Development of a Two-Step Hydrolysis Hypoallergenic Cow's Milk Formula and Evaluation of Residue Allergenicity by Peptidomics and Immunoreactivity Analysis.

Cow's milk allergy (CMA) is an abnormal immune response that severely affects the nutritional supplementation of allergic infants. Currently, only a limited number of hypoallergenic formulas are available on the market, and these are only categorized according to their degree of hydrolysis, which still poses an allergy risk and cannot be consumed by CMA patients, especially infants. To address this issue, we developed a two-step hydrolysis hypoallergenic formula targeting destruction of allergen epitope from whey protein. Then, a comprehensive evaluation system was constructed, including peptidomics analysis, in vivo and in vitro allergenicity assessments, revealing allergic changes in the product from the epitope structure level to the immunological level. The results showed that 97.14% of hydrolyzed peptides from α-lactalbumin and ß-lactoglobulin did not contain allergenic epitopes after treatment with trypsin and flavourzyme. In vitro and in vivo allergenicity assessment results confirmed that the two-step hydrolysis method effectively reduced the allergenicity of whey protein. Compared with the common milk powder, the hypoallergenic formula induced lower levels of basophil degranulation and relieved the body's anaphylactic symptoms caused by cow milk. This study provides a promising solution to the limited hypoallergenic formula problem and may benefit allergic infants who require nutritional supplements.

Viral Capsid, Antibody, and Receptor Interactions: Experimental Analysis of the Antibody Escape Evolution of Canine Parvovirus.

Canine parvovirus (CPV) is a small nonenveloped single-stranded DNA virus that causes serious diseases in dogs worldwide. The original strain of the virus (CPV-2) emerged in dogs during the late 1970s due to a host range switch of a virus similar to the feline panleukopenia virus that infected another host. The virus that emerged in dogs had altered capsid receptor and antibody binding sites, with some changes affecting both functions. Further receptor and antibody binding changes arose when the virus became better adapted to dogs or to other hosts. Here, we used in vitro selection and deep sequencing to reveal how two antibodies with known interactions select for escape mutations in CPV. The antibodies bound two distinct epitopes, and one largely overlapped the host receptor binding site. We also generated mutated antibody variants with altered binding structures. Viruses were passaged with wild-type (WT) or mutated antibodies, and their genomes were deep sequenced during the selective process. A small number of mutations were detected only within the capsid protein gene during the first few passages of selection, and most sites remained polymorphic or were slow to go to fixation. Mutations arose both within and outside the antibody binding footprints on the capsids, and all avoided the transferrin receptor type 1 binding footprint. Many selected mutations matched those that have arisen in the natural evolution of the virus. The patterns observed reveal the mechanisms by which these variants have been selected in nature and provide a better understanding of the interactions between antibody and receptor selections. IMPORTANCE Antibodies protect animals against infection by many different viruses and other pathogens, and we are gaining new information about the epitopes that induce antibody responses against viruses and the structures of the bound antibodies. However, less is known about the processes of antibody selection and antigenic escape and the constraints that apply in this system. Here, we used an in vitro model system and deep genome sequencing to reveal the mutations that arose in the virus genome during selection by each of two monoclonal antibodies or their mutated variants. High-resolution structures of each of the Fab:capsid complexes revealed their binding interactions. The wild-type antibodies or their mutated variants allowed us to examine how changes in antibody structure influence the mutational selection patterns seen in the virus. The results shed light on the processes of antibody binding, neutralization escape, and receptor binding, and they likely have parallels for many other viruses.

The effect of decellularization protocols on characterizations of thermoresponsive and light-curable corneal extracellular matrix hydrogels.

