Analytical Isoelectric Focusing Research Articles

Implementation of a Newborn Screening for Sickle Cell Disease, at the Hospital Materno Infantil Dr Manuel Pedro Azancot De Menezes, Angola

Sickle Cell Disease (SCD) is a hereditary disease, and a worldwide public health problem, with a higher incidence in sub-Saharan Africa, where the prevalence is very high (about 80%). SCD prevalence can reach 3,3 % in Angola, while carriers of the sickle cell trait account for more than 25% of the population. The early identification of patients and their follow-up in consultation are an added value for the reduction of morbidity and mortality of children with SCD. The implementation of early infant screening and follow-up programs for children with SCD showed excellent results in increasing the survival of these children. With this project we intend to implement a Newborn screening in Hospital Materno Infantil Dr Manuel Pedro Azancot de Menezes, a referral of the main maternity in Angola, located in Luanda, the country capital, that could be replicated in other hospitals, giving training to the health professionals, and to the laboratory technicians, in order to diagnose the SCD patients. The objective of this project is to establish a neonatal screening for SCD at a main Hospital in Angola, contributing to reduce the under 5 mortality, and to support for pediatric follow-up consultations of children diagnosed with SCD. Specific aims include: record the data and medical history of babies diagnosed with SCD up to 5 years of age at regular appointments; estimate the prevalence of SCD in the municipality of Camama, Luanda; and to evaluate the costs of neonatal screening and early interventions in Angola, by supporting a registry of SCD. The newborn screening has started in June 2023, with the training of the team at the hospital, and with the creation of all documents and forms needed. The project received ethical approval in June 2023. The study includes every child born in the maternity ward of the Hospital, and also every child that take vaccinations there (in this way we can include newborns delivered at home), that the legal guardians authorize the collection of blood sample by prick heel for screening for SCD. The samples of filter paper duly identified with the sample number and date of collection are being sent to Lisbon (Portugal), for hemoglobin electrophoresis by isoelectric focusing (IEF) carried out in the Migele equipment (acquired with the support of Perkin Elmer, and the ARISE project) (Figure 1). All SS samples are being genotyped for the HbS mutation by PCR-RFLP. Since the start of the newborn screening (June 28 th) we have collected 1000 samples in 4 weeks and have started the IEF analysis with MIGELE (Perkin Elmer). The preliminary results (221 samples, 177 AA, 41 AS and 3 SS) showed a 1.4% prevalence of SS, and 19.5% of AS, confirmed by PCR-RFLP (in all AS and SS). The frequencies were in Hardy-Weinberg equilibrium. Moreover, we are searching for other possible hemoglobinopathies. More results will be presented. All children diagnosed as SS (SCD) are being contacted by the Hospital Materno Infantil Dr Manuel Pedro Azancot de Menezes, Angola, to be followed in consultation. Newborn screening and early interventions for SCD should be a health priority and implemented at national level. It is our plan to catalyze progress toward this aim by demonstrating the high prevalence of neonatal SCD in the Luanda Hospital and demonstrate the feasibility of this screening in reducing under 5 mortalities. The present project has the support of ARISE project “African Research and Innovative initiative for Sickle cell Education: Improving Research Capacity for Service Improvement” project (GA 824021 - ARISE - H2020-MSCA-RISE-2018) by supporting the secondments of Angolan Laboratory technicians in Lisbon (Portugal) to perform the IEF and genetic analysis and be trained. Until now 5 Angolan secondees, and 3 Nigerian secondees have been trained in Lisbon. Perkin Elmer (Revvity) is giving llogistical support by kindly support the low-cost acquisition, installation and training in MIGELE equipment for IEF, and being always available to technical support. Figure 1 - Flow diagram of the methodology followed in the present study.

Marker-Free Isoelectric Focusing Patterns for Identification of Meat Samples via Deep Learning.

