A simple and rapid protein array based method for the simultaneous detection of biowarfare agents

A protein chip has been developed that allows the simultaneous detection of a multitude of different biowarfare agents. The chip was developed for the ArrayTube platform providing a cheap and easy to handle technology solution that combines a microtube-integrated protein chip with the classical procedure of a sandwich-enzyme-linked immunosorbent assay and signal amplification by streptavidin-poly-horseradish peroxidase. Specific immunoassays for Staphylococcus enterotoxin B, ricin, Venezuelan equine encephalitis virus, St. Louis encephalitis virus, West Nile virus, Yellow fever virus, Orthopox virus species, Francisella tularensis, Yersinia pestis, Brucella melitensis, Burkholderia mallei and Escherichia coli EHEC O157:H7 were developed and optimized. All assays could be completed within 1 to 1 1/2 h and detection levels were demonstrated to be as low as in well established ELISAs. Most interesting, as a result of careful antibody screening and testing, it is currently possible to analyse at least five of the "dirty dozen" agents on one single protein chip in parallel.     

High resolution ion chamber array delivery quality assurance for robotic radiosurgery: Commissioning and validation

PurposeHigh precision radiosurgery demands comprehensive delivery-quality-assurance techniques. The use of a liquid-filled ion-chamber-array for robotic-radiosurgery delivery-quality-assurance was investigated and validated using several test scenarios and routine patient plans.Methods and materialPreliminary evaluation consisted of beam profile validation and analysis of source–detector-distance and beam-incidence-angle response dependence. The delivery-quality-assurance analysis is performed in four steps: (1) Array-to-plan registration, (2) Evaluation with standard Gamma-Index criteria (local-dose-difference ⩽ 2%, distance-to-agreement ⩽ 2 mm, pass-rate ⩾ 90%), (3) Dose profile alignment and dose distribution shift until maximum pass-rate is found, and (4) Final evaluation with 1 mm distance-to-agreement criterion. Test scenarios consisted of intended phantom misalignments, dose miscalibrations, and undelivered Monitor Units. Preliminary method validation was performed on 55 clinical plans in five institutions.ResultsThe 1000SRS profile measurements showed sufficient agreement compared with a microDiamond detector for all collimator sizes. The relative response changes can be up to 2.2% per 10 cm source–detector-distance change, but remains within 1% for the clinically relevant source–detector-distance range. Planned and measured dose under different beam-incidence-angles showed deviations below 1% for angles between 0° and 80°. Small-intended errors were detected by 1 mm distance-to-agreement criterion while 2 mm criteria failed to reveal some of these deviations. All analyzed delivery-quality-assurance clinical patient plans were within our tight tolerance criteria.ConclusionWe demonstrated that a high-resolution liquid-filled ion-chamber-array can be suitable for robotic radiosurgery delivery-quality-assurance and that small errors can be detected with tight distance-to-agreement criterion. Further improvement may come from beam specific correction for incidence angle and source–detector-distance response.     

Proteomic analysis of interactors for yeast protein arginine methyltransferase Hmt1 reveals novel substrate and insights into additional biological roles

Protein arginine methylation is a PTM catalyzed by an evolutionarily conserved family of enzymes called protein arginine methyltransferases (PRMTs), with PRMT1 being the most conserved member of this enzyme family. This modification has emerged to be an important regulator of protein functions. To better understand the role of PRMTs in cellular pathways and functions, we have carried out a proteomic profiling experiment to comprehensively identify the physical interactors of Hmt1, the budding yeast homolog for human PRMT1. Using a dual‐enzymatic digestion linear trap quadrupole/Orbitrap proteomic strategy, we identified a total of 108 proteins that specifically copurify with Hmt1 by tandem affinity purification. A reverse coimmunoprecipitation experiment was used to confirm Hmt1's physical association with Bre5, Mtr4, Snf2, Sum1, and Ssd1, five proteins that were identified as Hmt1‐specific interactors in multiple biological replicates. To determine whether the identified Hmt1‐interactors had the potential to act as an Hmt1 substrate, we used published bioinformatics algorithms that predict the presence and location of potential methylarginines for each identified interactor. One of the top hits from this analysis, Snf2, was experimentally confirmed as a robust substrate of Hmt1 in vitro. Overall, our data provide a feasible proteomic approach that aid in the better understanding of PRMT1's roles within a cell.     

