Quantifying Positional Isomers (QPI) by Top-Down Mass Spectrometry.

Proteomics has exposed a plethora of posttranslational modifications, but demonstrating functional relevance requires new approaches. Top-down proteomics of intact proteins has the potential to fully characterize protein modifications in terms of amount, site(s), and the order in which they are deposited on the protein; information that so far has been elusive to extract by shotgun proteomics. Data acquisition and analysis of intact multimodified proteins have however been a major challenge, in particular for positional isomers that carry the same number of modifications at different sites. Solutions were previously proposed to extract this information from fragmentation spectra, but these have so far mainly been limited to peptides and have entailed a large degree of manual interpretation. Here, we apply high-resolution Orbitrap fusion top-down analyses in combination with bioinformatics approaches to attempt to characterize multiple modified proteins and quantify positional isomers. Automated covalent fragment ion type definition, detection of mass precision and accuracy, and extensive use of replicate spectra increase sequence coverage and drive down false fragment assignments from 10% to 1.5%. Such improved performance in fragment assignment is key to localize and quantify modifications from fragment spectra. The method is tested by investigating positional isomers of Ubiquitin mixed in known concentrations, which results in quantification of high ratios at very low standard errors of the mean (<5%), as well as with synthetic phosphorylated peptides. Application to multiphosphorylated Bora provides an estimation of the so far unknown stoichiometry of the known set of phosphosites and uncovers new sites from hyperphosphorylated Bora.

To Cleave or Not To Cleave in XL-MS?

Cross-linking mass spectrometry (XL-MS) is an efficient technique for uncovering structural features and interactions of the in-solution state of the proteins under investigation. Distance constraints obtained by this technique are highly complementary to classical structural biology approaches like X-ray crystallography and cryo-EM and have successfully been leveraged to shed light on protein structures of increasing size and complexity. To accomplish this, small reagents are used that typically incorporate two amine reactive moieties connected by a spacer arm and that can be applied in solution to protein structures of any size. Over the years, many reagents initially developed for different applications were adopted, and others were specifically developed for XL-MS. This has resulted in a vast array of options, making it difficult to make the right choice for specific experiments. Here, we delve into the previous decade of published XL-MS literature to uncover which workflows have been predominantly applied. We focus on application papers as these represent proof that biologically valid results can be extracted. This ignores some more recent approaches that did not have sufficient time to become more widely applied, for which we supply a separate discussion. From our selection, we extract information on the types of samples, cross-linking reagent, prefractionation, instruments, and data analysis, to highlight widely used workflows. All of the results are summarized in an easy-to-use flow chart defined by selection points resulting from our analysis. Although potentially biased by our own experiences, we expect this overview to be useful for novices stepping into this rapidly expanding field.

Cell Colonization Ability of a Commercialized Large Porous Alveolar Scaffold.

The use of filling biomaterials or tissue-engineered large bone implant-coupling biocompatible materials and human bone marrow mesenchymal stromal cells seems to be a promising approach to treat critical-sized bone defects. However, the cellular seeding onto and into large porous scaffolds still remains challenging since this process highly depends on the porous microstructure. Indeed, the cells may mainly colonize the periphery of the scaffold, leaving its volume almost free of cells. In this study, we carry out an in vitro study to analyze the ability of a commercialized scaffold to be in vivo colonized by cells. We investigate the influence of various physical parameters on the seeding efficiency of a perfusion seeding protocol using large manufactured bone substitutes. The present study shows that the velocity of the perfusion fluid and the initial cell density seem to impact the seeding results and to have a negative effect on the cellular viability, whereas the duration of the fluid perfusion and the nature of the flow (steady versus pulsed) did not show any influence on either the fraction of seeded cells or the cellular viability rate. However, the cellular repartition after seeding remains highly heterogeneous.

A correlational study between signature, writing abilities and decision-making capacity among people with initial cognitive impairment.