Isoelectric focusing (IEF) is a powerful tool for resolving complex protein samples, which generates IEF patterns consisting of multiplex analyte bands. However, the interpretation of IEF patterns requires the careful selection of isoelectric point (pI) markers for profiling the pH gradient and a trivial process of pI labeling, resulting in low IEF efficiency. Here, we for the first time proposed a marker-free IEF method for the efficient and accurate classification of IEF patterns by using a convolutional neural network (CNN) model. To verify our method, we identified 21 meat samples whose IEF patterns comprised different bands of meat hemoglobin, myoglobin, and their oxygen-binding variants but no pI marker. Thanks to the high throughput and short assay time of the microstrip IEF, we efficiently collected 1449 IEF patterns to construct the data set for model training. Despite the absence of pI markers, we experimentally introduced the severe pH gradient drift into 189 IEF patterns in the data set, thereby omitting the need for profiling the pH gradient. To enhance the model robustness, we further employed data augmentation during the model training to mimic pH gradient drift. With the advantages of simple preprocessing, a rapid inference of 50 ms, and a high accuracy of 97.1%, the CNN model outperformed the traditional algorithm for simultaneously identifying meat species and cuts of meat of 105 IEF patterns, suggesting its great potential of being combined with microstrip IEF for large-scale IEF analyses of complicated protein samples.

Isoelectric point barcode and similarity analysis with the earth mover’s distance for identification of species origin of raw meat

In this work, by combining the microcolumn isoelectric focusing (mIEF) and similarity analysis with the earth mover’s distance (EMD) metric, we proposed the concept of isoelectric point (pI) barcode for the identification of species origin of raw meat. At first, we used the mIEF to analyze 14 meat species, including 8 species of livestock and 6 species of poultry, to generate 140 electropherograms of myoglobin/hemoglobin (Mb/Hb) markers. Secondly, we binarized the electropherograms and converted them into the pI barcodes that only showed the major Mb/Hb bands for the EMD analysis. Thirdly, we efficiently developed the barcode database of 14 meat species and successfully used the EMD method to identify 9 meat products thanks to the high throughput of mIEF and the simplified format of the barcode for similarity analysis. The developed method had the merits of facility, rapidity and low cost. The developed concept and method had evident potential to the facile identification of meat species.

Yield and Composition Variations of the Milk from Different Camel Breeds in Saudi Arabia

With the increasing interest in the identification of differences between camel breeds over the last decade, this study was conducted to estimate the variability of milk production and composition of four Saudi camel breeds during different seasons. Milk records were taken two days per week from females of Majahem, Safra, Wadha, and Hamra breeds distributed over Saudi Arabia. The milk yield during winter indicated that the weekly average of the Wadha breed was significantly lower (27.13 kg/week) than Majahem and Hamra breeds. The Safra breed had the lowest milk yield (30.7 kg/week) during summer. During winter, the Hamra breed had a lower content of all analyzed milk components except proteins and was characterized by a lower pH than the milk of the other breeds. However, the Hamra breed had significantly higher contents of milk fat and lactose than the other breeds during summer, with the corresponding values of 3.87 and 4.86%, respectively. Milk collected during winter from Majahem, Safra, and Wadha breeds was characterized by a significant increase in all milk components and milk pH. Finally, the isoelectric focusing analysis revealed noticeable variability of casein purified from camel milk within the different Saudi breeds, with the highest significant value of 2.29 g per 100 mL recorded for the Wadha breed.

Purification and Characterization of Novel Antihypertensive and Antioxidative Peptides From Whey Protein Fermentate: In Vitro, In Silico, and Molecular Interactions Studies

