Advances in Hemoglobinopathies and Thalassemia Evaluation.

Hemoglobin (Hb) disorders are among the most prevalent inherited diseases. Despite a limited number of involved genes, these conditions represent a broad clinical and prognostic spectrum. The menu of laboratory tests is extensive. From widely available modalities, for example, complete blood count to rather sophisticated molecular technologies, the investigation of Hb disorders recapitulates an increasing complexity of laboratory workup in other medical fields. This review highlights a current state of biochemical and molecular investigation of Hb disorders and offers a glimpse on technologies that are yet to be fully embraced in clinical practice.

Proper application of DNA dyes in agarose gel electrophoresis.

Various dyes are used to visualize DNA bands in agarose gel electrophoresis (AGE) by the methods of pre- or post-staining. The DNA dye user's guides generally state that the binding of the dye to DNA will affect DNA mobility in electrophoresis, thus recommending post-staining for accurate measurement of DNA size. However, many AGE performers prefer pre-staining procedures for reasons such as convenience, real-time observation of DNA bands, and/or the use of a minimal amount of dye. The detrimental effect of the dye on DNA mobility and the associated risk for inaccurate measurement of DNA size are often overlooked by AGE performers. Here we quantitatively determine the impact on DNA migration imposed by frequently used dyes, including GelRed, ethidium bromide (EB), and Gold View. It was observed that pre-staining with GelRed and EB significantly slowed down DNA migration to cause as much as 39.1% overestimation on the size of sample DNA, whereas Gold View had little effect. The slowdown of DNA migration increased with dye concentration until it plateaued when the dye concentration reached a saturated level. Thus, to take advantage of pre-staining, saturated levels of DNA dyes should always be applied for both DNA samples and DNA markers to ensure a fair comparison of DNA sizes. In addition, GelRed and EB display much higher sensitivity than Gold View in the detection of DNA bands in post-staining. The saturated concentrations, cost considerations, and other useful features of these frequently used dyes are summarized for the information of AGE performers.

Short-term impact of vitamin K2 supplementation on biochemical parameters and lipoprotein fractions.

This study explored the short-term effects of vitamin K2 (VK2) supplementation on biochemical parameters (vitamin D, vitamin E, vitamin A, alkaline phosphatase, calcium, phosphorus (P), magnesium, metallothionein, triglycerides, cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and lipoprotein fractions (albumin, HDL, very low-density lipoprotein (VLDL), LDL, and chylomicrons). A short-term experiment (24 h, six probands) was performed to track changes in VK2 levels after a single-dose intake (360 µg/day). Liquid chromatography-tandem mass spectrometry was used to monitor vitamin K levels (menaquinone-4 (MK-4), menaquinone-7 (MK-7), and vitamin K1 [VK1]) with a limit of detection of 1.9 pg/mL for VK1 and 3.8 pg/mL for the two forms of VK2. Results showed that MK-7 levels significantly increased within 2-6 h post-administration and then gradually declined. MK-4 levels were initially low, showing a slight increase, whereas VK1 levels rose initially and then decreased. Biochemical analyses indicated no significant changes in sodium, chloride, potassium, calcium, magnesium, albumin, or total protein levels. A transient increase in P was observed, peaking at 12 h before returning to baseline. Agarose gel electrophoresis of lipoprotein fractions revealed distinct chylomicron bands and variations in VLDL and HDL mobility, influenced by dietary lipids and VK2 supplementation. These findings suggest effective absorption and metabolism of MK-7 with potential implications for bone metabolism and cardiovascular health.

An enhanced and rapid method for von Willebrand factor multimer analysis for mechanical circulatory device testing.

