Native Plasma Research Articles

A Novel Broad-Spectrum Elastase-Like Serine Protease From the Predatory Bacterium Bdellovibrio bacteriovorus Facilitates Elucidation of Site-Specific IgA Glycosylation Pattern.

The increased interest in predatory bacteria due to their ability to kill antibiotic resistant bacteria has also highlighted their inherent plethora of hydrolytic enzymes, and their potential as natural sources of novel therapeutic agents and biotechnological tools. Here, we have identified and characterized a novel protease from the predatory bacterium Bdellovibrio bacteriovorus: BspE (Bdellovibrio elastase-like serine protease). Mapping preferential sites of proteolytic activity showed a single proteolytic cleavage site of native plasma IgA (pIgA) in the Fc-tail; as well as in the secretory component (SC) of secretory IgA (SIgA). Proteolysis of other native immunoglobulins and plasma proteins was either absent (IgG1 and 2, IgM, albumin and orosomucoid) or unspecific with multiple cleavage sites (IgG3 and 4, IgE, IgD). BspE displayed a broad activity against most amino acid bonds in shorter peptides and denatured proteins, with a slight preference for hydrolysis C-terminal of Y, V, F, S, L, R, P, E, and K. BspE autoproteolysis results in numerous cleavage products sustaining activity for more than 6 h. The enzymatic activity remained stable at pH 5.0–9.0 but was drastically reduced in the presence of MnCl2 and completely inhibited by ZnCl2. The hydrolysis of pIgA was subsequently utilized for the specific glycan characterization of the released pIgA Fc-tail (Asn459). Besides contributing to the basic knowledge of Bdellovibrio biology and proteases, we propose that BspE could be used as a potential tool to investigate the importance, and biological function of the pIgA Fc-tail.IMPORTANCEAntibodies are well-established as key components of the immune system, and the importance of antibody glycosylation is steadily gaining recognition. Modifications of antibodies by glycosylation creates a vast repertoire of antibody glycovariants with distinctive and diverse functions in the immune system. Most of the available information regarding antibody glycosylation is based on studies with IgG, which have contributed greatly to the advance of therapeutic antibody treatments. However, much is still unknown regarding the importance of glycosylation and the Fc-structure for the remaining antibody classes. Such research has proven to be technically challenging and demonstrates a need for novel tools to facilitate such investigations. Here we have identified and characterized a novel protease from B. bacteriovorus, facilitating the study of plasma IgA by cleaving the Fc-tail, including the Asn459 N-glycan. This further highlights the potential of B. bacteriovorus as a source to identify potential novel biotechnological tools.

Use of virus-like particles as a native membrane model to study the interaction of insulin with the insulin receptor

There is emerging evidence of the utility of virus-like particles (VLPs) as a novel model for the study of receptor-ligand interactions in a native plasma membrane environment. VLPs consist of a viral core protein encapsulated by portions of the cell membrane with membrane proteins and receptors expressed in their native conformation. VLPs can be generated in mammalian cells by transfection with the retroviral core protein (gag). In this study, we used Chinese hamster ovary (CHO T10) cells stably overexpressing the insulin receptor (IR) to generate IR bearing VLPs. The diameter and size uniformity of VLPs were estimated by dynamic light scattering and morphological features examined by scanning electron microscopy. The presence of high affinity IR on VLPs was demonstrated by competitive binding assays (KD: 2.3 ± 0.4 nM, n = 3), which was similar to that on the parental CHO T10 cells (KD: 2.1 ± 0.4 nM, n = 3). We also report that increases or decreases in membrane cholesterol content by treatment with methyl-β-cyclodextrin (MBCD) or cholesterol pre-loaded methyl-β-cyclodextrin (cMBCD), respectively, substantially decreased insulin binding (> 30%) to both VLPs and cells, and we speculate this is due to a change in receptor disposition. We suggest that this novel finding of decreases in insulin binding in response to changes in membrane cholesterol content may largely account for the unexplained decreases in insulin signalling events previously reported elsewhere. Finally, we propose VLPs as a viable membrane model for the study of insulin-IR interactions in a native membrane environment.

