Protein Peptide Research Articles

Effect of microbial fermentation on the quality of soybean meal

SummarySoybean meal is an important and inexpensive source of protein in food and animal feed, and contains anti‐nutritional factors such as trypsin inhibitors, antigenic proteins, oligosaccharides, phytic acids and lectins, which limit its application. Microbial fermentation has the capability to improve the nutritional value and biological activities of soybean meal by changing its natural composition. Fermentation results in the degradation of anti‐nutritional factors in soybean meal, an increased amount of small molecular protein peptides and improved content of isoflavone aglycone. However, the nutritional composition and biological activities of the resulting fermented products differ due to the different microbial species and conditions used for fermentation. Compared to soybean meal, fermented soybean meal has several beneficial effects such as digestibility, antioxidant and hypoallergenicity. The objective of this review is to discuss the fermentation processes of soybean meal, as well as outlines some biological activities associated with it. Furthermore, the current application of fermented soybean meal is also summarised.

Immunotherapy targeting isoDGR‐protein damage extends lifespan in a mouse model of protein deamidation

Aging results from the accumulation of molecular damage that impairs normal biochemical processes. We previously reported that age‐linked damage to amino acid sequence NGR (Asn‐Gly‐Arg) results in “gain‐of‐function” conformational switching to isoDGR (isoAsp‐Gly‐Arg). This integrin‐binding motif activates leukocytes and promotes chronic inflammation, which are characteristic features of age‐linked cardiovascular disorders. We now report that anti‐isoDGR immunotherapy mitigates lifespan reduction of Pcmt1−/− mouse. We observed extensive accumulation of isoDGR and inflammatory cytokine expression in multiple tissues from Pcmt1−/− and naturally aged WT animals, which could also be induced via injection of isoDGR‐modified plasma proteins or synthetic peptides into young WT animals. However, weekly injection of anti‐isoDGR mAb (1 mg/kg) was sufficient to significantly reduce isoDGR‐protein levels in body tissues, decreased pro‐inflammatory cytokine concentrations in blood plasma, improved cognition/coordination metrics, and extended the average lifespan of Pcmt1−/− mice. Mechanistically, isoDGR‐mAb mediated immune clearance of damaged isoDGR‐proteins via antibody‐dependent cellular phagocytosis (ADCP). These results indicate that immunotherapy targeting age‐linked protein damage may represent an effective intervention strategy in a range of human degenerative disorders.

Multicomponent Peptide-Based Hydrogels Containing Chemical Functional Groups as Innovative Platforms for Biotechnological Applications.

Multicomponent hydrogels (HGs) based on ultrashort aromatic peptides have been exploited as biocompatible matrices for tissue engineering applications, the delivery of therapeutic and diagnostic agents, and the development of biosensors. Due to its capability to gel under physiological conditions of pH and ionic strength, the low molecular-weight Fmoc-FF (Nα-fluorenylmethoxycarbonyl-diphenylalanine) homodimer is one of the most studied hydrogelators. The introduction into the Fmoc-FF hydrogel of additional molecules like protein, organic compounds, or other peptide sequences often allows the generation of novel hydrogels with improved mechanical and functional properties. In this perspective, here we studied a library of novel multicomponent Fmoc-FF based hydrogels doped with different amounts of the tripeptide Fmoc-FFX (in which X= Cys, Ser, or Thr). The insertion of these tripeptides allows to obtain hydrogels functionalized with thiol or alcohol groups that can be used for their chemical post-derivatization with bioactive molecules of interest like diagnostic or biosensing agents. These novel multicomponent hydrogels share a similar peptide organization in their supramolecular matrix. The hydrogels' biocompatibility, and their propensity to support adhesion, proliferation, and even cell differentiation, assessed in vitro on fibroblast cell lines, allows us to conclude that the hybrid hydrogels are not toxic and can potentially act as a scaffold and support for cell culture growth.

Cytoprotective E-WE thrombin reduces disease severity in a murine model of relapsing-remitting multiple sclerosis.

