Physiological and molecular changes of onion (Allium cepa L.) seeds under different aging conditions.

BACKGROUND: Onion seeds have limited storage capacity compared to other vegetable seeds. It is crucial to identify the mechanisms that induce tolerance to storage conditions and reduce seed deterioration. To address this goal, an experiment was conducted to evaluate changes in germination, biochemical, physiological, and molecular characteristics of onion seed landraces (Horand, Kazerun landraces and Zargan cultivar) at different aging levels (control, three-days and six-days accelerated aging, and natural aging for one year). RESULTS: The findings suggest that there was an increase in glucose, fructose, total sugar, and electrolyte leakage in the Horand (HOR), Kazerun (KAZ) landraces, and Zarghan (ZAR) cultivar, with Kazerun exhibiting the greatest increase. The percentage and rate of germination of Kazerun decreased by 54% and 33%, respectively, in six-day accelerated aging compared to the control, while it decreased by 12% and 14%, respectively, in Horand. Protein content decreased with increasing levels of aging, with a decrease of 26% in Kazerun landrace at six days of aging, while it was 16% in Horand landrace. The antioxidant activities of catalase, superoxide dismutase, and glutathione peroxidase decreased more intensively in Kazerun. The expression of AMY1, BMY1, CTR1, and NPR1 genes were lower in Kazerun landraces than in Horand and Zargan at different aging levels. CONCLUSIONS: The AMY1, BMY1, CTR1, and NPR1 genes play a pivotal role in onion seed germination, and their downregulation under stressful conditions has been shown to decrease germination rates. In addition, the activity of CAT, SOD, and GPx enzymes decreased by seed aging, and the amount of glucose, fructose, total sugar and electrolyte leakage increased, which ultimately led to seed deterioration. Based on the results of this experiment, it is recommended to conduct further studies into the molecular aspects involved in onion seed deterioration. More research on the genes related to this process is suggested, as well as investigating the impact of different priming treatments on the genes expression involved in the onion seed aging process.

Improving Process-Based Modelling to Simulate the Effects of Low-Temperature Stress During Pre-Anthesis on the Quality Characteristics of Wheat Grains.

Low temperatures in late spring pose a potential threat to the maintenance of grain yield and quality. Despite the importance of protein and starch in wheat quality, they are often overlooked in models addressing climate change effects. In this study, we conducted multiyear environment-controlled phytotron experiments and observed adverse effects resulting from low-temperature stress (LTS) on plant carbon and nitrogen dynamics, grain protein and starch formation, and sink capacity. We quantified the relationships between low temperature during the jointing and booting stages and plant nitrogen uptake, grain nitrogen accumulation, grain starch accumulation, grain setting, and potential grain weight using source-sink relationship-based methods. The LTS factor was introduced to account for the cultivar-specific to LTS at different growth stages. Compared with the original model, the improved model produced fewer errors when simulating aboveground nitrogen accumulation, grain protein concentration, grain starch concentration, grain starch yield, grain number, and grain weight under LTS, with reductions of 60%, 71%, 73%, 58%, 50% and 65%, respectively. The improvements in the model enhance its mechanism and applicability in assessing short-term successive frost effects on wheat grain quality. Furthermore, when using the improved model, special attention should be given to the low-temperature sensitivity parameters.

Quantifying Protein Shape to Elucidate Its Influence on Solution Viscosity in High-Concentration Electrolyte Solutions.