BACKGROUND: Von Willebrand factor (VWF) is a critical glycoprotein in hemostasis and is an important factor in diagnosing bleeding disorders. Albeit the analysis of VWF is often compromised by inconsistent methodologies and challenges quantifying multimeric size. Current VWF multimer analysis methods are costly, time-consuming, and often inconsistent; thus, demanding skilled professionals. This study aimed to streamline and optimize the VWF multimer analysis technique, making it more efficient and reproducible, particularly for identifying or predicting mechanical circulatory support (MCS) induced bleeding disorders. METHODS: Blood samples from healthy volunteers were exposed to high shear forces via a Medtronic HeartWare ventricular assist device. VWF multimers were analyzed using vertical-gel agarose electrophoresis and Western blotting. Differences in VWF distribution were determined using densitometry, and two methods of densitometric analysis were compared: proprietary software against open-source software. RESULTS: Using the developed method: (i) protocol duration was accelerated from three days (in classical methods) to ~ eight hours; (ii) the resolution of the high molecular weight (HMW) VWF multimers were substantially improved; and (iii) densitometric analysis tools were validated. Additionally, the densitometry analysis using two software types showed a strong correlation between results, with the proprietary software reporting slightly higher HMW VWF percentages. CONCLUSION: This methodology is recommended for affordable, accurate, and reproducible VWF multimer evaluations during MCS use and testing. Further research comparing this method with semi-automated methods would provide additional insight and improve inter-laboratory comparisons.

Discordance of Penta D x.4 microvariant alleles results between three capillary electrophoresis and one massively parallel sequencing short tandem repeat kits.

Forensic Biology is contingent upon matching DNA profiles between a crime sample and a reference sample. There are several capillary electrophoresis kits available to generate a short tandem repeat (STR) profile from DNA samples, while newer methods using massively parallel sequencing are slowly being implemented in forensic laboratories worldwide. During evaluation of a newer capillary electrophoresis kit, Applied Biosystems™ VeriFiler™ Plus, a discordance was observed in the Penta D locus. The previous kit, Promega PowerPlex 21® System produced a 13.4,14 genotype, whilst VeriFiler™ Plus produced a 14,14 genotype. An expanded investigation into Penta D microvariant alleles revealed that multiple discordances were observed for DNA profiles containing larger x.4 variants. There was full concordance between PowerPlex® 21 and QIAGEN Investigator® 26plex, however discordances were observed between VeriFiler™ Plus and the other three kits tested, including the massively parallel sequencing kit, Verogen ForenSeq® MainstAY. Notably, four of these discordances resulted in null alleles with the VeriFiler™ Plus kit. A review of the Penta D DNA sequences in MainstAY revealed fully concordant microvariant alleles involved deletions within the repeat region, whilst variability in the discordances observed were dependent on the location of the variation outside the repeat region and the analysis method used. Variations observed within the 5' flanking region produced the same allele designation across all capillary electrophoresis kits. However, deletions within the 3' region either produced a null allele for VeriFiler™ Plus where the deletion is thought to overlap the primer binding site, or microvariant alleles for the PowerPlex® 21 and Investigator 26plex kits, which produced longer Penta D amplicons. The discovery of these variations in the Penta D flanking sequences is informative as it increases the awareness of Penta D discordances between different kit chemistries in nominated reference DNA profile comparisons and DNA database searching and matching alike, and provides support for this phenomenon when providing evidence as to the admissibility of such results in trial proceedings.

Comparison of multiplex PCR capillary electrophoresis assay and PCR-reverse dot blot assay for human papillomavirus DNA genotyping detection in cervical cancer tissue specimens.

Background: The study aimed to evaluate the positivity rates and genotype distribution of the multiplex PCR capillary electrophoresis (MPCE) and PCR-Reverse Dot Blot (PCR-RDB) assays for human papillomavirus (HPV) detection in cervical cancer tissue specimens, and to explore their detection principles and applications in large-scale population screening. Methods: The MPCE and PCR-RDB assays were performed separately on 425 diagnosed cervical cancer tissue specimens. Subsequently, the results of both assays were compared based on the HPV infection positivity rates and genotype distribution. Results: The overall positive rates of HPV genotypes for the MPCE and PCR-RDB assays were 97.9% and 92.9%, respectively. A p-value < 0.001 indicated a statistically significance difference in consistency between the two assays. The kappa value was 0.390, indicating that the consistency between both assays was fair. HPV16 was the most common single-genotype infection type, with infection rates detected via MPCE and PCR-RDB assays being 75.7% and 68.3%, respectively. In the age group >50 years, the HPV multiple-type infection rate detected via MPCE assay was significantly higher than that detected by the PCR-RDB assay, with a statistically significant difference (p = 0.002). Conclusion: To reduce the false-negative rate and improve screening efficiency, the MPCE assay, which targets the oncogenic gene E6/E7 segments, can be extended to the general female population for the early detection, diagnosis, and treatment of cervical cancer.

