Complex Protein Mixtures Research Articles

A Glycopolymer Sensor Array That Differentiates Lectins and Bacteria.

Identification of bacterial lectins offers an attractive route to the development of new diagnostics, but the design of specific sensors is complicated by the low selectivity of carbohydrate-lectin interactions. Here we describe a glycopolymer-based sensor array which can identify a selection of lectins with similar carbohydrate recognition preferences through a pattern-based approach. Receptors were generated using a polymer scaffold functionalized with an environmentally sensitive fluorophore, along with simple carbohydrate motifs. Exposure to lectins induced changes in the emission profiles of the receptors, enabling the discrimination of analytes using linear discriminant analysis. The resultant algorithm was used for lectin identification across a range of concentrations and within complex mixtures of proteins. The sensor array was shown to discriminate different strains of pathogenic bacteria, demonstrating its potential application as a rapid diagnostic tool to characterize bacterial infections and identify bacterial virulence factors such as production of adhesins and antibiotic resistance.

B-060 Prevalence of CCD and profilins antibodies in a seasonal allergy study

Abstract Background Allergen sources are complex and heterogeneous mixtures of proteins, which may contain from innocuous IgE-binding structures such as CCDs (cross-reactive carbohydrate determinants), which are not associated with symptoms, to a panallergen such as profilin, which is associated with multiple sensitizations to pollen and pollen-food-latex syndromes. CCDs are present in many plant and animal allergens and cause strong cross-reactivity due to the high similarity of their structure. They complicate the interpretation of positive in vitro diagnostic results because they do not associate symptomatology and, thats why, the determination of specific IgE antibodies against CCDs can provide useful information when positive IgE results are not consistent with the clinical picture. Profilins are ubiquitous proteins present in all eukaryotic cells and identified as allergens in pollen, latex and plant foods. The highly conserved structure explains the cross-reactivity of IgE antibodies against plant profilins and their designation as panallergens. As a panallergen, profilin is associated with multiple pollen sensitization and pollen-food-latex syndromes that the allergist must be aware of for accurate diagnosis and successful treatment of allergic diseases. We intend to evaluate the prevalence of these two markers with a new diagnostic tool, since profilins and CCDs are the substances most likely to cause in vitro cross-reactivity. Methods In our study 8,242 adults who presented rhinoconjunctivitis and visited their primary care center were included. The samples were analyzed in serum samples which were routinely processed in our laboratory with the EUROLINE Mediterranean Inhalation 2 (IgE) assay from Euroimmun® from February to July 2023, according to the manufacturer’s protocols. Patients with specific IgE levels ≥ 0.35 kU/L were identified as sensitized to allergen. Results A total of 8,242 patients were enrolled in this study, 5,173 women and 3,069 men, ranging in age from 15 to 65 years. Of these, 279 patients (3.39%) had antibodies against CCD, 570 (6.92%) antibodies against profilins and 183 (2.22%) had both. A higher prevalence of these antibodies was observed in men (CCD, n = 168, 5.47%; Profilins, n = 308, 10.04%) than in women (CCD, n = 111, 2.15%; Profilins, n = 262, 5.06%). Both markers were more frequently found in the presence of specific IgE antibodies to grass and weed pollens. Conclusions The possibility of detecting the presence of IgE antibodies against CCD and panallergenic profilins is useful to detect cross-reactivity in vitro, which could explain the differences found between laboratory results and patients' symptoms, helping the allergist to make a diagnosis with greater certainty and accuracy.