Therapeutic proteins with a high concentration and low viscosity are highly desirable for subcutaneous and certain local injections. The shape of a protein is known to influence solution viscosity; however, the precise quantification of protein shape and its relative impact compared to other factors like charge-charge interactions remains unclear. In this study, we utilized seven model proteins of varying shapes and experimentally determined their shape factors (v) based on Einstein's viscosity theory, which correlate strongly with the ratios of the proteins' surface area to the 2/3 power of their respective volumes, based on protein crystal structures resolved experimentally or predicted by AlphaFold. This finding confirms the feasibility of computationally estimating protein shape factors from amino acid sequences alone. Furthermore, our results demonstrated that, in high-concentration electrolyte solutions, a more spherical protein shape increases the protein's critical concentration (C*), the transition concentration beyond which protein viscosity increases exponentially relative to concentration increases. In summary, our work elucidates protein shape as a key determinant of solution viscosity through quantitative analysis and comparison with other contributing factors. This provides insights into molecular engineering strategies to optimize the molecular design of therapeutic proteins, thus optimizing their viscosity.

Comparative Assessment of Mitochondria Isolation Buffers for Optimizing Tissue-Specific Yields in Buffalo.

INTRODUCTION: Mitochondrial studies are crucial for assessing livestock health and performance. While extensive research has been done on cattle and pigs, the influence of mitochondria in Indian buffalo remains unexplored. Therefore, in order to understand functions of mitochondria, their energy-related processes, or any additional mitochondrial traits in buffaloes, it is imperative to isolate high-yield mitochondria with purity and functionality. Mitochondria are extracted by few conventional buffers. These buffers were previously characterized for their effectiveness in isolating mitochondria from rodent and human tissues. Therefore, the present study is to assess the performance of mitochondria isolation buffers specifically in buffalo tissues. METHODS: The study involved isolation of mitochondria from four different tissues, i.e., liver, brain, heart and muscles of slaughtered buffalo (n = 3), using: (i) Tris-Mannitol buffer (ii) Tris-Sucrose buffer, and (iii) MOPS-Sucrose buffer. Buffer efficiency in preserving high fidelity during mitochondria isolation was assessed by comparison with Cayman's MitoCheck® Mitochondrial Isolation Kit (control). Further mitochondrial purity and functionality was assessed through comparative estimation of protein concentration and marker enzyme assays, respectively. RESULTS: Our results revealed insights into the suitability of specific buffer for functional mitochondria isolation from specific type of buffalo tissue. Notably for obtaining high quality functional mitochondria from buffalo, MOPS-Sucrose buffer appeared optimal for soft tissues (liver and brain), while Tris-Mannitol buffer was efficient for hard tissues (muscles and heart). CONCLUSIONS: Thus, our research highlights the influence of buffer composition and tissue-specific variations in buffer effectiveness on mitochondrial activity in different tissues, leading to improved mitochondrial isolation in buffalo.

Multiple Reaction Monitoring-Mass Spectrometry Enables Robust Quantitation of Plasma Proteins Regardless of Whole Blood Processing Delays That May Occur in the Clinic.

Plasma is an important biofluid for clinical research and diagnostics. In the clinic, unpredictable delays-from minutes to hours-between blood collection and plasma generation are often unavoidable. These delays can potentially lead to protein degradation and modification and might considerably affect intact protein measurement methods such as sandwich enzyme-linked immunosorbent assays that bind proteins on two epitopes to increase specificity, thus requiring largely intact protein structures. Here, we investigated, using multiple reaction monitoring mass spectrometry (MRM-MS), how delays in plasma processing affect peptide-centric "bottom-up" proteomics. We used validated assays for proteotypic peptide surrogates of 270 human proteins to analyze plasma generated after whole blood had been kept at room temperature from 0 to 40 h to mimic delays that occur in the clinic. Moreover, we evaluated the impact of different plasma-thawing conditions on MRM-based plasma protein quantitation. We demonstrate that >90% of protein concentration measurements were unaffected by the thawing procedure and by up to 40-h delayed plasma generation, reflected by relative standard deviations (RSDs) of <30%. Of the 159 MRM assays that yielded quantitative results in 60% of the measured time points, 139 enabled a stable protein quantitation (RSD <20%), 14 showed a slight variation (RSD 20-30%), and 6 appeared unstable/irreproducible (RSD > 30%). These results demonstrate the high robustness and thus the potential for MRM-based plasma-protein quantitation to be used in a clinical setting. In contrast to enzyme-linked immunosorbent assay, peptide-based MRM assays do not require intact three-dimensional protein structures for an accurate and precise quantitation of protein concentrations in the original sample.

