Serum or Plasma (and Which Plasma), That Is the Question

Blood derivatives are the biofluids of choice formetabolomic clinical studies since blood can be collected with lowinvasiveness and is rich in biological information. However, the choiceof the blood collection tubes has an undeniable impact on the plasmaand serum metabolic content. Here, we compared the metabolomicand lipoprotein profiles of blood samples collected at the same timeand place from six healthy volunteers but using different collectiontubes (each enrolled volunteer provided multiple blood samples at adistance of a few weeks/months): citrate plasma, EDTA plasma, andserum tubes. All samples were analyzed via nuclear magnetic resonancespectroscopy. Several metabolites showed statistically significantalterations among the three blood matrices, and also metabolites'correlations were shown to be affected. The effects of blood collectiontubes on the lipoproteins'profiles are relevant too, but less marked. Overcoming the issue associated with different blood collectiontubes is pivotal to scale metabolomics and lipoprotein analysis at the level of epidemiological studies based on samples frommulticenter cohorts. We propose a statistical solution, based on regression, that is shown to be efficient in reducing the alterationsinduced by the different collection tubes for both the metabolomic and lipoprotein profile

Three-Dimensional Solution Structure of Saccharomyces cerevisiae Reduced Iso-l-cytochromec

Two-dimensional ^1H NMR spectra of Saccharomyces cerevisiae reduced iso-1-cytochrome c have been used to confirm and slightly extend the assignment available in the literature. 1702 NOESY cross-peaks have been assigned, and their intensities have been measured. Through the program DIANA and related protocols (Güntert, 1992), a solution structure has been obtained by using 1442 meaningful NOEs and 13 hydrogen-bond constraints. The RMSD values with respect to the mean structure for the backbone and all heavy atoms for a family of 20 structures are 0.61 ± 0.09 and 0.98 ± 0.09 Å, the average target function value being as small as 0.57 Å^2. The larger number of slowly exchanging amide NHs observed in this system compared to that observed in the cyanide derivative of oxidized Ala 80 cytochrome c suggests that the oxidized form is much more flexible and that the backbone protons are more solvent accessible. Comparison of the present structure with the crystal structures of reduced yeast cytochrome c and of the complex between cytochrome c peroxidase and oxidized yeast cytochrome c reveals substantial similarity among the backbone conformations but differences in the residues located in the region of protein−protein interaction. Interestingly, in solution the peripheral residues involved in the interaction with cytochrome c peroxidase are on average closer to the position found in the crystal structure of the complex than to the solid state structure of the isolated reduced form

Diauxie and co-utilization of carbon sources can coexist during bacterial growth in nutritionally complex environments

It is commonly thought that when multiple carbon sources are available, bacteria metabolize them either sequentially (diauxic growth) or simultaneously (co-utilization). However, this view is mainly based on analyses in relatively simple laboratory settings. Here we show that a heterotrophic marine bacterium, Pseudoalteromonas haloplanktis, can use both strategies simultaneously when multiple possible nutrients are provided in the same growth experiment. The order of nutrient uptake is partially determined by the biomass yield that can be achieved when the same compounds are provided as single carbon sources. Using transcriptomics and time-resolved intracellular 1H-13C NMR, we reveal specific pathways for utilization of various amino acids. Finally, theoretical modelling indicates that this metabolic phenotype, combining diauxie and co-utilization of substrates, is compatible with a tight regulation that allows the modulation of assimilatory pathways