Objective The goal of this research was to purify and characterize the novel angiotensin-converting enzyme (ACE)–inhibitory and antioxidant peptides from fermented whey protein concentrate produced by Lactobacillus paracasei and Saccharomyces cerevisiae in a co-fermentation system. Method Whey protein fermented with lactic acid bacteria and yeast culture was analyzed for antioxidative, ACE inhibition, as well as anti-inflammatory activity followed by SDS-PAGE, isoelectric focusing, and 2-dimensional (2D) analysis. Anti-inflammatory activity of whey protein fermentate was also studied on the RAW 264.7 cell line. The bioactive peptides were separated from the whey protein fermentate using reverse-phase high-performance liquid chromatography (RP-HPLC) and reverse-phase liquid chromatography mass spectrometry (RPLC/MS), and thus identification and characterization of purified bioactive peptide was performed. Results Whey protein fermentate samples’ bioactivity was analyzed at specific time intervals at 12, 24, 36, and 48 hours at 37 °C for M11 and at 25 °C for WBS2A. The development settings (incubation time [12, 24, 36, and 48 hours) and inoculation rates [1.5%, 2.0%, and 2.5%]) were optimized for peptide synthesis via the o-phthaldialdehyde (OPA) method (proteolytic activity). Maximum proteolytic activity was observed at 37 °C for M11 (6.50 mg/mL) and at 25 °C for WBS2A (8.59 mg/mL) for 48 hours of incubation. Protein profiling was carried out using SDS-PAGE and 2D gel electrophoresis, in which Sodium dodecyl-sulfate (SDS) exhibited protein bands in the 10- to 55-kDa range, while 2D showed protein bands varying from 10 to 70 kDa. Every spot from 2D was digested by trypsin and identified by RPLC/MS. Protein fractionations (3- and 10-kDa permeates) were carried out employing RP-HPLC. Whey protein fermentate has anti-inflammatory action in RAW 264.7 macrophages that have been exposed to lipopolysaccharide. A molecular docking system was also used to investigate the interactions of peptides (AFLDSRTR, ILGAFIQIITFR) with human myeloperoxidase enzyme. Conclusions The antihypertensive and antioxidative peptides discovered from whey protein fermentate may be helpful in the design of pharmacologically active healthy ingredients in the upcoming years.

Oligoclonal Band Straightening Based on Optimized Hierarchical Warping for Multiple Sclerosis Diagnosis.

The detection of immunoglobulin G (IgG) oligoclonal bands (OCB) in cerebrospinal fluid (CSF) by isoelectric focusing (IEF) is a valuable tool for the diagnosis of multiple sclerosis. Over the last decade, the results of our clinical research have suggested that tears are a non-invasive alternative to CSF. However, since tear samples have a lower IgG concentration than CSF, a sensitive OCB detection is therefore required. We are developing the first automatic tool for IEF analysis, with a view to speeding up the current visual inspection method, removing user variability, reducing misinterpretation, and facilitating OCB quantification and follow-up studies. The removal of band distortion is a key image enhancement step in increasing the reliability of automatic OCB detection. Here, we describe a novel, fully automatic band-straightening algorithm. The algorithm is based on a correlation directional warping function, estimated using an energy minimization procedure. The approach was optimized via an innovative coupling of a hierarchy of image resolutions to a hierarchy of transformation, in which band misalignment is corrected at successively finer scales. The algorithm’s performance was assessed in terms of the bands’ standard deviation before and after straightening, using a synthetic dataset and a set of 200 lanes of CSF, tear, serum and control samples on which experts had manually delineated the bands. The number of distorted bands was divided by almost 16 for the synthetic lanes and by 7 for the test dataset of real lanes. This method can be applied effectively to different sample types. It can realign minimal contrast bands and is robust for non-uniform deformations.

Evaluation of Six Protocols for Protein Extraction from Rice Young Panicles byTwo-Dimensional Electrophoresis

ABSTRACT For most reported proteomics approaches, protein extraction and sample preparation are of crucial importance for optimal results. However, extraction of protein from crop plant tissues remains a great challenge due to low protein content and abundant secondary metabolites that prominently interfere with isoelectric focusing and subsequent proteomic analysis. Up until now, no attempts are focused on comparison of protein extraction procedures from rice young panicles. To establish a high-efficiency protein extraction protocol suitable for two-dimensional electrophoresis (2-DE) in rice young panicles, six protocols for protein preparation were evaluated: (1) Phenol extraction; (2) Mg/ Nonidet P-40 (NP-40) extraction; (3) Tris-Base/acetone precipitation; (4) SDS extraction; (5) trichloroacetic acid (TCA)/acetone/phenol extraction; (6) TCA/acetone precipitation. The study explicitly demonstrates that TCA/ acetone/phenol method provides a high-enhanced protein extraction efficacy from rice young panicles than other protocols in terms of the higher quality of 1-DE gel, the maximum number (450) of well-resolved protein spots, greater resolution and spot abundance. In addition, these methods also generated remarkably different twodimensional gel electrophoresis protein spot patterns. Twenty-nine of 30 visible differential extracted proteins were identified by MALDI-TOF-MS/MS analysis and were divided into eight categories according to molecular function. Accumulated data suggested that different extraction methods respectively necessitate certain special plant tissues due to different physicochemical properties of each protocol. Overall, this study, which is presented in this paper, will facilitate to providing a cornerstone of comparative proteomic analysis from rice young panicles, including other complicated plant tissues.