The blood-brain barrier is composed of microvascular endothelial cells, immune cells, and astrocytes that work in concert with the coagulation cascade to control inflammation and immune cell infiltration into the central nervous system. Endothelial cell dysfunction leading to increased permeability and compromised barrier function are hallmarks of neuroinflammatory and autoimmune disorders, including multiple sclerosis (MS). Therapeutic strategies that improve or protect endothelial barrier function may be beneficial in the treatment or prevention of neuroinflammatory diseases. We therefore tested the hypothesis that biasing thrombin toward anticoagulant and cytoprotective activities would provide equivalent or even additive benefit compared with standard-of-care therapeutic strategies, including corticosteroids. In a mouse model of relapsing-remitting MS, treatment with the thrombin mutant, E-WE thrombin, an engineered thrombin mutant with cytoprotective activities that is biased toward anticoagulant and cytoprotective activity, reduced neuroinflammation and extracellular fibrin formation in SJL mice inoculated with proteolipid protein (PLP) peptide. When administered at the onset of detectable disease, E-WE thrombin significantly improved the disease severity of the initial attack as well as the relapse and delayed the onset of relapse to a similar extent as observed with methylprednisolone. Both methylprednisolone and E-WE thrombin reduced demyelination and immune cell recruitment. These results provide rationale for considering engineered forms of thrombin biased toward anticoagulant and cytoprotective activity as a therapeutic strategy and perhaps an effective alternative to high-dose methylprednisolone for the management of acute relapsing MS attacks.NEW & NOTEWORTHY There are limited treatment options for mitigating acute relapsing attacks for patients with multiple sclerosis. We tested the hypothesis that harnessing the cytoprotective activity of the blood coagulation enzyme, thrombin, would provide benefit and protection against relapsing disease in a mouse model of MS. Our results provide rationale for considering engineered forms of thrombin biased toward cytoprotective activity as a therapeutic strategy and perhaps an alternative to steroids for the management of relapsing MS attacks.

Interleukin-6 plasma levels are elevated in patients with acute myocardial infarction and predict death or recurrent infarction within 1 year - a case-control study

Abstract Background Acute myocardial infarction (AMI) harbours marked inflammatory components. While cytokines of the same family have been causally related to patient outcome, the role of interleukin (IL)-6 in AMI remains incompletely understood. Recent trials suggest benefits of inhibiting IL-6 in patients with ST-segment elevation myocardial infarction (STEMI). In patients at high atherosclerotic risk, IL-6 blockade reduces markers of inflammation and thrombosis. Purpose This study aimed to assess the levels of IL-6 in AMI patients and their predictive value. Methods 160 cases (recurrent AMI or death within 1 year) and 160 matched controls were selected from the prospective, multicentric SPUM-ACS Cohort. All patients had an AMI (STEMI or NSTEMI) as initial event and underwent percutaneous coronary intervention (PCI) within 24h of symptom onset. Blood samples were drawn before PCI and biomarker plasma levels were assessed (mesoscale). Comparisons were made with age- and sex-matched healthy blood donors (HD). IL-6 levels are presented as medians and interquartile ranges. Cases, controls and HD were compared using the Kruskal-Wallis test. Correlations of baseline variables to IL-6 levels within cases and controls were assessed by linear regression analyses. Patients were divided into those with higher or lower IL-6 levels according to the median IL-6 level. Kaplan-Meier (KM) analyses for the combined endpoint of death or recurrent AMI were performed. Results The overall median IL-6 level was 21.3pg/ml. Patients with higher IL-6 had more previous AMI (n=22, 14%) than patients with lower IL-6 (n=12, 7%, p=0.045). Patients with increased IL-6 had more previous PCI (n=30, 20%) than patients with lower IL6 (n=19, 11%, p=0.045). Both cases and controls showed higher levels of IL-6 than HD (cases: 25.7pg/ml [14.8–81.6], controls: 20.0pg/ml [11.3–40.1], HD: 5.9pg/ml [3.6–9.9]; cases/controls vs. HD: p<0.0001). Among AMI patients, cases showed higher IL-6 levels than controls (p=0.030) (Figure 1). Within cases and controls, IL-6 was related to IL-1beta (r=0.628, p<0.001), IL-1alpha (r=0.174, p=0.002) and IL-1 receptor antagonist (r=0.422, p<0.001). IL-6 was related to neutrophil/lymphocyte ratio (r=0.147, p=0.016), but not to high-sensitivity C-reactive protein (r=0.087, p=0.142) or N-terminal pro B-type natriuretic peptide (r=0.091, p=0.118). IL-6 was related to Killip Class upon presentation (r=0.190, p<0.001). KM analyses showed significantly higher rates of death or recurrent AMI within 1 year for patients with higher IL-6 (log-rank p=0.006, Figure 2), with early divergence of the curves. Conclusions IL-6 is markedly higher in patients with AMI compared to healthy controls and predicts death or recurrent AMI within 1 year. IL-6 might be a valuable target for early anti-inflammatory therapy to patients with AMI. A deeper understanding of factors influencing IL-6 levels may help select those patients that will benefit most from such treatment.Figure 1:Cases vs. Controls vs. HDFigure 1:Kaplan-Meier Analysis

Improving the antioxidant activity of microbial canthaxanthin by its encapsulation with bioactive peptides of soybean protein