To compare the effects of two decellularization protocols on the characteristics of fabricated COrnea Matrix (COMatrix) hydrogels. Porcine corneas were decellularized with Detergent (De) or Freeze-Thaw (FT)-based protocols. DNA remnant, tissue composition and α-Gal epitope content were measured. The effect of α-galactosidase on α-Gal epitope residue was assessed. Thermoresponsive and light-curable (LC) hydrogels were fabricated from decellularized corneas and characterized with turbidimetric, light-transmission and rheological experiments. The cytocompatibility and cell-mediated contraction of the fabricated COMatrices were assessed. Both protocols reduced the DNA content to < 0.1 µg/mg (native, > 0.5 µg/mg), and preserved the collagens and glycosaminoglycans. The α-Gal epitope remnant decreased by > 50% following both decellularization methods. We observed more than 90% attenuation in α-Gal epitope after treatment with α-galactosidase. The thermogelation half-time of thermoresponsive COMatrices derived from De-Based protocol (De-COMatrix) was 18 min, similar to that of FT-COMatrix (21 min). The rheological characterizations revealed significantly higher shear moduli of thermoresponsive FT-COMatrix (300.8 ± 22.5 Pa) versus De-COMatrix 178.7 ± 31.3 Pa, p < 0.01); while, this significant difference in shear moduli was preserved after fabrication of FT-LC-COMatrix and De-LC-COMatrix (18.3 ± 1.7 vs 2.8 ± 2.6 kPa, respectively, p < 0.0001). All thermoresponsive and light-curable hydrogels have similar light-transmission to human corneas. Lastly, the obtained products from both decellularization methods showed excellent in vitro cytocompatibility. We found that FT-LC-COMatrix was the only fabricated hydrogel with no significant cell-mediated contraction while seeded with corneal mesenchymal stem cells (p < 0.0001). The significant effect of decellularization protocols on biomechanical properties of hydrogels derived from porcine corneal ECM should be considered for further applications.

Evaluating the influence of cold plasma bubbling on protein structure and allergenicity in sesame milk.

BACKGROUND: Sesame is a traditional oilseed comprising essential amino acids. However, the presence of allergens in sesame is a significant problem in its consumption; thus, this study attempted to reduce these allergens in sesame oilseeds. OBJECTIVE: The present study aimed to evaluate the effect of cold plasma processing on structural changes in proteins, and thereby the alteration of allergenicity in sesame milk. Method: Sesame milk (300 mL) was processed using atmospheric pressure plasma bubbling unit (dielectric barrier discharge, power: 200 V, and airflow rate: 16.6 mL/min) at different exposure times (10, 20, and 30 min). RESULTS: The efficiency of plasma-bubbling unit as measured by electron paramagnetic resonance in terms of producing reactive hydroxyl (OH) radicals proved that generation of reactive species increased with exposure time. Further, the plasma-processed sesame milk subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and differential scanning calorimetery analysis revealed that plasma bubbling increased the oxidation of proteins with respect to bubbling time. The structural analysis by Fourier transform infrared spectroscopy and circular dichroism revealed that the secondary structure of proteins was altered after plasma application. This change in the protein structure helped in changing the immunoglobulin E (IgE)-binding epitopes of the protein, which in turn reduced the allergen-binding capacity by 23% at 20-min plasma bubbling as determined by the sandwich-type enzyme-linked immunosorbent assay. However, 30-min plasma bubbling intended to increase allergenicity, possibly because of increase in IgE binding due to the generation of neo epitopes. CONCLUSION: These changes proved that plasma bubbling is a promising technology in oxidizing protein structure, and thereby reducing the allergenicity of sesame milk. However, increase in binding at 30-min bubbling is to be studied to facilitate further reduction of the binding capacity of IgE antibodies.

Characterization of antibody clones that bind exclusively to insoluble fibrin.