BACKGROUND: Some clinical conditions, including dementia, compromise cognitive functions involved in decision-making processes, with repercussions on the ability to subscribe a will. Because of the increasing number of aged people with cognitive impairment there is an acute and growing need for decision-making capacity evidence-based assessment. AIMS: Our study investigates the relationship between writing abilities and cognitive integrity to see if it is possible to make inferences on decision-making capacity through handwriting analysis. We also investigated the relationship between signature ability and cognitive integrity. METHODS: Thirty-six participants with diagnosis of MCI and 38 participants with diagnosis of initial dementia were recruited. For each subject we collected two samples of signature-an actual and a previous one-and an extract of spontaneous writing. Furthermore, we administered a neuropsychological battery to investigate cognitive functions involved in decision-making. RESULTS: We found significant correlations between spontaneous writing indexes and neuropsychological test results. Nonetheless, the index of signature deterioration does not correlate with the level of cognitive decline. DISCUSSION: Our results suggest that a careful analysis of spontaneous writing can be useful to make inferences on decision-making capacity, whereas great caution should be taken in attributing validity to handwritten signature of subjects with MCI or dementia. CONCLUSIONS: The analysis of spontaneous writing can be a reliable aid in cases of retrospective evaluation of cognitive integrity. On the other side, the ability to sign is not an index of cognitive integrity.

A fine structural modification of glycosaminoglycans is correlated with the progression of muscle regeneration after ischaemia: towards a matrix-based therapy?

Critical limb ischaemia often leads to amputation of the limb and potential mortality. Moreover, there are still significant problems with current therapeutic treatments, according to poor revascularisation of degenerated tissue probably due to modifications within the microenvironment. This study is focused on the changes of structure and bioactivity of glycosaminoglycans (GAGs), especially heparan sulphate (HS) and chondroitin sulphate (CS) in rat Extensor Digitorum Longus (EDL) muscle after ischaemia. Male Wistar rats were subjected to ischaemic-injury by ligation of the neurovascular trunk accompanying EDL-tendon. After 4, 8, 15, 21, 60 and 90 d, the rats were sacrificed and the muscles were collected and submitted to histological, biochemical and gene expression assays. We demonstrated that ischaemia induced modification of expression of enzymes involved in GAG biosynthesis which correlated with significant changes in HS and CS structural features such as size and sulphation pattern. These major structural changes are associated to modifications of GAG abilities to bind growth factors and to modulate cell activity. Moreover, a CS hallmark of injury is maintained as well after the regeneration process. Finally, we showed the relevance of the role of this glycanic matrix remodelling, since a GAG mimetic treatment accelerated muscle repair after ischaemia.

Forearm post-traumatic deformities: classification and treatment.

INTRODUCTION: There is no classification for acquired forearm deformities. A clinical-radiographic study was conducted to classify these deformities and evaluate the results. MATERIALS AND METHODS: Thirteen patients with forearm deformities following traumas or their treatment were included (11 men and two women, from 2000 to 2010). Mean age was 31 years (range 10-75 years). Initial treatment was conservative in five patients and surgical in eight patients. One segment was affected in seven patients (the radius in four patients, the ulna in three), and both segments were affected in six patients. Location assessment: 2 projections X-rays, including wrist and elbow. Deformity location: proximal, diaphisary, distal, defined with the abbreviation, in distal sense, R1, R2, R3 for the radius, and U1, U2, U3 for the ulna. Primary and secondary deformities were distinguished: secondary deformities occurred later in a different location than the primary one. Six patients were treated with plate and screws. An external fixator was used in six patients. One patient was treated with bone resection. Iliac crest bone graft was used in 10 patients, and vascularised fibula graft in one patient. RESULTS: The primary deformity affecting the radial diaphysis (R2) determined a secondary deformity in four patients: in the distal ulna (U3) with ulnocarpal dislocation in three patients and in the distal radius (R3) in one patient. Results of osteosynthesis treatment were excellent in one patient, satisfactory in four and unsatisfactory in one. External fixation was excellent in one patient and satisfactory in five. Bone resection was satisfactory in one patient. DISCUSSION: Surgical treatments with osteosynthesis are the major cause of acquired forearm deformities in adults. Location and aetiology of the deformities are essential for the surgical indication and the result. It is important to restore the length of the deformed segment, realigning the anatomical axis. X-rays enable clinicians to distinguish between primary and secondary forearm deformities. CONCLUSION: Characteristics and locations of post-traumatic deformities were identified. The major location is diaphisary and distal, the elbow is rarely affected. The functional consequence is a limitation in the range of motion of the hand. The best results are achieved with short-term treatment.

Operative treatment of tibiofibular diastasis: a comparative study between transfixation screw and reabsorbable cerclage. Preliminary result.