Hypersensitivity transfusion reactions to platelet concentrate: a retrospective analysis of the French hemovigilance network.

Among labile blood products, platelet concentrates (PCs) are the leading cause of hypersensitivity transfusion reactions (HTRs). These reactions often lead to interruption of PC transfusion and can result in a prolonged transfusion process leading to significant morbidity and use of premedication and close monitoring for patients with a history of allergic transfusion reactions. The French hemovigilance database is one of the largest standardized databases providing information on HTRs following administration of labile blood products. In this study, we analyzed this database to assess the relative risk of HTR for each type of PC. HTRs following PC transfusion were retrospectively extracted from the e-Fit Hemovigilance database of the French National Agency for Medicines and Health Products Safety (ANSM). Frequencies were calculated using the number of specific PCs transfused. Between 2008 and 2014, the overall estimated incidence of HTRs following PC administration was calculated at 232 HTRs per 100,000 PCs transfused. The rate of HTRs was significantly higher with apheresis PC (337/100,000) than with buffy-coat PC (94/100,000). Platelets in additive solutions (PAS) were associated with a significantly lower frequency of HTRs when compared with PCs in native plasma. Amotosalen/UVA- PCs (APCs and BCPCs) which are always in PAS in France, exhibited the lowest frequency of HTRs when compared with their corresponding PCs in native plasma or in PAS (p < 10-7 in all comparisons). Our results showed that the type of PC and its processing may have an impact on the risk of HTR.

Lateral distribution of phosphatidylinositol 4,5-bisphosphate in membranes regulates formin- and ARP2/3-mediated actin nucleation

Spatial and temporal control of actin polymerization is fundamental for many cellular processes, including cell migration, division, vesicle trafficking, and response to agonists. Many actin-regulatory proteins interact with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and are either activated or inactivated by local PI(4,5)P2 concentrations that form transiently at the cytoplasmic face of cell membranes. The molecular mechanisms of these interactions and how the dozens of PI(4,5)P2-sensitive actin-binding proteins are selectively recruited to membrane PI(4,5)P2 pools remains undefined. Using a combination of biochemical, imaging, and cell biologic studies, combined with molecular dynamics and analytical theory, we test the hypothesis that the lateral distribution of PI(4,5)P2 within lipid membranes and native plasma membranes alters the capacity of PI(4,5)P2 to nucleate actin assembly in brain and neutrophil extracts and show that activities of formins and the Arp2/3 complex respond to PI(4,5)P2 lateral distribution. Simulations and analytical theory show that cholesterol promotes the cooperative interaction of formins with multiple PI(4,5)P2 headgroups in the membrane to initiate actin nucleation. Masking PI(4,5)P2 with neomycin or disrupting PI(4,5)P2 domains in the plasma membrane by removing cholesterol decreases the ability of these membranes to nucleate actin assembly in cytoplasmic extracts.

Immunoregulatory properties of cell-free DNA in plasma of celiac disease patients – A pilot study

