Variation In Protein Concentration Research Articles

Effect of fermentation time on the molecular weight distribution of ACE inhibitory peptide from jack bean tempe

This research examined the variation in concentration of protein derived from jack bean throughout mold fermentation and the distribution of the molecular weight (MW) of the resultant peptides. In addition, the effects of peptide size on ACE inhibitory action were investigated. The jack bean was soaked and boiled before fermentation, then inoculated using 0.2% of commercial tempe inoculum. Furthermore, the inoculated seeds were incubated at room temperature for 48 h (T48), and 72 h (T72), and unfermented jack bean (T0) was used as a control. The total protein, soluble protein, MW distribution of peptides, and ACE inhibitory action were all examined. The results demonstrated that the concentration of soluble proteins rose as fermentation duration increased (p < 0.05). Meanwhile, the total crude protein in all samples did not change significantly (p > 0.05). The resultant peptides were then separated by dialysis to generate four peptide fractions, namely; fractions A (MW of <1 kDa), B (MW of 1-3.5 kDa), C (MW of 3.5-14 kDa), and D (MW of > 14 kDa). The peptide fractions of the three samples demonstrated that the number of peptides in fraction D decreased significantly during the fermentation, followed by an increase in peptide fractions with smaller MW (Fractions A, B, and C). The number of peptides in fraction A of T0, T48, and T72 was 9.51; 49.87; and 56.31%, respectively. The more significant amount of smaller peptides indicated a higher possibility to have the capacity to inhibit ACE. Fractions A, B, C, and D of T72 exhibited ACE inhibitory action with the value of 83.21; 62.53; 61.07; and 49.64%, respectively. In this investigation, the ACE inhibitory action was stronger as the MW of the peptides decreased. Fraction A had the greatest inhibitory action with an IC50 of 0.60 mg/mL. In conclusion, the fermentation duration enhanced the concentration of soluble protein. Consequently, jack bean protein degraded into various sizes of peptides during fermentation. Furthermore, the smallest size of peptides fraction demonstrated the best ACE inhibitory activity.

Intracellular variation and membrane binding requires high protein expression levels to maintain the fidelity of cyanobacterial circadian rhythms in clonal populations.

Giant unilamellar vesicles (GUVs) can bridge the gap between complex in vivo cell studies and in vitro bulk studies which occur in volumes considerably larger (∼10∧10 times) than cellular volumes. We demonstrate the benefits of in vitro GUV studies by functionally reconstituting the core oscillator from circadian clock of the cyanobacterium, Synechococcus elongatus, and show we can introduce intercellular variation to the GUV system. Unlike in bulk experiments, we discovered the fidelity of the circadian rhythms were highly dependent on protein levels in the vesicles and the size of the vesicles. By quantifying the encapsulation of proteins into vesicles, we show that the intercellular variation seen in cells can be mimicked through our diffusive loading process for our population of vesicles. We also show KaiB can bind to vesicle membranes, matching reports of binding to cellular membranes in vivo. We built a computational model to show how the fidelity of the circadian rhythms is modulated by the cell-like variation in protein concentrations and the membrane binding of proteins. When cell-like variation in introduced, we show the core oscillators of the clock is unable to achieve near ∼100% fidelity seen in the cell, which show why the transcriptional feedback and other complementary systems are necessary along with the core oscillator. We also show how membrane binding of proteins causes the clock fidelity to be modulated by the vesicle radius, r, due to the free protein reduction that is a function of the surface area to volume ratio (3/r). This work shows the potential for in vitro GUVs studies to mimic the cellular environment while maintaining the benefits of an in vitro system.

Changing mechanical properties of photopolymerized, dityrosine-crosslinked protein-based hydrogels.