OBJECTIVES: The tibiofibular diastasis requires an appropriate surgical treatment in order not to run into negative results such as decreased range of motion and chronic instability. We have conducted a comparative study between the transfixation screw and a new technique based upon the reabsorbable cerclage, a less invasive technique. MATERIALS AND METHODS: We enrolled 30 patients affected by tibiotarsal distortion with an acute lesion of the syndesmosis, and we divided them in 2 groups randomly. The first group of patients (15 cases) has been treated with a tricortical, or quadricortical syndesmotic screw, and the second group of patients (15 cases) has been treated with a tibiofibular cerclage with reabsorbing wires. The evaluation of the lesions was documented through comparative radiographies of the ankle in the AP, LL and mortise projections. RESULTS: In group 1, we observed an excellent outcome in 4 patients, while in the remaining 11 cases there was evidence of alterations in the evaluated parameters. In group 2, we observed an excellent outcome in 6 patients, and only in half of the remaining cases it was found a slight alteration only when the articulation is stressed. CONCLUSIONS: The main indications for the tibiofi bular cerclage are the syndesmotic lesions not associated to fi bular fractures. The achieved results support the validity of the cerclage technique, showing evidence of advantages concerning the functional recovery. The cerclage also allows to avoid the subsequent surgery required for the screw removal. Therefore the tibiofi bular cerclage represents a valid alternative to the treatment with the syndesmotic screw.

[Implication of hyaluronic acid in normal and pathological angiogenesis. Application for cellular engineering]. / Implication de l'acide hyaluronique dans l'angiogenèse normale et pathologique, application à l'ingénierie cellulaire.

Angiogenesis is a physiological process that allows the formation of new blood vessels, either from the local vascular structures, or from circulating endothelial progenitor cells, mobilized from the bone marrow, and attracted to the neovascularization site. This mechanism is controlled by pro-angiogenic molecules. It is crucial to supply oxygen and nutrients to tissues during growth, embryonic development or tissue regeneration in response to injuries. Thus, the dermis part of the skin is highly vascularized by a dense network of small and medium arteries and of capillaries and venules. In case of injury, rapid tissue repair is possible through this vascular network. However, once the vascularization is restored in tissue repair, the process of angiogenesis is negatively regulated by anti-angiogenic molecules. Controling the balance between pro-and anti-angiogenic agents is crucial and its deregulation leads to serious disease. The extracellular matrix plays an important role in controlling angiogenesis, allowing at least, the distribution of growth factors and the regulation of endothelial cell migration. Among these matrix components, hyaluronic acid plays a major role in the mechanical properties of connective tissues in ensuring their hydration. This glycosaminoglycan is a large size polymer, whose breakdown products strongly act on angiogenesis, especially in pathological situations (cancer, inflammation). Regarding its biological and mechanical properties, hyaluronic acid is used as matrix in tissue engineering, for improving the revascularization of tissues like skin.

Insights on a new path of pre-mitochondrial apoptosis regulation by a glycosaminoglycan mimetic.

Lysosomal cathepsins have recently been reported to play crucial roles in the regulation of the mitochondrial death cascade by an unclear mechanism leading to mitochondrial membrane permeabilization. Glycosaminoglycans (GAG) are a family of ionic polysaccharides present at the lysosomal compartment and shown to inhibit lysosomal cathepsin activities. The implication of this family of polysaccharides in the regulation of the pre-mitochondrial death cascade has still not been considered. Here, we demonstrate in a model of skin fibroblasts submitted to oxidative stress that a GAG-mimetic protects the lysosome from membrane disruption, reduces intracellular ROS levels, and inhibits mitochondrial membrane potential collapse, cytochrome c release and caspases-9 and -3 activations without affecting the extrinsic pathway of apoptosis. Heparan sulfate and chondroitin sulfate, but not heparin, showed also protecting effects when assessing key points of the intrinsic pathway of apoptosis. We suggest the existence of molecular links between endogenous GAGs and the regulation of apoptosis.

Identification of a GATA-overlapping sequence within the enhancer of the murine GPIIb promoter that induces transcriptional deregulation in human K562 cells.