The elevated plasma cell-free DNA (cfDNA) concentrations were repeatedly reported in association with the process of inflammation. The qualitative and quantitative characteristics of plasma cfDNA in active (newly diagnosed) celiac disease patients (CD) have not yet been studied despite the fact that cfDNA of healthy individuals is able to regulate immune response. We determined the total cfDNA concentration and relative content of telomeric sequences in plasma cfDNA in CD (n = 10) and healthy age- and sex-matched controls (HC, n = 10) by quantitative PCR. To obtain the evidence that the observed biological effects are caused solely by cfDNA molecules, we applied the treatment of paired plasma samples with DNase. Using paired samples of plasma (non-treated/native and treated by DNase), we analyzed the contribution of cfDNA to the activation of TLR9 and TNF-α mRNA expression in THP1 monocytic cell line. There were no significant differences in the quantities of plasma cfDNA and relative contents of telomeric sequences in their pools. When we compared the levels of TNF-α mRNA expression in THP1 cells achieved after stimulation with native CD and HC plasma samples, we found significantly (p = .031) higher expression after stimulation with CD samples. We documented also the ability of cfDNA contained in CD plasma samples to stimulate the production of TLR9 mRNA. The TLR9 mRNA expression levels were significantly (p = .014) lowered after cfDNA removal from CD plasma samples. The design of our experiments allowed us to study the effects of cfDNA without its isolation from plasma. cfDNA contained in CD plasma samples differs significantly in its immunoregulatory capacity from cfDNA in HC plasma. The differences are caused neither by different concentrations of cfDNA in plasma samples nor by different relative abundance of telomeric sequences. Further studies are needed to elucidate the role of plasma cfDNA in celiac disease pathogenesis.

Early Stage Alpha-Synuclein Amyloid Fibrils are Reservoirs of Membrane-Binding Species

The presence of αSN fibrils indisputably associates with the development of synucleinopathies. However, while certain fibril morphologies have been linked to downstream pathological phenotypes, others appear less harmful, leading to the concept of fibril strains, originally described in relation to prion disease. Indeed, the presence of fibrils does not associate directly with neurotoxicity. Rather, it has been suggested that the toxic compounds are soluble amyloidogenic oligomers, potentially co-existing with fibrils. Here, combining synchrotron radiation circular dichroism, transmission electron microscopy and binding assays on native plasma membrane sheets, we reveal distinct biological and biophysical differences between initial and matured fibrils, transformed within the timespan of few days. Immature fibrils are reservoirs of membrane-binding species, which in response to even gentle experimental changes release into solution in a reversible manner. In contrast, mature fibrils, albeit macroscopically indistinguishable from their less mature counterparts, are structurally robust, shielding the solution from the membrane active soluble species. We thus show that particular biological activity resides transiently with the fibrillating sample, distinct for one, but not the other, spontaneously formed fibril polymorph. These results shed new light on the principles of fibril polymorphism with consequent impact on future design of assays and therapeutic development.

Molecular Simulations Reveal the Dynamics of the Band 3 Anion Transporter in a Model Native Red Blood Cell Membrane

Band 3, the red blood cell anion exchanger (AE1/SLC4A1), is responsible for the rapid transport of bicarbonate and chloride across the red blood cell plasma membrane, a process necessary for efficient respiration. Human Band 3 is comprised of a cytosolic domain and a membrane domain that contains 14 transmembrane helices. Although structural data are available for both isolated domains, the structure of the complete Band 3 remains elusive. This is a limiting factor in the study of Band 3 anion transport and of its interactions with cytosolic proteins. By integrating molecular modelling and molecular dynamics simulations at the coarse-grained and all-atom resolutions, we have constructed a model of Band 3 that consists of both the transmembrane and the cytosolic domains. This model enabled us to identify the orientation of the cytosolic domain relative to the transmembrane domain and the role of the linker regions that connect these two domains in their interactions. Our model was validated using functional data and molecular simulations. Simulations were performed in complex bilayers that resemble the native red cell plasma membrane, containing a full complement of phospholipids, sphingomyelin and cholesterol. Our results provide novel molecular insights into the interactions of the lipid environment with Band 3. Specific lipids, e.g. cholesterol, have been found to localize in the Band 3 dimer interface possibly stabilizing the dimer. Specific lipid head groups were also found to interact with the cytosolic domain regulating its orientation relative to the transmembrane domain. Moreover, our modeling approach identified key residues involved in anion binding. Understanding the dynamics and interactions of Band 3 in a model native red cell membrane provides new insights into the function of this important human transporter.