Modelling hCDKL5 heterologous expression in bacteria

hCDKL5 refers to the human cyclin-dependent kinase like 5 that is primarily expressed in the brain. Mutations in its coding sequence are often causative of hCDKL5 deficiency disorder, a devastating neurodevelopmental disorder currently lacking a cure. The large-scale recombinant production of hCDKL5 is desirable to boost the translation of preclinical therapeutic approaches into the clinic. However, this is hampered by the intrinsically disordered nature of almost two-thirds of the hCDKL5 sequence, making this region more susceptible to proteolytic attack, and the observed toxicity when the enzyme is accumulated in the cytoplasm of eukaryotic host cells. The bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) is the only prokaryotic host in which the full-length production of hCDKL5 has been demonstrated. To date, a system-level understanding of the metabolic burden imposed by hCDKL5 production is missing, although it would be crucial for upscaling of the production process. Here, we combined experimental data on protein production and nutrients assimilation with metabolic modelling to infer the global consequences of hCDKL5 production in PhTAC125 and to identify potential overproduction targets. Our analyses showed a remarkable accuracy of the model in simulating the recombinant strain phenotype and also identified priority targets for optimised protein production

The anti-apoptotic effect of ASC-exosomes in an in vitro ALS model and their proteomic analysis

Stem cell therapy represents a promising approach in the treatment of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). The beneficial effect of stem cells is exerted by paracrine mediators, as exosomes, suggesting a possible potential use of these extracellular vesicles as non-cell based therapy. We demonstrated that exosomes isolated from adipose stem cells (ASC) display a neuroprotective role in an in vitro model of ALS. Moreover, the internalization of ASC-exosomes by the cells was shown and the molecules and the mechanisms by which exosomes could exert their beneficial effect were addressed. We performed for the first time a comprehensive proteomic analysis of exosomes derived from murine ASC. We identified a total of 189 proteins and the shotgun proteomics analysis revealed that the exosomal proteins are mainly involved in cell adhesion and negative regulation of the apoptotic process. We correlated the protein content to the anti-apoptotic effect of exosomes observing a downregulation of pro-apoptotic proteins Bax and cleaved caspase-3 and upregulation of anti-apoptotic protein Bcl-2 \u3b1, in an in vitro model of ALS after cell treatment with exosomes. Overall, this study shows the neuroprotective effect of ASC-exosomes after their internalization and their global protein profile, that could be useful to understand how exosomes act, demonstrating that they can be employed as therapy in neurodegenerative diseases

Three-Dimensional Solution Structure of the Cyanide Adduct of a Variant of Saccharomyces cerevisiae Iso-1-cytochrome c Containing the Met80Ala Mutation. Identification of Ligand-Residue Interactions in the Distal Heme Cavity

The ^1H NMR spectrum of the the cyanide adduct of a triply mutated Saccharomyces cerevisiae iso-1-cytochrome c (His39Gln/Met80Ala/CyslO2Seri)n the oxidized form has been assigned through 1D NOE and 2D COSY, TOCSY, NOESY, and NOE-NOESY experiments; 562 protons out of a total of 683 have been assigned. The solution structure, the first of a paramagnetic heme protein, was determined using 1426 meaningful NOE constraints out of a total of 1842 measured NOES. The RMSD values at the stage of restrained energy minimization of 17 structures obtained from distance geometry calculations are 0.68 ± 0.11 and 1.32 ± 0.14 Å for the backbone and all heavy atoms, respectively. The quality, in terms of RMSD, of the present structure is the same as that obtained for the solution structure of the diamagnetic horse heart ferrocytochrome c [Qi, P. X., et al. (1994) Biochemistry 33, 6408-64191. The secondary structure elements and the overall folding in the variant are observed to be the same as those of the wild-type protein for which the X-ray structure is available. However, the replacement of the methionine axial ligand with an alanine residue creates a ligand-binding “distal cavity.” The properties of the distal cavity seen in this solution structure are compared to those of other heme proteins