In this study, the pH-shifting method was used for the encapsulation of microbial canthaxanthin with soy protein hydrolysates to increase its antioxidant activity. Enzymatic hydrolysis improved the functional and structural properties of soy protein hydrolysates in comparison to native ones. Reducing power and radical quenching activity of hydrolysates were raised respectively from 14.6 and 0.05 to 92.9 and 0.19 and their bioactive loading properties were significantly increased more than five times. Encapsulation parameters, structural attributes, and antioxidant activity were also studied after complexation. Fourier transform infrared spectroscopy findings indicated the formation of hydrogen bonds between canthaxanthin and hydrolysates. The crystalline nature of canthaxanthin was changed to amorphous, according to the X-ray diffraction assay. Reducing power and radical quenching activity increased (respectively 2.3-fold and 1.5-fold). The results showed the positive effects of enzymatic hydrolysis of soy protein and the pH-driven method of encapsulation on improvement in the antioxidant activity of canthaxanthin.

The Efficacy of Budesonide, Levocabastine, and Their Combination in Treatment of Vasomotor Rhinitis

Introduction: Relevant studies have demonstrated that glucocorticoids and antihistamines, such as budesonide and azelastine, are effective in the treatment of vasomotor rhinitis, with their combined use being more effective than that of a single drug. The aim of this study was to assess the improvement in the symptoms of patients following the combined administration of these drugs. Methods: We conducted a single-center randomized study on 42 patients. Participants were randomly treated with budesonide, levocabastine hydrochloride, or their combination for 2 weeks. The visual analog scale (VAS) score and levels of eosinophil cationic protein (ECP), histamine (HA), leukotriene B4 (LTB4), and vasoactive intestinal peptide (VIP) in nasal secretions were evaluated before and after treatment. Results: The symptoms of patients were improved in all 3 treatment groups compared with those before treatment. Following combined treatment, the improvement in symptoms of nasal obstruction, runny nose, nasal itching, and sneezing was much greater than those in the groups treated with budesonide or levocabastine hydrochloride alone (p = 0.04, 0.004, 0.005, 0.004, respectively). The decreased levels of these inflammatory mediators were significantly different between the different treatment groups. Conclusions: Budesonide or levocabastine hydrochloride alone improved the nasal symptoms of patients with vasomotor rhinitis and reduced the levels of ECP, HA, LTB4, and VIP in nasal secretions. However, their combination improved the symptoms of patients more significantly than each drug alone.

A protein chimera for dsRNA binding and delivery into oocytes for large-scale gene silencing in crustacean aquaculture

RNA interference (RNAi) is one of the most intensively used gene silencing techniques for gene function studies and biotechnology applications. However, the gonads, specifically the ovary, of arthropods appear to be intractable to efficient gene silencing. In this article, we report the development of a tool for specific binding and delivery of double-stranded (ds) RNA to the crustacean oocyte for gene silencing. The tool, an oocyte-specific silencing protein chimera, which we designated OSSCot, is composed of a dsRNA-binding domain retrieved from a specific Macrobrachium rosenbergii dsRNA binding protein and 24-amino acid peptide derived from the yolk protein, vitellogenin. The results of the in vitro and in vivo examinations carried out in this study indicate that an OSSCot-piggybacked dsRNA specifically enters the oocytes. The results also indicate that the OSSCot protein protects the dsRNA from degradation by endonucleases in the ovary. When OSSCot was linked to the specific PAX6-like dsRNA of M. rosenbergii (where PAX6 is the master gene for eye development) and injected into the hemolymph of vitellogenic females, the expression of the RNAi enzyme machinery was enhanced, and eye development was impaired in the embryos of the treated females. These findings validate the concept of exploiting OSSCot to piggyback a functional dsRNA into crustacean oocytes for gene silencing. The newly developed tool will find application for oocyte-specific gene silencing in other crustacean species and for research and biotechnology applications.

Molecular docking, simulations of animal peptides against the envelope protein of Dengue virus

Peptides are biologically active, small molecules with high specificity in its mode of action that can be effective at nanomolar concentrations. Peptide-based antiviral medicines have already been licensed and used to treat human immunodeficiency virus (HIV), influenza virus and hepatitis C virus. So far, no peptide drug has been approved for antiviral treatment against Dengue virus, and many are under clinical trials. Therefore, developing a reasonable peptide against the Dengue virus Envelope protein structure will be a successful strategy for treating Dengue. Hence, we investigated protein-protein docking interactions between 215 peptides retrieved from the AVP database against the envelope protein using Cluspro and HADDOCK followed by the evaluation of their allegenicity, toxicity and physicochemical characteristics investigation. Further validation of the protein–peptide complexes was performed with Molecular dynamics simulations and Molecular Mechanics Poisson–Boltzmann surface area (MMPBSA) analysis on the hit inhibitors. This study revealed that Indolicidin (−75.026 ± 1.54 KJ/mol) and Human Neutrophil peptide-1 (−71.6551 ± 2.112 KJ/mol) shows higher negative ΔG binding implicating their relative stabilization in the protein–peptide interactions during 100 ns of dynamic simulations. Also, both the peptides exhibited desirable physicochemical properties and were nonallergenic. Hence, we further aim to test these peptides by in vitro and in vivo studies to confirm their efficacy against Dengue virus. Communicated by Ramaswamy H. Sarma