FISIK: Framework for the Inference of In Situ Interaction Kinetics from Single-Molecule Imaging Data

Receptor interactions in the plasma membrane are critical for transmembrane signal transduction. Using live-cell single-molecule imaging it is possible to monitor inter-receptor interactions with high spatiotemporal resolution in their native environment, although only for the labeled subset of receptors, which tends to be a small fraction (often <10% of the full population). As a result, it has remained a challenge to calculate receptor interaction kinetics, especially association rates, from single-molecule data. To overcome this challenge, we developed a mathematical modeling-based Framework for the Inference of in Situ Interaction Kinetics from single-molecule imaging data (termed “FISIK”). FISIK consists of (1) devising a mathematical model of receptor dynamics and interactions, mimicking the biological system and data acquisition setup, and (2) estimating the unknown model parameters, including receptor association and dissociation rates, by fitting the model to experimental single-molecule data. Due to the stochastic nature of model and data, we adapted the method of indirect inference for model calibration. To validate FISIK, we simulated trajectories of diffusing receptors that interact with each other, considering a wide range of model parameters, and including tracking errors that result in noisy data for model calibration. Our tests demonstrated that FISIK has the sensitivity to determine association and dissociation rates; its accuracy depends on the labeled fraction of receptors as well as the extent of tracking errors. For conditions where the labeled fraction was relatively low (e.g. to afford accurate tracking), we found that combining dynamic single-molecule imaging data with static localization microscopy data improves FISIK's performance. All in all, FISIK is a promising novel approach for the derivation of receptor interaction kinetics in their native plasma membrane from single-molecule data.

Application of Native Plasma to the Treatment of Knee Joint Injury

One of the most common injuries in the structure of joint injuries is trauma of the knee joint, which is largely due to its anatomical features. At the present stage of a knee joint injury treating, as a rule, is the use of surgical methods of correction. However, even surgical intervention is not a guarantee of complete recovery of such patients.The objective: to investigate the efficacy and safety of PAP as part of integrated treatment early in the post-traumatic knee joint surgery in younger patients compared with standard treatment.Materials and methods. The study was conducted on the basis of the KZ KOR «Kyiv Regional Clinical Hospital» and included 60 patients in the early post-traumatic period of the knee joint. The patients were divided into two groups. All patients included in the study received standard treatment (NSAIDs, complex physiotherapy) for 10 days. In agreement with the patients, 30 patients (Gr.1) received 3 intraarticular injections of PAP in addition to standard treatment. Within each group, the patients did not differ significantly in their age, sex, and duration of the disease. All patients included in the study had a comprehensive clinical and laboratory examination before treatment, after 2 and 4 weeks, as well as 6 and 12 months.Results. During the treatment period, the patients of both groups showed a positive result both in the general index of KOOS and in the indices of all subscales, with statistically significant differences between the groups. Moreover, the most revealing changes in each group were for the two subchalates – sport and activity on rest and assessment of quality of life. The analysis of the safety of complex therapy with the use of PAP in the form of intra-articular injections in knee joint injuries did not reveal any case of development of undesirable events, except for a temporary painful sensation at the injection site.Conclusions. The use of PAP in the treatment of knee joint injuries reduce pain, stiffness, improves the function of the knee joint, and also reduces the need for use of NSAIDs.

Provisional standardization of hepcidin assays: creating a traceability chain with a primary reference material, candidate reference method and a commutable secondary reference material.