Hydrogels based on renewable resources are a promising class of materials for future applications in pharmaceutics, drug delivery and personalized medicine. Thus, optional adjustments of mechanical properties such as swelling behavior, elasticity and network strength are desired. In this context, hydrogels based on the biological raw materials bovine serum albumin and casein were prepared by dityrosine-crosslinking of their tyrosine residues through visible light-induced photopolymerization. Changing the tyrosine accessibility by urea addition before photopolymerization increased the storage modulus of the hydrogels by 650% while simultaneously being more elastic. Furthermore, contributions of the buffer system composition, variation of protein concentration and storage medium towards mechanical properties of the hydrogel such as storage moduli, elasticity, fracture strain, compressive strength and relative weight swelling ratio are discussed. It could be shown, that changes in precursor solution and storage medium characteristics are crucial parameters towards tuning the mechanical properties of protein-based hydrogels.

Seed to seed variation of proteins of the yellow pea (Pisum sativum L.).

The existing variation among pea protein isolates’ functionality limits their application in food formulations. The source and extent of variations among yellow pea protein profiles was assessed in 10 single seeds of two varieties with different size and weight. A new approach was developed to analyze proteins of yellow pea combining three analytical methods of size exclusion chromatography (SEC), reverse phase high performance liquid chromatography (RP-HPLC), and microfluidic SDS-PAGE, to achieve the highest separation resolution. A high variation of protein concentration was observed not only between varieties, but also among seeds of the same variety. Vicilin to legumin ratio was between 2.72–4.19, and 1.70–2.22 among the individual seeds of AC Agassiz and CDC Saffron varieties, respectively. V/L ratio was significantly different among the individual seeds for both varieties. The amount of some protein fractions/subunits were correlated with seeds’ size and weight for AC Agassiz, while such correlations were not observed for CDC Saffron.

Protein Biofortification in Lentils (Lens culinaris Medik.) Toward Human Health.

Lentil (Lens culinaris Medik.) is a nutritionally dense crop with significant quantities of protein, low-digestible carbohydrates, minerals, and vitamins. The amino acid composition of lentil protein can impact human health by maintaining amino acid balance for physiological functions and preventing protein-energy malnutrition and non-communicable diseases (NCDs). Thus, enhancing lentil protein quality through genetic biofortification, i.e., conventional plant breeding and molecular technologies, is vital for the nutritional improvement of lentil crops across the globe. This review highlights variation in protein concentration and quality across Lens species, genetic mechanisms controlling amino acid synthesis in plants, functions of amino acids, and the effect of antinutrients on the absorption of amino acids into the human body. Successful breeding strategies in lentils and other pulses are reviewed to demonstrate robust breeding approaches for protein biofortification. Future lentil breeding approaches will include rapid germplasm selection, phenotypic evaluation, genome-wide association studies, genetic engineering, and genome editing to select sequences that improve protein concentration and quality.

Microfluidic protein detection and quantification using droplet morphology

Sensitive, inline detection of proteins is required for post-chromatographic analyses in proteomics, cell-based assays, and drug discovery workflows. Among the common inline methods, post-column derivatization requires chemical labels, while label-free methods are either expensive (mass spectrometry) or have limited sensitivity at small length scales (UV–Vis). This paper presents a label-free detection technique based on the concept that dissolved proteins can function as surfactants and decrease the dynamic interfacial tension (IFT) of an immiscible (water–oil) interface. Existing methods for measuring IFT, such as axisymmetric drop shape analysis (ADSA), operate in batch mode and are not suitable for continuous detection. Here we show that a microfluidic flow-focusing droplet generator operating at a frequency of > 100 Hz can track IFT changes continuously, with high temporal resolution and small detection volumes. Variations in protein concentration alter the size and shape of the drops/plugs formed, and these changes can be quantified in time using a high-speed camera and in-house image processing software. Moreover, the continuously refreshing interface alleviates issues related to surface aging. Two applications are demonstrated: (1) direct injection of a single protein into a microfluidic chip. (2) post-column detection of protein mixtures separated by high performance size exclusion chromatography (SEC HPLC). Of interest, the dynamic range of protein (bovine serum albumin, BSA) was 50 -104 μg/ml without using HPLC unit. The lowest limit of detection without HPLC unit was ~ 1 μg/ml of thyroglobulin protein in a 1 nl droplet, which equates to 1 fg of total protein. When used as a detector, the aforementioned detection method offered a sensitivity of six orders of magnitude higher than conventional UV–VIS detectors.

