Electrophoretic Analysis Research Articles

A Fluorescence Cross-Correlation-Spectroscopy-Based Immunoassay for Rapid, Selective, and Accurate Protein Sizing in Human Plasma, Applied to the von Willebrand Factor.

Multimeric abnormalities in plasma von Willebrand factor (VWF) cause bleeding or clotting disorders. Electrophoretic analysis of multimers is used to detect such abnormalities but is qualitative, slow, and difficult to standardize. Fluorescence correlation spectroscopy (FCS) is a good alternative but is affected by low selectivity and concentration bias. Here, we report the development of a homogeneous immunoassay based on dual-color fluorescence cross-correlation spectroscopy (FCCS) that overcomes these challenges. By performing a mild denaturation treatment followed by reacting with polyclonal antibodies, the concentration bias was drastically reduced. The use of a dual antibody assay improved selectivity. Diffusion times of immunolabeled VWF were measured with FCCS and standardized relative to calibrator measurements. The assay measures size changes in VWF using 1 μL of plasma and less than 10 ng of antibody per measurement and was validated over a 16-fold range of VWF antigen concentration (VWF:Ag), with a sensitivity of VWF:Ag 0.8%. Concentration bias and imprecision were less than 10%. Measurements were unaffected by hemolytic, icteric, or lipemic interference. Strong correlations were obtained with reference densitometric readouts (0.97 for calibrators, 0.85 for clinical samples), and significant differences were found between normal (n = 10), type 2A (n = 5), and type 2B (n = 5) von Willebrand's disease and acquired thrombotic thrombocytopenic purpura (n = 10) samples (p < 0.01). This FCCS based immunoassay accurately and selectively determines changes in the multimeric status of plasma VWF and may be used as a simpler, faster, and a standardizable alternative for multimer analysis, following further clinical validation in larger cohorts.

A combination of Sephadexes for the milk whey protein fractions isolation

Whey proteins perform important biological functions. They can also be a source of numerous natural bioactive peptides that have a positive effect on various physiological systems of the body. For research and usage of this phenomenon, the question of these proteins isolating with saving of their structure and biological functions is relevant. Considering the significant difference in molecular weights, gel filtration is a promising method. The purpose of the study was the fractionation of milk whey proteins using gel filtration with a combination of different dextran gels. We used in the work milk whey proteins after isoelectric precipitation of casein and isolation of low molecular weight peptides by gel filtration on Sephadex G-25. The first gel filtration of the whey protein preparation was performed on a column with Sephadex G-150. For further fractionation were used three sectors from the first two chromatographic peaks obtained by gel filtration on Sephadex G-150. The first peak contains high molecular fractions of proteins eluting with a volume close to the free volume of the chromatographic column. The second asymmetric peak consists mainly of β-lactoglobulin and α-lactalbumin mixture. The third peak includes low molecular weight polypeptides and peptides that are not fixed in a polyacrylamide gel. The combined chromatographic fractions of three sectors from the first two chromatographic peaks were re-fractionated on a G-100 column. Electrophoretic analysis of chromatographic fractions after repeated chromatography of milk whey proteins confirmed the feasibility of choosing a certain sequence of sephadex. At the same time, the yield of β-lactoglobulin fraction increases by 12 ± 1 %, and α-lactalbumin by 9 ± 1 % compared to the results obtained by repeated gel filtration on one Sephadex (G-100). The degree of purification of β-lactoglobulin and α-lactalbumin, according to the densitometry, was 93 ± 5 % and 77 ± 4 %, respectively.

SEMI-QUANTITATIVE ANALYSISOF NUCLEIC ACIDS ON AGAROSE GELS WITH CHOICE OF BEVERAGES AS ELECTROPHORETIC BUFFER

The pH of beverages is known to be low, and has therefore been involved in the increasing acidity incidence. In this study,to report on a rapid and cost-effective approach for nucleic acid characterization using different running buffers for agarose gel electrophoresis.Commercial DNA is used in electrophoretic analysis using a variety of beverages by optimizing its pH value.The results suggest that Royal Stag has little effect on nucleic acid research, while Peter Scot shows high concentration and sensitivity in nucleic acid characterization.Royal Stag drink has revealed that concentration, time, and voltage have little effect on nucleic acid research, although Peter Scot has reported high concentration and sensitivity in nucleic acid characterization. We conclude that this approach is simple, easy, and cost-effective compared to using TAE or TBE as running buffers in electrophoresis of the agarose gel.