The assessment of Dickkopf-1 and Dickkopf-2 protein concentration in different subtypes of non-small cell lung cancer subtypes.

Introduction: Lung cancer is one of the most prevalent cancers worldwide. Dickkopf-1 (DKK-1) and -2 (DKK-2) are important proteins for the regulated Wnt signalling pathway. Alternations in the Wnt pathway are associated with tumour progression. The aim of the study was to analyse the concentration of DKK-1 and DKK-2 in tumour and matched non-tumour (NT) samples of 65 patients with non-small cell lung cancer (NSCLC), including 3 subtypes: adenocarcinoma (AC), squamous cell carcinoma (SCC), and large cell carcinoma (LCC). Material and methods: The protein concentration was measured by enzyme-linked immunosorbent assay (ELISA) in homogenates. Results: The difference between the level of DKK-1 in tumour and NT specimens was not significant for the whole NSCLC group and SCC and LCC subtype, while in AC samples they were significantly higher (p = 0.028). The highest concentration of DKK-1 was found in the advanced NSCLC samples, with the T4 parameter as well as stage III. Significantly decreased DKK-2 concentrations were detected in all NSCLC subtypes (p < 0.05). Moreover, the DKK-2 level was higher in non-smokers than in smokers. The results indicate that concentrations of DKKs were different in relation to subtypes as well as clinical and socio-demographic parameters. The concentration of DKKs could be associated with the progression of NSCLC. Conclusions: We suggest that DKK-1 could play an oncogenic role in AC, while DKK-2 could be a tumour suppressor in all NSCLC subtypes. Dickkopf-1 and DKK-2 proteins could have differential roles in the Wnt signalling pathway, which is important in many cellular processes, such as proliferation and apoptosis.

The Potential Association between E2F2, MDM2 and p16 Protein Concentration and Selected Sociodemographic and Clinicopathological Characteristics of Patients with Oral Squamous Cell Carcinoma.

BACKGROUND: E2F transcription factor 2 (E2F2), murine double minute 2 (MDM2) and p16 are some of the key proteins associated with the control of the cell cycle. The aim of this study was to evaluate E2F2, MDM2 and p16 concentrations in the tumour and margin samples of oral squamous cell carcinoma and to assess their association with some selected sociodemographic and clinicopathological characteristics of the patients. METHODS: The study group consisted of 73 patients. Protein concentrations were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: There were no statistically significant differences in the levels of E2F2, MDM2 or p16 in the tumour samples as compared to the margin specimens. We found that patients with N0 showed significantly lower E2F2 concentrations than patients with N1 in the tumour samples and the median protein concentration of E2F2 was higher in HPV-negative patients in the tumour samples. Moreover, the level of p16 in the margin samples was lower in alcohol drinkers as compared to non-drinkers. Similar observations were found in concurrent drinkers and smokers compared to non-drinkers and non-smokers. CONCLUSIONS: E2F2 could potentially promote tumour progression and metastasis. Moreover, our results showed a differential level of the analysed proteins in response to alcohol consumption and the HPV status.

Personalized diagnostic approach and indirect quantification of extravasation in human anaphylaxis.