The human and the murine glycoprotein platelet IIb (GPIIb) promoters are megakaryocyte specific in human and murine cell systems, respectively. Here we show that the murine promoter is, however, highly active when transfected in K562 human cells in which the human promoter is almost inactive. A murine promoter, in which the enhancer element was replaced by the human, retrieves its megakaryocytic specificity in human cell lines. The human and murine GATA-binding sites located in the enhancer region display slight sequence divergence next to the consensus GATA core sequence. Gel shift experiments show that, although the murine and the human GATA sequences both bind GATA-1, the murine sequence alone forms an additional complex (B) not detected with the human sequence. When the murine GATA-containing region is replaced by the human in the context of the murine GPIIb promoter, megakaryocyte specificity is restored in the human cell lines. A G nucleotide 3 to GATA appears crucial because its substitution abrogates B but not GATA-1 binding and restores megakaryocyte specificity to the murine promoter. Conversely, substitution of the human GATA-1 binding sequence by its murine homologue that binds both GATA-1 and complex B induces an abnormal activity for the human promoter in K562 cells. Altogether, our data suggest that limited changes in the GATA-containing enhancer of the GPIIb promoter can induce the recruitment of accessory proteins that could be involved in alteration of a megakaryocyte-restricted gene activation program. (Blood. 2000;96:1348-1357)

Road traffic accidents with vehicular entrapment: incidence of major injuries and need for advanced life support.

Road traffic accidents (RTAs) with entrapment are perceived as a challenge to emergency systems because of the severity of the ensuing traumas and the inherent complexity of the rescue procedures. To clarify these two aspects this prospective cohort study enrolling 244 entrapped trauma patients was conducted by a Regional Medical Helicopter Service. Forty-six victims (18.9%) were found dead, 101 (51%) of the 198 patients who reached the hospital alive had an injury severity score (ISS) > or = 16. The use of seat belts was associated with lower trauma severity. Out of the 101 severely traumatized patients (ISS > or = 16), 46 (42.6%) were intubated at road side, 12 required decompression of a tension pneumothorax on the scene and in 15 cases a pneumothorax was drained during the early intrahospital phase. Thirty-six (34.7%) patients had the first systolic blood pressure (SBP) < or = 90 mmHg and were then aggressively infused: in 75% of these cases, the SBP on arrival at the emergency department increased. The first SBP was significantly correlated with mortality. There was no correlation of extrication time, total rescue time and mortality. Fourteen patients (13.9%) died during hospitalization. These data demonstrate that a high percentage of entrapped patients require advanced life support (ALS), including on scene intubation and chest decompression. Aggressive field resuscitation and immediate transport to a level 1 trauma centre is associated with a mortality lower than that predicted by TRISS in spite of the prolonged prehospital time.

Phase I clinical and pharmacologic study of weekly cisplatin combined with weekly irinotecan in patients with advanced solid tumors.

PURPOSE: In vitro synergy between cisplatin and irinotecan (CPT-11) has been reported. We designed a combination schedule of these agents to maximize the potential for synergistic interaction. PATIENTS AND METHODS: To maximize the opportunity for synergy, we divided the cisplatin into four consecutive weekly treatments, followed by a 2-week rest. Each dose of cisplatin was immediately followed by a dose of irinotecan. The dose of cisplatin was fixed at 30 mg/m2/wk. The initial irinotecan dose was 50 mg/m2/wk and this was escalated by 30% increments in successive cohorts of three to six patients to establish the maximum-tolerated dose (MTD). Pharmacokinetics of irinotecan and its metabolites, SN-38 and SN-38 glucuronide (SN-38G), were analyzed. RESULTS: Of 35 patients with solid tumors enrolled onto this trial, 30 were assessable for toxicity and response. The MTD for this regimen was 30 mg/m2/wk of cisplatin plus 50 mg/m2/wk of irinotecan in previously treated patients and 30 mg/m2/wk of cisplatin plus 65 mg/m2/wk of irinotecan in chemotherapy-naive patients. Neutropenia was the dose-limiting toxicity (DLT) encountered in this trial. Diarrhea was infrequent and rarely dose-limiting. Seven of 30 assessable patients achieved a partial response. No alteration in irinotecan, SN-38, or SN-38G pharmacokinetics resulted from the administration of cisplatin with irinotecan. CONCLUSION: The administration of cisplatin and irinotecan on this weekly schedule provides a practical and well-tolerated regimen that has the potential to maximize any clinical synergy between the two agents. Evidence of substantial clinical activity was seen in this phase I study.

The formation and change of fertility preferences: a case study of the generational change in the size of the Italian family in the United States.

PIP: The process of fertility preference formation and change is examined from the psychological angle, using the example of changes over time in the size of families of Italian origin in the United States. The relationship between psychological and economic factors in this process of change is considered. The author develops a theory of personality development in which these changes in fertility preference can be seen as an outgrowth of adaptation and cultural change over time.^ieng