GelBox: Open-source software to improve rigor and reproducibility when analyzing gels and immunoblots.

GelBox is open-source software that was developed with the goal of enhancing rigor, reproducibility, and transparency when analyzing gels and immunoblots. It combines image adjustments (cropping, rotation, brightness, and contrast), background correction, and band-fitting in a single application. Users can also associate each lane in an image with metadata (for example, sample type). GelBox data files integrate the raw data, supplied metadata, image adjustments, and band-level analyses in a single file to improve traceability. GelBox has a user-friendly interface and was developed using MATLAB. The software, installation instructions, and tutorials, are available at https://campbell-muscle-lab.github.io/GelBox/.

Separation of Lysozyme-Ovotransferrin Complexes and the Cooperative Role of Their Components in Egg White.

A complex of ovotransferrin and lysozyme was directly isolated from egg white using an anti-transferrin antibody-immobilized membrane after antiserum proteins were separated by non-denaturing two-dimensional electrophoresis and transferred onto a membrane. The complex retained lysozyme activity that catalyzes the breakdown of peptidoglycans in the bacterial cell wall at the ß1-4 bond between N-acetylmuramic acid and N-acetylglucosamine residues. The activity of the purified lysozyme was suppressed to 6.4% in the presence of 1 µmol Fe2+, whereas that of the mixture of the purified lysozyme and ovotransferrin was maintained at 58%. The activity of the purified lysozyme was suppressed to 35% in the presence of 10 nmol Fe3+, whereas that of the mixture of the purified lysozyme and ovotransferrin was maintained at 66%. Furthermore, the bacteriolytic activity against Bacillus subtilis of egg white with reduced glycoproteins such as ovotransferrin was assessed, and the bacteriolytic activity was found to be suppressed in the presence of Fe2+ and Fe3+. This suppression was ions, thereby alleviating the inhibition of lysozyme activity by iron ions. A complex of ovotransferrin and lysozyme is efficient because ovotransferrin effectively captures iron ions near lysozyme. Thus, protein complexes containing enzymes can be applied to control their activity.

Prevalence of Pulmonary Hypertension among Sudanese Patients with Sickle Cell Disease.

Background: Sickle Cell Disease (SCD) is a hereditary condition characterized by aberrant red blood cell morphology, leading to persistent hemolytic anemia. The consequential impact of SCD on the pulmonary vasculature can result in pulmonary hypertension (PHT), a severe complication that detrimentally affects the well-being and survival of individuals with SCD. The prevalence and risk determinants of PHT in SCD patients exhibit variations across diverse geographical regions and populations. This study aims to ascertain the prevalence of PHT among Sudanese SCD patients and identify associated factors. Methods: A cohort of thirty-one adult sickle cell disease (SCD) patients, as confirmed by hemoglobin electrophoresis, were recruited for participation in this cross-sectional study. Comprehensive data encompassing demographic, clinical, and laboratory parameters were collected. Doppler echocardiography was employed to quantify pulmonary arterial systolic pressure (PASP) and evaluate right ventricular size and function. Results: Within our cohort, the prevalence of PHT was 29%. Active cigarette smoking demonstrated a significant association with PHT (P=0.042), while hydroxyurea therapy exhibited no noticeable impact on PHT (P=0.612). Conclusion: Our investigation revealed a PHT prevalence of less than one-third in our SCD patient population, aligning with prior studies. Notably, independent of other factors, cigarette smoking emerged as a distinct risk factor for PHT in SCD patients. This highlights the potential utility of smoking cessation as an intervention to delay the onset of this condition. However, further research is imperative to elucidate the mechanisms through which smoking contributes to PHT development in individuals with SCD.

[Familial bisalbuminemia]. / Familiäre Bisalbuminämie.