Previously, we established an antibody, termed 102-10, which recognizes insoluble fibrin exclusively, unlike the previously established anti-insoluble fibrin antibodies that also cross-reacted with fibrinogen. We established that the epitope of this antibody is on the ß chain that lines an indented structure that becomes exposed only when insoluble fibrin is formed. The amino acid sequence of the epitope is completely conserved from mouse to humans. This study attempted to determine the most suitable insoluble fibrin clone for future diagnostic and therapeutic development. Binding kinetics and properties of antibodies were evaluated by the surface plasmon resonance analysis (SPR) and ELISA among 1101, 99, 443, and 102-10. Immunohistochemical staining for mouse and human pancreatic cancer tissues were also performed. For frozen sections, visually appropriate staining results were observed at an antibody concentration of 1-10 µg/ml, while for paraffin sections, 10 µg/ml was required. From immunohistochemistry and ELISA analyses, clone 99 and clone 1101 showed almost no nonspecific binding in normal pancreatic tissues. Hybridoma production for 1101 yielded more antibodies than that of 99 and demonstrated good long-term stability. It was, therefore, concluded that clone 1101 would be useful for future clinical development as well as basic research.

Identifying the Epitopes of Bacillus thuringiensis Cry2Aa Toxin Involved in Cadherin Interaction by a Monoclonal Antibody.

Antibodies are a useful tool for assistance to map the binding epitopes in Bacillus thuringiensis Cry toxins and their receptors, and even determine how receptors promote toxicity. In this work, a monoclonal antibody (mAb-1D2) was produced by the hybridoma cell line raised against Cry2Aa toxins, with a half inhibition concentration (IC50) of 9.16 µg/mL. The affinity constant of two recombinant toxin-binding fragments derived from Helicoverpa armigera and Plutella xylostella cadherin-like protein (HaCad-TBR or PxCad-TBR) to Cry2Aa toxin was measured to be 1.21 µM and 1.24 µM, respectively. Competitive ELISA showed that mAb-1D2 competed with HaCad-TBR or PxCad-TBR binding to Cry2Aa. Meanwhile, the toxicity of the Cry2Aa toxin to the H. armigera and P. xylostella larvae were greatly reduced when the toxin was mixed with mAb-1D2, which indicated that cadherin may play an important functional role in the toxicity of Cry2Aa. After transforming mAb-1D2 to a single-chain variable fragment (scFv), the hot spot residues of Cry2Aa with 1D2-scFv, PxCad-TBR, and HaCad-TBR were analyzed by molecular docking. It was demonstrated that the hot spot residues of Cry2Aa involving with 1D2-scFv interaction were mainly in Domain II, and some residues in Domain I. Moreover, mAb-1D2 and the two cadherin fragments shared the common hot spot residues on Cry2Aa, which could explain mAb-1D2 inhibited Cry2Aa binding with cadherin fragments. This monoclonal antibody could be a useful tool for identifying the binding epitopes between Cry2Aa and cadherin, and even assist to analyze the roles of cadherin in Cry2Aa toxicity.

Resolving SARS-CoV-2 CD4+ T cell specificity via reverse epitope discovery.

The current strategy to detect immunodominant T cell responses focuses on the antigen, employing large peptide pools to screen for functional cell activation. However, these approaches are labor and sample intensive and scale poorly with increasing size of the pathogen peptidome. T cell receptors (TCRs) recognizing the same epitope frequently have highly similar sequences, and thus, the presence of large sequence similarity clusters in the TCR repertoire likely identify the most public and immunodominant responses. Here, we perform a meta-analysis of large, publicly available single-cell and bulk TCR datasets from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals to identify public CD4+ responses. We report more than 1,200 αßTCRs forming six prominent similarity clusters and validate histocompatibility leukocyte antigen (HLA) restriction and epitope specificity predictions for five clusters using transgenic T cell lines. Collectively, these data provide information on immunodominant CD4+ T cell responses to SARS-CoV-2 and demonstrate the utility of the reverse epitope discovery approach.

Immunoglobin G Sero-Dynamics Aided Host Specific Linear Epitope Identification and Differentiation of Infected from Vaccinated Hosts.

