Interaction of Supramolecular Congo Red and Congo Red-Doxorubicin Complexes with Proteins for Drug Carrier Design

Targeted immunotherapy has expanded to simultaneous delivery of drugs, including chemotherapeutics. The aim of the presented research is to design a new drug carrier system. Systems based on the use of proteins as natural components of the body offer the chance to boost safety and efficacy of targeted drug delivery and excess drug removal. Congo red (CR) type supramolecular, self-assembled ribbon-like structures (SRLS) were previously shown to interact with some proteins, including albumin and antibodies complexed with antigen. CR can intercalate some chemotherapeutics including doxorubicin (Dox). The goal of this work was to describe the CR-Dox complexes, to analyze their interaction with some proteins, and to explain the mechanism of this interaction. In the present experiments, a model system composed of heated immunoglobulin light chain Lλ capable of CR binding was used. Heat aggregated immunoglobulins (HAI) and albumin were chosen as another model system. The results of experiments employing methods such as gel filtration chromatography and dynamic light scattering confirmed the formation of the CR-Dox complex of large size and properties different from the free CR structures. Electrophoresis and chromatography experiments have shown the binding of free CR to heated Lλ while CR-Dox mixed structures were not capable of forming such complexes. HAI was able to bind both free CR and CR-Dox complexes. Albumin also bound both CR and its complex with Dox. Additionally, we observed that albumin-bound CR-Dox complexes were transferred from albumin to HAI upon addition of HAI. DLS analyses showed that interaction of CR with Dox distinctly increased the hydrodynamic diameter of CR-Dox compared with a free CR supramolecular structure. To our knowledge, individual small proteins such as Lλ may bind upon heating a few molecules of Congo red tape penetrating protein body due to the relatively low cohesion of the dye micelle. If, however, the compactness is high (in the case of, e.g., CR-Dox) large ribbon-like, micellar structures appear. They do not divide easily into smaller portions and cannot attach to proteins where there is no room for binding large ligands. Such binding is, however, possible by albumin which is biologically adapted to form complexes with different large ligands and by tightly packed immune complexes and heat aggregated immunoglobulin-specific protein complex structures of even higher affinity for Congo red than albumin. The CR clouds formed around them also bind the CR-Dox complexes. The presented research is essential in the search for optimum solutions for SRLS application in immuno-targeting therapeutic strategies, especially with the use of chemotherapeutics.

Spike protein multiorgan tropism suppressed by antibodies targeting SARS-CoV-2

While there is clinical evidence of severe acute respiratory syndrome coronavirus 2 multiorgan tropism in severely infected coronavirus 19 patients, it is unclear if there is differential multiorgan biodistribution and organ uptake in healthy young individuals, a group that usually has asymptomatic to moderate coronavirus 19 symptoms. In addition, for antibody therapies and vaccines that target the spike protein, it is unclear if these reduce severe acute respiratory syndrome coronavirus 2 or spike protein multiorgan tropism equally. We used fluorescently labeled spike protein near infrared fluorescence to study viral behavior, using an in vivo dynamic imaging system, in young mice. We found a spike protein body-wide biodistribution followed by a slow regional elimination, except for the liver, which showed an accumulation. Spike protein uptake was highest for the lungs, and this was followed by kidney, heart and liver, but, unlike the choroid plexus, it was not detected in the brain parenchyma or cerebrospinal fluid. Thus, the brain vascular barriers were effective in restricting the entry of spike protein into brain parenchyma in young healthy mice. While both anti-angiotensin converting enzyme 2 and anti-spike protein antibodies suppressed spike protein biodistribution and organ uptake, anti-spike protein antibody was more effective. By extension, our data support the efficacy of these antibodies on severe acute respiratory syndrome coronavirus 2 biodistribution kinetics and multiorgan tropism that could determine coronavirus 19 organ-specific outcomes.

EFFECTS OF GUANIDINOACETIC ACID SUPPLEMENTATION IN BROILER CHICKEN DIET ON CARCASS CHARACTERISTICS

Guanidinoacetic acid (GAA) is a natural organic acid in the body that acts as a precursor of creatine which plays the role of energy carrier in the cell This study was conducted to determine the effects of supplementing broiler chicken feed with GAA on carcass characteristics. Two hundred-and forty, day old Cornish breeds were kept under a deep litter system. Six treatments with four replicates of 10 birds per replica were used. The experimental diets comprised of six treatments with varying levels of feed additive (GAA) supplemented in the diets D1 (control) with no GAA, D2 was supplemented with (0.003% GAA/kg), D3 (0.006% GAA/kg), D4 (0.009% GAA/kg), D5 (0.012% GAA/kg), and D6 (0.015% GAA/kg). The treatments were laid down in a complete randomized design (CRD) with four replicates. Carcass characteristics were determined in the laboratory by analyzing the (abdominal fat, tissue protein, tissue fat and pH). There were statistically significant effects observed on abdominal fat, body tissue protein and body tissue fat of the carcass. The study concluded that GAA supplementation increased carcass characteristics, and provided better economic returns. The study recommends utilization of GAA supplementation at level of 0.12% GAA/Kg for rapid growth of broiler, enhanced abdominal fat, body tissue protein, body tissue fat, and utilization of GAA for better economic returns to the farmers.