BACKGROUND: Anaphylaxis is the most acute and life-threatening manifestation of allergic disorders. Currently, there is a need to improve its medical management and increase the understanding of its molecular mechanisms. This study aimed to quantify the extravasation underlying human anaphylactic reactions and propose new theragnostic approaches. METHODS: Molecular determinations were performed in paired serum samples obtained during the acute phase and at baseline from patients presenting with hypersensitivity reactions. These were classified according to their severity as Grades 1, 2 and 3, the two latter being considered anaphylaxis. Tryptase levels were measured by ImmunoCAP, and serum protein concentration was quantified by Bradford assay. Human serum albumin (HSA) and haemoglobin beta subunit (HBB) levels were determined by Western blot and polyacrylamide gel electrophoresis, respectively. RESULTS: A total of 150 patients were included in the study. Of them, 112 had experienced anaphylaxis (83 and 29 with Grade 2 and 3 reactions, respectively). Tryptase diagnostic efficiency substantially improved when considering patients' baseline values (33%-54%) instead of the acute value threshold (21%). Serum protein concentration and HSA significantly decreased in anaphylaxis (p < .0001). HSA levels dropped with the severity of the reaction (6% and 15% for Grade 2 and 3 reactions, respectively). Furthermore, HBB levels increased during the acute phase of all hypersensitivity reactions (p < .0001). CONCLUSIONS: For the first time, the extravasation underlying human anaphylaxis has been evaluated based on the severity of the reaction using HSA and protein concentration measurements. Additionally, our findings propose new diagnostic and potential therapeutic approaches for this pathological event.

Initial heat analysis in dissociation isothermal titration calorimetry: An analytical tool for thermodynamic dissection of biomolecular condensates.

Multi-domain proteins or intrinsically disordered proteins (IDPs) often undergo liquid-liquid phase separation (LLPS) and form membraneless organelles or protein condensates. Such compartmentalization is considered critical in many cellular processes dynamically modulated by various external signals. However, molecular mechanisms underlying potential regulatory functions of the protein condensates remain obscure due to a limited understanding of the driving forces for their assembly. Here we propose isothermal titration calorimetry (ITC) as an efficient analytical tool to dissociate condensates and measure the corresponding dissociation heat. Subsequent analysis of the initial dissociation heat as a function of total protein concentration allows simple and accurate determination of the thermodynamic parameters for cooperative condensate formations including the dissociation (or condensation) enthalpy and the critical protein concentration. By performing systematic simulations, we further demonstrate that the initial heat analysis is sufficiently robust to quantitatively dissect protein condensates with a broad range of thermodynamic properties. Therefore, our proposed method analyzing the initial heat measured in dissociation ITC provides opportunities to further scrutinize the thermodynamic quantities as functions of solution variables to explore the molecular driving forces of LLPS.

Morphogen gradient formation in partially absorbing media.

Morphogen gradients play an essential role in the spatial regulation of cell patterning during early development. The classical mechanism of morphogen gradient formation involves the diffusion of morphogens away from a localized source combined with some form of bulk absorption. Morphogen gradient formation plays a crucial role during early development, whereby a spatially varying concentration of morphogen protein drives a corresponding spatial variation in gene expression during embryogenesis. In most models, the absorption rate is taken to be a constant multiple of the local concentration. In this paper, we explore a more general class of diffusion-based model in which absorption is formulated probabilistically in terms of a stopping time condition. Absorption of each particle occurs when its time spent within the bulk domain (occupation time) exceeds a randomly distributed thresholda; the classical model with a constant rate of absorption is recovered by taking the threshold distributionΨ(a)=e-κ0a. We explore how the choice of Ψ(a) affects the steady-state concentration gradient, and the relaxation to steady-state as determined by the accumulation time. In particular, we show that the more general model can generate similar concentration profiles to the classical case, while significantly reducing the accumulation time.

Plasma-induced nanoparticle aggregation for stratifying COVID-19 patients according to disease severity.

Stratifying patients according to disease severity has been a major hurdle during the COVID-19 pandemic. This usually requires evaluating the levels of several biomarkers, which may be cumbersome when rapid decisions are required. In this manuscript we show that a single nanoparticle aggregation test can be used to distinguish patients that require intensive care from those that have already been discharged from the intensive care unit (ICU). It consists of diluting a platelet-free plasma sample and then adding gold nanoparticles. The nanoparticles aggregate to a larger extent when the samples are obtained from a patient in the ICU. This changes the color of the colloidal suspension, which can be evaluated by measuring the pixel intensity of a photograph. Although the exact factor or combination of factors behind the different aggregation behavior is unknown, control experiments demonstrate that the presence of proteins in the samples is crucial for the test to work. Principal component analysis demonstrates that the test result is highly correlated to biomarkers of prognosis and inflammation that are commonly used to evaluate the severity of COVID-19 patients. The results shown here pave the way to develop nanoparticle aggregation assays that classify COVID-19 patients according to disease severity, which could be useful to de-escalate care safely and make a better use of hospital resources.