Oligomeric State Transitions of Pore Forming Toxins Induce Non-Monotonic Lipid Dynamics in Biomembranes

Membrane-bound protein complexes involving PFTs, released by virulent bacteria are known to form pores leading to host cell lysis. However, membrane remodelling and related repair processes during attack by PFTs remain largely unexplored. in our recent experiments [1] with GUVs exposed to prototypical cholesterol dependent cytolysin, Listeriolysin O (LLO), we were able to correlate changes in lipid dynamics to membrane bound conformational states of LLO oligomers. We now report non-monotonic variations in lipid diffusion due to interplay of lipid ejection and crowding by membrane bound oligomers of LLO. Our experiments monitor lipid dynamics and membrane micro-rheology over a broad range of protein-to-lipid ratios encompassing the dilute, intermediate as well as the crowded regime. This systematic variation of protein concentration reveals for the first time a unique non-monotonic behaviour of lipid diffusivity as a function of PFT concentration which falls outside the purview of existing membrane dynamics models. by monitoring the fluorescent intensity of lipids using confocal microscopy and number of lipids in FCS we provide direct evidence of lipid loss during pore formation. Not only do we observe the anticipated crowding induced decreasing lipid diffusivities at high protein concentration, but our results reveal a counter-intuitive increase in lipid diffusivity at intermediate LLO concentrations. We quantitatively capture these trends using an established model based on the dependence of membrane lipidxdiffusion on the available free area. We correlate changes in observed lipid diffusivity to the free area contributions from different PFT oligomeric states, thus providing unique insights connecting lipid dynamics to bound protein distributions. 1. Ilanila IP, Ramesh Cheerla, K Ganapathy Ayappa and Jaydeep K Basu, “Correlated protein conformational states and membrane dynamics during attack by pore-forming toxins”, Proc Nat Acad Sci. 116, 12839 (2019).

Abstract PO-086: Local quantification of protein expression on frozen tissue sections to evaluate tumor heterogeneity

Abstract The discernment of intra-tumor heterogeneity is critical to understand therapy failure and differences of prognosis between patients with similar disease states. Part of the heterogeneity can be appreciated at the protein level, where quantitation can aid the elucidation of complex biological processes. We propose a workflow to quantify protein expression on a “local” level on frozen breast tumor tissue sections, providing a measure and easy visualization of the phenotypical heterogeneity present in the tissue. In this workflow, we extract 8-10 regions of interest (ROIs) of ~100 µm in diameter from frozen tissue sections of 5 patients using a microfluidic probe (MFP). The MFP is a microfluidic scanning probe device that confines a biochemical on a selected region of a tissue, with the capacity of working in liquid environments, and which allows the extraction of individual regions (footprints) in less than 3 min. The rapid extraction together with the liquid environment in which the tissue is present are key for minimizing protein degradation. The extracted proteins are analyzed through an antibody microarray with a panel of 13 proteins that aids a proteomic-based classification of molecular subtypes of breast cancer. Protein expression is quantified based on the greyscale intensity of the corresponding spot in the microarray and normalized by the area of the extracted region. Further analysis determines the presence and extension of phenotypical clusters. Using local protein expression, we generated an expression map of the 13 analyzed proteins. We observe large variations of protein concentrations in the case of beta-actin, estrogen receptor (ER) and EGFR. Cytokeratins (CK)5, CK8, CK17, progesterone receptor and GAPDH presented lower variations in concentration between the analyzed regions in this set of patients. We then evaluated whether the observed differences were statistically significant using Cohen size D effect, showing higher heterogeneity among some proteins (beta-actin, ER, or androgen receptor) and some patients (Patient 1). Finally, we performed a hierarchical clustering analysis across the patients to understand the presence and distribution of phenotypical variants. Patients 3 and 5 presented one single phenotypical variant, while the other patients had up to four. Quantitative methods to describe heterogeneity on the proteomic level are essential to improve our comprehension of tumor biology and thus develop more precise cancer diagnostic approaches. Techniques such as the one presented here can advance our understanding of the spatial location of phenotypical variants within the tumor mass, hinting towards a more accurate choice of treatment modalities. Citation Format: Anna Fomitcheva Khartchenko, Peter Schraml, Govind V. Kaigala. Local quantification of protein expression on frozen tissue sections to evaluate tumor heterogeneity [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-086.