Rabbit Carcasses for Use in Feline Diets: Amino Acid Concentrations in Fresh and Frozen Carcasses With and Without Gastrointestinal Tracts

Whole-prey diets for exotic feline species are common, and this practice has also increased in popularity for domestic cats. However, prior analyses of prey indicate possible essential amino acid inadequacy, and dilated cardiomyopathy from taurine deficiency was reported in cats fed whole ground rabbit. Crude protein, body water, and amino acid concentrations were evaluated in fresh and frozen ground rabbits with (n=10) or without (n = 10) gastrointestinal tracts. Amino acids were greater in fresh samples without gastrointestinal tracts (p < 0.05) except taurine, glycine, and cysteine. When normalized for protein content, only glutamate, alanine, methionine, isoleucine, tyrosine, lysine, histidine, and arginine were greater in fresh rabbits without gastrointestinal tracts (g/16 g N basis; p < 0.05). Freezing at −18°C for 30 days had no effect on crude protein or body water content. After freezing, only methionine was lower and only proline was higher when gastrointestinal tracts were omitted (g/16 g N basis; p < 0.05). Regardless, all essential amino acids except taurine exceeded Association of American Feed Control Officials and National Research Council nutrient recommendations for all feline life stages. In contrast, there was minimal impact of treatment on taurine concentrations. However, although feline taurine requirements for prey and other raw or fresh food diets remain undefined, none of the rabbit samples met any recommendation for taurine concentrations for commercial canned or dry extruded diets, ranging from 20 to 90% of the minimum values. Taurine supplementation is recommended when feeding rabbit to cats. Determination of taurine requirements of cats fed whole-prey diets is warranted.

Comparing the educational gradients in three cardiovascular disease-specific health measures

Less-educated persons have worse cardiovascular health. We compare the educational gradients in three disease-specific health measures (biomarkers, self-reported doctors’ diagnoses and cause-specific mortality) in order to compare their relevance in different stages of the disease process. We study 14,102 people aged 50–89 from the US Health Retirement Study (HRS) in the period 2006–17. We use six CVD biomarkers (systolic/ diastolic blood pressure, ratio total/HDL cholesterol, C-reactive protein, body mass index, HbA1c) and two self-reported doctors’ diagnoses (stroke, heart attack). We estimate the gradient in biomarkers using log-binomial regression and the hazard of diagnoses and CVD mortality with Cox survival models.Among those without pre-diagnosed CVD conditions, the educational gradient in mortality is highest (RR 1.97), the gradient for those who receive a CVD diagnosis is in the middle (RR 1.46), and the gradient in biomarkers is lowest (RR 1.32). Among those with recent/ older diagnoses, the biomarker gradient is comparable to levels among the non-diagnosed, while the mortality gradient is much lower (RR 1.35). The gradients in diagnoses and mortality are only slightly explained by differences in biomarkers.The comparison of the three gradients and the mediation analysis suggest that in each of the steps to diagnosis and death there are social factors involved that increase the gradient and go beyond what biomarkers can predict. Having a CVD diagnosis leads to smaller mortality gradients, presumably because of the convergence of educational differences in behaviour and during treatment and monitoring. Our findings support prevention as a strategy against social inequalities in CVD.<br />Key messages<br /><ul><li>The educational gradient is highest for mortality; next highest is diagnoses; lowest is biomarkers.</li><br /><li>The gradients in diagnoses and mortality are only slightly explained by differences in biomarkers.</li><br /><li>CVD progression is subject to social factors that widen the gradient beyond biomarkers’ predictivity.</li><br /><li>Among diagnosed people, changes in behaviour and treatment seem to lower the mortality gradient.</li></ul>

Aspartate Residues in a Forisome-Forming SEO Protein Are Critical for Protein Body Assembly and Ca2+ Responsiveness

Forisomes are protein bodies known exclusively from sieve elements of legumes. Forisomes contribute to the regulation of phloem transport due to their unique Ca2+-controlled, reversible swelling. The assembly of forisomes from sieve element occlusion (SEO) protein monomers in developing sieve elements and the mechanism(s) of Ca2+-dependent forisome contractility are poorly understood because the amino acid sequences of SEO proteins lack conventional protein–protein interaction and Ca2+-binding motifs. We selected amino acids potentially responsible for forisome-specific functions by analyzing SEO protein sequences in comparison to those of the widely distributed SEO-related (SEOR), or SEOR proteins. SEOR proteins resemble SEO proteins closely but lack any Ca2+ responsiveness. We exchanged identified candidate residues by directed mutagenesis of the Medicago truncatula SEO1 gene, expressed the mutated genes in yeast (Saccharomyces cerevisiae) and studied the structural and functional phenotypes of the forisome-like bodies that formed in the transgenic cells. We identified three aspartate residues critical for Ca2+ responsiveness and two more that were required for forisome-like bodies to assemble. The phenotypes observed further suggested that Ca2+-controlled and pH-inducible swelling effects in forisome-like bodies proceeded by different yet interacting mechanisms. Finally, we observed a previously unknown surface striation in native forisomes and in recombinant forisome-like bodies that could serve as an indicator of successful forisome assembly. To conclude, this study defines a promising path to the elucidation of the so-far elusive molecular mechanisms of forisome assembly and Ca2+-dependent contractility.