Electron microscopy holdings of the Protein Data Bank: the impact of the resolution revolution,new validation tools,and implications for the future

As a discipline, structural biology has been transformed by the three-dimensional electron microscopy (3DEM) “Resolution Revolution” made possible by convergence of robust cryo-preservation of vitrified biological materials, sample handling systems, and measurement stages operating a liquid nitrogen temperature, improvements in electron optics that preserve phase information at the atomic level, direct electron detectors (DEDs), high-speed computing with graphics processing units, and rapid advances in data acquisition and processing software. 3DEM structure information (atomic coordinates and related metadata) are archived in the open-access Protein Data Bank (PDB), which currently holds more than 11,000 3DEM structures of proteins and nucleic acids, and their complexes with one another and small-molecule ligands (~ 6% of the archive). Underlying experimental data (3DEM density maps and related metadata) are stored in the Electron Microscopy Data Bank (EMDB), which currently holds more than 21,000 3DEM density maps. After describing the history of the PDB and the Worldwide Protein Data Bank (wwPDB) partnership, which jointly manages both the PDB and EMDB archives, this review examines the origins of the resolution revolution and analyzes its impact on structural biology viewed through the lens of PDB holdings. Six areas of focus exemplifying the impact of 3DEM across the biosciences are discussed in detail (icosahedral viruses, ribosomes, integral membrane proteins, SARS-CoV-2 spike proteins, cryogenic electron tomography, and integrative structure determination combining 3DEM with complementary biophysical measurement techniques), followed by a review of 3DEM structure validation by the wwPDB that underscores the importance of community engagement.     

Protein sorting and expression of a unique soybean cotyledon protein,GmSBP, destined for the protein storage vacuole

The initial biochemical characterization of the soybean sucrose-binding protein, GmSBP, within our lab and others produced several incongruous characteristics that required a re-characterization of GmSBP via sequence homology, cell biology, immunolocalization, and semi-quantitative analysis. The GmSBP proteins share amino acid sequence homology as well as putative structural homology with globulin-like seed storage proteins. A comparison to the major soybean seed storage proteins, glycinin and -conglycinin established several storage protein-like characteristics for GmSBP. All three proteins were present in a prevacuolar compartment and protein storage vacuole. All three proteins increased in expression during seed development and are remobilized during germination. Quantitatively, the relative concentrations of GmSBP, -conglycinin (/ subunits), and glycinin (acidic subunits) indicated that GmSBP contributes 19-fold less to the stored nitrogen. The quantitative differences between GmSBP and glycinin may be attributed to the unconserved order and spacing of cis-acting regulatory elements present within the promoter regions. Ultimately, GmSBP is transported to the mature protein storage vacuole. The biological function of GmSBP within the protein storage vacuole remains uncertain, but its localization is a remnant of its evolutionary link to a globulin-like or vicilin-like ancestor that gave rise to the 7S family of storage proteins.     

The estimation of micro-algal protein content and its meaning to the evaluation of algal biomass I. Comparison of methods for extracting protein

The spectrophotometric evaluation of micro-algal protein needs a prior extraction from cells in order to liberate protein for measurement. The conditions of extraction (temperature, duration, normality of sodium hydroxide, pretreatment) which yield optimal protein content are tested with three algal cultures (Scenedesmus, Synechococcus, Asterionella). A standard method of extraction is presented. Comparison of this method with nine published methods reveals markedly lower protein yields for easy extractable (43–100%) and hard extractable (5–75%) algal species, relative to this method, depending on ease of cell wall breakage. The application of this standard method to field investigations is demonstrated and compared to other biochemical parameters. The advantages of this method over other protein extraction methods, with respect to field material, are discussed.     

Distinct retrieval and retention mechanisms are required for the quality control of endoplasmic reticulum protein folding.