Management and environmental factors explaining soybean seed protein variability in central Argentina

Soybean production is challenged to increase yield while maintaining seed protein concentration levels. Variability in protein concentration has been poorly described in many regions, and management options targeting their levels are not available. Our objectives were (i) to describe soybean seed protein in the central production systems of Argentina, (ii) to explore spatial patterns across the region, (iii) to quantify the importance of management and environment on protein concentration, and (iv) to explore correlations between seed protein concentration, seed yield, and total canopy N uptake. We sampled 1721 farm fields across the region, and conducted 52 cultivar trials from 2012 to 2016. Average seed protein concentration was 36.6 and 37.6% for soybean grown as a single crop in the season and as a second crop after a winter one, respectively. Seed protein concentration showed a spatial pattern in soybean as single crop, allowing us to identify well-delimited areas with contrasting protein values. Regression trees of farm fields explained 38 and 48% of total variation in seed protein for soybean as single and second crop, respectively. Relative to this explained variation, management variables accounted for ∼73% of seed protein concentration variation in both crops, where cultivar selection was the most relevant (71.5 and 68.9% for soybean as single and second crop, respectively), followed by planting date (2.5%), and maturity group (1.8%). Despite its minor importance compared to management practices, temperature and rainfall were also associated to protein variability. The response to temperature differed between soybeans as single and second crop. Reduced rainfall was associated to increased seed protein concentration only for soybean as a second crop. Cultivar trials identified consistent high seed protein cultivars. Sites with high yield and quality were evident, but results showed clear cultivar trade-off correlations between yield and protein concentration. Results highlight the need to investigate ways to improve total canopy N uptake for allowing both yield and seed protein to increase simultaneously.

A proteome-wide screen of Campylobacter jejuni using protein microarrays identifies novel and conformational antigens.

Campylobacter jejuni (C. jejuni) is a foodborne intestinal pathogen and major cause of gastroenteritis worldwide. C. jejuni proteins that are immunogenic have been sought for their potential use in the development of biomarkers, diagnostic assays, or subunit vaccines for humans or livestock. To identify new immunogenic C. jejuni proteins, we used a native protein microarray approach. A protein chip, with over 1400 individually purified GST-tagged C. jejuni proteins, representing over 86% of the proteome, was constructed to screen for antibody titers present in test sera raised against whole C. jejuni cells. Dual detection of GST signals was incorporated as a way of normalizing the variation of protein concentrations contributing to the antibody staining intensities. We detected strong signals to 102 C. jejuni antigens. In addition to antigens recognized by antiserum raised against C. jejuni, parallel experiments were conducted to identify antigens cross-reactive to antiserum raised against various serotypes of E. coli or Salmonella or to healthy human sera. This led to the identification of 34 antigens specifically recognized by the C. jejuni antiserum, only four of which were previously known. The chip approach also allowed identification of conformational antigens. We demonstrate in the case of Cj1621 that antigen signals are lost to denaturing conditions commonly used in other approaches to identify immunogens. Antigens identified in this study include those possessing sequence features indicative of cell surface localization, as well as those that do not. Together, our results indicate that the unbiased chip-based screen can help reveal the full repertoire of host antibodies against microbial proteomes.