Background Hepcidin concentrations measured by various methods differ considerably, complicating interpretation. Here, a previously identified plasma-based candidate secondary reference material (csRM) was modified into a serum-based two-leveled sRM. We validated its functionality to increase the equivalence between methods for international standardization. Methods We applied technical procedures developed by the International Consortium for Harmonization of Clinical Laboratory Results. The sRM, consisting of lyophilized serum with cryolyoprotectant, appeared commutable among nine different measurement procedures using 16 native human serum samples in a first round robin (RR1). Harmonization potential of the sRM was simulated in RR1 and evaluated in practice in RR2 among 11 measurement procedures using three native human plasma samples. Comprehensive purity analysis of a candidate primary RM (cpRM) was performed by state of the art procedures. The sRM was value assigned with an isotope dilution mass spectrometry-based candidate reference method calibrated using the certified pRM. Results The inter-assay CV without harmonization was 42.1% and 52.8% in RR1 and RR2, respectively. In RR1, simulation of harmonization with sRM resulted in an inter-assay CV of 11.0%, whereas in RR2 calibration with the material resulted in an inter-assay CV of 19.1%. Both the sRM and pRM passed international homogeneity criteria and showed long-term stability. We assigned values to the low (0.95±0.11 nmol/L) and middle concentration (3.75±0.17 nmol/L) calibrators of the sRM. Conclusions Standardization of hepcidin is possible with our sRM, which value is assigned by a pRM. We propose the implementation of this material as an international calibrator for hepcidin.

Current and Emerging Reconstituted HDL-apoA-I and HDL-apoE Approaches to Treat Atherosclerosis.

Atherosclerosis affects millions of people worldwide. However, the wide variety of limitations in the current therapeutic options leaves much to be desired in future lipid-lowering therapies. For example, although statins, which are the first-line treatment for coronary heart disease (CHD), reduce the risk of cardiovascular events in a large percentage of patients, they lead to optimal levels of low density lipoprotein-cholesterol (LDL-C) in only about one-third of patients. A new promising research direction against atherosclerosis aims to improve lipoprotein metabolism. Novel therapeutic approaches are being developed to increase the levels of functional high density lipoprotein (HDL) particles. This review aims to highlight the atheroprotective potential of the in vitro synthesized reconstituted HDL particles containing apolipoprotein E (apoE) as their sole apolipoprotein component (rHDL-apoE). For this purpose, we provide: (1) a summary of the atheroprotective properties of native plasma HDL and its apolipoprotein components, apolipoprotein A-I (apoA-I) and apoE; (2) an overview of the anti-atherogenic functions of rHDL-apoA-I and apoA-I-containing HDL, i.e., natural HDL isolated from transgenic Apoa1−/− × Apoe−/− mice overexpressing human apoA-I (HDL-apoA-I); and (3) the latest developments and therapeutic potential of HDL-apoE and rHDL-apoE. Novel rHDL formulations containing apoE could possibly present enhanced biological functions, leading to improved therapeutic efficacy against atherosclerosis.

The Use of Natural Platelet Autoplasma and Its Thermally Processed Modifications for Correction of Age-Related Changes of Skin

The article describes the results of a comparative prospective study of the results of correcting age-related changes in the skin of the face and neck in 72 patients using native platelet auto plasma and a new method for obtaining its thermally processed modifications based on the denaturation of protein plasma fractions when heated in a thermostat to different temperatures. (Patent No. 2629527/August 29, 2017.). The results obtained suggest that the use of a combination of native platelet auto plasma (PAP) with its thermally processed modifications to correct age-related changes in the skin of the face and neck is more effective than monotherapy of native PAP. The absence of complications during the observation period and the autologous nature of the material allow us to speak about the high level of safety of this method of treatment.

P1.11-13 Noninvasive Detection of Early Stage NSCLC EGFR Mutations Using Electric Field Induced Release and Measurement (EFIRM)