Role of pulse globulins and albumins in air-water interface and foam stabilization

Pulse protein isolates are promising substitutes for animal protein in foam preparation, but they typically are complex mixtures of multiple proteins, and the contribution of the individual proteins to the behavior of the mixture in air-water interface stabilization is largely unknown. The major protein fractions in isolates are the globulins and albumins, and this study systematically investigated the molecular properties, and interfacial and foaming properties of these two fractions for three different pulses: lentils, faba beans and chickpeas. The key parameters of these pulse proteins in air-water interface and foam stabilization were investigated by correlation analysis. Based on this, low denaturation enthalpy tends to result in both high foamability and foam stability, most likely due to a greater conformational flexibility that allows for a faster adsorption to the air-water interface, and higher degree of structural rearrangement at the interface, which increases interfacial network stiffness. For globulin-rich pulse protein fractions, vicilins and convicilins tend to introduce high surface hydrophobicity, which increases the affinity of the proteins for the interface, and increases protein-protein in-plane interactions through hydrophobic interactions, resulting in the enhancement of interfacial network connectivity and the level of network branching. These factors increase the interfacial resistance to large deformations and increase foam stability. Vicilins and convicilins also tend to have a high value of the surface charge and further promote foam stability by increasing electrostatic repulsion between air bubbles. Legumins tend to reduce foamability since they adsorb to the air-water interface slowly and tend to disrupt the interfacial network structure. These findings provide deeper insights in the role of pulse globulins and albumins in air-water interface and foam stabilization. The proposed key parameters will benefit the predictability of the interfacial and foaming behavior of pulse proteins.

A method for producing protease pS273R of the African swine fever virus

The pS273R protease of the African swine fever virus (ASFV) is responsible for the processing of the viral polyproteins pp220 and pp62, precursors of the internal capsid of the virus. The protease is essential for a productive viral infection and is an attractive target for antiviral therapy. This work presents a method for the production of pS273R in E. coli cells by fusing the protease with the SlyD chaperone. The chimeric protein pS273R protease, during expression, is formed in a soluble form possessing enzymatic activity. Subsequently, pS273R separates from SlyD through autocatalytic cleavage at the TEV protease site in vivo. This work devised a straightforward protocol for chromatographic purification, resulting in the production of a highly purified viral protease. Additionally, we suggest using a fluorescence method to assess the activity of pS273R. This method is predicated on a shift in the chimeric protein thioredoxin-EGFP's electrophoretic mobility following its protease cleavage. It was shown that thioredoxin-EGFP substrate is effectively and selectively cleaved by the pS273R protease, even in complex protein mixtures such as mammalian cell lysates.

Oxidation-Controlled, Strain-Promoted Tellurophene-Alkyne Cycloaddition (OSTAC): A Bioorthogonal Tellurophene-Dependent Conjugation Reaction.

Tellurophene-bearing small molecules have emerged as valuable tools for localizing cellular activities in vivo using mass cytometry. To broaden the utility of tellurophenes in chemical biology, we have developed a bioorthogonal reaction to facilitate tagging of tellurophene-bearing conjugates for downstream applications. Using TePhe, a tellurophene-based phenylalanine analogue, labeled recombinant proteins were generated for reaction development. Using these proteins, we demonstrate an oxidation-controlled, strain-promoted tellurophene-alkyne cycloaddition (OSTAC) reaction. Mild oxidation of the tellurophene ring with N-chlorosuccinimide produces a reactive Te(IV) species which undergoes rapid (k > 100 M-1 s-1) cycloaddition with bicyclo[6.1.0]nonyne (BCN) yielding a benzo-fused cyclooctane. Selective labeling of TePhe-containing proteins can be achieved in complex protein mixtures and on fixed cells. OSTAC reactions can be combined with strain-promoted azide alkyne cycloaddition (SPAAC) and copper-catalyzed azide alkyne click (CuAAC) reactions. Demonstrating the versatility of this approach, we observe the expected staining patterns for 5-ethynyl-2'-deoxyuridine (DNA synthesis-CuAAC) and immunohistochemistry targets in combination with TePhe (protein synthesis-OSTAC) in fixed cells. The favorable properties of the OSTAC reaction suggest its broad applicability in chemical biology.

