Cloning and sequencing of a cDNA encoding 2,3-bisphosphoglycerate-independent phosphoglycerate mutase from maize. Possible relationship to the alkaline phosphatase family.

The primary sequence of maize 2,3-bisphosphoglycerate-independent phosphoglycerate mutase was deduced from cDNAs isolated from maize cDNA libraries by screening with specific antibodies to the cofactor-independent enzyme and from a maize genomic clone. The genomic clone provided the 5'-nucleotide sequence encoding the N-terminal amino acids which could not be obtained from the cDNA. Confirmation that the nucleotide sequence was for the cofactor-independent phosphoglycerate mutase was obtained by sequencing the peptides generated from cyanogen bromide cleavage of the purified protein. This is the first report of the amino acid sequence of a 2,3-bisphosphoglycerate cofactor-independent phosphoglycerate mutase, which consists of 559 amino acids and is twice the molecular size of the mammalian cofactor-dependent enzyme subunit. Analysis of the cofactor-independent phosphoglycerate mutase amino acid sequence revealed no identity with the cofactor-dependent mutase types. Northern blot analysis confirmed this difference since the maize cofactor-independent phosphoglycerate mutase cDNA did not hybridize with mRNA of the cofactor-dependent mutase. The lack of amino acid identity between cofactor-dependent and -independent enzymes is consistent with their different catalytic mechanisms and suggests that both enzymes are unrelated evolutionarily and arose from two independent ancestral genes. However, a constellation of residues which are involved in metal ion binding in various alkaline phosphatases is conserved in the maize cofactor-independent phosphoglycerate mutase, which suggests that the enzyme is a member of the alkaline phosphatase family of enzymes.     

9-Dihydroerythromycin ethers as motilin agonists—Developing structure–activity relationships for potency and safety

A series of derivatives of the amine of 9-dihydro-9-O-ethylamino-N-desmethyl-N-isopropyl erythromycin A derivatives were synthesized as motilin agonists. The compounds were developed for potency without showing antibacterial activity and inhibition of the hERG potassium channel. The formamide of the amide series was found to show the optimal combination of properties relative to carbamates, ureas, thioureas, and amines. This prompted an investigation of heterocyclic isosteres for the amide. In this series the triazole had the optimal combination of properties. From the study, two compounds met the criteria for detailed pharmacokinetic studies.     

Defibrotide inhibits donor leucocyte‐endothelial interactions and protects against acute graft‐versus‐host disease

Allogeneic hematopoietic stem cell transplantation (allo‐HCT) is an effective therapy for the treatment of high‐risk haematological malignant disorders and other life‐threatening haematological and genetic diseases. Acute graft‐versus‐host disease (aGvHD) remains the most frequent cause of non‐relapse mortality following allo‐HCT and limits its extensive clinical application. Current pharmacologic agents used for prophylaxis and treatment of aGvHD are not uniformly successful and have serious secondary side effects. Therefore, more effective and safe prophylaxis and therapy for aGvHD are an unmet clinical need. Defibrotide is a multi‐target drug successfully employed for prophylaxis and treatment of veno‐occlusive disease/sinusoidal obstruction syndrome. Recent preliminary clinical data have suggested some efficacy of defibrotide in the prevention of aGvHD after allo‐HCT. Using a fully MHC‐mismatched murine model of allo‐HCT, we report here that defibrotide, either in prophylaxis or treatment, is effective in preventing T cell and neutrophil infiltration and aGvHD‐associated tissue injury, thus reducing aGvHD incidence and severity, with significantly improved survival after allo‐HCT. Moreover, we performed in vitro mechanistic studies using human cells revealing that defibrotide inhibits leucocyte‐endothelial interactions by down‐regulating expression of key endothelial adhesion molecules involved in leucocyte trafficking. Together, these findings provide evidence that defibrotide may represent an effective and safe clinical alternative for both prophylaxis and treatment of aGvHD after allo‐HCT, paving the way for new therapeutic approaches.     