The ability of liquid biopsy to detect circulating tumor mutations has proved especially valuable in the treatment of patients with NSCLC since well characterized variants inform small molecule chemotherapeutic options. EFIRM is a signal amplification technology that allows direct detection of mutations in native plasma and saliva without amplification or sequencing. Previously published work demonstrated a near perfect correlation between biopsy and EFIRM results for the Exon19 del and p.L858R mutations in EGFR in late stage NSCLC patients. In this study we investigated the performance of EFIRM in patients early stage (I and II) NSCLC patients. Plasma samples were collected from 33 healthy controls with biopsy proven benign lung masses and 21 stage I and II NSLC patients with biopsy proven EGFR mutations The samples were immediately centrifuged and frozen. The samples were then blinded and sent for EFIRM analysis. There were 12 biopsies positive for p.L858R (11 stage 1 and 1 stage II) and 9 positive for Exon 19 del (7 stage 1A, and 2 stage II). Using statistically derived cut-offs to optimize sensitivity and specificity, there were 2 false positives (both for p.L858R) in the controls yielding an overall specificity of 91% for the 2 SNP Mutation assay. The sensitivity was 92% for the p.L858R with only one negative in 12 samples in a patient with stage 1 and 77% for Exon 19 del with 2 negatives in 9 samples in a single patient each for stage 1 and 2. The preponderance of patients in this study had stage I NSCLC. The sensitivity of 94% and 77% respectively for the 2 most common mutations is remarkable for patients with Stage 1 lung cancer and a correspondingly low tumor burden and more importantly at a potentially curable stage. We are in the process of improving the technical performance of this assay and adding additional variants to the panel in order to increase clinical utility. These data are promising for the potential use of the EFIRM platform for the purposes of drug selection, disease recurrence, follow-up of indeterminate lung nodules from spiral CT screening programs, and / or population screening.

Feasibility of a plasma bioassay to assess oxidative protection of low-density lipoproteins by high-density lipoproteins

Traditionally, the impact of lipoproteins on vascular disease has been evaluated in light of their quantity, that is, cholesterol content, in plasma. However, recent studies of high-density lipoproteins (HDLs) have focused on functionality with regard to atheroprotection. For example, bioassays have emerged to assess the ability of HDL, in its near native plasma environment, to promote cholesterol removal (efflux) from cells. As a result, attention has focused on developing plasma-based assays for other putative HDL protective functions including protecting low-density lipoproteins (LDLs) from oxidative damage. To determine the feasibility of such an assay in a complex sample such as plasma, we evaluated the contribution of HDL vs other plasma factors in preventing LDL oxidation. We separated normolipidemic human plasma by gel filtration chromatography and assessed each fraction for its ability to prevent LDL modification by water soluble radical and copper-initiated oxidation mechanisms. Using proteomics and selective precipitation methods, we identified major antioxidative contributions for fibrinogen, immunoglobulin G, albumin, and small soluble molecules like uric acid and ascorbate, with albumin being especially dominant in copper-initiated mechanisms. HDL particles were minor contributors (∼1%-2%) to the antioxidant capacity of plasma, irrespective of oxidation mechanism. Given the overwhelming background of antioxidant capacity inherent to highly abundant plasma proteins, specific bioassays of HDL antioxidative function will likely require its complete separation from plasma.

Emicizumab-mediated haemostatic function in patients with haemophilia A is down-regulated by activated protein C through inactivation of activated factor V.

Activated protein C (APC) inactivates activated factor V (FVa) and moderates FVIIIa by restricting FV cofactor function. Emicizumab is a humanized anti-FIXa/FX bispecific monoclonal antibody that mimicks FVIIIa cofactor function. In recent clinical trials in haemophilia A patients, once-weekly subcutaneous administration of emicizumab was remarkably effective in preventing bleeding events, but the mechanisms controlling the regulation of emicizumab-mediated haemostasis remain to be explored. We investigated the role of APC-mediated reactions in these circumstances. APC dose-dependently depressed thrombin generation (TG) initiated by emicizumab in FVIII-deficient plasmas, and in normal plasmas preincubated with an anti-FVIII antibody (FVIII-depleted). FVIIIa-independent FXa generation with emicizumab was not affected by the presence of APC, protein S and FV. The results suggested that APC-induced down-regulation of emicizumab-dependent TG was accomplished by direct inactivation of FVa. The addition of APC to emicizumab mixed with FVIII-depleted FV-deficient plasma in the presence of various concentrations of exogenous FV demonstrated similar attenuation of TG, irrespective of specific FV concentrations. Emicizumab-related TG in FVIII-depleted FVLeiden plasma was decreased by APC more than that observed with native FVLeiden plasma. The findings indicated that emicizumab-driven haemostasis was down regulated by APC-mediated FVa inactivation in plasma from haemophilia A patients without or with FV defects.