Characterization and comparative analysis of sericin protein 150 in Bombyx mori

Lepidopteran silk is a complex mixture of proteins, consisting mainly of fibroins and sericins. Sericins are a small family of highly divergent proteins that serve as adhesives and coatings for silk fibers. So far, five genes encoding sericin proteins have been identified in Bombyx mori. Having previously identified sericin protein 150 (SP150) as a major sericin-like protein in the cocoons of the pyralid moths Galleria mellonella and Ephestia kuehniella, we describe the identification of its homolog in B. mori. Our refined gene model shows that it consists of four exons and a long open reading frame with a conserved motif, CXCXCX, at the C-terminus, reminiscent of the structure observed in a class of mucin proteins. Notably, despite a similar expression pattern, both mRNA and protein levels of B. mori SP150 were significantly lower than those of its pyralid counterpart. We also discuss the synteny of homologous genes on corresponding chromosomes in different moth species and the possible phylogenetic relationships between SP150 and certain mucin-like proteins. Our results improve our understanding of silk structure and the evolutionary relationships between adhesion proteins in the silk of different lepidopteran species.

Comparative in vitro and in silico analysis of the ability of basic Asp49 phospholipase A2 and Lys49-phospholipase A2-like myotoxins from Bothrops diporus venom to inhibit the metastatic potential of murine mammary tumor cells and endothelial cell tubulogenesis: Asp49 vs Lys49 phospholipases A2: Inhibition of metastasis and angiogenesis

Snake venoms are a complex mixture of proteins and polypeptides that represent a valuable source of potential molecular tools for understanding physiological processes for the development of new drugs. In this study two major PLA2s, named PLA2-I (Asp49) and PLA2-II (Lys49), isolated from the venom of Bothrops diporus from Northeastern Argentina, have shown cytotoxic effects on LM3 murine mammary tumor cells, with PLA2-II-like exhibiting a stronger effect compared to PLA2-I. At sub-cytotoxic levels, both PLA2s inhibited adhesion, migration, and invasion of these adenocarcinoma cells. Moreover, these toxins hindered tubulogenesis in endothelial cells, implicating a potential role in inhibiting tumor angiogenesis. All these inhibitory effects were more pronounced for the catalytically-inactive toxin. Additionally, in silico studies strongly suggest that this PLA2-II-like myotoxin could effectively block fibronectin binding to the integrin receptor, offering a dual advantage over PLA2-I in interacting with the αVβ3 integrin. In conclusion, this study reports for the first time, integrating both in vitro and in silico approaches, a comparative analysis of the antimetastatic and antiangiogenic potential effects of two isoforms, an Asp49 PLA2-I and a Lys49 PLA2-II-like, both isolated from Bothrops diporus venom.

Realization of Higher Resolution Charge Detection Mass Spectrometry.

Charge detection mass spectrometry (CD-MS) allows mass distributions to be measured for heterogeneous samples that cannot be analyzed by conventional MS. With CD-MS, the m/z and charge are measured for individual ions using a detection cylinder embedded in an electrostatic linear ion trap (ELIT). Imprecision in both the m/z and charge measurements contribute to the mass resolution. However, if the charge can be measured with a precision of <0.2 e the charge state can be assigned with a low error rate and the mass resolving power only depends on the m/z resolution. Prior to this work, the best resolving power demonstrated experimentally for CD-MS was 700. Here we demonstrate a resolving power of >14,600, 20-times higher than the previous best. Trajectory simulations were used to optimize the geometry and electrostatic potentials of the ELIT. We found conditions where the energy dependence of the oscillation frequency becomes parabolic, and then operated with a nominal ion energy at the minimum of the parabola. The 14,600 resolving power was obtained with a beam collimator before the ELIT. With the collimator removed, the resolving power of the optimized ELIT is 7300, which is still an order of magnitude higher than the previous best. The resolving power was demonstrated by resolving the isotope distributions for peptides and proteins. High resolution CD-MS measurements were then used to resolve the glycans on a monoclonal antibody and applied to the analysis of hepatitis B virus capsids. The results indicate that procedures for adduct removal need to be improved for the full benefit of the higher resolving power to be realized for higher mass species. However, these results represent a key step toward using CD-MS to analyze very complex protein mixtures where charge states are not well resolved in the m/z spectrum because of congestion from numerous overlapping peaks.