Endocrine disrupting effects of binary mixtures of 17ß-estradiol and testosterone in adult female western mosquitofish (Gambusia affinis).

Androgens and estrogens often co-exist in aquatic environments and pose potential risks to fish populations. However, little is known about the endocrine disrupting effects of the mixture of androgens and estrogens in fish. In this study, transcriptional level of target genes related to the hypothalamic-pituitary-gonadal-liver (HPGL) axis, sex hormone level, VTG protein concentration, histology and secondary sex characteristic were assessed in the ovaries and livers of adult female western mosquitofish (Gambusia affinis) exposed to 17ß-estradiol (E2), testosterone (T), and mixtures of E2 and T for 91 days. The results showed that the transcriptional expression of cytochrome P450, family 19, subfamily A, polypeptide 1a (Cyp19a1a) was suppressed in the 200 ng/L T treatment and the 50 ng/L E2 + 200 ng/L T treatment in the ovaries. Steroidogenic acute regulatory protein (Star) and Cyp11a1 showed a similar expression pattern in the T treatment to its corresponding T + E2 mixtures. In the ovaries, the concentrations of 17ß-estradiol and testosterone were decreased in most treatments compared with the solvent control. VTG protein was induced in all steroid treatment. However, exposure to T or E2 + T mixture did not cause the abnormal cells of the ovaries and livers and an extension of the anal fins in female G. affinis. This study demonstrates that chronic exposure to E2, T and their mixtures affects the transcripts of genes in the HPGL axis, steroid hormone level and VTG protein concentration in the ovaries and livers, but fails to cause the histopathological effect of the ovaries and livers and alter the morphology of the anal fins in G. affinis.

Inhibitory effect of thiourea derivatives on the growth of blue-green algae.

High nutrient loading in aquatic environment has become the main causative of harmful algae blooms (HABs) in water resources particularly pond, lake and river. HABs are mostly dominated by microalgae derived from the group of blue-green algae which are capable of releasing harmful toxins. Therefore, this study aims to investigate the inhibitory effects of thiourea derivatives on the growth of such blue-green algae. Thiourea derivatives have been proven to exhibit antifungal and antibacterial effects. However, there is still limited study had been conducted on the effect of thiourea derivatives toward blue-green algae species in recent years. In this research, a species of blue-green algae from Kenyir Lake, Terengganu, Malaysia was successfully isolated using morphological characters and molecularly identified as Synechoccus elongatus. Four new thiourea derivative compounds were also successfully synthesised. The compounds were designed with variation on different R-substitution group and characterised using Nuclear Magnetic Resonance (NMR) to confirm their molecular structure. Those compounds were characterised as 1-Benzyl-3-(3,5-dimethoxy-benzoyl)-thiourea (C1), 1-(3-Chloro-benzyl)-3-(3,5-dimethoxy-benzoyl)-thiourea (C2), 1-(3,5-Dimethoxy-benzoyl)-3-(3-methyl-benzyl)-thiourea (C3) and 1-(3,5-Dimethoxy-benzoyl)-3-(3-trifluoromethyl-benzyl)-thiourea (C4). For the inhibition assessment,S. elongatus were treated with C1-C4 for 5 day at concentration of 2, 5, 10 and 20 µg/ml, respectively. C3 compound showed the highest inhibition percentage with 98% of inhibition after 5 days treatment. By using Bradford method, protein extraction of S. elongatus was conducted at the highest inhibition percentage. Protein concentration of treated species was observed with 3.28 µg/ml as compared to protein concentration of control with 6.48 µg/ml. This result indicated the reduction of protein content after the treatment. Protein band pattern was identified intensed after the treatment SDS PAGE was carried out. The thiourea derivatives compound proved to have successfully inhibited the growth of blue-green algae. Hence, further study should be carried out to ensure the compound can be practically utilized in the pond and in natural environment.

