Nanoparticle-based capillary electrochromatography

In this work, lipid nanoparticles were used as pseudostationary phase, PSP, for protein separation in capillary electrochromatography, CEC. The aim of the work was to use a new analytical separation technique for future analysis of protein drugs by using lipid nanoparticles as a pseudo-stationary phase with conventional UV-detection. PSP-CEC was combined with UV-detection to study proteins elution. The tested proteins BSA, cytochrome c and insulin have been selected according to different hydrophobicities, sizes and isoelectric points (pI). A zwitter ionic buffer, MOPS, was used as an electrolyte at pH 7.4. The nanoparticles were tested for their compatibility with MALDI-TOF/MS (Matrix assisted laser desorption/ionization-time of flight). MADI-TOF/MS works with a suspension of lipid nanoparticles in MOPS that contains insulin and it can be a future possible detection method for PSP-CEC. All three proteins were successfully eluted with PSP-CEC combined with UV-detection. Nanoparticles coated with silver might be studied with “surface enhanced Raman spectroscopy”-detection.Popular Science Summery Capillary electrophoresis, CE is a separation technique in which analytes are separated according to their size to charge ratio in the interior of the capillary. Capillary electrochromatography, CEC is a combination of CE and HPLC. The fundamental difference between CE and CEC is that the separation is not based on the electrophoretic mobility difference only but on the interaction with the stationary phase as well. Lipid nanoparticles were used as pseudostationary phase (moving phase). The aim of current work is to separate different proteins, BSA, cytochrome c and insulin have been separated according to their charge and size. They have been successfully separated. It was found that PSP-CEC in combination with UV-detection is a good methodology to elute Proteins. An identification technique named MALDI-TOF/MS was used to identify the insulin that has been dissolved in nanoparticles. Insulin has been successfully identified. MADI-TOF/MS works well with lipid nanoparticles and it can be used as a future possible detection of PSP-CEC

Proteome changes in autosomal recessive primary microcephaly

Background/aim: : Autosomal recessive primary microcephaly (MCPH) is a rare and genetically heterogeneous group of disorders characterized by intellectual disability and microcephaly at birth, classically without further organ involvement. MCPH3 is caused by biallelic variants in the cyclin-dependent kinase 5 regulatory subunit-associated protein 2 gene CDK5RAP2. In the corresponding Cdk5rap2 mutant or Hertwig's anemia mouse model, congenital microcephaly as well as defects in the hematopoietic system, germ cells and eyes have been reported. The reduction in brain volume, particularly affecting gray matter, has been attributed mainly to disturbances in the proliferation and survival of early neuronal progenitors. In addition, defects in dendritic development and synaptogenesis exist that affect the excitation-inhibition balance. Here, we studied proteomic changes in cerebral cortices of Cdk5rap2 mutant mice. Material and methods: : We used large-gel two-dimensional gel (2-DE) electrophoresis to separate cortical proteins. 2-DE gels were visualized by a trained observer on a light box. Spot changes were considered with respect to presence/absence, quantitative variation and altered mobility. Result: : We identified a reduction in more than 30 proteins that play a role in processes such as cell cytoskeleton dynamics, cell cycle progression, ciliary functions and apoptosis. These proteome changes in the MCPH3 model can be associated with various functional and morphological alterations of the developing brain. Conclusion: : Our results shed light on potential protein candidates for the disease-associated phenotype reported in MCPH3

MMP-13 substrate specificity in cartilage breakdown

MMP13 (Collagenase 3) is a member of the matrix metalloproteinase (MMP) family. In pathology it is overexpressed in rheumatoid arthritis (RA), osteoarthritis (OA) and human carcinomas. It is secreted in its inactive proforms from which it can be activated. The project studies how MMP13 can degrade/digest the normal femoral head human articular cartilage. Peptides were separated using reversed phase chromatography coupled on-line with various mass spectrometry techniques including ion trap, quadropole Time-Of-Flight (Q-TOF) and triple quadropole (QQQ) instruments. Guanidine hydrochloride (GuHCl) was used to extract proteins from the cartilage tissue. Sodium dodecyl sulfate poly acrylamide gel electrophoresis was used also to give visualize similarities and differences between the control and the MMP13 treated sample. MMP13 showed an effect on both media (released proteins from the cartilage tissue via the buffer solution) and a little effect on the cartilage tissue (pellet). The main result showed that the tissue sample preparation was critical in order to obtain sufficient release of proteins. The powderisation of tissue was much better in releasing proteins than intact tissue plugs probably due to larger contact area and shorter diffusion distance