The path forward for protein footprinting, covalent labeling, and mass spectrometry-based protein conformational analyses.

Mass spectrometry-based approaches to assess protein conformation have become widely utilized due to their sensitivity, low sample requirements, and broad applicability to proteins regardless of size and environment. Their wide applicability and sensitivity also make these techniques suitable for the analysis of complex mixtures of proteins, and thus, they have been applied at the cell and even the simple organism levels. These works are impressive, but they predominately employ "bottom-up" workflows and require proteolytic digestion prior to analysis. Once digested, it is not possible to distinguish the proteoform from which any single peptide is derived and therefore, one cannot associate distal-in primary structure-concurrent post-translational modifications (PTMs) or covalent labels, as they would be found on separate peptides. Thus, analyses via bottom-up proteomics report the average PTM status and higher-order structure (HOS) of all existing proteoforms. Second, these works predominately employ promiscuous reagents to probe protein HOS. While this does lead to improved conformational resolution, the formation of many products can divide the signal associated with low-copy number proteins below signal-to-noise thresholds and complicate the bioinformatic analysis of these already challenging systems. In this perspective, I further detail these limitations and discuss the positives and negatives of top-down proteomics as an alternative.

A novel ACE inhibitory peptide from Pelodiscus sinensis Wiegmann meat water-soluble protein hydrolysate

Pelodiscus sinensis meat is a nutritional food and tonic with angiotensin-converting enzyme (ACE) inhibitory activities. To identify the bioactive substances responsible, several bioinformatics methods were integrated to enable a virtual screening for bioactive peptides in proteins identified within a water-soluble protein fraction of Pelodiscus sinensis meat by Shotgun proteomics. The peptides were generated from the identified proteins by in silico proteolysis using six proteases. A comparison of the numbers of proteins suitable for digestion with each enzyme and the iBAQ (intensity-based absolute quantification) values for these proteins revealed that bromelain and papain were the most suitable proteases for this sample. Next, the water solubility, toxicity, and ADMET (absorption/distribution/metabolism/excretion/toxicity) properties of these peptides were evaluated in silico. Finally, a novel ACE inhibitory peptide IEWEF with an IC50 value of 41.33 µM was identified. The activity of the synthesized peptide was verified in vitro, and it was shown to be a non-competitive ACE inhibitor. Molecular docking revealed that IEWEF could tightly bind to C-ACE, and N-ACE with energies less than 0 kJ mol−1, and the peptide IEWEF can form hydrogen bonds with C-ACE and N-ACE respectively. These results provide evidence that bioactive peptides in the water-soluble protein fraction account for (at least) some of the ACE inhibitory activities observed in Pelodiscus sinensis meat. Furthermore, our research provides a workflow for the efficient identification of novel ACE inhibitory peptides from complex protein mixtures.

Transcriptome profiling of the posterior salivary glands of the cuttlefish Sepia officinalis from the Portuguese West coast

Cephalopods like octopuses and cuttlefishes are known to secrete a ‘toxic saliva’ to inject into their prey, especially crustaceans since the XIX century. However, only in the mid-XX century were the first coleoid-specific toxins successfully isolated. Motivated by the growing interest on the global ocean as an almost inexhaustible source of novel bioactive compounds, we used RNA-Seq – based transcriptomics and de novo assembly of transcriptomes to screen the posterior salivary glands of Sepia officinalis (the common cuttlefish) from the Portuguese West coast for toxins and other bioactive proteins and peptides. Supported by microanatomical analyses, the posterior salivary glands constitute indeed the ‘venom gland’ whereas the more elusive anterior salivary glands (embedded in the buccal mass) are responsible for the production of mucin-rich saliva that is effectively the vehicle that transports the toxins as the venom is injected into the prey. Indeed, the transcriptomic profiling suggests that the cuttlefish venom is complex mixture of bioactive proteins, among which neurotoxins are major players, together with enzymes whose function is to digest the extracellular matrix to facilitate diffusion of the toxins. Nonetheless, by comparing with previous RNA-Seq data obtained from S. officinalis collected from other biogeographical areas, it may be suggested that significant inter-populational variation in venom composition can occur, which may potentially increase the span of bioactives secreted by these animals. We isolated and validated the full coding sequences for three important toxins, a cysteine-rich venom protein (CRVP), a venom insulin (VIns) and a cephalotoxin (CTX). The toxins seem to be relatively conserved among coleoids but diverging from other venomous mollusks such as cone snails. Their properties as potent modulators of glucose (in the case of VIns) and as potential neurotoxins (like CRVP and CTX) can render them primer targets for drug development.