Metabolizable Energy Levels for Free-Range Broiler Chickens

The aim of this study was to evaluate the effect of dietary metabolizable energy levels on the performance and carcass yield of free-range broiler chickens from 1 to 84 days of age. A total of 900 male day-old naked neck lineage chicks were distributed in a completely randomized design between six levels of metabolizable energy (2,700; 2,800; 2,900; 3,000; 3,100 and 3,200 kcal.kg-1 diet) with six replications of 25 birds each. The increase in levels of dietary metabolizable energy resulted in a linear reduction of the feed intake, crude protein and digestible lysine intakes, as well as in the protein body deposition and protein efficiency and linear improvements in the feed conversion ratio of chickens in all experimental phases. The carcass yield, wing and abdominal fat weight and percentage of abdominal fat reduced linearly by increasing the level of dietary metabolizable energy. The diet including 2700 kcal.kg-1 of metabolizable energy in the diet of free-range broiler chickens in phases 1 to 28, 28 and 56 and 57 to 84 days of age does not interfere in the broilers performance and results in a better carcass yield in the final period of production.

Increased Nitro-Oxidative Stress Toxicity as a Major Determinant of Increased Blood Pressure in Mood Disorders

Background: Hypertension, atherogenicity and insulin resistance are major risk factors of cardiovascular disorder (CVD), which shows a strong comorbidity with major depression (MDD) and bipolar disorder (BD). Activated oxidative and nitrosative stress (O&amp;NS), inflammatory pathways, and increased atherogenicity are shared pathways underpinning CVD and mood disorders. Methods: The current study examined the effects of lipid hydroperoxides (LOOH), superoxide dismutase (SOD), nitric oxide metabolites (NOx), advanced oxidation protein products (AOPP), and malondialdehyde (MDA) on systolic (SBP) and diastolic (DBP) blood pressure in 96 mood disordered patients and 60 healthy controls. Results: A large part of the variance in SBP (31.6%) was explained by the regression on a z unit-weighted composite score (based on LOOH, AOPP, SOD, NOx) reflecting nitro-oxidative stress toxicity (NOSTOX), coupled with highly sensitive C-reactive protein, body weight and use of antihypertensives. Increased DBP was best predicted (23.8%) by body mass index and NOSTOX. The most important O&amp;NS biomarkers predicting an increased SBP were in descending order of significance: LOOH, AOPP and SOD. Higher levels of the atherogenic index of plasma, HOMA2 insulin resistance index and basal thyroid-stimulating hormone also contributed to increased SBP independently from NOSTOX. Although there were no significant changes in SBP/DBP in mood disorders, the associations between NOSTOX and blood pressure were significant in patients with mood disorders but not in healthy controls. Conclusions: Activated O&amp;NS pathways including increased lipid peroxidation and protein oxidation, which indicates hypochlorous stress, are the most important predictors of an increased BP, especially in patients with mood disorders.

Fatty acid metabolism in newborns with sepsis; Report 1 — New clinical outcome prediction scale

The aim of our study was to develop a new scale for predicting clinical outcome in newborns with sepsis, taking into account the information about depth of metabolic disorders.Methods. Design of study — retrospective observational single-center study. Medical cards data of 163 newborns with sepsis were included in analysis. To measure the predictive value of the analyzed clinical and laboratory signs we used the Kullback measure. The clinical outcome of the disease was determined as the response function: survived or died.Results. An analysis of the predictive value of the clinical and laboratory parameters of in newborns with sepsis was made, the threshold values of the most informative indicators were developed, which were: the number of blood platelets, the level of total blood protein, body weight and the number of blood neutrophils. The Clinical and Laboratory Condition Index for Newborns scale has been developed, which takes into account the depth of metabolic disorders. ROC analysis (Area Under Curve — 0,723) and the information method (information coefficient 0.992) showed that the Clinical and Laboratory Condition Index score can be used in the intensive care unit to predict the risk negative clinical outcome and evaluate the effectiveness of treatment in infants with organ dysfunction caused by systemic infection.Conclusion. New Clinical and Laboratory Condition Index score scale allows to predict the development of negative outcome of sepsis in newborns taking into account the depth of metabolic disorders in newborns with sepsis.