Differentiation of infected from vaccinated hosts (DIVH) is a critical step in virus eradication programs. DIVH-compatible vaccines, however, take years to develop, and are therefore unavailable for fighting the sudden outbreaks that typically drive pandemics. Here, we establish a protocol for the swift and efficient development of DIVH assays, and show that this approach is compatible with any type of vaccines. Using porcine circovirus 2 (PCV2) as the experimental model, the first step is to use Immunoglobin G (IgG) sero-dynamics (IsD) curves to aid epitope discovery (IsDAED): PCV2 Cap peptides were categorized into three types: null interaction, nonspecific interaction (NSI), and specific interaction (SI). We subsequently compared IsDAED approach and traditional approach, and demonstrated identifying SI peptides and excluding NSI peptides supports efficient diagnostic kit development, specifically using a protein-peptide hybrid microarray (PPHM). IsDAED directed the design of a DIVH protocol for three types of PCV2 vaccines (while using a single PPHM). Finally, the DIVH protocol successfully differentiated infected pigs from vaccinated pigs at five farms. This IsDAED approach is almost certainly extendable to other viruses and host species. IMPORTANCE Sudden outbreaks of pandemics caused by virus, such as SARS-CoV-2, has been determined as a public health emergency of international concern. However, the development of a DIVH-compatible vaccine is time-consuming and full of uncertainty, which is unsuitable for an emergent situation like the ongoing COVID-19 pandemic. Along with the development and public health implementation of new vaccines to prevent human diseases, e.g., human papillomavirus vaccines for cervical cancer; enterovirus 71 vaccines for hand, foot, and mouth disease; and most recently SARS-CoV-2, there is an increasing demand for DIVH. Here, we use the IsDAED approach to confirm SI peptides and to exclude NSI peptides, finally to direct the design of a DIVH protocol. It is plausible that our IsDAED approach is applicable for other infectious disease.

A Specific Calprotectin Neo-epitope [CPa9-HNE] in Serum from Inflammatory Bowel Disease Patients Is Associated with Neutrophil Activity and Endoscopic Severity.

BACKGROUND AND AIMS: Endoscopy and the use of faecal calprotectin [faecal CP] are among the least-favoured methods for assessing disease activity by inflammatory bowel disease [IBD] patients; the handling/processing of faecal samples is also impractical. Therefore, we sought to develop a novel neo-epitope serum calprotectin enzyme-linked immunosorbent assay [ELISA], CPa9-HNE, with the aim of quantifying neutrophil activity and neutrophil extracellular trap [NET]-osis and proposing a non-invasive method for monitoring disease activity in IBD patients. METHODS: In vitro cleavage was performed by mixing calprotectin [S100A9/S100A8] with human neutrophil elastase [HNE], and a novel HNE-derived calprotectin neo-epitope [CPa9-HNE] was identified by mass spectrometry for ELISA development. The CPa9-HNE ELISA was quantified in supernatants from ex vivo activated neutrophils and serum samples from patients with ulcerative colitis [UC, n = 43], Crohn's disease [CD, n = 93], and healthy subjects [HS, n = 23]. For comparison, faecal CP and MRP8/14 biomarkers were also measured. RESULTS: CPa9-HNE was specific for activated neutrophils ex vivo. Serum CPa9-HNE levels were 4-fold higher in CD [p <0.0001] and UC [p <0.0001] patients than in HS. CPa9-HNE correlated well with the Simple Endoscopic Score [SES]-CD score [r = 0.61, p <0.0001], MES [r = 0.46, p = 0.0141], and the full Mayo score [r = 0.52, p = 0.0013]. CPa9-HNE was able to differentiate between CD and UC patients in endoscopic remission and moderate/severe disease activity (CD: area under the curve [AUC] = 0.82 [p = 0.0003], UC: AUC = 0.87 [p = 0.0004]). The performance of CPa9-HNE was equipotent or slightly better than that of faecal CP. CONCLUSIONS: Serum CPa9-HNE levels were highly associated with CD and UC patients. CPa9-HNE correlated with the SES-CD score and the full Mayo score, indicating a strong association with disease activity.

Identification and Affinity Determination of Protein-Antibody and Protein-Aptamer Epitopes by Biosensor-Mass Spectrometry Combination.