An Investigation to Examine the Effect of the Elastomeric Surface Treatment on Protein Stability.

Various kinds of treatments on the surface of the elastomeric components can have negative impacts on the quality of protein therapeutics. We compared the effects of bare (non-siliconized and nonlaminated), siliconized, and fluoropolymer-laminated elastomeric components on the stability of ß-lactoglobulin, human serum albumin, adalimumab, abatacept, and immunoglobulin antibodies. The study was conducted in two main parts. Part I was to evaluate the stability of proteins under agitation-induced stress. Protein aggregate formation, turbidity, and protein recovery were analyzed using dynamic FI, absorbance at 350 nm, and size-exclusion high-performance liquid chromatography, respectively. Proteins were found to be more stable with laminated stoppers as compared with bare or siliconized stoppers. Part II was to identify the chemical modifications when the proteins were stored in contact with the same three stoppers. Capillary isoelectric focusing analysis of the adalimumab samples showed formation of acidic variants in siliconized and bare stoppers. Reverse-phase high-performance liquid chromatography suggested chemical changes to the human serum albumin. Analysis of tryptic digest of human serum albumin by liquid chromatography/mass spectrometry/mass spectrometry indicated that the amino acids most susceptible to oxidation (cysteine, tryptophan, and methionine) were also the ones that were modified. Part III of this study investigated the barrier property of the fluoropolymer film with no drug product. Our results were consistent with the suggestion that the fluoropolymer lamination provides a barrier that prevents leachables from the elastomeric components into the protein therapeutics. Our work provided an in-depth understanding of the effects of elastomeric surface treatments on the biophysical and chemical stability of protein drugs.

A straightforward gel-free proteomics pipeline assisted by liquid isoelectric focusing (OFFGEL) and mass spectrometry analysis to study bovine meat proteome.

Bovine sarcoplasmic sub-proteome was studied through a straightforward gel-free pipeline supported by liquid isoelectric focusing (OFFGEL) protein fractionation coupled to liquid chromatography-mass spectrometry (LC-MS) analysis. Full-MS and data-dependent MS/MS analyses were simultaneously performed by a conventional three-dimensional ion-trap addressing targeted quantitative and untargeted qualitative research, respectively. There were unambiguously identified 47 proteins distributed along 12 OFFGEL fractions assayed. Regarding intermediate- and high-abundant peptides, bulky quantitative data processing performed by MZmine 2 freeware yielded a satisfactory linearity and coefficient of variation with r2 in the 0.95-0.99 range and about 25%, respectively. Up to 41 peptides from 20 identified proteins were relatively quantified throughout OFFGEL fractions. This reliable, flexible and affordable gel-free proteomic approach could be readily implemented by industry to improve quality assessment of protein-based food products.

A novel approach enables imaged capillary isoelectric focusing analysis of PEGylated proteins.

PEGylation has been used as a strategy to enhance pharmacokinetic properties of therapeutic proteins by pharmaceutical industry. Imaged CIEF (iCIEF) is the current industry standard technology for pI determination and charge variant quantification of proteins and antibodies. However, the charge variants of PEGylated proteins merge into one broad peak during iCIEF, most likely due to masking of proteins by the surrounding PEG chain as well as the increased hydrodynamic volume due to PEGylation. Here, we report our novel matrix formula with a combination of glycine and taurine that significantly improved the separation of charge variants in PEGylated proteins. As a result, it is no longer necessary to conduct IEF of proteins prior to PEGylation, which does not reflect the changes caused by PEGylation and purification processes. The novel matrix (glycine and taurine) enables iCIEF analysis of PEGylated proteins in their real conjugated states.