Linkage of Diabetic Retinopathy with Blood Antioxidants and Gut Microbiota in Type Two Diabetes Mellitus Saudi Patients

Background: The retina is a light-sensitive nerve layer located at the back of the eye that creates images of objects. These cells kept alive by getting oxygen and nutrients from tiny blood vessels in the eye. Retinopathy is a disease of the retina that is more prevalent in type 2 diabetes mellitus patients. Diabetic retinopathy is a leading cause of blindness because hyperglycemia weakens retinal capillaries, resulting in leakage of blood into the surrounding space. This bleeding can result in formation of scar tissue, which can cause traction retinal detachment and maculopathy. The development of a panel of blood biomarkers to monitor diabetic retinopathies is essential for both diagnosis and prognosis. Proteomics as a powerful tool for the analysis of complex mixtures of proteins and the identification of biomarkers can be of great importance. Purpose: To detect early nerve fiber layer changes around macula and optic disc in diabetic patients and to correlate diabetic retinopathy with blood antioxidants and gut microbiota in T2DM in Saudi patients. Materials and Methods: In this cross-sectional case-control study, a total of 77 eyes of 39 subjects aged 40-60 years who did not have any history of eye injuries or eye diseases affecting fundus viewing, were recruited from King Saud University Campus and the department of ophthalmology in King Abdul Aziz university hospital in Riyadh. All subjects underwent full ophthalmic examination including Peripapillary retinal nerve fiber layer thickness and macular profile, Proteomic approach of collected overnight fasting plasma and Microbial stool examination. Results: The nerve fiber layer thickness around the optic disc was measured for all groups and there was no statistically significant difference in all quadrants between groups. The total retinal thickness at the macular area was different among all groups and tends to increase in group 3 due to diabetic retinopathy. The macular thickness in the 4 quadrants revealed no statistical difference except in the inferior quadrant. Glutathione S transferase and lipid peroxides showed no significant difference between the three studied groups; vitamin C and Glutathione were surprisingly higher in controlled diabetic patients relative to controls. Moreover, over growth of bacteroids participated to the evolution of retinopathy in diabetic patients. Conclusion: As hyperglycemia and oxidative stress are implicated in the pathogenesis of diabetic retinopathy, the present study certified that the progressive damage can be delayed in controlled type 2 diabetic patients using different treatment modalities that subside oxidative stress.

Impact of PEDV infection on the biological characteristics of porcine intestinal exosomes.

Porcine epidemic diarrhea (PED) is a highly contagious intestinal infection primarily affecting pigs. It is caused by the porcine epidemic diarrhea virus (PEDV). PEDV targets the villus tissue cells in the small intestine and mesenteric lymph nodes, resulting in shortened intestinal villi and, in extreme cases, causing necrosis of the intestinal lining. Moreover, PEDV infection can disrupt the balance of the intestinal microflora, leading to an overgrowth of harmful bacteria like Escherichia coli. Exosomes, tiny membrane vesicles ranging from 30 to 150 nm in size, contain a complex mixture of RNA and proteins. MicroRNA (miRNA) regulates various cell signaling, development, and disease progression processes. This study extracted exosomes from both groups and performed high-throughput miRNA sequencing and bioinformatics techniques to investigate differences in miRNA expression within exosomes isolated from PEDV-infected porcine small intestine tissue compared to healthy controls. Notably, two miRNA types displayed upregulation in infected exosomes, while 12 exhibited downregulation. These findings unveil abnormal miRNA regulation patterns in PEDV-infected intestinal exosomes, shedding light on the intricate interplay between PEDV and its host. This will enable further exploration of the relationship between these miRNA changes and signaling pathways, enlightening PEDV pathogenesis and potential therapeutic targets.