Analytical methods for molecular characterization of diagnostic or therapeutic targets have recently gained high interest. This review summarizes the combination of mass spectrometry and surface plasmon resonance (SPR) biosensor analysis for identification and affinity determination of protein interactions with antibodies and DNA-aptamers. The binding constant (KD) of a protein-antibody complex is first determined by immobilizing an antibody or DNA-aptamer on an SPR chip. A proteolytic peptide mixture is then applied to the chip, and following removal of unbound material by washing, the epitope(s) peptide(s) are eluted and identified by MALDI-MS. The SPR-MS combination was applied to a wide range of affinity pairs. Distinct epitope peptides were identified for the cardiac biomarker myoglobin (MG) both from monoclonal and polyclonal antibodies, and binding constants determined for equine and human MG provided molecular assessment of cross immunoreactivities. Mass spectrometric epitope identifications were obtained for linear, as well as for assembled ("conformational") antibody epitopes, e.g., for the polypeptide chemokine Interleukin-8. Immobilization using protein G substantially improved surface fixation and antibody stabilities for epitope identification and affinity determination. Moreover, epitopes were successfully determined for polyclonal antibodies from biological material, such as from patient antisera upon enzyme replacement therapy of lysosomal diseases. The SPR-MS combination was also successfully applied to identify linear and assembled epitopes for DNA-aptamer interaction complexes of the tumor diagnostic protein C-Met. In summary, the SPR-MS combination has been established as a powerful molecular tool for identification of protein interaction epitopes.

Identification of a Common Epitope in Nucleocapsid Proteins of Euro-America Orthotospoviruses and Its Application for Tagging Proteins.

The NSs protein and the nucleocapsid protein (NP) of orthotospoviruses are the major targets for serological detection and diagnosis. A common epitope of KFTMHNQIF in the NSs proteins of Asia orthotospoviruses has been applied as an epitope tag (nss-tag) for monitoring recombinant proteins. In this study, a monoclonal antibody TNP MAb against the tomato spotted wilt virus (TSWV) NP that reacts with TSWV-serogroup members of Euro-America orthotospoviruses was produced. By truncation and deletion analyses of TSWV NP, the common epitope of KGKEYA was identified and designated as the np sequence. The np sequence was successfully utilized as an epitope tag (np-tag) to monitor various proteins, including the green fluorescence protein, the coat protein of the zucchini yellow mosaic virus, and the dust mite chimeric allergen Dp25, in a bacterial expression system. The np-tag was also applied to investigate the protein-protein interaction in immunoprecipitation. In addition, when the np-tag and the nss-tag were simultaneously attached at different termini of the expressed recombinant proteins, they reacted with the corresponding MAbs with high sensitivity. Here, we demonstrated that the np sequence and TNP MAb can be effectively applied for tagging and detecting proteins and can be coupled with the nss-tag to form a novel epitope-tagging system for investigating protein-protein interactions.

Paper-based ELISA diagnosis technology for human brucellosis based on a multiepitope fusion protein.

BACKGROUND: Brucellosis, as a serious zoonotic infectious disease, has been recognized as a re-emerging disease in the developing countries worldwide. In china, the incidence of brucellosis is increasing each year, seriously threatening the health of humans as well as animal populations. Despite a quite number of diagnostic methods currently being used for brucellosis, innovative technologies are still needed for its rapid and accurate diagnosis, especially in area where traditional diagnostic is unavailable. METHODOLOGY/PRINCIPAL FINDINGS: In this study, a total of 22 B cell linear epitopes were predicted from five Brucella outer membrane proteins (OMPs) using an immunoinformatic approach. These epitopes were then chemically synthesized, and with the method of indirect ELISA (iELISA), each of them displayed a certain degree of capability in identifying human brucellosis positive sera. Subsequently, a fusion protein consisting of the 22 predicted epitopes was prokaryotically expressed and used as diagnostic antigen in a newly established brucellosis testing method, nano-ZnO modified paper-based ELISA (nano-p-ELISA). According to the verifying test using a collection of sera collected from brucellosis and non-brucellosis patients, the sensitivity and specificity of multiepitope based nano-p-ELISA were 92.38% and 98.35% respectively. The positive predictive value was 98.26% and the negative predictive value was 91.67%. The multiepitope based fusion protein also displayed significantly higher specificity than Brucella lipopolysaccharide (LPS) antigen. CONCLUSIONS: B cell epitopes are important candidates for serologically testing brucellosis. Multiepitope fusion protein based nano-p-ELISA displayed significantly sensitivity and specificity compared to Brucella LPS antigen. The strategy applied in this study will be helpful to develop rapid and accurate diagnostic method for brucellosis in human as well as animal populations.