Studies on the Thiol Components of Isolated Nuclei

The thiol components of the nonhistone proteins prepared from isolated nuclei from rat liver, regenerating liver and hepatoma 223 cells have been investigated after reaction with radio labelled N-ethylmaleimide and 5-5’-dithiobis-(2-nitrobenzoic acid) (DTNB). The labelled adducts formed were examined by isoelectric focusing analysis in polyacrylamide gel and the distribution of the radiolabels within sliced portions of the gels determined. In the case of the 14C labelled NEM adduct the label was found to be spread amongst numerous protein components within the gel however, in the case of the 35S labelled DTNB adducts, only a small proportion of the label was found in the protein material which was retained in the acidic isoelectric point (pI) region of the gel. The bulk of the 35S labelled adduct (56% - 60%) was found to have migrated into the anode solution (10 mM phosphoric acid). This could be adsorbed onto a hydrophobic resin (XAD2) resin and eluted with methanol. Gel filtration chromatographic analysis of this adduct material on BioGel P2, (exclusion limit 1500 daltons) showed low molecular weight components to be present. Slightly different patterns were obtained for these nuclei, each containing several 35S components with molecular weights greater than the Ellman reagent itself. These 35S labelled thiol components did not contain any protein, peptide or amino acid components indicating strongly that a novel species of thiols could be present in these nuclei bound within the non-histone protein matrices.

Overexpression of an alternative allele of carboxyl/choline esterase 4 (CCE04) of Tetranychus urticae is associated with high levels of resistance to the keto-enol acaricide spirodiclofen.

Spirodiclofen is an acaricide that targets lipid biosynthesis by inhibiting acetyl-coenzyme A carboxylase. Spirodiclofen resistance in spider mites has been previously documented and was associated with overexpression of CYP392E10, a cytochrome P450 mono-oxygenase that metabolizes spirodiclofen. However, additional mechanisms have been suggested in several studies and a carboxyl/choline esterase gene, CCE04, was shown to be overexpressed in two genetically different strains, SR-VP and SR-TK, both exhibiting high spirodiclofen resistance levels. We identified two different CCE04 alleles in both resistant strains, CCE04SR-VP and CCE04London , with CCE04SR-VP being highly overexpressed. Isoelectric focusing analysis confirmed the overexpression of a single esterase isozyme, while copy number and random fragment length polymorphism analysis revealed that CCE04SR-VP overexpression was more likely due to selection for the CCE04SR-VP allele rather than gene amplification. Both CCE04 alleles were functionally expressed using the Pichia expression system. Functional enzyme assays revealed only limited kinetic differences between CCE04 isoforms for model substrates. In addition, inhibition/competition experiments with spirodiclofen suggested a similar interaction with both enzymes, whereas its active metabolite, spirodiclofen enol, did not inhibit enzyme activity. Our study suggests that selection with spirodiclofen results in enrichment of a specific allele of CCE04 (CCE04SR-VP ) in two genetically independent strains, which is highly overexpressed. Based on kinetic enzyme data, however, quantitative rather than qualitative differences between CCE04SR-VP and CCE04London seem more likely to be involved in resistance. Our findings are discussed in the light of a possible spirodiclofen resistance mechanism, with sequestration of spirodiclofen by CCE04SR-VP being a likely hypothesis. © 2019 Society of Chemical Industry.

Milk protein polymorphism in Amiata donkey

Interest in donkey milk for human nutrition has been growing steadily, especially in the therapeutic-diet field, due to its similarity to human milk. Caseins are one of the main allergenic components of milk. Whereas cow milk has a high casein content, in donkey milk the casein concentration is lower and is characterized by a greater digestibility. In fact, recent studies have shown that donkey milk can be consumed by children with cow milk protein allergy. The aim of this study was to evaluate the protein profile of individual samples of Amiata donkey milk and to quantify the different protein fractions in order to better understand the characteristics of donkey milk. Protein fractions of 57 individual whole milk samples were analysed by isoelectric focusing (IEF). The main milk protein fractions were quantified both by IEF gel image analysis and directly on milk using the Agilent 2100 Bioanalyzer. The IEF analysis identified 10 different electrophoretic patterns, which differ from each other in terms of the presence of bands that correspond to homozygous or heterozygous genotypes, as well as the presence/absence of specific protein bands. In addition the quantification of different caseins (αS1, β and κ-CN) and whey proteins (β-LG, α-LA and LZ), highlighted a significant phenotypic variability among individuals which seems to be related to the patterns identified. In particular, different casein:whey protein ratios were identified among jennies and this is known to affect the nutritional, allergenic and technological properties of milk. These findings could be useful in the selection of donkeys with specific milk protein polymorphisms.

Ultrasensitive Electrical Detection of Hemagglutinin for Point-of-Care Detection of Influenza Virus Based on a CMP-NANA Probe and Top-Down Processed Silicon Nanowire Field-Effect Transistors.