Vaccination with antigenically complex hemagglutinin mixtures confers broad protection from influenza disease.

Current seasonal influenza virus vaccines induce responses primarily against immunodominant but highly plastic epitopes in the globular head of the hemagglutinin (HA) glycoprotein. Because of viral antigenic drift at these sites, vaccines need to be updated and readministered annually. To increase the breadth of influenza vaccine-mediated protection, we developed an antigenically complex mixture of recombinant HAs designed to redirect immune responses to more conserved domains of the protein. Vaccine-induced antibodies were disproportionally redistributed to the more conserved stalk of the HA without hindering, and in some cases improving, antibody responses against the head domain. These improved responses led to increased protection against homologous and heterologous viral challenges in both mice and ferrets compared with conventional vaccine approaches. Thus, antigenically complex protein mixtures can at least partially overcome HA head domain antigenic immunodominance and may represent a step toward a more universal influenza vaccine.

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System.

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. The Fast Protein Liquid Chromatography (FPLC) system allows high-resolution separation of complex protein mixtures. By adjusting various parameters in FPLC, such as selecting the appropriate purification matrix, regulating the protein sample's temperature, and managing the sample's flow rate onto the matrix and the elution rate, it is possible to ensure the protein's stability and functionality. In this protocol, we will demonstrate the versatility of the FPLC system to purify 6X-His-tagged flap endonuclease 1 (FEN1) protein, produced in bacterial cultures. To improve protein purification efficiency, we will focus on multiple considerations, including proper column packing and preparation, sample injection using a sample loop, flow rate of sample application to the column, and sample elution parameters. Finally, the chromatogram will be analyzed to identify fractions containing high yields of protein and considerations for proper recombinant protein long-term storage. Optimizing protein purification methods is crucial for improving the precision and reliability of protein analysis.

Abstract 1838: Advancing cancer research with Next-Generation Protein Sequencing™ on Platinum™ by Quantum-Si

Abstract Protein sequencing has the potential of playing a crucial role in advancing cancer research by providing insights into the specific proteins involved in cancer pathways. New discoveries from protein sequencing advance our understanding of cancer and potential targeted therapies. Here, we demonstrate the power of next-generation protein sequencing™ using the insight-generating Platinum™ platform. Individual peptides are digested from proteins and immobilized on a semiconductor chip and probed by dye-labeled N-terminal amino acid (NAA) recognizers, followed by subsequent aminopeptidase cleavage to expose each NAA in the peptide for recognition. By recording and analyzing fluorescent intensity, lifetime, and binding kinetics of each NAA binding event, we can decipher single amino acid substitutions and modifications. To demonstrate the versatility of this methodology across proteins associated with cellular biology, we sequenced a series of interleukins: IL4, IL6, IL7, IL13, IL20, and IL34. Interleukins, a group of cytokines that play a vital role in cancer by modulating immune responses, promoting inflammation, and influencing tumor microenvironment, impacting tumor growth and the efficacy of treatments. We have successfully sequenced all the mentioned interleukins individually or within protein mixtures. Furthermore, we were able to sequence IL6 that was enriched through immunoprecipitation from human serum. Finally, we demonstrated the unique potential of Platinum in detecting post-translational modifications (PTMs) relevant to cancer, as exemplified by the successful identification of arginine citrullination in vimentin, an onco-protein that promotes cancer cell mobility and invasion into surround tissues. These results demonstrate the transformative potential of next-generation protein sequencing using Platinum™ in enabling the identification of proteins in complex mixtures and biofluids, as well as the detection of critical PTMs implicated in human pathophysiology, offering valuable insights into cancer research. Citation Format: Khanh Dinh Nguyen. Advancing cancer research with Next-Generation Protein Sequencing™ on Platinum™ by Quantum-Si [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1838.