Intraspecific divergence of diploid grass Aegilopscomosa is associated with structural chromosome changes.

Aegilopscomosa Smith in Sibthorp et Smith, 1806 is diploid grass with MM genome constitution occurring mainly in Greece. Two morphologically distinct subspecies - Ae.c.comosa Chennaveeraiah, 1960 and Ae.c.heldreichii (Holzmann ex Boissier) Eig, 1929 are discriminated within Ae.comosa, however, genetic and karyotypic bases of their divergence are not fully understood. We used Fluorescence in situ hybridization (FISH) with repetitive DNA probes and electrophoretic analysis of gliadins to characterize the genome and karyotype of Ae.comosa to assess the level of their genetic diversity and uncover mechanisms leading to radiation of subspecies. We show that two subspecies differ in size and morphology of chromosomes 3M and 6M, which can be due to reciprocal translocation. Subspecies also differ in the amount and distribution of microsatellite and satellite DNA sequences, the number and position of minor NORs, especially on 3M and 6M, and gliadin spectra mainly in the a-zone. Frequent occurrence of hybrids can be caused by open pollination, which, along with genetic heterogeneity of accessions and, probably, the lack of geographic or genetic barrier between the subspecies, may contribute to extremely broad intraspecific variation of GAAn and gliadin patterns in Ae.comosa, which are usually not observed in endemic plant species.

Validation of ELISA assays for the calculation of FLC indices for the diagnosis of intrathecal immunoglobulin synthesis.

Define the cutoff thresholds of the Kappa (K) and Lambda (L) free light chains (FLC) indices for the detection of intrathecal immunoglobulin synthesis (IIS) using the new K and L FLC ELISA from SEBIA. The reference technique, which is not readily standardized between laboratories, is based on the demonstration of oligoclonal banding (OCB) in cerebrospinal fluid (CSF) which is absent in serum. For the past 6 years, we have also routinely calculated the K FLC index using The Binding Site (TBS) reagents on an Optilite instrument, an approach increasingly used as an alternative and/or a complement to electrophoretic analysis. We analyzed 391 serum/CSF pairs divided into three groups. The first group were cases without OCB and with normal albumin CSF/serum ratio (n=174). The second group were cases with specific OCB (n=73). The last group included patients with increased albumin CSF/sera ratio without OCB (n=142). Analysis of the first group determined that the cutoffs for detection of IIS are respectively 2.55 and 1.02 forthe K FLC and L FLC indices. Of the 73 cases with IIS, only2 had a K FLC index below this threshold (sensitivity of 97.26%), while 16 out of 73 cases (78.08%) and 13 out of 72 cases (81.94%) had an IgG and L FLC index below the cutoffs, respectively. Additionally, we illustrate equivalent performances for prediction of the presence of OCB between SEBIA and TBS methods. Sebia K FLC and L FLC assays are adequate alternative methods for the diagnosis of IIS.

Alpha-thiol deoxynucleotide triphosphates (S-dNTPs) as radioprotective agents: A novel approach

In this study, the ability of a mixture of four different alpha-thiol deoxynucleotide triphosphates (S-dNTPs) each at a concentration of 10μM when incorporated into the genomic DNA of proliferating human HL-60 and Mono-Mac-6 (MM-6) cells in vitro to provide protection from 2, 5, and 10 Gy of gamma radiation was investigated. Incorporation of the four different S-dNTPs into nuclear DNA at 10 μM concentration for five days was validated by agarose gel electrophoretic band shift analysis. S-dNTP-treated genomic DNA reacted with BODIPY-iodoacetamide demonstrated a band shift to higher molecular weight to confirm the presence of sulfur moieties in the resultant phosphorothioate DNA backbones. No overt signs of toxicity or obvious morphologic cellular differentiation were noted in the presence of 10 μM S-dNTPs even after 8 days in culture. Significantly reduced radiation-induced persistent DNA damage measured at 24 and 48 h post-exposure by γ-H2AX histone phosphorylation using FACS analysis in S-dNTP incorporated HL-60 and MM6 cells indicated protection against radiation-induced direct and indirect DNA damage. Statistically significant protection by S-dNTPs was noted at the cellular level by CellEvent™ Caspase-3/7 assay, which assess the extent of apoptotic events, and by trypan blue dye exclusion to assed cell viability. The results appear to support an innocuous antioxidant thiol radioprotective effect built into genomic DNA backbones as the last line of defense against ionizing radiation and free radical-induced DNA damage.