Rather than the internal genome nucleic acids, the biomolecules on the surface of the influenza virus itself should be detected for a more exact and rapid point-of-care yes/no decision for influenza virus-induced infectious diseases. This work demonstrates the ultrasensitive electrical detection of the HA1 domain of hemagglutinin (HA), a representative viral surface protein of the influenza virus, using the top-down complementary metal oxide semiconductor (CMOS) processed silicon nanowire (SiNW) field-effect transistor (FET) configuration. Cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-NANA) was employed as a probe that specifically binds both to the aldehyde self-aligned monolayer on the SiNWs and to HA1 simultaneously. CMP-NANA was serially combined with two kinds of linkers, namely 3-aminopropyltriethoxysilane and glutaraldehyde. The surface functionalization used was verified using the purification of glutathione S-transferase-tagged HA1, contact angle measurement, enzyme-linked immunosorbent assay test, and isoelectric focusing analysis. The proposed functionalized SiNW FET showed high sensitivities of the threshold voltage shift (ΔVT) ~51 mV/pH and the ΔVT = 112 mV (63 mV/decade) with an ultralow detectable range of 1 fM of target protein HA1.

"Switching Partners": Piperacillin-Avibactam Is a Highly Potent Combination against Multidrug-Resistant Burkholderia cepacia Complex and Burkholderia gladioli Cystic Fibrosis Isolates.

In persons with cystic fibrosis (CF), airway infection with Burkholderia cepacia complex (Bcc) species or Burkholderia gladioli presents a significant challenge due to inherent resistance to multiple antibiotics. Two chromosomally encoded inducible β-lactamases, a Pen-like class A and AmpC are produced in Bcc and B. gladioli Previously, ceftazidime-avibactam demonstrated significant potency against Bcc and B. gladioli isolated from the sputum of individuals with CF; however, 10% of the isolates tested resistant to ceftazidime-avibactam. Here, we describe an alternative antibiotic combination to overcome ceftazidime-avibactam resistance. Antimicrobial susceptibility testing was performed on Bcc and B. gladioli clinical and control isolates. Biochemical analysis was conducted on purified PenA1 and AmpC1 β-lactamases from Burkholderia multivorans ATCC 17616. Analytic isoelectric focusing and immunoblotting were conducted on cellular extracts of B. multivorans induced by various β-lactams or β-lactam-β-lactamase inhibitor combinations. Combinations of piperacillin-avibactam, as well as piperacillin-tazobactam plus ceftazidime-avibactam (the clinically available counterpart), were tested against a panel of ceftazidime-avibactam nonsusceptible Bcc and B. gladioli The piperacillin-avibactam and piperacillin-tazobactam-ceftazidime-avibactam combinations restored susceptibility to 99% of the isolates tested. Avibactam is a potent inhibitor of PenA1 (apparent inhibitory constant [Kiapp] = 0.5 μM), while piperacillin was found to inhibit AmpC1 (Kiapp = 2.6 μM). Moreover, piperacillin, tazobactam, ceftazidime, and avibactam, as well as combinations thereof, did not induce expression of blapenA1 and blaampC1 in the B. multivorans ATCC 17616 background. When ceftazidime-avibactam is combined with piperacillin-tazobactam, the susceptibility of Bcc and B. gladioli to ceftazidime and piperacillin is restored in vitro Both the lack of blapenA1 induction and potent inactivation of PenA1 by avibactam likely provide the major contributions toward susceptibility. With in vivo validation, piperacillin-tazobactam-ceftazidime-avibactam may represent salvage therapy for individuals with CF and highly drug-resistant Bcc and B. gladioli infections.

Low-molecular-mass nitrophenol-based compounds suitable for the effective tracking of pH gradient in isoelectric focusing

Low-molecular-mass isoelectric point (pI) markers are a reasonable alternative to commonly used peptide and protein pI markers in the field of isoelectric focusing. We present a comparative study dealing with characterization of 44 nitrophenol-based compounds synthesized by the Mannich reaction which pI values cover a pH range from 3.2 to 10.4. The values of pIs of all the presented nitrophenol-based compounds were precisely determined by capillary isoelectric focusing technique (cIEF) when a standard deviation of measurement reached a value of a few hundredths of a pH unit (n ≥ 3). Differences have been found between in silico analysis of acid-base properties of the investigated compounds and the experimental cIEF data especially at the basic pH region. The data from the acidic pH range showed better correlation between these methods. Only three compounds were excluded from the group of pI markers because they contained impurities originating from the synthesis or long-term storage. Based on the presented results we identified 41 nitrophenol-based pI markers (NPIMs) which can fulfill requirements of the most applications in a field of isoelectric focusing analyses.