Bridging Offline Functional Model Carrying Aging-Specific Growth Rate Information and Recombinant Protein Expression: Entropic Extension of Akaike Information Criterion.

This study presents a mathematical model of recombinant protein expression, including its development, selection, and fitting results based on seventy fed-batch cultivation experiments from two independent biopharmaceutical sites. To resolve the overfitting feature of the Akaike information criterion, we proposed an entropic extension, which behaves asymptotically like the classical criteria. Estimation of recombinant protein concentration was performed with pseudo-global optimization processes while processing offline recombinant protein concentration samples. We show that functional models including the average age of the cells and the specific growth at induction or the start of product biosynthesis are the best descriptors for datasets. We also proposed introducing a tuning coefficient that would force the modified Akaike information criterion to avoid overfitting when the designer requires fewer model parameters. We expect that a lower number of coefficients would allow the efficient maximization of target microbial products in the upstream section of contract development and manufacturing organization services in the future. Experimental model fitting was accomplished simultaneously for 46 experiments at the first site and 24 fed-batch experiments at the second site. Both locations contained 196 and 131 protein samples, thus giving a total of 327 target product concentration samples derived from the bioreactor medium.

Evaluation of concentration process of bovine, goat and buffalo whey proteins by ultrafiltration.

In this research, the protein concentration, the permeate flux, and the predominant fouling mechanisms were investigated during ultrafiltration of different whey samples. The research was carried out at different values of transmembrane pressure and temperature using an experimental design, and a protein concentration of approximately 37 g L-1 was obtained for the bovine whey powder solution, at 60 kPa and 40 °C. The maximum flux observed was 8.9 and 7.9 kg m-2 h-1, respectively, for the bovine whey powder solution and bovine whey, at 50 kPa and 30 °C. Although goat and buffalo whey presented lower permeate flux, probably due to high solutes and calcium contents, protein concentrates of around 40 g L-1 were obtained using the ultrafiltration process. This demonstrates the potential of ultrafiltration to obtain non-bovine protein concentrates. The best fit, verified by Ho and Zydney model, suggests that the fouling for all analyzed whey occurs due to pore blocking and subsequent deposit on the membrane surface.

Establishment of a Protein Concentration Gradient in the Outer Membrane Requires Two Diffusion-Limiting Mechanisms.

OmpA-like proteins are involved in the stabilization of the outer membrane, resistance to osmotic stress, and pathogenesis. In Caulobacter crescentus, OmpA2 forms a physiologically relevant concentration gradient that forms by an uncharacterized mechanism, in which the gradient orientation depends on the position of the gene locus. This suggests that OmpA2 is synthesized and translocated to the periplasm close to the position of the gene and that the gradient forms by diffusion of the protein from this point. To further understand how the OmpA2 gradient is established, we determined the localization and mobility of the full protein and of its two structural domains. We show that OmpA2 does not diffuse and that both domains are required for gradient formation. The C-terminal domain binds tightly to the cell wall and the immobility of the full protein depends on the binding of this domain to the peptidoglycan; in contrast, the N-terminal membrane ß-barrel diffuses slowly. Our results support a model in which once OmpA2 is translocated to the periplasm, the N-terminal membrane ß-barrel is required for an initial fast restriction of diffusion until the position of the protein is stabilized by the binding of the C-terminal domain to the cell wall. The implications of these results on outer membrane protein diffusion and organization are discussed.IMPORTANCE Protein concentration gradients play a relevant role in the organization of the bacterial cell. The Caulobacter crescentus protein OmpA2 forms an outer membrane polar concentration gradient. To understand the molecular mechanism that determines the formation of this gradient, we characterized the mobility and localization of the full protein and of its two structural domains an integral outer membrane ß-barrel and a periplasmic peptidoglycan binding domain. Each domain has a different role in the formation of the OmpA2 gradient, which occurs in two steps. We also show that the OmpA2 outer membrane ß-barrel can diffuse, which is in contrast to what has been reported previously for several integral outer membrane proteins in Escherichia coli, suggesting a different organization of the outer membrane proteins.