Temperature dependency of whole blood viscosity and red cell properties in desert ungulates: Studies on scimitar-horned oryx and dromedary camel.

The dromedary camel and the oryx antelope are exposed to excessive heat and solar radiation in their desert habitat. Desertification of areas with by now little rainfall may occur eventually. Well-adapted large animal species show us what is needed to survive in scorching regions. Four scimitar-horned oryx antelopes (Oryx dammah), 10 camels (Camelus dromedarius), nine South African Merino sheep, and 17 Nguni cows were tested for RBC aggregation, RBC elongation, and plasma viscosity. The temperature dependency of blood viscosity was tested in 10 camels and compared to human reference values. Unlike sheep, Nguni cow, and dromedary camel, oryx RBCs aggregate in native plasma (M0:5.2 (3.3/6.7); M1:18.1 (16.7/27.9); Myrenne MA1). Elongation indices of oryx RBCs were intermediate to low (EImax: 22.6 (19.2/25.3); SS1/2 3.67 (2.52/4.95); Rheodyn SSD). Camel RBCs did not display the typical SS/EI curve by rotational ektacytometry. In-vitro blood viscosity (Physica MCR302) was lower in camels than in human blood at equal hematocrit. A decrease of temperature had only little effect on camel blood. At 10 s−1, blood viscosity in camel increased from 2.18mPa*s (2.01/2.37) at 42◦C to 4.39mPa*s (4.22/4.51) at 12◦C. In human blood, viscosity ranged from 8.21mPa*s (6.95/8.25) at 37◦C to 15.52mPa*s (14.25/16.03) at 12◦C. At 1000 s−1, blood viscosity in camel ranged from 2.00mPa*s (1.95/2.04) at 42◦C to 3.98mPa*s (3.88/4.08) at 12◦C. In human blood, viscosity ranged from 5.35mPa*s (4.96/5.87) at 37◦C to 11.24mPa*s (10.06/11.17) at 12◦C. Desert ungulates may need RBC membranes, which are fortified to withstand changes in osmolality during dehydration-rehydration cycles. This reduces RBC deformability. Dromedary camel blood does not undergo stark changes in viscosity with changes in temperature. Therefore, blood fluidity could be rather maintained during the day and night cycle. This should reduce the need of the vascularity to rhythmically adapt to changing shear forces when camels experience heterothermy.

Mutual interaction of red blood cells assessed by optical tweezers and scanning electron microscopy imaging.

The adhesion of red blood cells (RBC) has been studied extensively in frame of cell-to-cell interaction induced by dextran macromolecules, whereas the data are lacking for native plasma solution. We apply optical tweezers to investigate the induced adhesion of RBC in plasma and in dextran solution. Two hypotheses, cross-bridges and depletion layer, are typically used to describe the mechanism of cell interaction; however, both mechanisms need to be confirmed experimentally. These interactions in fact are very much dependent on the size and concentration of dextran and proteins in plasma. The results show that in different dextran solutions, the interaction of adhering RBC agrees well with the quantitative predictions obtained based on the depletion-induced cells adhesion model, whereas the migrating cross-bridges model is more appropriate for plasma. Despite the different mechanisms of RBC interaction in a mixture of dextran with the size ranges and volume fraction proportional to plasma proteins, the dependence of RBC adhering tends to be close to the cross-bridges model. The induced aggregation of RBC in the dextran solutions and in native plasma are observed by direct visualization utilizing scanning electron microscopy.

Isolation of native plasma membrane H+-ATPase (Pma1p) in both the active and basal activation states.