Impact of Hydrocolloids and Homogenization Treatment on the Foaming Properties of Raspberry Fruit Puree

The production of healthy, ready-to-eat fruit snacks by means of foam-mat drying has to face the challenge of ensuring high foam stability throughout the entire process. Foaming properties of raspberry puree whipped for 10 min with varying concentrations of potato protein isolate (2.5, 5, 10% w/w) as foaming agent were studied. Furthermore, the impact of hydrocolloids as foam stabilizers, i.e., maltodextrin (5, 15, 30% w/w) and pectin (0.1, 0.25, 0.5, 0.75, 1.0, 2.5% w/w), in combination with the variation of protein concentration as well as the impact of a homogenization treatment of fruit puree on the foam characteristics and puree properties were investigated. Foam stability was enhanced by the increase of protein and hydrocolloid concentration. Maltodextrin and pectin showed similar behavior even though pectin was used at considerably smaller concentrations. Compared to single hydrocolloid systems, the addition of pectin and maltodextrin in combination led to a further enhancement of foam stability, which offered the opportunity to simultaneously decrease the concentration of both hydrocolloids. The destruction of fruit tissue by homogenization treatment resulted in a reduction of mean particle diameters (D[4,3], D[3,2]) and a decrease of viscosity. Despite the increase in the specific surface area (ASF) and thus, expected enhanced oxidative processes, no changes were observed for ascorbic acid and anthocyanin contents. However, the structural changes induced by the homogenization treatment significantly affected foaming properties.

Approach to Classification of Cavitary Effusion and Comparison between Manual and Automatic Methods for Total Nucleated Cell Count

Background: Two classifications are used to categorize cavitary effusions using total nucleated cell count (TNCC): protein concentration and pathophysiology of its formation. The aims of the present study were to evaluate the correlation between the TNCC values of cavitary effusions obtained in the automatic and the manual method, and also evaluating the classification methodology. Materials, Methods &amp; Results: Cavitary effusions were analyzed for physical, chemical and cytological aspects, as well as manual and automatic cell counts for the correlation between the traditional methods and those suggested by Stockham &amp; Scott. Bland-Altman regression and Spearman correlation analysis were performed. Of the total, 44 were abdominal effusions (73.3%), 15 thoracic (25%) and 1 pericardial (1.7%). According to the traditional classification, most of the effusions were classified as modified transudates (40%) and according to the classification of Stockham and Scott, as transudates poor in protein (31.7%). The correlation between cell counting techniques between pure, modified and exudate transudates was 0.94, 0.97 and 0.94, respectively, indicating an excellent correlation between the parameters (p = 0.95%).Discussion: Considering the concentration of proteins and CCNT, the effusions classified as modified transudate were mainly caused by neoplastic processes (carcinomas/adenocarcinomas), since there are several mechanisms of their formation, such as large variation of protein concentration. According to the Stockham &amp; Scott classification a unique classification is considered for exfoliative neoplastic effusions, the variation of the protein concentration of the effusion does not alter its classification. In neoplastic effusions, classified as exudates, lymphomas were the most prevalent, and hypercellularity (approximately 150,000 cells / μL) allowed this classification. When considering low-protein transudates, the findings related to low concentrations did not differ much from the traditional classification. In the ruptures of viscera and vessels, the hemorrhagic ones were the most frequent, thus, the cytological diagnosis is essential, since it can give information about the contamination with blood during the collection. Most of these were due to neoplasia as the underlying cause. A case of chylotorax was diagnosed by comparing cholesterol and triglyceride values of effusion and serum. In cases of uroperitoneum, the presence of urine in the abdominal cavity promotes the dilution of the fluid from the cavity, being initially classified as pure transudate and, with its permanence in the cavity, increasing the CCNT, becomes an exudate. As in cases of exfoliative neoplastic effusions, the classification of the uroperitoneum, according to Stockham &amp; Scott, is classified directly into effusion due to rupture of the viscera, giving a quick and clear diagnosis. According to Stockham &amp; Scott, cases classified as nonseptic exudates (n = 3), two of which resulted from feline infectious peritonitis (PIF). The effusive form of PIF presents with accumulations of fluid in the abdomen, having an inflammatory character, but according to the traditional classification, they enter the category of modified transudates, because, despite containing protein concentrations close to or above the serum level, they present a CCNT lower than an exudate. Cavitary effusions were classified as septic exudates when intracellular bacteria were present and in the present study, two effusions were classified as such in two patients, one with septic peritonitis and in the other the final diagnosis was not found. The high values of Spearman correlation coefficients found when comparing the automatic counts with the manual demonstrate that there is an excellent correlation between the methods and, the Bland-Altman test showed significant agreement between them.