Methodology of adipose tissue type detection in mammals

Nowadays, an interest in studying the composition, properties and functions of adipose tissue (AT) is growing among researchers, which is conditioned by its important role in the normal functioning of the body. Due to different types of adipose tissue (AT) in mammals (white, beige, brown and pink) and different physiological tasks performed by each type of AT, rapid, correct and effective detection of an AT type is highly topical. Methods used today are labor consuming and in the case of NMR and CT expensive, which limits possibilities of scientists. In this connection, the aim of this research was to develop a methodological approach allowing rapid and effective detection of an adipose tissue type. A methodology was formed based on the concept, formalized requirements for the method, step-wise structure of investigations and interpretation of results. The concept is based on differences in the structure of the adipose cell (adipocyte) of different AT types. The method is based on extraction of heme containing proteins. To this end, solvents and parameters of extraction that facilitate their better extraction have been chosen. An AT type has been determined by the total content of iron contained in the cytochrome fragment. Our own modification was selected. This modification includes preliminary mincing of a sample with the ice-cold TES buffer (pH 8.5) in a ratio of 1:5 (g: mL), homogenization at 9,000 rpm for 2 min with the following centrifugation at 10,000 g and 4 °C for 15 min. Effectiveness of the proposed method was confirmed by the histological and electrophoretic analyses. Therefore, the new methodology of identification and differentiation of adipocytes was proposed for rapid and effective detection of an adipose tissue type.

Center backbone-rigidified DNA polygonal nanostructures and bottom face-templated polyhedral pyramids with structural stability in a complex biological medium.

Due to the sequence programmability, good biocompatibility, versatile functionalities and vast sequence space, DNA oligonucleotides are considered to be ideal building blocks for the assembly of diverse nanostructures in one, two and three dimensions that are capable of engineering of multiple functional nucleic acids into a useful tool to implement intended tasks in biological and medical field. However, the construction of wireframe nanostructures consisting of only a few DNA strands remains quite challenging mainly because of the molecular flexibility-based uncontrollability of size and shape. In this contribution, utilizing gel electrophoretic analysis and atomic force microscopy, we demonstrate the modeling assembly technique for the construction of wireframe DNA nanostructures that can be divided into two categories: rigid center backbone-guided modeling (RBM) and bottom face-templated assembly (BTA) that are responsible for the construction of DNA polygons and polyhedral pyramids, respectively. The highest assembly efficiency (AE) is about 100%, while the lowest AE is not less than 50%. Moreover, when adding one edge for polygons or one side face for pyramids, we only need to add one oligonucleotide strand. Especially, the advanced polygons (e.g., pentagon and hexagon) of definite shape are for the first time constructed. Along this line, introduction of cross-linking strands enables the hierarchical assembly of polymer polygons and polymer pyramids. These wireframe DNA nanostructures exhibit the substantially enhanced resistance to nuclease degradation and maintain their structural integrity in fetal bovine serum for several hours even if the vulnerable nicks are not sealed. The proposed modeling assembly technique represents important progress toward the development of DNA nanotechnology and is expected to promote the application of DNA nanostructures in biological and biomedical fields. STATEMENT OF SIGNIFICANCE: DNA oligonucleotides are considered to be ideal building blocks for the assembly of diverse nanostructures. However, the construction of wireframe nanostructures consisting of only a few DNA strands remains quite challenging. In this contribution, we demonstrate the modeling technique for the construction of different wireframe DNA nanostructures: rigid center backbone-guided modeling (RBM) and bottom face-templated assembly (BTA) that are responsible for the assembly of DNA polygons and polyhedral pyramids, respectively. Moreover, cross-linking strands enables the hierarchical assembly of polymer polygons and polymer pyramids. These wireframe DNA nanostructures exhibit the substantially enhanced resistance to nuclease degradation and maintain their structural integrity in fetal bovine serum for several hours, promoting the application of DNA nanostructures in biological and biomedical fields.