Application of Magnetic Resonance to Assess Lyophilized Drug Product Reconstitution.

PURPOSE: Dynamic in-situ proton (1H) magnetic resonance imaging (MRI) and 1H T2-relaxometry experiments are described in an attempt to: (i) understand the physical processes, that occur during the reconstitution of lyophilized bovine serum albumin (BSA) and monoclonal antibody (mAb) proteins; and (ii) objectify the reconstitution time. METHODS: Rapid two-dimensional 1H MRI and diffusion weighted MRI were used to study the temporal changes in solids dissolution and characterise water mass transport characteristics. One-shot T2 relaxation time measurements were also acquired in an attempt to quantify the reconstitution time. Both MRI data and T2-relaxation data were compared to standard visual observations currently adopted by industry. The 1H images were further referenced to MRI calibration data to give quantitative values of protein concentration and, percentage of remaining undissolved solids. RESULTS: An algorithmic analysis of the 1H T2-relaxation data shows it is possible to classify the reconstitution event into three regimes (undissolved, transitional and dissolved). Moreover, a combined analysis of the 2D 1H MRI and 1H T2-relaxation data gives a unique time point that characterises the onset of a reconstituted protein solution within well-defined error bars. These values compared favourably with those from visual observations. Diffusion weighted MRI showed that low concentration BSA and mAb samples showed distinct liquid-liquid phase separation attributed to two liquid layers with significant density differences. CONCLUSIONS: T2 relaxation time distributions (whose interpretation is validated from the 2D 1H MR images) provides a quick and effective framework to build objective, quantitative descriptors of the reconstitution process that facilitate the interpretation of subjective visual observations currently adopted as the standard practice industry.

Changes in Total Protein Concentration Due to Fluid Removal During and Shortly after Hemodialysis.

BACKGROUND: Changes in plasma volume during hemodialysis are complex and have been shown to depend on the rate of fluid removal and the degree of fluid overload. We examined changes in total protein concentration during and shortly after a dialysis treatment in archived data from the HEMO study. METHODS: During follow-up months 4 and 36 of the HEMO study, additional blood samples were obtained during a typical dialysis session at 30 and 60 min after dialysis. In 315 studies from 282 patients where complete data were available, we calculated the concentration change in total protein and compared it to the modeled change in both total body water and extracellular fluid space as derived from 2-pool urea kinetic modeling. RESULTS: The mean postdialysis modeled urea volume (V) was 31.1 ± 6.18 L. Mean fluid removal was 2.76 ± 1.27 kg, over a session length of 207 ± 28 min. The ratio of predialysis V to postdialysis V averaged 1.090 ± 0.040. The mean TP ratios (post/pre) at 0, 30, and 60 min postdialysis averaged 1.121 ± 0.070 (SD), 1.091 ± 0.090, and 1.091 ± 0.086. The dialysate to serum sodium gradient, studied in a different group of treatments where this information was available, had no impact on these findings, nor did the length of the interdialytic interval. CONCLUSIONS: On average, after equilibration, the change in plasma volume due to fluid removal is similar to the modeled change in total body water (urea space), irrespective of dialysate to serum sodium gradient. This supports previous observations that during dialysis with ultrafiltration, plasma volume contracts to a lesser degree than the interstitial volume and that some fluid may be removed from spaces other than the extracellular fluid.

Spectroscopic methods for assessing the molecular origins of macroscopic solution properties of highly concentrated liquid protein solutions.