Expression of endogenous proteins in maize hybrids in a multi-location field trial in India

Genetically modified (GM) crops undergo large scale multi-location field trials to characterize agronomics, composition, and the concentration of newly expressed protein(s) [herein referred to as transgenic protein(s)]. The concentration of transgenic proteins in different plant tissues and across the developmental stages of the plant is considered in the safety assessment of GM crops. Reference or housekeeping proteins are expected to maintain a relatively stable expression pattern in healthy plants given their role in cellular functions. Understanding the effects of genotype, growth stage and location on the concentration of endogenous housekeeping proteins may provide insight into the contribution these factors could have on transgenic protein concentrations in GM crops. The concentrations of three endogenous proteins (actin, elongation factor 1-alpha, and glyceraldehyde 3-phosphate dehydrogenase) were measured in several different maize hybrids grown across multiple field locations over 2 years. Leaf samples were collected from healthy plants at three developmental stages across the growing seasons, and protein concentrations were quantified by indirect enzyme-linked immunosorbent assay (ELISA) for each protein. In general, the concentrations of these three endogenous proteins were relatively consistent across hybrid backgrounds, when compared within one growth stage and location (2–26%CV), whereas the concentrations of proteins in the same hybrid and growth stage across different locations were more variable (12–64%CV). In general, the protein concentrations in 2013 and 2014 show similar trends in variability. Some degree of variability in protein concentrations should be expected for both transgenic and endogenous plant-expressed proteins. In the case of GM crops, the potential variation in protein concentrations due to location effects is captured in the current model of multi-location field testing.

Serum Biochemical Profile of Nile Tilapias (Oreochromis niloticus) Bred in Net Cages during Summer and Winter

Background: The growth of fish farming in Brazil has been noticeable over the past few years, and the Nile tilapia (Oreochromis niloticus) is the most bred species in the country. Studies on the physiology of these animals are necessary so that the production continues to grow and to improve its rates. However, very discrepant data are still found in the area of blood biochemistry. The aim of this study was to evaluate the physiological variations in serum concentrations of proteins, metabolites, electrolytes and serum enzymes in Nile tilapias bred in net cages in Cachoeira Dourada, state of Minas Gerais, Brazil, during summer and winter.Materials, Methods &amp; Results: During the year of 2016, samples from 100 Nile tilapias were collected during summer (January and February) and samples from 100 Nile tilapias were also collected during winter (July and August). Only one blood sample was collected by caudal venipuncture of each fish, just before the slaughter, always in the morning. The serum samples obtained were then analyzed for serum concentrations of proteins, metabolites, electrolytes, lipids and serum enzymes. Serum concentrations of total proteins, globulins, triglycerides, high density lipoprotein (HDL), alkaline phosphatase, sodium and chloride were significantly higher during winter in comparison to summer, while the serum concentration of uric acid was significantly lower during winter in comparison to summer.Discussion: Due to the lower temperature of water during winter, tilapias reduced food intake and became more susceptible to diseases, which led to an increase in circulating globulins. In that same season, there was an increase in serum concentrations of sodium and chloride as a result of adding sodium chloride to the water in order to stimulate mucus secretion on the fishes’ skin and gills to prevent pathologies. Triglycerides, HDL, and uric acid are considered indicators of nutritional status of animals. The increase in serum concentrations of triglycerides and HDL indicates that tilapias are mobilizing energy reserves, however, when the increase in serum concentrations of uric acid is observed, it is fair to assume there has been an increase in food intake, raising protein catabolism. The increase of serum concentration in alkaline phosphatase is caused by the presence of multiple forms of enzymes that are induced by the action of endogenous corticosteroids, which are produced as a result of the stress suffered by tilapias during the fishing process and during winter, when the water temperature is lower than the ideal for this species. It can be concluded that the season has influence on some serum biochemical constituents of Nile tilapias bred in net cages, and that the predominant variations happen during winter due to immunity decline and during summer due to the increase in food intake and, consequently, in the metabolism of tilapias.