Characterization and epitope prediction of phosphopyruvate hydratase from Penaeus monodon (black tiger shrimp).

Shellfish allergies constitute an important cause of food-induced anaphylactic reactions, which pose challenges to food safety and human health worldwide. In the present study, the specific IgE (sIgE) binding characteristics of different shrimp proteins of black tiger shrimp (Penaeus monodon) to the sera of eight shrimp-allergic patients from China were studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and nanoliquid chromatography time-of-flight mass spectrometry. According to the PLGS scores (>2000) and the sequence coverage (>40%), eight proteins with sIgE binding activity were identified, including myosin heavy chain type 1 (K4Q4N8), hemocyanin (G1AP69 and Q95V28), phosphopyruvate hydratase (O96656), arginine kinase (C7E3T4), tropomyosin (A1KYZ2), sarcoplasmic calcium binding protein (H7CHW2) and glyceraldehyde-3-phosphate dehydrogenase (A0A097BQP2). Among these eight proteins, phosphopyruvate hydratase was a prevalent IgE-binding protein among these Chinese patients with binding observed in 100% of sera. Moreover, 13 peptides were predicted as epitopes of phosphopyruvate hydratase. These new details help us to understand the crustacean IgE-binding proteins especially Penaeus monodon IgE-binding proteins, that would cause allergic reaction to Chinese patients. And our findings may provide essential information to improve allergy prevention and clinical treatment to shrimp allergy in China. PRACTICAL APPLICATION: This research may have diagnostic and therapeutic value for shrimp allergies in China.

Analysis of ORF5 sequences of Porcine Reproductive and Respiratory Syndrome virus (PRRSV) circulating within swine farms in Costa Rica.

BACKGROUND: Worldwide, Porcine Reproductive and Respiratory Syndrome (PRRS) is among the diseases that cause the highest economic impact in modern pig production. PRRS was first detected in Costa Rica in 1996 and has since then severely affected the local swine industry. Studies of the molecular characterization of circulating strains, correlation with clinical records, and associations with pathogens associated with Porcine Respiratory Disease Complex (PRDC) have not been done in Costa Rica. RESULTS: Sequencing and phylogenetic analysis of ORF5 proved that PRRSV-2 was the only species detected in all locations analyzed. These sequences were grouped into three clusters. When comparing samples from San Jose, Alejuela, and Puntarenas to historical isolates of the previously described lineages (1 to 9), it has been shown that these were closely related to each other and belonged to Lineage 5, along with the samples from Heredia. Intriguingly, samples from Cartago clustered in a separate clade, phylogenetically related to Lineage 1. Epitope analysis conducted on the GP5 sequence of field isolates from Costa Rica revealed seven peptides with at least 80% amino acid sequence identity with previously described and experimentally validated immunogenic regions. Previously described epitopes A, B, and C, were detected in the Santa Barbara-Heredia isolate. CONCLUSIONS: Our data suggest that the virus has three distinct origins or introductions to the country. Future studies will elucidate how recently introduced vaccines will shape the evolutionary change of circulating field strains.

Phage display demonstrates durable differences in serological profile by route of inoculation in primary infections of non-human primates with Dengue Virus 1.