Chondroitin Sulfate from Oreochromis niloticus Waste Reduces Leukocyte Influx in an Acute Peritonitis Model

Oreochromis niloticus (tilapia) is one of the most cultivated fish species worldwide. Tilapia farming generates organic waste from fish removal processes in nurseries. Visceral waste can damage natural ecosystems. Therefore, the use of this material as a source of biomolecules helps reduce environmental impacts and improve pharmacological studies. Tilapia viscera were subjected to proteolysis and complexation with an ion-exchange resin. The obtained glycosaminoglycans were purified using ion exchange chromatography (DEAE-Sephacel). The electrophoretic profile and analysis of 1H/13C nuclear magnetic resonance (NMR) spectra allowed for the characterization of the compound as chondroitin sulfate and its sulfation position. This chondroitin was named CST. We tested the ability of CST to reduce leukocyte influx in acute peritonitis models induced by sodium thioglycolate and found a significant reduction in leukocyte migration to the peritoneal cavity, similar to the polymorphonuclear population of the three tested doses of CST. This study shows, for the first time, the potential of CST obtained from O. niloticus waste as an anti-inflammatory drug, thereby contributing to the expansion of the study of molecules with pharmacological functions.

The XylR/NtrC-type regulator CbsR positively regulates upstream pathway of chlorobenzene degradation in Pandoraea pnomenusa.

Pandoraea pnomenusa MCB032 completely degrades chlorobenzene, whose metabolic pathway is encoded by cbs and clc gene clusters. The putative regulatory factors ClcR and CbsR are predicted to regulate the cbs and clc gene clusters. This research aims to understand the function of ClcR and CbsR. RT-PCR analyses demonstrated that the cbsFAaAbAcAdB operon that encodes catabolic pathways for the degradation of chlorobenzene to chlorocatechol is located on an operon. Moreover, the clcABCDE operon is involved in the 3-chlorocatechol pathway. Gene knockout and transcriptional analysis showed that the transcription of the cbsFAaAbAcAdB operon is positively regulated by CbsR, whereas the clcABCDE operon is activated by ClcR. Primer extension analysis was used to locate the transcription start sites of the cbsFAaAbAcAdB and cbsR operons. Electrophoretic mobility shift assay analyses showed that CbsR is bound to the sites in the promoter regions of cbsFAaAbAcAdB and cbsR operons. The XylR/NtrC-type regulator CbsR positively regulates the transcription of the cbsFAaAbAcAdB operon encoding the upstream pathway of chlorobenzene catabolism, while the LysR-type regulator ClcR activates the clcABCDE operon encoding the downstream pathway.

Identification and characterization of the Doublesex gene and its mRNA isoforms in the brine shrimp Artemia franciscana.

Doublesex (DSX) proteins are members of the Doublesex/mab-3-related (DMRT) protein family and play crucial roles in sex determination and differentiation among the animal kingdom. In the present study, we identified two Doublesex (Dsx)-like mRNA isoforms in the brine shrimp Artemia franciscana (Kellogg 1906), which are generated by the combination of alternative promoters, alternative splicing and alternative polyadenylation. The two transcripts exhibited sex-biased enrichment, which we termed AfrDsxM and AfrDsxF. They share a common region which encodes an identical N-terminal DNA-binding (DM) domain. RT-qPCR analyses showed that AfrDsxM is dominantly expressed in male Artemia while AfrDsxF is specifically expressed in females. Expression levels of both isoforms increased along with the developmental stages of their respective sexes. RNA interference with dsRNA showed that the knockdown of AfrDsxM in male larvae led to the appearance of female traits including an ovary-like structure in the original male reproductive system and an elevated expression of vitellogenin. However, silencing of AfrDsxF induced no clear phenotypic change in female Artemia. These results indicated that the male AfrDSXM may act as inhibiting regulator upon the default female developmental mode in Artemia. Furthermore, electrophoretic mobility shift assay analyses revealed that the unique DM domain of AfrDSXs can specifically bind to promoter segments of potential downstream target genes like AfrVtg. These data show that AfrDSXs play crucial roles in regulating sexual development in Artemia, and further provide insight into the evolution of sex determination/differentiation in sexual organisms.

Changes in the protein profile of beers with bilberry

In the present study, beers with an original extract 12°P, 14°P and 16°P were obtained. During the maturation process, on different stages, bilberries (Vaccinium myrtillus L.) from Bulgaria were added. Changes in the protein profile of the beers were investigated, as well as the influence of these changes on the body in the mouthfeel and the head retention of the resulting beers. The electrophoretic analysis of the proteins was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration was determined by the Bradford method. The sensory evaluation of the beer’s body and head retention was conducted by a trained five-membered testing panel. It was found that independently of the original extract, the bilberry addition led to a significant reduction of the protein concentration and the number of protein fractions. Beers with bilberry addition contained between 74% and 94% less protein, than control beers. The number of protein fractions decreased from 10 in the control beers to 4–6 in the beers with bilberry. The fractions with molecular weight 31 kDa and below 14 kDa were the most stable to the bilberry addition. Their part in the beers with bilberry remained the highest. Despite of the significant changes in the protein profiles, the beers with bilberry had very good mouthfeel body and head retention.