Thermo‐kinetic analysis space expansion for cyclophilin‐ligand interactions – identification of a new nonpeptide inhibitor using Biacore™ T200

We have established a refined methodology for generating surface plasmon resonance sensor surfaces of recombinant his‐tagged human cyclophilin‐A. Our orientation‐specific stabilisation approach captures his‐tagged protein under ‘physiological conditions’ (150 mm NaCl, pH 7.5) and covalently stabilises it on Ni2+‐nitrilotriacetic acid surfaces, very briefly activated for primary amine‐coupling reactions, producing very stable and active surfaces (≥ 95% specific activity) of cyclophilin‐A. Variation in protein concentration with the same contact time allows straightforward generation of variable density surfaces, with essentially no loss of activity, making the protocol easily adaptable for studying numerous interactions; from very small fragments, ~ 100 Da, to large protein ligands. This new method results in an increased stability and activity of the immobilised protein and allowed us to expand the thermo‐kinetic analysis space, and to determine accurate and robust thermodynamic parameters for the cyclophilin‐A–cyclosporin‐A interaction. Furthermore, the increased sensitivity of the surface allowed identification of a new nonpeptide inhibitor of cyclophilin‐A, from a screen of a fragment library. This fragment, 2,3‐diaminopyridine, bound specifically with a mean affinity of 248 ± 60 μm. The X‐ray structure of this 109‐Da fragment bound in the active site of cyclophilin‐A was solved to a resolution of 1.25 Å (PDB: 5LUD), providing new insight into the molecular details for a potential new series of nonpeptide cyclophilin‐A inhibitors.

Variations in Protein Concentration and Nitrogen Sources in Different Positions of Grain in Wheat.

The distribution patterns of total protein and protein components in different layers of wheat grain were investigated using the pearling technique, and the sources of different protein components and pearling fractions were identified using 15N isotope tracing methods. It was found that N absorbed from jointing to anthesis (JA) and remobilized to the grain after anthesis was the principal source of grain N, especially in the outer layer. For albumin and globulin, the amount of N absorbed during different stages all showed a decreasing trend from the surface layer to the center part. Whereas, for globulin and glutenin, the N absorbed after anthesis accounted for the main part indicating that for storage protein, the utilization of N assimilated after anthesis is greater than that of the stored N assimilated before anthesis. It is concluded that manipulation of the N application rate during different growth stages could be an effective approach to modulate the distribution of protein fractions in pearled grains for specific end-uses.

Protein Concentration in Milk Formula, Growth, and Later Risk of Obesity: A Systematic Review