Bisalbuminemia is characterized by two albumin peaks in the electrophoresis of serum. There are different forms of bisalbuminemia: inherited and acquired. The acquired form is mainly transitory, whereas the familial form is permanent. The frequency of bisalbuminemia in the general population has been reported to be between 0.0003 and 0.01%. This paper presents a case of familial bisalbuminemia as well as the family tree-to the extent obtainable. A married couple, in which the husband had bisalbuminemia, had seven children and 18 grandchildren. Bisalbuminemia was also found in two children and in two grandchildren.

Polysaccharides Analysis of Cell Wall Using Carbohydrate Gel Electrophoresis.

The plant cell wall is rich in polysaccharides with high heterogeneity. Investigating the composition and structure of cell wall polysaccharides is crucial for understanding the functionalities of plant cell walls. Carbohydrate electrophoresis is a sensitive and rapid method to analyze polysaccharides qualitatively and quantitatively. The process includes digesting the polysaccharides with appropriate cleavage enzymes, labeling the reducing ends of the released oligosaccharides with a highly charged fluorophore, and separating the labeled oligosaccharides in a polyacrylamide gel via high-voltage electrophoresis. The generated fluorescence can be calculated as compared to that of oligosaccharide standards. Therefore, this is a convenient method for polysaccharide characterization that can be performed in most laboratories. Here, we introduce the detailed operational steps and precautions, which are helpful for researchers to quickly obtain the structural information of polysaccharides.

Comprehensive Study of Total Nitrogen Content and Microfluidic Profiles in Additive-Enriched Plant-Based Drinks.

The growing consumption of plant-based milk substitutes raises important questions about their composition. The various additives used by manufacturers, including those employed as flavor enhancers, protein additives, and stabilizers, may contain both protein and non-protein nitrogen components. In our study, we examined not only popular milk alternatives but also other milk substitutes made from specific plants. We present a reproducible and rapid method for the simultaneous qualitative and quantitative determination of the total nitrogen content in milk alternatives, focusing on applicability. Using the microchip gel electrophoretic method, we determined that the total nitrogen content differed from the protein content indicated on the packaging. Our results, along with statistical evaluations, supported the hypothesis that different brands of products, derived from the same plant source, resulted in different microfluidic profiles, likely due to the presence of additives. As expected, the microfluidic profiles of additive-free products differed from those of fortified products made from the same plant-based milk replacer. Total nitrogen content provides crucial information for individuals with kidney disease, as is essential to reduce the burden on the kidneys to slow deterioration, alleviate symptoms and avoid complications.

Artificial chemotaxis under electrodiffusiophoresis.

HYPOTHESIS: Through a large parameter space, electric fields can tune colloidal interactions and forces leading to diverse static and dynamical structures. So far, however, field-driven interactions have been limited to dipole-dipole and hydrodynamic contributions. Nonetheless, in this work, we propose that under the right conditions, electric fields can also induce interactions based on local chemical fields and diffusiophoretic flows. EXPERIMENTS: Herein, we present a strategy to generate and measure 3D chemical gradients under electric fields. In this approach, faradaic reactions at electrodes induce global pH gradients that drive long-range transport through electrodiffusiophoresis. Simultaneously, the electric field induces local pH gradients by driving the particle's double layer far from equilibrium. FINDINGS: As a result, while global pH gradients lead to 2D focusing away from electrodes, local pH gradients induce aggregation in the third dimension. Evidence points to a mechanism of interaction based on diffusiophoresis. Interparticle interactions display a strong dependence on surface chemistry, zeta potential and diameter of particles. Furthermore, pH gradients can be readily tuned by adjusting the voltage and frequency of the electric field. For large Péclet numbers, we observed a collective chemotactic-like collapse of particles. Remarkably, such collapse occurs without reactions at a particle's surface. By mixing particles with different sizes, we also demonstrate, through experiments and Brownian dynamics simulations, the emergence of non-reciprocal interactions, where small particles are more drawn towards large ones.

Highly sensitive two-dimensional profiling of N-linked glycans by hydrophilic interaction liquid chromatography and dual stacking capillary gel electrophoresis.