Helping decision making for reliable and cost‐effective 2b‐RAD sequencing and genotyping analyses in non‐model species

High‐throughput sequencing has revolutionized population and conservation genetics. RAD sequencing methods, such as 2b‐RAD, can be used on species lacking a reference genome. However, transferring protocols across taxa can potentially lead to poor results. We tested two different IIB enzymes (AlfI and CspCI) on two species with different genome sizes (the loggerhead turtle Caretta caretta and the sharpsnout seabream Diplodus puntazzo) to build a set of guidelines to improve 2b‐RAD protocols on non‐model organisms while optimising costs. Good results were obtained even with degraded samples, showing the value of 2b‐RAD in studies with poor DNA quality. However, library quality was found to be a critical parameter on the number of reads and loci obtained for genotyping. Resampling analyses with different number of reads per individual showed a trade‐off between number of loci and number of reads per sample. The resulting accumulation curves can be used as a tool to calculate the number of sequences per individual needed to reach a mean depth ≥20 reads to acquire good genotyping results. Finally, we demonstrated that selective‐base ligation does not affect genomic differentiation between individuals, indicating that this technique can be used in species with large genome sizes to adjust the number of loci to the study scope, to reduce sequencing costs and to maintain suitable sequencing depth for a reliable genotyping without compromising the results. Here, we provide a set of guidelines to improve 2b‐RAD protocols on non‐model organisms with different genome sizes, helping decision‐making for a reliable and cost‐effective genotyping.     

IL-17RA in Non-Hematopoietic Cells Controls CXCL-1 and 5 Critical to Recruit Neutrophils to the Lung of Mycobacteria-Infected Mice during the Adaptive Immune Response

During chronic infection with Mycobacterium tuberculosis (Mtb), bacilli multiplication is constrained within lung granulomas until excessive inflammation destroys the lung. Neutrophils are recruited early and participate in granuloma formation, but excessive neutrophilia exacerbates the tuberculosis disease. Neutrophils thus appear as potential targets for therapeutic interventions, especially in patients for whom no antibiotic treatment is possible. Signals that regulate neutrophil recruitment to the lung during mycobacterial infection need to be better understood. We demonstrated here, in the mouse model, that neutrophils were recruited to the lung in two waves after intranasal infection with virulent Mtb or the live attenuated vaccine strain Bacillus Calmette Guérin (BCG). A first wave of neutrophils was swiftly recruited, followed by a subsequent adaptive wave that reached the lung together with IFN-γ- and IL-17A-producing T cells. Interestingly, the second neutrophil wave did not participate to mycobacteria control in the lung and established contacts with T cells. The adaptive wave was critically dependent on the expression of IL-17RA, the receptor for IL-17A, expressed in non-hematopoietic cells. In absence of this receptor, curtailed CXCL-1 and 5 production in the lung restrained neutrophil recruitment. CXCL-1 and 5 instillation reconstituted lung neutrophil recruitment in BCG-infected IL17RA-/- mice.     

Trophoblast–endothelium signaling involves angiogenesis and apoptosis in a dynamic bioprinted placenta model

Trophoblast invasion and remodeling of the maternal spiral arteries are required for pregnancy success. Aberrant endothelium–trophoblast crosstalk may lead to preeclampsia, a pregnancy complication that has serious effects on both the mother and the baby. However, our understanding of the mechanisms involved in this pathology remains elementary because the current in vitro models cannot describe trophoblast–endothelium interactions under dynamic culture. In this study, we developed a dynamic three-dimensional (3D) placenta model by bioprinting trophoblasts and an endothelialized lumen in a perfusion bioreactor. We found the 3D printed perfusion bioreactor system significantly augmented responses of endothelial cells by encouraging network formations and expressions of angiogenic markers, cluster of differentiation 31 (CD31), matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-9 (MMP9), and vascular endothelial growth factor A (VEGFA). Bioprinting favored colocalization of trophoblasts with endothelial cells, similar to in vivo observations. Additional analysis revealed that trophoblasts reduced the angiogenic responses by reducing network formation and motility rates while inducing apoptosis of endothelial cells. Moreover, the presence of endothelial cells appeared to inhibit trophoblast invasion rates. These results clearly demonstrated the utility and potential of bioprinting and perfusion bioreactor system to model trophoblast–endothelium interactions in vitro. Our bioprinted placenta model represents a crucial step to develop advanced research approach that will expand our understanding and treatment options of preeclampsia and other pregnancy-related pathologies.     