Natural dengue virus (DENV) infections occur by mosquito bite but how the inoculation route affects the humoral immune response is unknown. We serologically profiled 20 non-human primates (NHP) from a prior study of DENV1 infection where animals were inoculated by mosquito (N = 10) or subcutaneous injection (N = 10). Using a comprehensive, densely tiled and highly redundant pan-flavivirus programmable phage library containing 91,562 overlapping 62 amino acid peptides, we produced a high-resolution map of linear peptide sequences enriched during DENV seroconversion. Profiles in mosquito-inoculated and subcutaneously-inoculated animals were similar up to 90 days after primary infection, but diverged at 1 year with differences in sero-reactivity in the Envelope (E; residues 215-406; p < 0.08), and Nonstructural-3 (NS3; residues 549-615; p < 0.05) proteins in mosquito-inoculated versus subcutaneously-inoculated animals. Within the E protein, residues 339-384 in domain III accounted for > 99% of the observed sero-reactivity difference. Antibody breadth did not vary by mode of inoculation. The differential reactivity to E domain III seen by phage display validated orthogonally by ELISA, but did not correlate with late neutralization titers. Serological profiling of humoral immune responses to DENV infection in NHP by programmable phage display demonstrated durable differences in sero-reactivity by route of inoculation.

Glycan Epitopes on 201B7 Human-Induced Pluripotent Stem Cells Using R-10G and R-17F Marker Antibodies.

We developed two human-induced pluripotent stem cell (hiPSC)/human embryonic stem cell (hESC)-specific glycan-recognizing mouse antibodies, R-10G and R-17F, using the Tic (JCRB1331) hiPSC line as an antigen. R-10G recognizes a low-sulfate keratan sulfate, and R-17F recognizes lacto-N-fucopentaose-1. To evaluate the general characteristics of stem cell glycans, we investigated the hiPSC line 201B7 (HPS0063), a prototype iPSC line. Using an R-10G affinity column, an R-10G-binding protein was isolated from 201B7 cells. The protein yielded a single but very broad band from 480 to 1236 kDa by blue native gel electrophoresis. After trypsin digestion, the protein was identified as podocalyxin by liquid chromatography/mass spectrometry. According to Western blotting, the protein reacted with R-10G and R-17F. The R-10G-positive band was resistant to digestion with glycan-degrading enzymes, including peptide N-glycanase, but the intensity of the band was decreased significantly by digestion with keratanase, keratanase II, and endo-ß-galactosidase, suggesting the R-10G epitope to be a keratan sulfate. These results suggest that keratan sulfate-type epitopes are shared by hiPSCs. However, the keratan sulfate from 201B7 cells contained a polylactosamine disaccharide unit (Galß1-4GlcNAc) at a significant frequency, whereas that from Tic cells consisted mostly of keratan sulfate disaccharide units (Galß1-4GlcNAc(6S)). In addition, the abundance of the R-10G epitope was significantly lower in 201B7 cells than in Tic cells.

Partial DnaK protein expression from Coxiella-like endosymbiont of Rhipicephalus annulatus tick.

Q fever is one of the most important zoonotic diseases caused by the obligate intracellular bacteria, Coxiella burnetii. This bacterial infection has been frequently reported in both humans and animals, especially ruminants. Ticks are important ectoparasite and serve as reservoir hosts of Coxiella-like endosymbionts (CLEs). In this study, we have attempted to express chaperone-coding genes from CLEs of Rhipicephalus annulatus ticks collected fromcow path. The partial DnaK coding sequence has been amplified and expressed by Escherichia coli. Amino acid sequences have been analyzed by MS-MS spectrometry and the UniProt database. Despites nucleotide sequences indicating high nucleotide variation and diversity, many nucleotide substitutions are synonymous. In addition, amino acid substitutions compensate for the physicochemical properties of the original amino acids. Immune Epitope Database and Analysis Resource (IEDB-AR) was employed to indicate the antigenicity of the partial DnaK protein and predict the epitopes of B-and T-cells. Interestingly, some predicted HLA-A and B alleles of the MHC-I and HLA-DR alleles belonging to MHC-II were similar to T-cell responses to C. burnetii in Q fever patients. Therefore, the partial DnaK protein of CLE from R. annulatus could be considered a vaccine candidate and immunogenic marker with future prospects.

