sensitivity to genotoxic stress. In particular, irradiation induces a new isoform of the RNA helicase DHX9 in the more sensitive SK-N-MC cells, which is targeted to nonsense-mediated decay (NMD), causing its downregulation. DHX9 protein forms a complex with RNA polymerase II (RNAPII) and EWS-FLI1 to enhance transcription. Silencing of DHX9 in ES cells sensitizes them to UV treatment and impairs recruitment of EWS-FLI1 to target genes, whereas DHX9 overexpression protects ES cells from genotoxic stress. Mechanistically, we found that UV light irradiation leads to enhanced phosphorylation and decreased processivity of RNAPII in SK-N-MC cells, which in turn causes inclusion of DHX9 exon 6A. A similar effect on DHX9 splicing was also elicited by treatment with the chemotherapeutic drug etoposide, indicating a more general mechanism of regulation in response to DNA damage. Our data identify a new NMD-linked splicing event in DHX9 with impact on EWS-FLI1 oncogenic activity and ES cell viability

Current nanocarrier strategies improve vitamin B12 pharmacokinetics, ameliorate patients’ lives, and reduce costs

Vitamin B12 (VitB12) is a naturally occurring compound produced by microorganisms and an essential nutrient for humans. Several papers highlight the role of VitB12 deficiency in bone and heart health, depression, memory performance, fertility, embryo development, and cancer, while VitB12 treatment is crucial for survival in inborn errors of VitB12 metabolism. VitB12 is administrated through intramuscular injection, thus impacting the patients’ lifestyle, although it is known that oral administration may meet the specific requirement even in the case of malabsorption. Furthermore, the high-dose injection of VitB12 does not ensure a constant dosage, while the oral route allows only 1.2% of the vitamin to be absorbed in human beings. Nanocarriers are promising nanotechnology that can enable therapies to be improved, reducing side effects. Today, nanocarrier strategies applied at VitB12 delivery are at the initial phase and aim to simplify administration, reduce costs, improve pharmacokinetics, and ameliorate the quality of patients’ lives. The safety of nanotechnologies is still under investigation and few treatments involving nanocarriers have been approved, so far. Here, we highlight the role of VitB12 in human metabolism and diseases, and the issues linked to its molecule properties, and discuss how nanocarriers can improve the therapy and supplementation of the vitamin and reduce possible side effects and limits

De Finetti theorem on the CAR algebra

The symmetric states on a quasi local C*-algebra on the infinite set of indices J are those invariant under the action of the group of the permutations moving only a finite, but arbitrary, number of elements of J. The celebrated De Finetti Theorem describes the structure of the symmetric states (i.e. exchangeable probability measures) in classical probability. In the present paper we extend De Finetti Theorem to the case of the CAR algebra, that is for physical systems describing Fermions. Namely, after showing that a symmetric state is automatically even under the natural action of the parity automorphism, we prove that the compact convex set of such states is a Choquet simplex, whose extremal (i.e. ergodic w.r.t. the action of the group of permutations previously described) are precisely the product states in the sense of Araki-Moriya. In order to do that, we also prove some ergodic properties naturally enjoyed by the symmetric states which have a self--containing interest.Comment: 23 pages, juornal reference: Communications in Mathematical Physics, to appea

Conditional deletion of LRRC8A in the brain reduces stroke damage independently of swelling-activated glutamate release

The ubiquitous volume-regulated anion channels (VRACs) facilitate cell volume control and contribute to many other physiological processes. Treatment with non-specific VRAC blockers or brain-specific deletion of the essential VRAC subunit LRRC8A is highly protective in rodent models of stroke. Here, we tested the widely accepted idea that the harmful effects of VRACs are mediated by release of the excitatory neurotransmitter glutamate. We produced conditional LRRC8A knockout either exclusively in astrocytes or in the majority of brain cells. Genetically modified mice were subjected to an experimental stroke (middle cerebral artery occlusion). The astrocytic LRRC8A knockout yielded no protection. Conversely, the brain-wide LRRC8A deletion strongly reduced cerebral infarction in both heterozygous (Het) and full KO mice. Yet, despite identical protection, Het mice had full swelling-activated glutamate release, whereas KO animals showed its virtual absence. These findings suggest that LRRC8A contributes to ischemic brain injury via a mechanism other than VRAC-mediated glutamate release