Synthesis and characterization of transition metal carbonyl clusters with chiral ligands and their application in asymmetric catalysis

Triruthenium dodecacarbonyl was reacted with the non chiral ligand PSSP as well as chiral ferrocenyl diphosphines of the Josiphos (J008, J006 and J015) and Walphos (W009) families to give [Ru3(CO)10(µ-1,2-P-P)], (P-P = W009 1, J008 2, J006 3, J015 4 and PSSP 5. The X-ray structure of 3 shows that a Ru-Ru edge is bridged by a diphosphine ligand in an equatorial coordination mode. The corresponding clusters with the ligand in a chelating coordination mode were also obtained as minor products. The tetraruthenium cluster [H2Ru4(CO)13] was reacted with chiral diphosphine ligands of the Josiphos family (J007 and J015). Four products were isolated: [H2Ru4(CO)11(µ-1,2-J007)] 6 and [H2Ru4(CO)11(µ-1,2-J015)] 7, in which the diphosphine coordinates in a bridging mode, as well as [H2Ru4(CO)11(µ-1,1-J007)] 8 and [H2Ru4(CO)11(µ-1,1-J015)] 9, where the diphosphine coordinates in a chelating mode. Clusters 6 and 7 were found to give high conversion and good enantiomeric excess for the hydrogenation of tiglic acid. IR spectroscopy indicates that 6 is a precursor to the active catalyst, while 8 may be an active catalyst

Redox Proteomes in Human Physiology and Disease Mechanisms

Redox proteomics is a field of proteomics that is concerned with the characterization of the oxidation state of proteins to gain information about their modulated structure, function, activity, and involvement in different physiological pathways. Oxidative modifications of proteins have been shown to be implicated in normal physiological processes of cells as well as in pathomechanisms leading to the development of cancer, diabetes, neurodegenerative diseases, and some rare hereditary metabolic diseases, like classic galactosemia. Reactive oxygen species generate a variety of reversible and irreversible modifications in amino acid residue side chains and within the protein backbone. These oxidative post-translational modifications (Ox-PTMs) can participate in the activation of signal transduction pathways and mediate the toxicity of harmful oxidants. Thus the application of advanced redox proteomics technologies is important for gaining insights into molecular mechanisms of diseases. Mass-spectrometry-based proteomics is one of the most powerful methods that can be used to give detailed qualitative and quantitative information on protein modifications and allows us to characterize redox proteomes associated with diseases. This Review illustrates the role and biological consequences of Ox-PTMs under basal and oxidative stress conditions by focusing on protein carbonylation and S-glutathionylation, two abundant modifications with an impact on cellular pathways that have been intensively studied during the past decade

Pancreatic surgery outcomes: multicentre prospective snapshot study in 67 countries

Background: Pancreatic surgery remains associated with high morbidity rates. Although postoperative mortality appears to have improved with specialization, the outcomes reported in the literature reflect the activity of highly specialized centres. The aim of this study was to evaluate the outcomes following pancreatic surgery worldwide.Methods: This was an international, prospective, multicentre, cross-sectional snapshot study of consecutive patients undergoing pancreatic operations worldwide in a 3-month interval in 2021. The primary outcome was postoperative mortality within 90 days of surgery. Multivariable logistic regression was used to explore relationships with Human Development Index (HDI) and other parameters.Results: A total of 4223 patients from 67 countries were analysed. A complication of any severity was detected in 68.7 percent of patients (2901 of 4223). Major complication rates (Clavien-Dindo grade at least IIIa) were 24, 18, and 27 percent, and mortality rates were 10, 5, and 5 per cent in low-to-middle-, high-, and very high-HDI countries respectively. The 90-day postoperative mortality rate was 5.4 per cent (229 of 4223) overall, but was significantly higher in the low-to-middle-HDI group (adjusted OR 2.88, 95 per cent c.i. 1.80 to 4.48). The overall failure-to-rescue rate was 21 percent; however, it was 41 per cent in low-to-middle-compared with 19 per cent in very high-HDI countries.Conclusion: Excess mortality in low-to-middle-HDI countries could be attributable to failure to rescue of patients from severe complications. The authors call for a collaborative response from international and regional associations of pancreatic surgeons to address management related to death from postoperative complications to tackle the global disparities in the outcomes of pancreatic surgery (NCT04652271; ISRCTN95140761)