Two-Step Purification and Partial Characterization of Keratinolytic Proteases from Feather Meal Bioconversion by Bacillus sp. P45

This study aimed to purify and partially characterize a keratinolytic protease produced by Bacillus sp. P45 through bioconversion of feather meal. Crude protease extract was purified using a sequence of an aqueous two-phase system (ATPS) in large volume systems (10, 50, and 500 g) to increase obtaining purified enzyme, followed by a diafiltration (DF) step. Purified protease was characterized in terms of protein profile analysis by SDS-PAGE, optimum temperature and pH, thermal deactivation kinetics at different temperatures and pH, and performance in the presence of several salts (NaCl, CaCl2, MnCl2, CaO, C8H5KO4, MgSO4, CuSO4, ZnSO4, and FeCl3) and organic solvents (acetone, ethanol, methanol, acetic acid, diethyl ether, and formaldehyde). ATPS with high capacities resulted in purer protease extract without compromising purity and yields, reaching a purification factor up to 2.6-fold and 6.7-fold in first and second ATPS, respectively, and 4.0-fold in the DF process. Recoveries were up to 79% in both ATPS and reached 84.3% after the DF step. The electrophoretic analysis demonstrated a 25–28 kDa band related to keratinolytic protease. The purified protease’s optimum temperature and pH were 55 °C and 7.5, respectively. The deactivation energy (Ed) value was 118.0 kJ/mol, while D (decimal reduction time) and z (temperature interval required to reduce the D value in one log cycle) values ranged from 6.7 to 237.3 min and from 13.6 to 18.8 °C, respectively. Salts such as CaCl2, CaO, C8H5KO4, and MgSO4 increased the protease activity, while all organic solvents caused its decrease. The results are useful for future studies about ATPS scale-up for enzyme purification and protease application in different industrial processes.

Multilayer Coatings Based On Citrate-Stabilized Gold Nanoparticles and Polydiallyldimethylammonium Chloride for the Electrophoretic Separation of Carboxylic Acids

The conditions for the formation of physically adsorbed three-layer coatings of quartz capillary walls in capillary electrophoresis (CE) with successively deposited oppositely charged layers of polydiallyldimethylammonium chloride (PDADMAC) modifiers and citrate-stabilized gold nanoparticles (GNPs) are proposed. It was shown that three-layer PDADMAC–GNP–PDADMAC coatings favorably differ from monolayer coatings with PDADMAC by greater stability in a wide range of pH (2–10). The formed coatings were characterized by scanning electron microscopy, and the presence of a uniform dense layer of nanoparticles on the capillary surface was confirmed. The applicability of the modified capillaries under CE conditions was demonstrated by the separation of a mixture of 16 carboxylic acids. An increase in the separation selectivity achieved with the use of three-layer coatings based on GNPs was explained by the reversible exchange of citrate anions on the GNP surface with negatively charged analytes in the course of electrophoretic analysis.

Fourier Transform Infrared (FTIR) Spectroscopic Study of Biofilms Formed by the Rhizobacterium Azospirillum baldaniorum Sp245: Aspects of Methodology and Matrix Composition

Biofilms represent the main mode of existence of bacteria and play very significant roles in many industrial, medical and agricultural fields. Analysis of biofilms is a challenging task owing to their sophisticated composition, heterogeneity and variability. In this study, biofilms formed by the rhizobacterium Azospirillum baldaniorum (strain Sp245), isolated biofilm matrix and its macrocomponents have for the first time been studied in detail, using Fourier transform infrared (FTIR) spectroscopy, with a special emphasis on the methodology. The accompanying novel data of comparative chemical analyses of the biofilm matrix, its fractions and lipopolysaccharide isolated from the outer membrane of the cells of this strain, as well as their electrophoretic analyses (SDS-PAGE) have been found to be in good agreement with the FTIR spectroscopic results.