Proteins destined for the secretory pathway must first fold and assemble in the lumen of endoplasmic reticulum (ER). The pathway maintains a quality control mechanism to assure that aberrantly processed proteins are not delivered to their sites of function. As part of this mechanism, misfolded proteins are returned to the cytosol via the ER protein translocation pore where they are ubiquitinated and degraded by the 26S proteasome. Previously, little was known regarding the recognition and targeting of proteins before degradation. By tracking the fate of several mutant proteins subject to quality control, we demonstrate the existence of two distinct sorting mechanisms. In the ER, substrates are either sorted for retention in the ER or are transported to the Golgi apparatus via COPII-coated vesicles. Proteins transported to the Golgi are retrieved to the ER via the retrograde transport system. Ultimately, both retained and retrieved proteins converge at a common machinery at the ER for degradation. Furthermore, we report the identification of a gene playing a novel role specific to the retrieval pathway. The gene, BST1, is required for the transport of misfolded proteins to the Golgi, although dispensable for the transport of many normal cargo proteins.     

Accuracy, precision and quality control in age determination, including a review of the use and abuse of age validation methods

Many calcified structures produce periodic growth increments useful for age determination at the annual or daily scale. However, age determination is invariably accompanied by various sources of error, some of which can have a serious effect on age-structured calculations. This review highlights the best available methods for insuring ageing accuracy and quantifying ageing precision, whether in support of large-scale production ageing or a small-scale research project. Included in this review is a critical overview of methods used to initiate and pursue an accurate and controlled ageing program, including (but not limited to) validation of an ageing method. The distinction between validation of absolute age and increment periodicity is emphasized, as is the importance of determining the age of first increment formation. Based on an analysis of 372 papers reporting age validation since 1983, considerable progress has been made in age validation efforts in recent years. Nevertheless, several of the age validation methods which have been used routinely are of dubious value, particularly marginal increment analysis. The two major measures of precision, average percent error and coefficient of variation, are shown to be functionally equivalent, and a conversion factor relating the two is presented. Through use of quality control monitoring, ageing errors are readily detected and quantified; reference collections are the key to both quality control and reduction of costs. Although some level of random ageing error is unavoidable, such error can often be corrected after the fact using statistical ('digital sharpening)' methods.     

Effects of ractopamine hydrochloride and dietary protein content on performance,carcass traits and meat quality of Nellore bulls

Ractopamine hydrochloride (RH) alters protein metabolism and improves growth performance in Bos taurus cattle with high carcass fat. Our objective was to evaluate the effects of RH, dietary CP and RH×CP interaction on performance, blood metabolites, carcass characteristics and meat quality of young Nellore bulls. A total of 48 bulls were randomly assigned to four treatments in a 2×2 factorial arrangement. The factors were two levels of dietary CP (100% and 120% of metabolizable protein requirement, defined as CP100 and CP120, respectively), and two levels of RH (0 and 300 mg/animal·per day). Treated animal received RH for the final 35 days before slaughter. Animals were weighed at the beginning of the feedlot period (day 63), at the beginning of ractopamine supplementation (day 0), after 18 days of supplementation (day 18) and before slaughter (day 34). Animals were slaughtered and hot carcass weights recorded. After chilling, carcass data was collected and longissimus samples were obtained for determination of meat quality. The 9–11th rib section was removed for carcass composition analysis. Supplementation with RH increased ADG independently of dietary CP. There was a RH×CP interaction on dry matter intake (DMI), where RH reduced DMI at CP120, with no effect at CP100. Ractopamine improved feed efficiency, without RH×CP interaction. Ractopamine had no effect on plasma creatinine and urea concentration. Greater dietary CP tended to increase blood urea, and there was a RH×CP interaction for plasma total protein. Ractopamine supplementation increased plasma total protein at CP120, and had no effect at CP100. Ractopamine also decreased plasma glucose concentration at CP100, but had no effect at CP120. Ractopamine increased alkaline phosphatase activity at CP120 and had no effect at CP100. There was a tendency for RH to increase longissimus muscle area, independently of dietary CP. Ractopamine did not alter fat thickness; however, fat thickness was reduced by greater CP in the diet. Supplementation with RH decreased meat shear force, but only at day 0 of aging, having no effect after 7, 14 or 21 days. Greater dietary protein increased meat shear force after 0 and 7 days of aging, with no effect after 14 or 21 days. These results demonstrate for the first time the efficacy of ractopamine supplementation to improve gain and feed efficiency of intact Bos indicus males, with relatively low carcass fat content. Ractopamine effects were not further improved by increasing dietary protein content above requirements.     

