Characterization of the catalytic flexible loop in the dihydroorotase domain of the human multi-enzymatic protein CAD

The dihydroorotase (DHOase) domain of the multifunctional protein carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (CAD) catalyzes the third step in the de novo biosynthesis of pyrimidine nucleotides in animals. The crystal structure of the DHOase domain of human CAD (huDHOase) revealed that, despite evolutionary divergence, its active site components are highly conserved with those in bacterial DHOases, encoded as monofunctional enzymes. An important element for catalysis, conserved from Escherichia coli to humans, is a flexible loop that closes as a lid over the active site. Here, we combined mutagenic, structural, biochemical, and molecular dynamics analyses to characterize the function of the flexible loop in the activity of CAD's DHOase domain. A huDHOase chimera bearing the E. coli DHOase flexible loop was inactive, suggesting the presence of distinctive elements in the flexible loop of huDHOase that cannot be replaced by the bacterial sequence. We pinpointed Phe-1563, a residue absolutely conserved at the tip of the flexible loop in CAD's DHOase domain, as a critical element for the conformational equilibrium between the two catalytic states of the protein. Substitutions of Phe-1563 with Ala, Leu, or Thr prevented the closure of the flexible loop and inactivated the protein, whereas substitution with Tyr enhanced the interactions of the loop in the closed position and reduced fluctuations and the reaction rate. Our results confirm the importance of the flexible loop in CAD's DHOase domain and explain the key role of Phe-1563 in configuring the active site and in promoting substrate strain and catalysi

Samples and data accessibility in research biobanks. An explorative survey

Biobanks, which contain human biological samples and/or data, provide a crucial contribution to the progress of biomedical research. However, the effective and efficient use of biobank resources depends on their accessibility. In fact, making bio-resources promptly accessible to everybody may increase the benefits for society. Furthermore, optimizing their use and ensuring their quality will promote scientific creativity and, in general, contribute to the progress of bio-medical research. Although this has become a rather common belief, several laboratories are still secretive and continue to withhold samples and data. In this study, we conducted a questionnairebased survey in order to investigate sample and data accessibility in research biobanks operating all over the world. The survey involved a total of 46 biobanks. Most of them gave permission to access their samples (95.7%) and data (85.4%), but free and unconditioned accessibility seemed not to be common practice. The analysis of the guidelines regarding the accessibility to resources of the biobanks that responded to the survey highlights three issues: (i) the request for applicants to explain what they would like to do with the resources requested; (ii) the role of funding, public or private, in the establishment of fruitful collaborations between biobanks and research labs; (iii) the request of co-authorship in order to give access to their data. These results suggest that economic and academic aspects are involved in determining the extent of sample and data sharing stored in biobanks. As a second step of this study, we investigated the reasons behind the high diversity of requirements to access biobank resources. The analysis of informative answers suggested that the different modalities of resource accessibility seem to be largely influenced by both social context and legislation of the countries where the biobanks operate

Severe bloodstream infection due to KPC-producer e coli in a renal transplant recipient treated with the double-carbapenem regimen and analysis of in vitro synergy testing a case report

Transplant recipients are at high risk of infections caused by multidrug resistant microorganisms. Due to the limited thera- peutic options, innovative antimicrobial combinations against carbape- nem-resistant Enterobacteriaceae causing severe infections are necessary. A 61-year-old woman with a history of congenital solitary kidney underwent renal transplantation. The postoperative course was compli- cated by nosocomial pneumonia due to Stenotrophomonas maltophilia and pan-sensitive Escherichia coli, successfully treated with antimicrobial therapy. On postoperative day 22, diagnosis of surgical site infection and nosocomial pneumonia with concomitant bacteremia due to a Kle- bisella pneumoniae carbapenemase-producer E coli was made. The patient was treated with the double-carbapenem regimen (high dose of merope- nem plus ertapenem) and a potent synergistic and bactericidal activity of this un-conventional therapeutic strategy was observed in vitro. Despite a microbiological response with prompt negativity of blood cultures, the patient faced a worse outcome because of severe hemorrhagic shock. The double-carbapenem regimen might be considered as a rescue therapy in those subjects, including transplant recipients, in whom previous antimicrobial combinations failed or when colistin use might be discouraged. Performing in vitro synergy testing should be strongly encouraged in cases of infections caused by pan-drug resistant strains, especially in high-risk patients

Modelling complex bimolecular reactions in a condensed phase: the case of phosphodiester hydrolysis