BACKGROUND: N-Glycosylation is one of the most important post-translational modifications in proteins. As the N-glycan profiles in biological samples are diverse and change according to the pathological condition, various profiling methods have been developed, such as liquid chromatography (LC), capillary electrophoresis (CE), and mass spectrometry. However, conventional analytical methods have limitations in sensitivity and/or resolution, hindering the discovery of minor but specific N-glycans that are important both in the basic glycobiology research and in the medical application as biomarkers. Therefore, a highly sensitive and high-resolution N-glycan profiling method is required. RESULTS: In this study, we developed a novel two-dimensional (2D) separation system, which couples hydrophilic interaction liquid chromatography (HILIC) with capillary gel electrophoresis (CGE) via large-volume dual preconcentration by isotachophoresis and stacking (LDIS). Owing to the efficient preconcentration efficiency of LDIS, limit of detection reached 12 pM (60 amol, S/N = 3) with good calibration curve linearity (R2 > 0.999) in the 2D analysis of maltoheptaose. Finally, 2D profiling of N-glycans obtained from standard glycoproteins and cell lysates were demonstrated. High-resolution 2D profiles were successfully obtained by data alignment using triple internal standards. N-glycans were well distributed on the HILIC/CGE 2D plane based on the glycan size, number of sialic acids, linkage type, and so on. As a result, specific minor glycans were successfully identified in HepG2 and HeLa cell lysates. SIGNIFICANCE AND NOVELTY: In conclusion, the HILIC/CGE 2D analysis method showed sufficient sensitivity and resolution for identifying minor but specific N-glycans from complicated cellular samples, indicating the potential as a next-generation N-glycomics tool. Our novel approach for coupling LC and CE can also dramatically improve the sensitivity in other separation modes, which can be a new standard of 2D bioanalysis applicable not only to glycans, but also to other diverse biomolecules such as metabolites, proteins, and nucleic acids.

Characterization and Techno-Functional Properties of High Protein Walnut Flour from an Oil by-Product.

A high protein walnut flour (HPWF) was obtained by defatting walnut flour (WF), which is a by-product of the oil industry. The objective of this study was the chemical and techno-functional characterization of HPWF. Composition, amino acid content, protein secondary structure, protein solubility and thermal transitions were measured. Besides, the techno-functional properties, emulsion activity and stability, and water holding and oil absorption capacities, of HPWF were evaluated. Also, the molecular mass of proteins under denaturing conditions and the microstructure of HPWF were evaluated by electrophoresis and confocal scanning laser microscopy, respectively. HPWF had 55.4% protein content and 21.5% total dietary fibre. In terms of HPWF amino acid composition, the limiting amino acids were the sulphurated cysteine and methionine. By FTIR analysis, the main secondary structures were ß-sheet (49%) followed by α-helix (24%); both structures are considered to be ordered. Likewise, HPWF soluble proteins increased at basic pH and HPWF proteins were separated in 11 bands with molecular masses ranging from 97 kDa to 18 kDa by electrophoresis. With respect to techno-functional properties, HPWF presented good emulsion activity (51%) and high thermal emulsion stability (46%). In addition, HPWF retained 571% and 242% of water and oil by weight, respectively. Finally, the micrograph showed the predominance of protein structures and fibre fragments, and the presence of few lipids mostly trapped. These results showed that HPWF is an interesting source of plant-based proteins and walnut flour can be used to obtain high protein ingredients from non-traditional sources.

Assessment of the genetic diversity of the alleles of gliadin-coding loci in common wheat (Triticum aestivum L.) collections in Kazakhstan and Russia.