Solution Structure of Oxidized Saccharomyces cerevisiae Iso-1-cytochrome c

The solution structure of oxidized Saccharomyces cerevisiae Cys102Ser iso-1-cytochrome c has been determined using 1361 meaningful NOEs (of 1676 total) after extending the published proton assignment [Gao, Y., et al. (1990) Biochemistry 29, 6994−7003] to 77% of all proton resonances. The NOE patterns indicate that secondary structure elements are maintained upon oxidation in solution with respect to the solid state and solution structures of the reduced species. Constraints derived from the pseudocontact shifts [diamagnetic reference shift values are those of the reduced protein [Baistrocchi, P., et al. (1996) Biochemistry 35, 13788−13796]] were used in the final stages of structure calculations. After restrained energy minimization with constraints from NOEs and pseudocontact shifts, a family of 20 structures with rmsd values of 0.58 ± 0.08 and 1.05 ± 0.10 Å (relative to the average structure) for the backbone and all heavy atoms, respectively, was obtained. The solution structure is compared with the crystal structure and the structures of related systems. Twenty-six amide protons were detected in the NMR spectrum 6 days after the oxidized lyophilized protein was dissolved in D_2O (pH 7.0 and 303 K); in an analogous experiment, 47 protons were observed in the spectrum of the reduced protein. The decrease in the number of nonexchanging amide protons, which mainly are found in the loop regions 14−26 and 75−82, confirms the greater flexibility of the structure of oxidized cytochrome c in solution. Our finding of increased solvent accessibility in these loop regions is consistent with proposals that an early step in unfolding the oxidized protein is the opening of the 70−85 loop coupled with dissociation of the Met80−iron bond

Three-Dimensional Solution Structure of Saccharomyces cerevisiae Reduced Iso-l-cytochromec

Two-dimensional ^1H NMR spectra of Saccharomyces cerevisiae reduced iso-1-cytochrome c have been used to confirm and slightly extend the assignment available in the literature. 1702 NOESY cross-peaks have been assigned, and their intensities have been measured. Through the program DIANA and related protocols (Güntert, 1992), a solution structure has been obtained by using 1442 meaningful NOEs and 13 hydrogen-bond constraints. The RMSD values with respect to the mean structure for the backbone and all heavy atoms for a family of 20 structures are 0.61 ± 0.09 and 0.98 ± 0.09 Å, the average target function value being as small as 0.57 Å^2. The larger number of slowly exchanging amide NHs observed in this system compared to that observed in the cyanide derivative of oxidized Ala 80 cytochrome c suggests that the oxidized form is much more flexible and that the backbone protons are more solvent accessible. Comparison of the present structure with the crystal structures of reduced yeast cytochrome c and of the complex between cytochrome c peroxidase and oxidized yeast cytochrome c reveals substantial similarity among the backbone conformations but differences in the residues located in the region of protein−protein interaction. Interestingly, in solution the peripheral residues involved in the interaction with cytochrome c peroxidase are on average closer to the position found in the crystal structure of the complex than to the solid state structure of the isolated reduced form

Three-Dimensional Solution Structure of the Cyanide Adduct of a Variant of Saccharomyces cerevisiae Iso-1-cytochrome c Containing the Met80Ala Mutation. Identification of Ligand-Residue Interactions in the Distal Heme Cavity

The ^1H NMR spectrum of the the cyanide adduct of a triply mutated Saccharomyces cerevisiae iso-1-cytochrome c (His39Gln/Met80Ala/CyslO2Seri)n the oxidized form has been assigned through 1D NOE and 2D COSY, TOCSY, NOESY, and NOE-NOESY experiments; 562 protons out of a total of 683 have been assigned. The solution structure, the first of a paramagnetic heme protein, was determined using 1426 meaningful NOE constraints out of a total of 1842 measured NOES. The RMSD values at the stage of restrained energy minimization of 17 structures obtained from distance geometry calculations are 0.68 ± 0.11 and 1.32 ± 0.14 Å for the backbone and all heavy atoms, respectively. The quality, in terms of RMSD, of the present structure is the same as that obtained for the solution structure of the diamagnetic horse heart ferrocytochrome c [Qi, P. X., et al. (1994) Biochemistry 33, 6408-64191. The secondary structure elements and the overall folding in the variant are observed to be the same as those of the wild-type protein for which the X-ray structure is available. However, the replacement of the methionine axial ligand with an alanine residue creates a ligand-binding “distal cavity.” The properties of the distal cavity seen in this solution structure are compared to those of other heme proteins