The future of host cell protein (HCP) identification during process development and manufacturing linked to a risk‐based management for their control

The use of biological systems to synthesize complex therapeutic products has been a remarkable success. However, during product development, great attention must be devoted to defining acceptable levels of impurities that derive from that biological system, heading this list are host cell proteins (HCPs). Recent advances in proteomic analytics have shown how diverse this class of impurities is; as such knowledge and capability grows inevitable questions have arisen about how thorough current approaches to measuring HCPs are. The fundamental issue is how to adequately measure (and in turn monitor and control) such a large number of protein species (potentially thousands of components) to ensure safe and efficacious products. A rather elegant solution is to use an immunoassay (enzyme‐linked immunosorbent assay [ELISA]) based on polyclonal antibodies raised to the host cell (biological system) used to synthesize a particular therapeutic product. However, the measurement is entirely dependent on the antibody serum used, which dictates the sensitivity of the assay and the degree of coverage of the HCP spectrum. It provides one summed analog value for HCP amount; a positive if all HCP components can be considered equal, a negative in the more likely event one associates greater risk with certain components of the HCP proteome. In a thorough risk‐based approach, one would wish to be able to account for this. These issues have led to the investigation of orthogonal analytical methods; most prominently mass spectrometry. These techniques can potentially both identify and quantify HCPs. The ability to measure and monitor thousands of proteins proportionally increases the amount of data acquired. Significant benefits exist if the information can be used to determine critical HCPs and thereby create an improved basis for risk management. We describe a nascent approach to risk assessment of HCPs based upon such data, drawing attention to timeliness in relation to biosimilar initiatives. The development of such an approach requires databases based on cumulative knowledge of multiple risk factors that would require national and international regulators, standards authorities (e.g., NIST and NIBSC), industry and academia to all be involved in shaping what is the best approach to the adoption of the latest bioanalytical technology to this area, which is vital to delivering safe efficacious biological medicines of all types. Biotechnol. Bioeng. 2015;112: 1727–1737. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Protein accumulation,RNA and soluble amino nitrogen content in developing endosperm of two varieties of Triticum aestivum with high and low protein seed

Summary The kinetics of protein accumulation, the variation in RNA, the soluble amino nitrogen content of developing endosperm of two varieties of Triticum aestivum, with high and low protein content in the mature seed, suggest a possible relation between maintenance of the RNA content and the ability to synthesize protein. A sudden halt in protein accumulation is observed as the RNA starts to decrease. The hypothesis is also advanced that maintenance of the RNA content might, in turn, be dependent on the presence, in the endosperm of developing wheat seed, of a certain level of soluble amino nitrogen which could then play the role of limiting factor for protein synthesis.Publication No. 491 from the Divisione Applicazione delle Radiazioni del C.N.E.N., SCN Casaccia, S.M. di Galeria, Rome, Italy.     

Der1p, a protein required for degradation of malfolded soluble proteins of the endoplasmic reticulum: topology and Der1-like proteins

The endoplasmic reticulum (ER) contains a highly effective protein quality control system eliminating malfolded proteins by a mechanism called ER-associated protein degradation (ERAD). Here, we unravel the topology of Der1p, a previously identified component of the ERAD system. Der1p contains four transmembrane domains, its N- and C-terminus protrude into the cytoplasm and contribute to its function. Additionally, we describe a yeast homologue of Der1p, Dfm1p, which does not seem to be involved in ERAD. In contrast, a Caenorhabditis elegans orthologue of Der1p, R151.6, is capable of complementing der1-defective phenotypes in yeast.     