Fluorescent Immunolocalization of Arabinogalactan Proteins and Pectins in the Cell Wall of Plant Tissues.

Plant development involves constant adjustments of the cell wall composition and structure in response to both internal and external stimuli. Cell walls are composed of cellulose and non-cellulosic polysaccharides together with proteins, phenolic compounds and water. 90% of the cell wall is composed of polysaccharides (e.g., pectins) and arabinogalactan proteins (AGPs). The fluorescent immunolocalization of specific glycan epitopes in plant histological sections remains a key tool to uncover remodeling of wall polysaccharide networks, structure and components. Here, we report an optimized fluorescent immunolocalization procedure to detect glycan epitopes from AGPs and pectins in plant tissues. Paraformaldehyde/glutaraldehyde fixation was used along with LR-White embedding of the plant samples, allowing for a better preservation of the tissue structure and composition. Thin sections of the embedded samples obtained with an ultra-microtome were used for immunolocalization with specific antibodies. This technique offers great resolution, high specificity, and the chance to detect multiple glycan epitopes in the same sample. This technique allows subcellular localization of glycans and detects their level of accumulation in the cell wall. It also permits the determination of spatio-temporal patterns of AGP and pectin distribution during developmental processes. The use of this tool may ultimately guide research directions and link glycans to specific functions in plants. Furthermore, the information obtained can complement biochemical and gene expression studies.

Research advances in hydrogen-deuterium exchange mass spectrometry for protein epitope mapping.

With the development of biomedical technology, epitope mapping of proteins has become critical for developing and evaluating new protein drugs. The application of hydrogen-deuterium exchange for protein epitope mapping holds great potential. Although several reviews addressed the hydrogen-deuterium exchange, to date, only a few systematic reviews have focused on epitope mapping using this technology. Here, we introduce the basic principles, development history, and review research progress in hydrogen-deuterium exchange epitope mapping technology and discuss its advantages. We summarize the main hurdles in applying hydrogen-deuterium exchange epitope mapping technology, combined with relevant examples to provide specific solutions. We describe the epitope mapping of virus assemblies, disease-associated proteins, and polyclonal antibodies as examples of pattern introduction. Finally, we discuss the outlook of hydrogen-deuterium exchange epitope mapping technology. This review will help researchers studying protein epitopes to gain a more comprehensive understanding of this technology.

Antibody Epitope and Affinity Determination of the Myocardial Infarction Marker Myoglobin by SPR-Biosensor Mass Spectrometry.

Myoglobin (MG) is a biomarker for heart muscle injury, making it a potential target protein for early detection of myocardial infarction. Elevated myoglobin levels alone have low specificity for acute myocardial infarction (AMI) but in combination with cardiac troponin T have been considered highly efficient diagnostic biomarkers. Myoglobin is a monomeric heme protein with a molecular weight of 17 kDa that is found in skeletal and cardiac tissue as an intracellular storage unit of oxygen. MG consists of eight α-helices connected by loops and a heme group responsible for oxygen-binding. Monoclonal antibodies are widely used analytical tools in biomedical research and have been employed for immunoanalytical detection of MG. However, the epitope(s) recognized by MG antibodies have been hitherto unknown. Precise molecular identification of the epitope(s) recognized by antibodies is of key importance for the development of MG as a diagnostic biomarker. The epitope of a monoclonal MG antibody was identified by proteolytic epitope extraction mass spectrometry in combination with surface plasmon resonance (SPR) biosensor analysis. The MG antibody was immobilized both on an affinity microcolumn and a gold SPR chip. The SPR kinetic analysis provided an affinity-binding constant KD of 270 nM for MG. Binding of a tryptic peptide mixture followed by elution of the epitope from the SPR-MS affinity interface by mild acidification provided a single-epitope peptide located at the C-terminus [146-153] [YKELGFQG] of MG. The specificity and affinity of the epitope were ascertained by synthesis and affinity-mass spectrometric characterization of the epitope peptide.