Linkage of diabetic retinopathy with blood antioxidants and gut microbiota in type two diabetes mellitus Saudi patients

Background: The retina is a light-sensitive nerve layer located at the back of the eye that creates images of objects. These cells kept alive by getting oxygen and nutrients from tiny blood vessels in the eye. Retinopathy is a disease of the retina that is more prevalent in type 2 diabetes mellitus patients. Diabetic retinopathy is a leading cause of blindness because hyperglycemia weakens retinal capillaries, resulting in leakage of blood into the surrounding space. This bleeding can result in formation of scar tissue, which can cause traction retinal detachment and maculopathy. The development of a panel of blood biomarkers to monitor diabetic retinopathies is essential for both diagnosis and prognosis. Proteomics as a powerful tool for the analysis of complex mixtures of proteins and the identification of biomarkers can be of great importance. Purpose: To detect early nerve fiber layer changes around macula and optic disc in diabetic patients, and to correlate diabetic retinopathy with blood antioxidants and gut microbiota in T2DM in Saudi patients. Materials and Methods: In this cross-sectional case-control study, a total of 77 eyes of 39 subjects aged 40-60 years who did not have any history of eye injuries or eye diseases affecting fundus viewing, were recruited from King Saud University Campus and the department of ophthalmology in King Abdul Aziz university hospital in Riyadh. All subjects underwent full ophthalmic examination including Peripapillary retinal nerve fiber layer thickness and macular profile, Proteomic approach of collected overnight fasting plasma and Microbial stool examination. Results: The nerve fiber layer thickness around the optic disc was measured for all groups, and there was no statistically significant difference in all quadrants between groups. The total retinal thickness at the macular area was different among all groups and tends to increase in group 3 due to diabetic retinopathy. The macular thickness in the 4 quadrants revealed no statistical difference except in the inferior quadrant. Glutathione S transferase and lipid peroxides showed no significant difference between the three studied groups; vitamin C and Glutathione were surprisingly higher in controlled diabetic patients relative to controls. Moreover, over growth of bacteroids participated to the evolution of retinopathy in diabetic patients. Conclusion: As hyperglycemia and oxidative stress are implicated in the pathogenesis of diabetic retinopathy, the present study certified that the progressive damage can be delayed in controlled type 2 diabetic patients using different treatment modalities that subside oxidative stress.

Specific dairy foods and risk of frailty in older women: a prospective cohort study

BackgroundDairy contains a complex mixture of lipids, proteins, and micronutrients. Whether habitual dairy consumption is associated with health benefits is not well established. Since dairy is high in nutrients that are potentially protective against frailty, the association between dairy products and the risk of frailty is of interest.MethodsWe analyzed data from 85,280 women aged ≥ 60 years participating in the Nurses’ Health Study. Consumption of milk, yogurt, and cheese was obtained from repeated food frequency questionnaires administered between 1980 and 2010. Frailty was defined as having at least three of the following five criteria from the FRAIL scale: fatigue, low strength, reduced aerobic capacity, having ≥ 5 chronic illnesses, and a weight loss of ≥ 5%. The occurrence of frailty was assessed every four years from 1992 to 2018. Cox proportional hazard models were used to examine the association between the intake of dairy foods and frailty.ResultsDuring follow-up we identified 15,912 incident cases of frailty. Consumption of milk or yogurt was not associated with the risk of frailty after adjustment for lifestyle factors, medication use, and overall diet quality. Cheese consumption was positively associated with risk of frailty [relative risk (95% confidence interval) for one serving/day increment in consumption: 1.10 (1.05, 1.16)]. Replacing one serving/day of milk, yogurt, or cheese with one serving/day of whole grains, nuts, or legumes was associated with a significant lower risk of frailty, while replacing milk, yogurt, or cheese with red meat or eggs was associated with an increased risk. When milk was replaced with a sugar-sweetened or artificially sweetened beverage, a greater risk of frailty was observed, while replacing milk with orange juice was associated with a lower risk of frailty.ConclusionsThe results suggest that the association between milk, yogurt, and cheese and frailty partly depends on the replacement product. Habitual consumption of milk or yogurt was not associated with risk of frailty, whereas cheese consumption may be associated with an increased risk.