Background: the theoretical modelling of reactions occurring in liquid phase is a research line of primary importance both in theoretical-computational chemistry and in the context of organic and biological chemistry. Here we present the modelling of the kinetics of the hydroxide-promoted hydrolysis of phosphoric diesters. Method: the theoretical-computational procedure involves a hybrid quantum/classical approach based on the perturbed matrix method (PMM) in conjunction with molecular mechanics. Results: the presented study reproduces the experimental data both in the rate constants and in the mechanistic aspects (C-O bond vs. O-P bond reactivity). The study suggests that the basic hydrolysis of phosphodiesters occurs through a concerted ANDN mechanism, with no formation of penta-coordinated species as reaction intermediates. Conclusions: the presented approach, despite the approximations, is potentially applicable to a large number of bimolecular transformations in solution and therefore leads the way to a fast and general method to predict the rate constants and reactivities/selectivities in complex environments

an explorative survey

Biobanks, which contain human biological samples and/or data, provide a crucial contribution to the progress of biomedical research. However, the effective and efficient use of biobank resources depends on their accessibility. In fact, making bio-resources promptly accessible to everybody may increase the benefits for society. Furthermore, optimizing their use and ensuring their quality will promote scientific creativity and, in general, contribute to the progress of bio-medical research. Although this has become a rather common belief, several laboratories are still secretive and continue to withhold samples and data. In this study, we conducted a questionnaire-based survey in order to investigate sample and data accessibility in research biobanks operating all over the world. The survey involved a total of 46 biobanks. Most of them gave permission to access their samples (95.7%) and data (85.4%), but free and unconditioned accessibility seemed not to be common practice. The analysis of the guidelines regarding the accessibility to resources of the biobanks that responded to the survey highlights three issues: (i) the request for applicants to explain what they would like to do with the resources requested; (ii) the role of funding, public or private, in the establishment of fruitful collaborations between biobanks and research labs; (iii) the request of co-authorship in order to give access to their data. These results suggest that economic and academic aspects are involved in determining the extent of sample and data sharing stored in biobanks. As a second step of this study, we investigated the reasons behind the high diversity of requirements to access biobank resources. The analysis of informative answers suggested that the different modalities of resource accessibility seem to be largely influenced by both social context and legislation of the countries where the biobanks operate

Higher levels of osteoprotegerin and immune activation/immunosenescence markers are correlated with concomitant bone and endovascular damage in HIV-suppressed patients

HIV-infected patients appear to have a significantly greater risk of non-AIDS comorbidities such as osteoporosis and atherosclerosis. Subjects with osteoporosis are at a higher risk of developing cardiovascular disease than those with normal bone mass, therefore a possible relation between these two conditions can be hypothesized. In the setting of HIV infection, several factors might contribute to bone disease and endothelial dysfunction. The aim of our study was to evaluate the relationship between bone and cardiovascular disease and to investigate the role of traditional factors, T-cell phenotype and osteoprotegerin in HIV positive subjects on effective antiretroviral therapy. We included 94 HIV positive subjects on antiretroviral therapy with virological suppression and 41 healthy subjects matched for age and gender as a control group. Carotid-Intima Media Thickness (c-IMT) and bone mineral density (BMD) were performed by ultrasound and DEXA, respectively. CD4+/CD8+ T-cell activation, senescence and osteoprotegerin plasma levels were measured by flow-cytometry and ELISA, respectively. Among HIV positive patients, 56.4% had osteopenia/osteoporosis and 45.7% had pathological c-IMT (>0.9mm). Subjects with pathological c-IMT and BMD exhibited higher CD4+ and CD8+ activated, CD8+ senescent and osteoprotegerin than subjects with normal c-IMT and BMD. HIV positive subjects with osteopenia/osteoporosis had higher c-IMT than subjects with normal BMD, and linear regression analysis showed a negative correlation between BMD and c-IMT. Several factors are implicated in the pathogenesis of non-AIDS comorbidities in HIV positive patients. Osteoprotegerin together with inflammation and immunosenescence in HIV positive patients could affect bone and vascular system and could be considered as a possible common link between these two diseases