A Novel Filtering Tool for Detecting Sickle Cell Disease in the Pediatric Population

Human migration has reached an unprecedented level. Progressive globalization has integrated people and facilitated the introduction of deleterious genes into populations in which they were originally absent, thus impacting negatively on public health. A great challenge encountered by clinicians in the era of “migration hematology” lies in the hemoglobinopathies, a group of conditions representing the commonest, life-threatening, monogenic disorders globally. Of the ±400 000 newborn children affected annually, ~65% have sickle cell disease (SCD), a disorder that has spread far beyond its origins because of slave trade and contemporary population movements. More than 70% of all global SCD cases are due to autosomal recessive homozygous inheritance of a βS-mutation (missense Glu6Val) in the HBB gene, creating sickle hemoglobin (HbS), a structural variant of adult hemoglobin (HbA) and causing the most severe form of SCD, sickle cell anemia (SCA). Other forms result from inheritance of HbS in combination with different mutations, most commonly a second structural β-globin variant, βC (S/C) or one of many that lead to decreased β-globin synthesis (S/β-thalassemia). Clinical consequences are due to polymerization of deoxygenated HbS and its deleterious effects on erythrocytes. It is a debilitating syndrome characterized by chronic hemolysis and vaso-occlusive crises, manifesting as acute painful episodes, organ infarction and eventual multi-organ damage.Early diagnosis of SCD is vital so that appropriate management can be initiated timeously. Current diagnostic tests include hemoglobin electrophoresis (HE), high performance liquid chromatography (HPLC), isoelectric focusing (IEF) and DNA analysis. These specialized expensive modalities have been successfully incorporated into antenatal/neonatal screening programmes of some high-income countries such as in the UK. However, implementation of similar initiatives in various other regions has been difficult due to practical, political and economic challenges.The present study outlines a simple algorithm for use in the pediatric population to identify children at risk for SCD. Residual EDTA-anticoagulated capillary blood samples were analyzed in a tertiary hospital laboratory using an automated benchtop hematology instrument (Sysmex XN-450) that provides a CBC, differential white cell count and reticulocyte percentage (RET%). Patients with known SCD (85% SCA), confirmed by HE and HPLC, presenting for routine out-patient clinic review, were included (n=107; 1 month to 18 years old; 54% male). Samples were processed within 12 hours of collection and those who had received blood transfusions in the preceding 12 weeks were excluded.The mean ± SD was determined for each CBC parameter and RET%. Of these, 5 parameters were used to establish an algorithm with reference limits specific for each age category of patients with SCD (Table 1). When compared to the population-based normal reference ranges, these limits were statistically significantly different (p<0.05). The algorithm was validated against 107 random blood results previously obtained on routine follow up of pediatric patients with SCD. If a single outlying parameter was allowed, the algorithm demonstrated a sensitivity of 95.3% for SCD. The 4.7% of outlying cases (n=5) exhibited severe microcytosis and had a coexisting thalassemia component. The sensitivity with a complete match, i.e. 5/5 parameters, was 86.9%. Other CBC parameters were not informative.To combat the burden of SCD early and accurate diagnosis is critical. The algorithm described here is a cost-effective and sensitive filtering tool that can quickly be used to identify children at risk for SCD at any age and exclude those who do not require definitive testing, thereby limiting unnecessary expenditure. It will be beneficial in resource-constrained low-income countries; regions where SCD is not historically present and clinical suspicion is low; or in scenarios where it is difficult to obtain a medical history due to a language barrier and/or health-related stigma. Since using the algorithm only requires a CBC and RET%, it is suitable for application at any healthcare level. Assessment may be conveniently conducted in line with a child's immunization schedule prior to symptomatic presentation, and if combined with the Mentzer index and a family origin questionnaire, the sensitivity can be enhanced. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Loss of Ca2+/Calmodulin Dependent Protein Kinase Kinase 2 Leads to Aberrant Transferrin Phosphorylation and Trafficking: A Potential Biomarker for Alzheimer's Disease.

Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a serine/threonine kinase that is activated following an increase in the intracellular Ca2+ concentration and activates multiple signaling cascades that control physiologically important neuronal processes. CaMKK2 has been implicated in schizophrenia, bipolar disease, neurodegeneration, and cancer. Using isoelectric focusing (IEF) and mass spectrometry-based proteomic analysis, it was found that knockdown (KD) of CaMKK2 in cultured adult primary dorsal root ganglion (DRG) neurons resulted in the reduction of transferrin (TF) phosphorylation at multiple functionally relevant residues which corresponded to loss of an acidic fraction (pH~3-4) of TF. In vitro studies using CRISPR/Cas9 based CaMKK2 knockout (KO) HEK293 and HepG2 cells lines validated previous findings and revealed that loss of CaMKK2 interfered with TF trafficking and turnover. TF is an iron transporter glycoprotein. Abnormal accumulation of iron and/or deregulated Ca2+ homeostasis leads to neurodegeneration in Alzheimer's disease (AD). Therefore, it was hypothesized that aberrant CaMKK2 in AD may lead to aberrant phosphorylated transferrin (P-TF: pH~3-4 fraction) which may serve as a hallmark biomarker for AD. A significant reduction of P-TF in the brain and serum of CaMKK2 KO mice and a triple-transgenic mouse model of AD (3xTg-AD) supported this hypothesis. In addition, analysis of early (< 65 years) and late-stage (>65 years) postmortem human AD cerebrospinal fluid (CSF) and serum samples revealed that aberrant P-TF (pH~3-4 fraction) profile was associated with both early and late-stage AD compared to age-matched controls. This indicates P-TF (pH~3-4 fraction) profile may be useful as a minimally invasive biomarker for AD. In addition, this study provides a link between aberrant CaMKK2 with TF trafficking and turnover which provides a novel insight into the neurodegeneration process.

Combined immuno-purification and detection of recombinant erythropoietins and activin receptor type II-Fc fusion proteins by isoelectric focusing for application in doping control.

Iso-electric focusing (IEF) was the first method established to discriminate endogenous and recombinant erythropoietins (rEPOs). It is still approved by the World Anti-Doping Agency (WADA) as an initial testing procedure to detect erythropoiesis stimulating agents (ESAs) in doping control samples. However EPO-Fc, one of the prohibited rEPOs designated by WADA, is not detectable with the actual IEF conditions. Other newly developed ESAs - luspatercept and sotatercept, both activin receptor type II-Fc fusion proteins (ActRII-Fc) - are also now prohibited and could be used in combination with rEPOs. Methods of identification of ActRII-Fc in blood by SAR/SDS-PAGE have been described, but not by IEF. Here we detail improvements in blood sample preparation and IEF analysis: A combined immuno-purification of EPOs and ActRII-Fc proteins in a single procedure, an appropriate isoforms separation for all proteins using new pre-loading and gel conditions, and a single detection of all rEPOs and ActRII-Fc proteins after successive incubation with anti-EPO and anti-ActRII antibodies. With these changes, distinctive profiles for all the ESAs were obtained by IEF. Therefore, IEF could be used as a screening method to detect a wide spectrum of prohibited ESAs in blood samples prior to specific confirmation for the identified rEPO or ActRII-Fc.

Identification of bacterial uropathogens by preparative isoelectric focusing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

This study describes a new method for simultaneous identification of uropathogens in the case of polybacterial urinary tract infections. The method utilizes recently developed preparative isoelectric focusing (IEF) in cellulose-based separation medium with a subsequent analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Preparative IEF was successfully used for both purification and separation of bacteria, Escherichia coli (pI 4.6) and Staphylococcus aureus (pI 3.4), in urine samples. The focused zones of bacteria, localized by the positions of focused colored pI markers, were easily collected from the separation media after the IEF analysis and then unambiguously identified by MALDI-TOF MS. The proposed method enables the identification of bacteria in urine specimens when the concentration of individual bacteria is ≥104 cells mL−1. Another benefit is the viability of bacteria extracted from the collected fractions after preparative IEF.