Identification and semi-quantification of protein allergens in complex mixtures using proteomic and AllerCatPro 2.0 bioinformatic analyses: a proof-of-concept investigation

The demand for botanicals and natural substances in consumer products has increased in recent years. These substances usually contain proteins and these, in turn, can pose a risk for immunoglobulin E (IgE)-mediated sensitization and allergy. However, no method has yet been accepted or validated for assessment of potential allergenic hazards in such materials. In the studies here, a dual proteomic-bioinformatic approach is proposed to evaluate holistically allergenic hazards in complex mixtures of plants, insects, or animal proteins. Twelve commercial preparations of source materials (plant products, dust mite extract, and preparations of animal dander) known to contain allergenic proteins were analyzed by label-free proteomic analyses to identify and semi-quantify proteins. These were then evaluated by bioinformatics using AllerCatPro 2.0 (https://allercatpro.bii.a-star.edu.sg/) to predict no, weak, or strong evidence for allergenicity and similarity to source-specific allergens. In total, 4,586 protein sequences were identified in the 12 source materials combined. Of these, 1,665 sequences were predicted with weak or strong evidence for allergenic potential. This first-tier approach provided top-level information about the occurrence and abundance of proteins and potential allergens. With regards to source-specific allergens, 129 allergens were identified. The sum of the relative abundance of these allergens ranged from 0.8% (lamb’s quarters) to 63% (olive pollen). It is proposed here that this dual proteomic-bioinformatic approach has the potential to provide detailed information on the presence and relative abundance of allergens, and can play an important role in identifying potential allergenic hazards in complex protein mixtures for the purposes of safety assessments.

Changes in saliva protein profile throughout Rhipicephalus microplus blood feeding

BackgroundWhen feeding on a vertebrate host, ticks secrete saliva, which is a complex mixture of proteins, lipids, and other molecules. Tick saliva assists the vector in modulating host hemostasis, immunity, and tissue repair mechanisms. While helping the vector to feed, its saliva modifies the site where pathogens are inoculated and often facilitates the infection process. The objective of this study is to uncover the variation in protein composition of Rhipicephalus microplus saliva during blood feeding.MethodsTicks were fed on calves, and adult females were collected, weighed, and divided in nine weight groups, representing the slow and rapid feeding phases of blood feeding. Tick saliva was collected, and mass spectrometry analyses were used to identify differentially secreted proteins. Bioinformatic tools were employed to predict the structural and functional features of the salivary proteins. Reciprocal best hit analyses were used to identify conserved families of salivary proteins secreted by other tick species.ResultsChanges in the protein secretion profiles of R. microplus adult female saliva during the blood feeding were observed, characterizing the phenomenon known as “sialome switching.” This observation validates the idea that the switch in protein expression may serve as a mechanism for evading host responses against tick feeding. Cattle tick saliva is predominantly rich in heme-binding proteins, secreted conserved proteins, lipocalins, and protease inhibitors, many of which are conserved and present in the saliva of other tick species. Additionally, another remarkable observation was the identification of host-derived proteins as a component of tick saliva.ConclusionsOverall, this study brings new insights to understanding the dynamics of the proteomic profile of tick saliva, which is an important component of tick feeding biology. The results presented here, along with the disclosed sequences, contribute to our understanding of tick feeding biology and might aid in the identification of new targets for the development of novel anti-tick methods.Graphical