Background: Protein intake may influence important health outcomes in later life.Objective: The objective of this study was to investigate current evidence on the effects of infant formulas and follow-on formulas with different protein concentrations on infants’ and children’s growth, body composition, and later risk of overweight and obesity.Methods: In this systematic review, we searched electronic databases (including MEDLINE, Embase, and the Cochrane Library) up until November 2014 for randomized controlled trials (RCTs). Eligible studies had to include children aged 0–3 y who represented the general population and were fed cow milk–based infant formulas with variations in protein concentration. Control groups received lower-protein cow milk–based formulas (as defined by the authors). The primary outcomes were growth, overweight, obesity, and adiposity. Various time points for outcomes assessment were accepted for inclusion. If possible, a meta-analysis was performed.Results: Twelve RCTs met our inclusion criteria. Different formula protein concentrations did not affect linear growth other than a transient effect on mean length at 3 mo observed in a meta-analysis of 4 studies (mean difference, – 0.27 cm; 95% CI: −0.52, −0.02). Lower mean weight and weight z scores obtained from the infants fed lower-protein formulas were observed only from 6 to 12 mo of age. Data from one large RCT showed that consumption of a lower-protein infant formula may reduce body mass index at 12 mo of age and later (12 mo, 24 mo, and 6y) and the risk of obesity at 6 y. Effects on body composition remained unclear.Conclusions: The current evidence is insufficient for assessing the effects of reducing the protein concentration in infant formulas on long-term outcomes, but, if confirmed, this could be a promising intervention for reducing the risk of overweight and obesity in children. In view of the limited available evidence, more studies replicating effects on long-term health outcomes are needed.

Revealing Structural Features and Affinities of Protein Complexes in Living Cells by MFIS-FRET

Forster resonance energy transfer (FRET) due to its sensitivity of distance has been widely used to investigate the structure and interaction of biomolecules. Multiparameter fluorescence image spectroscopy (MFIS) provides particular advantages to FRET imaging because all the fluorescence parameters are monitored simultaneously with picosecond accuracy, which allows for a comprehensive analysis on biological systems. Traditionally, a reduction in average donor lifetime or an increase of average FRET efficiency was used as an indicator for molecular interaction. However, such changes observed in FRET-imaging can have two reasons: (1) the conformational change or (2) change in fraction of FRET-active species. To resolve this ambiguity, we introduce a new sub-ensemble analysis method to directly visualize and quantitatively analyze both factors. Characterization of true FRET efficiency enabled us to detect even subtle FRET variations and provided crucial information about the structural properties of molecular complexes. Furthermore, from determined fraction of FRET-active species, utilizing the intrinsic cell-to-cell variations of protein concentration, we show that dissociation constant (KD) of membrane-receptor interactions can be characterized in living cells.

Impact of the cell division cycle on gene circuits

In growing cells, protein synthesis and cell growth are typically not synchronous, and, thus, protein concentrations vary over the cell division cycle. We have developed a theoretical description of genetic regulatory systems in bacteria that explicitly considers the cell division cycle to investigate its impact on gene expression. We calculate the cell-to-cell variations arising from cells being at different stages in the division cycle for unregulated genes and for basic regulatory mechanisms. These variations contribute to the extrinsic noise observed in single-cell experiments, and are most significant for proteins with short lifetimes. Negative autoregulation buffers against variation of protein concentration over the division cycle, but the effect is found to be relatively weak. Stronger buffering is achieved by an increased protein lifetime. Positive autoregulation can strongly amplify such variation if the parameters are set to values that lead to resonance-like behaviour. For cooperative positive autoregulation, the concentration variation over the division cycle diminishes the parameter region of bistability and modulates the switching times between the two stable states. The same effects are seen for a two-gene mutual-repression toggle switch. By contrast, an oscillatory circuit, the repressilator, is only weakly affected by the division cycle.

Quantitative Profile Analysis of Mulberry Silkworm &lt;i&gt;Bombyx mori &lt;/i&gt;L. (CSR&lt;sub&gt;2&lt;/sub&gt;XCSR&lt;sub&gt;4&lt;/sub&gt;)

Bombyx mori. L. (CSR2XCSR4) is a bivolitne crossbreed that produces high quantity of silk. The weights of worms and glands are directly related to the yield of silk, higher larval weights leads to higher silk production. In the present study, quantitative parameters of 5th instar and pupal stages of silkworm larvae were observed. The analysis of quantitative estimation showed that the maximum weights and lengths of silk gland and silk worm were recorded on 7th day of 5th instar larval stage. The day dependent variation in protein concentration was observed in total silk gland 5th instar larval and pupal stages.