The yeast plasma membrane H+‐ATPase Pma1p is a P‐type ATPase that energizes the yeast plasma membrane. Pma1p exists in two activation states: an autoinhibited basal state and an activated state. Here we show that functional and stable Pma1p can be purified in native form and reconstituted in artificial liposomes without altering its activation state. Acetylated tubulin has previously been reported to maintain Pma1p in the basal state but, as this protein was absent from the purified preparations, it cannot be an essential component of the autoinhibitory mechanism. Purification of and reconstitution of native Pma1p in both activation states opens up for a direct comparison of the transport properties of these states, which allowed us to confirm that the basal state has a low coupling ratio between ATP hydrolysis and protons pumped, whereas the activated state has a high coupling ratio. The ability to prepare native Pma1p in both activation states will facilitate further structural and biochemical studies examining the mechanism by which plasma membrane H+‐ATPases are autoinhibited.

Quantification of cardiac troponin I in human plasma by immunoaffinity enrichment and targeted mass spectrometry.

Quantification of cardiac troponin I (cTnI), a protein biomarker used for diagnosing myocardial infarction, has been achieved in native patient plasma based on an immunoaffinity enrichment strategy and isotope dilution (ID) liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The key steps in the workflow involved isolating cTnI from plasma using anti-cTnI antibody coupled to magnetic nanoparticles, followed by an enzymatic digestion with trypsin. Three tryptic peptides from cTnI were monitored and used for quantification by ID-LC-MS/MS via multiple reaction monitoring (MRM). Measurements were performed using a matrix-matched calibration system. NIST SRM 2921 Human Cardiac Troponin Complex acted as the calibrant and a full-length isotopically labeled protein analog of cTnI was used as an internal standard. The method was successfully demonstrated on five patient plasma samples, with cTnI concentrations measuring between 4.86μg/L and 11.3μg/L (signifying moderate myocardial infarctions). LC-MS/MS measurement precision was validated by three unique peptides from cTnI and two MRM transitions per peptide. Relative standard deviation (CV) from the five plasma samples was determined to be ≤14.3%. This study has demonstrated that quantification of cTnI in native plasma from myocardial infarction patients can be achieved based on an ID-LC-MS/MS method. The development of an ID-LC-MS/MS method for cTnI in plasma is a first step for future certification of matrix-based reference materials, which may be used to help harmonize discordant cTnI clinical assays. Graphical abstract A schematic of the workflow for measuring cardiac troponin I (cTnI), a low-abundant protein biomarker used for diagnosing myocardial infarction, in human plasma by isotope-dilution LC-MS/MS analysis.

Expression, Purification, and Biochemical Characterization of Human Afamin.

Afamin is an 87 kDa glycoprotein with five predicted N-glycosylation sites. Afamin's glycan abundance contributes to conformational and chemical inhomogeneity presenting great challenges for molecular structure determination. For the purpose of studying the structure of afamin, various forms of recombinantly expressed human afamin (rhAFM) with different glycosylation patterns were thus created. Wild-type rhAFM and various hypoglycosylated forms were expressed in CHO, CHO-Lec1, and HEK293T cells. Fully nonglycosylated rhAFM was obtained by transfection of point-mutated cDNA to delete all N-glycosylation sites of afamin. Wild-type and hypo/nonglycosylated rhAFM were purified from cell culture supernatants by immobilized metal ion affinity and size exclusion chromatography. Glycan analysis of purified proteins demonstrated differences in micro- and macro-heterogeneity of glycosylation enabling the comparison between hypoglycosylated, wild-type rhAFM, and native plasma afamin. Because antibody fragments can work as artificial chaperones by stabilizing the structure of proteins and consequently enhance the chance for successful crystallization, we incubated a Fab fragment of the monoclonal anti-afamin antibody N14 with human afamin and obtained a stoichiometric complex. Subsequent results showed sufficient expression of various partially or nonglycosylated forms of rhAFM in HEK293T and CHO cells and revealed that glycosylation is not necessary for expression and secretion.