GCAT|Panel,a comprehensive structural variant haplotype map of the Iberian population from high-coverage whole-genome sequencing

The combined analysis of haplotype panels with phenotype clinical cohorts is a common approach to explore the genetic architecture of human diseases. However, genetic studies are mainly based on single nucleotide variants (SNVs) and small insertions and deletions (indels). Here, we contribute to fill this gap by generating a dense haplotype map focused on the identification, characterization, and phasing of structural variants (SVs). By integrating multiple variant identification methods and Logistic Regression Models (LRMs), we present a catalogue of 35 431 441 variants, including 89 178 SVs (≥50 bp), 30 325 064 SNVs and 5 017 199 indels, across 785 Illumina high coverage (30x) whole-genomes from the Iberian GCAT Cohort, containing a median of 3.52M SNVs, 606 336 indels and 6393 SVs per individual. The haplotype panel is able to impute up to 14 360 728 SNVs/indels and 23 179 SVs, showing a 2.7-fold increase for SVs compared with available genetic variation panels. The value of this panel for SVs analysis is shown through an imputed rare Alu element located in a new locus associated with Mononeuritis of lower limb, a rare neuromuscular disease. This study represents the first deep characterization of genetic variation within the Iberian population and the first operational haplotype panel to systematically include the SVs into genome-wide genetic studies.     

Effect of nitric oxide donors on oxygen-dependent cytotoxic responses mediated by neutrophils

We analyzed the effect of nitric oxide (NO) on oxygen-dependent cytotoxic responses mediated by neutrophils against unopsonized erythrocytes using three NO donors: S-nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP), and sodium nitroprusside (SNP). Neutrophils were treated with these compounds for 1-2 min at 37 degrees C and cytotoxicity was then triggered in the presence of NO donors by precipitating immune complexes, aggregated IgG, the chemotactic peptide FMLP, or opsonized zymosan. GSNO induced, in all cases, a marked increase in cytotoxic responses, while SNAP moderately increased cytotoxicity triggered by immune complexes, aggregated IgG, or Z, opsonized zymosen, without modifying those responses induced by FMLP. By contrast, SNP dramatically suppressed cytotoxicity triggered by all of the stimuli assessed. The enhancing effects mediated by GSNO and SNAP did not depend on the stimulation of guanylyl cyclase and were prevented by the NO scavengers hemoglobin and PTIO (2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide). The inhibitory activity of SNP, on the other hand, was not prevented by NO scavengers, suggesting that it cannot be ascribed to the release of NO. In another set of experiments, neutrophils were pretreated with GSNO or SNAP for different times. Then cells were washed to remove NO donors from the culture medium, and cytotoxicity was triggered by different stimuli. It was found that neutrophils must be pretreated with NO donors for at least 4 h to increase cytotoxic responses, and pretreatment for longer periods (i.e., 8 or 18 h) further increased cytotoxicity. Not only cytotoxic responses, but also the production of O2- and H2O2, and the release of myeloperoxidase were increased under these conditions.     

Nitric oxide, complex I, and the modulation of mitochondrial reactive species in biology and disease

Mitochondria are the specialized organelles for energy metabolism but also participate in the production of O(2) active species, cell cycle regulation, apoptosis and thermogenesis. Classically, regulation of mitochondrial energy functions was based on the ADP/ATP ratio, which dynamically stimulates the transition between resting and maximal O(2) uptake. However, in the last years, NO was identified as a physiologic regulator of electron transfer and ATP synthesis by inhibiting cytochrome oxidase. Additionally, NO stimulates the mitochondrial production of O(2) active species, primarily O(2)(-) and H(2)O(2), and, depending on NO matrix concentration, of ONOO(-), which is responsible for the nitrosylation and nitration of mitochondrial components. By this means, alteration in mitochondrial complexes restricts energy output, further increases O(2) active species and changes cell signaling for proliferation and apoptosis through redox effects on specific pathways. These mechanisms are prototypically operating in prevalent generalized diseases like sepsis with multiorgan failure or limited neurodegenerative disorders like Parkinson's disease. Complex I appears to be highly susceptible to ONOO(-) effects and nitration, which defines an acquired group of mitochondrial disorders, in addition to the genetically induced syndromes. Increase of mitochondrial NO may follow over-expression of nNOS, induction and translocation of iNOS, and activation and/or increased content of the newly described mtNOS. Likewise, mtNOS is important in the modulation of O(2) uptake and cell signaling, and in mitochondrial pathology, including the effects of aging, dystrophin deficiency, hypoxia, inflammation and cancer.     

Conserved asparagine residue 54 of alpha-sarcin plays a role in protein stability and enzyme activity

Asparagine 54 of alpha-sarcin is a conserved residue within the proteins of the ribotoxin family of microbial ribonucleases. It is located in loop 2 of the protein, which lacks repetitive secondary structure elements but exhibits a well-defined conformation. Five mutant variants at this residue have been produced and characterized. The spectroscopic characterization of these proteins indicates that the overall conformation is not changed upon mutation. Activity and denaturation assays show that Asn-54 largely contributes to protein stability, and its presence is a requirement for the highly specific inhibitory activity of these ribotoxins on ribosomes.