Molecular genetic characteristics of a family which coinheritance of rare-88 C>G (HBB:c.-138 C>G) β-thalassemia mutation with α-thalassemia and review of the literature

The molecular genetic characteristics of a family with rare -88 C>G (HBB: c.-138 C>G) β-thalassemia gene mutation were studied using cohort study. The cohort study was conducted from June to August 2022 by Prenatal Diagnosis Center of Sanya Women and Children's Hospital Managed by Shanghai Children's Medical Center. The phenotype and genotype were analyzed by hematological cytoanalyzer, automatic electrophoretic analysis system, and next-generation sequencing (NGS). And then, Sanger sequencing was used to verify the rare gene results. The results showed that the proband, her father, her uncle and her younger male cousin had discrete microcytosis (MCV 70.1 fl, 71.9 fl, 73.1 fl and 76.6 fl, respectively) and hypochromia (MCH 21.5 pg,22.0 pg,22.6 pg and 23.5 pg, respectively), elevated hemoglobin A2 level (5.3%, 5.4%, 5.4% and 5.5%, respectively), slightly elevated or normal fetal hemoglobin (Hb F), but no anemia. The proband was identified to have co-inherited ɑ-thalassemia (Hb Westmead gene heterozygous mutation, ɑwsɑ/ɑɑ) and β-thalassemia with a rare -88 C>G (HBB: c.-138 C>G) heterozygous mutation (β-88 C>G/βN). Her mother had the same α-thalassemia as the proband. Her father, her uncle and her younger male cousin had the same rare -88 C>G heterozygous mutations as the proband. While her grandmother and younger brother were not carrier of thalassemia. In conclusion, 4 cases of rare -88 C>G(HBB:c.-138 C>G) heterozygous mutation had been detected in a Chinese family. Carriers of this beta-thalassemia are clinically asymptomatic. This study enriches the knowledge of the thalassemia mutation spectrum in Chinese people and provides valuable information for genetic counseling, prenatal diagnosis, and prevention of thalassemia, providing a scientific basis for improving the quality of birth population and preventing birth defects.

Tau liquid-liquid phase separation is modulated by the Ca2+ -switched chaperone activity of the S100B protein.

Aggregation of the microtubule-associated protein tau is implicated in several neurodegenerative tauopathies including Alzheimer's disease (AD). Recent studies evidenced tau liquid-liquid phase separation (LLPS) into droplets as an early event in tau pathogenesis with the potential to enhance aggregation. Tauopathies like AD are accompanied by sustained neuroinflammation and the release of alarmins at early stages of inflammatory responses encompass protective functions. The Ca2+ -binding S100B protein is an alarmin augmented in AD that was recently implicated as a proteostasis regulator acting as a chaperone-type protein, inhibiting aggregation and toxicity through interactions of amyloidogenic clients with a regulatory surface exposed upon Ca2+ -binding. Here we expand the regulatory functions of S100B over protein condensation phenomena by reporting its Ca2+ -dependent activity as a modulator of tau LLPS induced by crowding agents (PEG) and metal ions (Zn2+ ). We observe that apo S100B has a negligible effect on PEG-induced tau demixing but that Ca2+ -bound S100B prevents demixing, resulting in a shift of the phase diagram boundary to higher crowding concentrations. Also, while incubation with apo S100B does not compromise tau LLPS, addition of Ca2+ results in a sharp decrease in turbidity, indicating that interactions with S100B-Ca2+ promote transition of tau to the mixed phase. Further, electrophoretic analysis and FLIM-FRET studies revealed that S100B incorporates into tau liquid droplets, suggesting an important stabilizing and chaperoning role contributing to minimize toxic tau aggregates. Resorting to Alexa488-labeled tau we observed that S100B-Ca2+ reduces the formation of tau fluorescent droplets, without compromising liquid-like behavior and droplet fusion events. The Zn2+ -binding properties of S100B also contribute to regulate Zn2+ -promoted tau LLPS as droplets are decreased by Zn2+ buffering by S100B, in addition to the Ca2+ -triggered interactions with tau. Altogether this work uncovers the versatility of S100B as a proteostasis regulator acting on protein condensation phenomena of relevance across the neurodegeneration continuum.