The study of genetic resources using prolamin polymorphism in wheat cultivars from countries with different climatic conditions makes it possible to identify and trace the preference for the selection of the alleles of gliadine-coding loci characteristic of specific conditions. The aim of the study was to determine the "gliadin profile" of the collection of common wheat (Triticum aestivum L.) from breeding centers in Russia and Kazakhstan by studying the genetic diversity of allelic variants of gliadin-coding loci. Intrapopulation (µ ± Sµ) and genetic (H) diversity, the proportion of rare alleles (h ± Sh), identity criterion (I) and genetic similarity (r) of common wheat from eight breeding centers in Russia and Kazakhstan have been calculated. It has been ascertained that the samples of common wheat bred in Kostanay region (Karabalyk Agricultural Experimental Station, Kazakhstan) and Chelyabinsk region (Chelyabinsk Research Institute of Agriculture, Russia) had the highest intrapopulation diversity of gliadin alleles. The proportion of rare alleles (h) at Gli-B1 and Gli-D1 loci was the highest in the wheat cultivars bred by the Federal Center of Agriculture Research of the South-East Region (Saratov region, Russia), which is explained by a high frequency of occurrence of Gli-B1e (86 %) and Gli-D1a (89.9 %) alleles. Based on identity criterion (I), the studied samples of common wheat from different regions of Kazakhstan and Russia have differences in gliadin-coding loci. The highest value of I = 619.0 was found when comparing wheat samples originated from Kostanay and Saratov regions, and the lowest I = 114.4, for wheat cultivars from Tyumen and Chelyabinsk regions. Some region-specific gliadin alleles in wheat samples have been identified. A combination of Gli-A1f, Gli-B1e and Gli-Da alleles has been identified in the majority of wheat samples from Kazakhstan and Russia. Alleles (Gli-A1f, Gli-A1i, Gli-A1m, Gli-A1o, Gli-B1e, Gli-D1a, Gli-D1f, Gli-A2q, Gli-B2o, and Gli-D2a) turned out to be characteristic and were found with varying frequency in wheat cultivars in eight regions of Russia and Kazakhstan. The highest intravarietal polymorphism (51.1 %) was observed in wheat cultivars bred in Omsk region (Russia) and the lowest (16.6 %), in Pavlodar region (Kazakhstan). On the basis of the allele frequencies, a "gliadin profile" of wheat from various regions and breeding institutions of Russia and Kazakhstan was compiled, which can be used for the selection of parent pairs in the breeding process, the control of cultivars during reproduction, as well as for assessing varietal purity.

Baseline health parameters of rhinoceros auklets (Cerorhinca monocerata) using serum protein electrophoresis, acute phase proteins, and biochemistry.

Clinical metrics of baseline health in sentinel seabird species can offer insight into marine ecosystem dynamics, individual and population health, and assist in wildlife rehabilitation and conservation efforts. Protein electrophoresis is useful for detecting changes in acute phase proteins and immunoglobulin levels that may indicate subtle inflammatory responses and/or infectious disease. Serum biochemistry can highlight nutritional status, metabolic derangements, and organ injury and function. However, baseline values for such health parameters are largely unknown for many seabird species. Therefore, the objective of this study is to establish baseline clinical health reference intervals for serum protein electrophoresis, acute phase proteins including serum amyloid A and haptoglobin, and biochemistry parameters in the rhinoceros auklet (Cerorhinca monocerata), a key sentinel species in the North Pacific. From 2013 to 2019, 178 wild, apparently healthy breeding adult rhinoceros auklets were captured across four breeding colonies in British Columbia, Canada (Lucy Island, Pine Island, Triangle Islands, and SGang Gwaay) and from one colony in Washington, United States (Protection Island). Reference intervals were calculated for protein electrophoresis fractions and acute phase proteins (n = 163), and serum biochemistry (n = 35) following established guidelines by the American Society of Veterinary Clinical Pathology. Animals were also assessed for the presence of antibodies to the influenza A virus. Approximately 48% (70/147) of sampled birds were seropositive for influenza A virus, with a prevalence of 50% (6/12) in 2013, 75% (47/63) in 2014, and 24% (17/72) in 2019. This work provides clinical baseline health metrics of a key North Pacific sentinel species to help inform marine ecosystem monitoring, recovery, and rehabilitation efforts in the Pacific Northwest.

Proteomic profile in plasma as a biomarker of pregnancy in cows.