Mapping quantitative trait loci for milling quality, protein content and color characteristics of rice using a recombinant inbred line population derived from an elite rice hybrid

Milling properties, protein content, and flour color are important factors in rice. A marker-based genetic analysis of these traits was carried out in this study using recombinant inbred lines (RILs) derived from an elite hybrid cross ’Shanyou 63’, the most-widely grown rice hybrid in production in China. Correlation analysis shows that the traits were inter-correlated, though the coefficients were generally small. Quantitative trait locus (QTL) analysis with both interval mapping (IM) and composite interval mapping (CIM) revealed that the milling properties were controlled by the same few loci that are responsible for grain shape. The QTL located in the interval of RM42-C734b was the major locus for brown rice yield, and the QTL located in the interval of C1087-RZ403 was the major locus for head rice yield. These two QTLs are the loci for grain width and length, respectively. The Wx gene plays a major role in determining protein content and flour color, and is modified by several QTLs with minor effect. The implications of the results in rice breeding were discussed. Received: 15 September 2000 / Accepted: 31 March 2001     

PAK4 is required for regulation of the cell-cycle regulatory protein p21, and for control of cell-cycle progression

The serine/threonine kinase PAK4 regulates cytoskeletal architecture, and controls cell proliferation and survival. In most adult tissues PAK4 is expressed at low levels, but overexpression of PAK4 is associated with uncontrolled proliferation, inappropriate cell survival, and oncogenic transformation. Here we have studied for the first time, the role for PAK4 in the cell cycle. We found that PAK4 levels peak dramatically but transiently in the early part of G1 phase. Deletion of Pak4 was also associated with an increase in p21 levels, and PAK4 was required for normal p21 degradation. In serum-starved cells, the absence of PAK4 led to a reduction in the amount of cells in G1, and an increase in the amount of cells in G2/M phase. We propose that the transient increase in PAK4 levels at early G1 reduces p21 levels, thereby abrogating the activity of CDK4/CDK6 kinases, and allowing cells to proceed with the cell cycle in a precisely coordinated way.     

Effect of irradiation on the protein profile,protein content,and quality of agar from <Emphasis Type="Italic">Gracilaria asiatica</Emphasis> Zhang et Xia (Rhodophyta)

There are about 15 species of Gracilaria reported in Vietnam. Of these, Gracilaria asiatica Zhang et Xia is being cultivated on a large scale in Northern Vietnam, which has a subtropical climate. During the rainy season, from May to October, the growth of G. asiatica is drastically reduced or even ceases due to very low salinity and high temperature. Therefore, it is important to improve the tolerance of G. asiatica to a wide range of salinity and temperatures. This paper presents the results of research on strain improvement of G. asiatica using irradiation and selection media. Three irradiation doses of 20, 60, and 100 krad were tested against the control (with no irradiation). Afterward, the seaweed biomass was cultivated on a selected medium, ESS-1, containing NaCl in concentrations of 23‰ (C1) and 0‰ (C2). The results showed that a higher survival rate of G. asiatica was observed with the 20- and 60-krad doses. The protein content and composition of selected seaweeds were analyzed and compared with the control. SDS-PAGE showed no remarkable difference in the protein composition between the control and irradiated samples. However, the 67-kDa protein band of seaweed treated with 20 and 60 krad, then grown on ESS-1 medium with 23% NaCl, had a higher density than other samples. This protein was reported to play an important role in G. asiatica, by enhancing its tolerance to variable salinity and temperature. Although the organic and inorganic content of all samples remained almost the same, the content and quality of agar extracted from irradiated seaweeds were higher than those of the controls. Due to the high amount of 3.6 anhydro-α-L-galactose combined with low amounts of sulfate found in irradiated seaweeds, the freezing and melting points of extracted agar were lower. Eventually, this resulted in higher condensation and better quality of agar, such as in its gel-forming ability. The quality of fluid agar extracted from selected seaweeds improved as shown in the remarkable decrease in Ca²⁺, Mg²⁺, and total Fe ion content, thus lowering its melting point compared with the control. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Development,validation and characterization of an analytical method for the quantification of hydrolysable urinary metabolites and plasma protein adducts of 2,4- and 2,6-toluene diisocyanate, 1,5-naphthalene diisocyanate and 4,4'-methylenediphenyl diisocyanate