PtrbZIP3 transcription factor regulates drought tolerance of Populus trichocarpa

Plant basic leucine zipper (bZIP) proteins participate in plant development and a variety of abiotic stress responses. However, their roles in response to abiotic stress in Populus are still unclear. In this study, we characterized a bZIP protein, PtrbZIP3, in Populus trichocarpa which belongs to the sub-family A. PtrbZIP3 expression was highly induced by treatment with NaCl, PEG6000, or abscisic acid (ABA). PtrbZIP3 is a nuclear protein with transcriptional activation activity. Yeast one hybrid and electrophoretic mobility shift analysis showed that PtrbZIP3 can specifically bind to abscisic acid-responsive element (ABRE) to regulate the expression of ABA-dependent genes and increase the amount of ABA in P. trichocarpa overexpressing PtrbZIP3. Under PEG-simulated drought conditions, overexpression of PtrbZIP3 increases the activities of antioxidant enzymes, scavenging of reactive oxygen species, maintains cell membrane integrity, reduces stomatal apertures and water loss, and increases proline accumulation. Thus PtrbZIP3 is a transcriptional activator that plays a role in an ABA-dependent pathway to improve osmotic tolerance in P. trichocarpa.

Lipase Production by Yarrowia lipolytica in Solid-State Fermentation Using Amazon Fruit By-Products and Soybean Meal as Substrate

The production of polyunsaturated fatty acids from fish oil, which is related to various health benefits including effects against cardiovascular diseases, antihypertensive, anticancer, antioxidant, antidepression, anti-aging, and anti-arthritis effects, among others, can be advantageously performed through the application of lipase. However, the high cost associated with enzyme production can make the process unfeasible and thus alternative substrates should be investigated to solve these problems. This research aimed to produce lipase by Yarrowia lipolytica IMUFRJ50682 in solid-state fermentation using by-products of the food processing industry (andiroba oil cake and soybean meal) and verify the potential application in the initial hydrolysis of fish oil to further produce polyunsaturated fatty acids in a suitable process. A screening was carried out for the analysis of andiroba oil cake and soybean meal combinations in different proportions (0:100 to 100:0, respectively) at 48 h of the fermentation process. Afterward, the solid matrix composed by soybean meal and andiroba oil cake was supplemented with soy oil and Tween 80 to improve the lipase activity. The enzymatic extract was characterized in relation to the protein profile by electrophoresis. Finally, the enzymatic extract and the solid biocatalyst produced were applied to evaluate the potential hydrolysis of the fish oil in an initial study. Maximum lipolytic activity (63.7 U·g−1) was achieved using andiroba oil cake and soybean meal (50:50) after 24 h of fermentation. Soybean oil 1.5% and Tween 80 (0.001%) in an emulsion provided an increase of 1.5-fold (82.52 U·g−1) in the enzyme activity. The electrophoretic analysis demonstrated a band between 37 and 40 kDa that may be related to lipase and a band of 75 kDa referring to the α subunit of the β-conglycinin present in soybean meal. After 48 h, the solid biocatalyst showed a higher degree of hydrolysis (DH) (71.0%) than the enzymatic extract (61.5%). The solid biocatalyst was stable during storage at room temperature for 7 months. The production of lipases using Amazon fruit by-product and soybean meal in solid-state fermentation is viable as well as the application of the extract and solid biocatalyst in the initial application for the hydrolysis of fish oil to further produce polyunsaturated fatty acids in an industrially suited process.

Sample Stacking-Capillary Electrophoretic Analysis of Nitrate and Nitrite in Organic- and Conventional-Originated Baby Food Formulas from Turkey.

Commercially available baby food formulas represent a convenient alternative to homemade meals especially in the recent years. The main purpose of this study is the determination of nitrate and nitrite levels by a sample stacking-capillary electrophoresis technique in the baby foods. The baby foods analyzed were organic-originated, vegetable-based, fruit-based, mixed puree, and a vegetable soup. Vegetables and fruits have high nitrate and nitrite concentrations. Nitrate itself is not actually hazardous. However, nitrite has negative health effects. Thus, baby foods have to be strictly controlled due to the potential health risk of nitrite. In this study, the sample stacking method enhanced the sensitivities of both anions. Nitrate contents ranged from 16.1 to 285 mg/kg with a mean concentration of 149 mg/kg for all samples. The lowest nitrate amount belonged to red fruity milky baby food whereas the highest nitrate was found in organic pumpkin, banana, and carrot mixed puree. The nitrite levels in all the samples were below the LOQ value of the analyzed method. As a conclusion, there is no health risk of the analyzed baby foods regarding nitrate and nitrite levels considering the regulations.