Pregnancy has its own protein dynamics, reflecting the hormonal profile. Quantitative and qualitative changes in plasma protein profile may provide useful information about this condition. Any alterations may be a signal heralding clinical or subclinical pathology. The objective of our study was to compare the plasma protein profile between selected months of pregnancy in cows for a better understanding gestation course. For this purpose, we collected blood from healthy pregnant (n = 30; n = 6 for each pregnancy stage) and non-pregnant (C; n = 6) Holstein-Friesian cows during a routine veterinary examination. Collected samples were selected according to pregnancy month (first, second, third, sixth, and ninth), prepared, and separated by two-dimensional electrophoresis. The Delta-2D program compared and statistically evaluated scanned gel images from the appropriate months. The mean volume of the spots was considered. The MALDI TOF/TOF spectrometer was used to identify statistically significant proteins. There were 11 distinct proteins found, including peptidyl-prolyl cis-trans isomerase F, oligoribonuclease, and PRELI domain-containing protein 3B (all of them have the lowest abundance in the C group), alpha-1B-glycoprotein, L-gulonolactone oxidase, hemopexin (first month with higher abundance than control), alpha-2-HS-glycoprotein (significantly higher abundance in the first month than in remaining groups), ermin (absent in the first month and lower abundance in the third and sixth months than in the remaining groups and control), endophilin-A2 (significant differences between the control and the second, third, sixth, and ninth months), apolipoprotein A-I (significant difference between control and the first and sixth months), alpha-1-antiproteinase (significant difference between control and the ninth month). The study demonstrated the distinctions between plasma protein composition and alterations during the pregnancy course which may potentially serve as diagnostic tools.

Atypical applications of transverse diffusion of laminar flow profiles methodology for in-capillary reactions in capillary electrophoresis.

Capillary electrophoresis (CE) is a powerful separation technique offering quick and efficient analyses in various fields of bioanalytical chemistry. It is characterized by many well-known advantages, but one, which is perhaps the most important for this application field, is somewhat overlooked. It is the possibility to perform chemical and biochemical reactions at the nL scale inside the separation capillary. There are two basic formats applicable for this purpose, heterogeneous and homogeneous. In the former, one reactant is immobilized onto a particle or monolithic support or directly on the capillary wall, and the other is injected. In the latter, the reactant mixing inside a capillary is based on electromigration or diffusion. One of the diffusion-based methodologies, termed Transverse Diffusion of Laminar Flow Profiles, is the subject of this review. Since most studies utilizing in-capillary reactions in CE focus on enzymes, which are being continuously and exhaustively reviewed, this review covers the atypical applications of this methodology, but still in the bioanalytical field. As can be seen from the demonstrated applications, they are not limited to reactions, but can also be utilized for other biochemical systems.

Virulence genes and pulsed-field gel electrophoresis profiles of Shiga toxin-producing Escherichia coli isolated from different food samples and patients with acute diarrhea.

Background and Objectives: Escherichia coli O157: H7 is one of the most important causes of hemorrhagic colitis, and hemolytic uremic syndrome. The present study aimed to isolate E. coli O157: H7 from foods and patients with hemorrhagic colitis, and identify Shiga toxin genes, phylogenetic comparison, and antibiotic resistance of the isolates. Materials and Methods: In total 400 samples, including patients stool and food were taken in Isfahan-Iran province. Phenotypic tests and PCR were performed to identify Shiga toxin-producing E. coli. The isolated strains were compared phylogenetically by PFGE. Agar disk diffusion was performed to identify the antibiotic resistance of the isolates. Results: Totally, 5 isolates of fecal samples were E. coli O157, but only 2 isolates carried H7 gene. Also, 9 isolates of E. coli O157 were isolated from food samples that 3 isolates were E. coli O157: H7. The isolates carried stx1, stx2, hlyA and eaeA genes. Also, E. coli non-O157: H7 identified from samples that contained stx1, stx2, hlyA genes. The highest susceptibility to imipenem and the highest resistance to ampicillin and ciprofloxacin were observed. There was a similarity of 100% between the E. coli O157: H7 strains isolated from patients and raw milk and minced beef samples. Conclusion: Serotypes other than the O157 of E. coli are more prevalent in patients and food. The E. coli O157: H7 isolates from patients had 100% genetic similarity with minced meat and cow milk isolates, which indicates cattle are the most important reservoir of this bacterium in Iran.