Molecular dynamics study of naturally existing cavity couplings in proteins

Couplings between protein sub-structures are a common property of protein dynamics. Some of these couplings are especially interesting since they relate to function and its regulation. In this article we have studied the case of cavity couplings because cavities can host functional sites, allosteric sites, and are the locus of interactions with the cell milieu. We have divided this problem into two parts. In the first part, we have explored the presence of cavity couplings in the natural dynamics of 75 proteins, using 20 ns molecular dynamics simulations. For each of these proteins, we have obtained two trajectories around their native state. After applying a stringent filtering procedure, we found significant cavity correlations in 60% of the proteins. We analyze and discuss the structure origins of these correlations, including neighbourhood, cavity distance, etc. In the second part of our study, we have used longer simulations (≥100ns) from the MoDEL project, to obtain a broader view of cavity couplings, particularly about their dependence on time. Using moving window computations we explored the fluctuations of cavity couplings along time, finding that these couplings could fluctuate substantially during the trajectory, reaching in several cases correlations above 0.25/0.5. In summary, we describe the structural origin and the variations with time of cavity couplings. We complete our work with a brief discussion of the biological implications of these results

Non-specific protein-DNA interactions control I-CreI target binding and cleavage

Homing endonucleases represent protein scaffolds that provide powerful tools for genome manipulation, as these enzymes possess a very low frequency of DNA cleavage in eukaryotic genomes due to their high specificity. The basis of protein-DNA recognition must be understood to generate tailored enzymes that target the DNA at sites of interest. Protein-DNA interaction engineering of homing endonucleases has demonstrated the potential of these approaches to create new specific instruments to target genes for inactivation or repair. Protein-DNA interface studies have been focused mostly on specific contacts between amino acid side chains and bases to redesign the binding interface. However, it has been shown that 4 bp in the central DNA sequence of the 22-bp substrate of a homing endonuclease (I-CreI), which do not show specific protein-DNA interactions, is not devoid of content information. Here, we analyze the mechanism of target discrimination in this substrate region by the I-CreI protein, determining how it can occur independently of the specific protein-DNA interactions. Our data suggest the important role of indirect readout in this substrate region, opening the possibility for a fully rational search of new target sequences, thus improving the development of redesigned enzymes for therapeutic and biotechnological applications

Understanding the indirect DNA read-out specificity of I-CreI Meganuclease

The high DNA specificity of homing endonucleases makes them a powerful protein scaffold to engineer enzymes for genome manipulation. Understanding their molecular recognition of DNA is an important prerequisite to generate engineered enzymes able to cleave DNA in specific desired genome sites. Protein-DNA recognition studies have been mostly focused on specific direct contacts between amino acid side chains and bases to redesign the binding interface. However, the important role of indirect readout in the central region of the target DNA of the homing endonuclease I-CreI suggested that indirect readout may play a key role in the redesign of protein-DNA interactions. The sequences of the I-CreI central substrate region, 2NN, along with the adjacent 5NNN, are key for substrate cleavage. Here, we analyse the mechanism of target discrimination at the 5NNN region by the I-CreI protein, revealing its critical role in the location and occupancy of the catalytic metal ions, which is crucial for cleavage. Our data highlight the importance of indirect readout for target DNA cleavage, thus aiding I-CreI engineering when targeting new DNA sequences.e thank the Swiss Light Source (SLS) and ALBA beamline staff for their support. This work was supported by the Spanish MINECO (JCI-2011-09308 to R.M and CTQ2017-83810-R to F.J.B.), the Severo Ochoa Excellence Accreditation (SEV-2016-0644) and the Novo Nordisk Foundation (Grant NNF14CC0001 to G.M.),S

Exploiting Reaction-Diffusion Conditions to Trigger Pathway Complexity in the Growth of a MOF

Coordination polymers (CPs), including metal-organic frameworks (MOFs), are crystalline materials with promising applications in electronics, magnetism, catalysis, and gas storage/separation. However, the mechanisms and pathways underlying their formation remain largely undisclosed. Herein, we demonstrate that diffusion-controlled mixing of reagents at the very early stages of the crystallization process (i.e., within ≈40 ms), achieved by using continuous-flow microfluidic devices, can be used to enable novel crystallization pathways of a prototypical spin-crossover MOF towards its thermodynamic product. In particular, two distinct and unprecedented nucleation-growth pathways were experimentally observed when crystallization was triggered under microfluidic mixing. Full-atom molecular dynamics simulations also confirm the occurrence of these two distinct pathways during crystal growth. In sharp contrast, a crystallization by particle attachment was observed under bulk (turbulent) mixing. These unprecedented results provide a sound basis for understanding the growth of CPs and open up new avenues for the engineering of porous materials by using out-of-equilibrium conditions