Occupational exposure to diisocyanates within the plastic industry causes irritation and disorders in the airway. The aim of this study was to develop, validate and characterize a method for the determination of 2,4-toluenediamine (2,4-TDA), 2,6-toluenediamine (2,6-TDA), 1,5-diaminonaphthalene (1,5-NDA) and 4,4′-methylenedianiline (4,4′-MDA) in hydrolysed urine and plasma, and to study the correlation between the plasma and urinary levels of these potential biomarkers of 2,4-toluene diisocyanate (2,4-TDI), 2,6-toluene diisocyanate (2,6-TDI), 1,5-naphthalene diisocyanate (1,5-NDI) and 4,4′-methylenediphenyl diisocyanate (4,4′-MDI), respectively. Samples were hydrolysed with 0.3 M NaOH at 100°C for 24 h. The diamines were extracted, derivatized with pentafluoropropionic acid anhydride, and quantified by selected ion monitoring on gas chromatography-mass spectrometry. The repeatability and reproducibility of the method were 7-18% and 7-19%, respectively. Dialysis experiments showed that the metabolites of 2,4-TDI, 2,6-TDI, 1,5-NDI and 4,4′-MDI in plasma were exclusively protein adducts. No free diamines were found in urine, indicating that all diisocyanate-related metabolites were in a conjugated form. For each diisocyanate-related biomarker, there were strongly significant correlations (p&lt;0.001) between individual levels of metabolites in plasma and urine, with Spearman's rank correlation coefficient (r_s) values of 0.74-0.90. The methods presented here will be valuable for the development of biological monitoring methods for diisocyanates.     

Identification of an endocytosis motif in an intracellular loop of Wntless protein, essential for its recycling and the control of Wnt protein signaling

The secretion of Wnt signaling proteins is dependent upon the transmembrane sorting receptor, Wntless (Wls), which recycles between the trans-Golgi network and the cell surface. Loss of Wls results in impairment of Wnt secretion and defects in development and homeostasis in Drosophila, Caenorhabditis elegans, and the mouse. The sorting signals for the internalization and trafficking of Wls have not been defined. Here, we demonstrate that Wls internalization requires clathrin and dynamin I, components of the clathrin-mediated endocytosis pathway. Moreover, we have identified a conserved YXXφ endocytosis motif in the third intracellular loop of the multipass membrane protein Wls. Mutation of the tyrosine-based motif YEGL to AEGL (Y425A) resulted in the accumulation of human mutant Wls on the cell surface of transfected HeLa cells. The cell surface accumulation of Wls^AEGL was rescued by the insertion of a classical YXXφ motif in the cytoplasmic tail. Significantly, a Drosophila Wls^AEGL mutant displayed a wing notch phenotype, with reduced Wnt secretion and signaling. These findings demonstrate that YXXφ endocytosis motifs can occur in the intracellular loops of multipass membrane proteins and, moreover, provide direct evidence that the trafficking of Wls is required for efficient secretion of Wnt signaling proteins.     

Two methods for proteomic analysis of formalin‐fixed,paraffin embedded tissue result in differential protein identification,data quality,and cost

Formalin‐fixed paraffin‐embedded (FFPE) tissue is a rich source of clinically relevant material that can yield important translational biomarker discovery using proteomic analysis. Protocols for analyzing FFPE tissue by LC‐MS/MS exist, but standardization of procedures and critical analysis of data quality is limited. This study compared and characterized data obtained from FFPE tissue using two methods: a urea in‐solution digestion method (UISD) versus a commercially available Qproteome FFPE Tissue Kit method (Qkit). Each method was performed independently three times on serial sections of homogenous FFPE tissue to minimize pre‐analytical variations and analyzed with three technical replicates by LC‐MS/MS. Data were evaluated for reproducibility and physiochemical distribution, which highlighted differences in the ability of each method to identify proteins of different molecular weights and isoelectric points. Each method replicate resulted in a significant number of new protein identifications, and both methods identified significantly more proteins using three technical replicates as compared to only two. UISD was cheaper, required less time, and introduced significant protein modifications as compared to the Qkit method, which provided more precise and higher protein yields. These data highlight significant variability among method replicates and type of method used, despite minimizing pre‐analytical variability. Utilization of only one method or too few replicates (both method and technical) may limit the subset of proteomic information obtained.