In cases of subcutaneous injection of therapeutic monoclonal antibodies, high protein concentrations (>50 mg/ml) are often required. During the development of these high concentration liquid formulations (HCLF), challenges such as aggregation, gelation, opalescence, phase separation, and high solution viscosities are more prone compared to low concentrated protein formulations. These properties can impair manufacturing processes, as well as protein stability and shelf life. To avoid such unfavourable solution properties, a detailed understanding about the nature of these properties and their driving forces are required. However, the fundamental mechanisms that lead to macroscopic solution properties, as above mentioned, are complex and not fully understood, yet. Established analytical methods for assessing the colloidal stability, i.e. the ability of a native protein to remain dispersed in solution, are restricted to dilute conditions and provide parameters such as the second osmotic virial coefficient, B22, and the diffusion interaction coefficient, kD. These parameters are routinely applied for qualitative estimations and identifications of proteins with challenging solution behaviours, such as high viscosities and aggregation, although the assays are prepared for low protein concentration conditions, typically between 0.1 and 20 mg/ml ("ideal" solution conditions). Quantitative analysis of samples of high protein concentration is difficult and it is hard to obtain information about the driving forces of such solution properties and corresponding protein-protein self-interactions. An advantage of using specific spectroscopic methods is the potential of directly analysing highly concentrated protein solutions at different solution conditions. This allows for collecting/gaining valuable information about the fundamental mechanisms of solution properties of the high protein concentration regime. In addition, the derived parameters might be more predictive as compared to the parameters originating from assays which are optimized for the low protein concentration range. The provided information includes structural data, molecular dynamics at various timescales and protein-solvent interactions, which can be obtained at molecular resolution. Herein, we provide an overview about spectroscopic techniques for analysing the origins of macroscopic solution behaviours in general, with a specific focus on pharmaceutically relevant high protein concentration and formulation conditions.

Influence of protein concentration and quality in a canned diet on urine composition, apparent nutrient digestibility and energy supply in adult cats.

BACKGROUND: Protein concentration and quality in cat food can vary considerably, and the impact on feline urine composition and nutrient supply is of high practical relevance. In the present study, 6 canned diets with varying protein concentrations and qualities were fed to 10 healthy adult cats. Protein quality in the diet differed depending on the amount of collagen-rich ingredients. Hydroxyproline concentrations were 2.56-4.45 g/kg dry matter in the high quality and 3.76-9.44 g/kg dry matter in the low quality diets. Protein levels were 36.2, 43.3 and 54.9% in the high quality and 36.7, 45.0 and 56.1% in the low quality groups. Each diet was fed for 6 weeks, using a randomized cross-over design. In the last 2 weeks of each feeding period, urine and faeces of the cats were collected. RESULTS: Renal calcium (Ca), oxalate (Ox) and citrate excretion were unaffected by the dietary protein concentration, possibly mediated by a high urine volume (24.2-34.2 ml/kg bodyweight (BW)/day) in all groups. However, renal Ox excretion was lower when the high quality diets were fed (P = 0.013). Urinary relative supersaturation (RSS) with calcium oxalate (CaOx) was low in general, but reduced in the high quality groups (P = 0.031). Urinary RSS values for magnesium ammonium phosphate (MAP) were high (2.64-5.00) among all groups. Apparent digestibility of crude protein and most minerals was unaffected by the different diets. Feed intake was higher in the low quality groups (P = 0.026), but BW of the cats did not differ depending on dietary protein quality. BW of the cats increased with increasing dietary protein concentrations (P = 0.003). CONCLUSION: In conclusion, a high protein canned diet might not be a specific risk factor for CaOx urolith formation in cats. In contrast, all diets resulted in high RSS MAP values, which might be critical concerning MAP crystallization. Protein quality had a minor, but significant impact on urine composition, necessitating further research on this subject. A lower energy supply when feeding a low protein quality can be assumed. Changes in BW were only small and require a careful interpretation.