Characterization of add-on testing before and after automation at a core laboratory

ObjectivesAdd-on testing refers to the process that occurs in clinical laboratories when clinicians request that additional tests be performed on a previously analysed specimen. This is a common but inefficient procedure, highly time-consuming, especially at core laboratories and could be optimised by automating these procedures. The aims of this study are: 1) To describe patterns of add-on testing at a core laboratory at a tertiary hospital, 2) To evaluate turnaround time (TAT) before and after automation of the pre-, post- and analytical phases. MethodsRetrospective, observational study conducted at the biochemistry area of a core laboratory of all add-on orders received in two different months (pre-automation and post-automation). ResultsA total of 2464 add-on orders were analysed, representing around 5 % of total requests. Most orders were for either one (>50 %) or two (≈20 %) tests. Most orders were received during the week (from Monday to Friday), particularly during the morning shift (>50 %). More than 50 % of requests were made by the Emergency Department. The two most common add-on parameters were C-reactive protein and N-terminal pro-brain natriuretic peptide. After automation, the median TAT decreased by 42.3 % (from 52 to 22 min). The largest decreases in TAT were observed for routine samples (58.89 %) and fully automated analyses (56.86 %). ConclusionsAutomation of our core laboratory substantially reduced turnaround time for add-on testing, indicating an increase in efficiency. Automation eliminated several manual steps in the process, leading to a mean reduction of 15 work hours per day (more than 2 full-time equivalents).

Receptor-based approaches and therapeutic targets in Alzheimer’s disease along with role of AI in drug designing: Unraveling pathologies and advancing treatment strategies

Alzheimer’s disease (AD) is a prevalent cause of dementia in the elderly, characterized by progressive cognitive decline and neurodegeneration. This review focuses on the etiology of AD, the role of various receptors [TNF (Tumor necrosis factor) receptor, nAChR (Neuronal nicotinic acetylcholine receptors), NMDARs (N-Methyl-D-aspartate receptors), APOE (Apolipoprotein E) receptor, and amyloid-beta receptor], and risk factors contributing to its development. AD progresses through mild, moderate, and severe stages, each exhibiting distinct symptoms. The hallmark pathologies are neurofibrillary tangles and amyloid plaques, comprised of hyperphosphorylated tau protein and amyloid-beta peptides, respectively. Current pharmacotherapeutic options alleviate symptoms but lack a complete cure. To address the challenges in developing effective AD treatments, researchers have turned to artificial intelligence (AI) and computational approaches in drug design. AI techniques, including machine learning and molecular docking, enable the analysis of large datasets and prediction of molecular interactions between potential drug candidates and target receptors. Virtual screening and molecular modeling aid in identifying novel therapeutic compounds. Predictive modeling and optimization algorithms optimize drug properties and predict efficacy. AI also facilitates the repurposing of existing drugs by analyzing their interactions with AD-related receptors and pathways. Clinical trial optimization using AI algorithms enhances patient selection, treatment monitoring, and outcome prediction. Integrating AI into AD drug design holds tremendous promise for accelerating the discovery of effective interventions. By leveraging AI’s capabilities, researchers can efficiently analyze extensive data, predict drug-target interactions, and optimize drug properties, leading to the identification of novel AD treatments. However, further research and validation are needed to translate AI-driven drug design approaches into clinically viable solutions for AD patients. Through continued advancements in AI and collaborative efforts, the development of targeted and advanced therapies for AD is within reach.

Transcriptomic response of Mytilus coruscus mantle to acute sea water acidification and shell damage

Mytilus coruscus is an economically important marine calcifier living in the Yangtze River estuary sea area, where seasonal fluctuations in natural pH occur owing to freshwater input, resulting in a rapid reduction in seawater pH. In addition, Mytilus constantly suffers from shell fracture or injury in the natural environment, and the shell repair mechanisms in mussels have evolved to counteract shell injury. Therefore, we utilized shell-complete and shell-damaged Mytilus coruscus in this study and performed transcriptomic analysis of the mantle to investigate whether the expression of mantle-specific genes can be induced by acute seawater acidification and how the mantle responds to acute acidification during the shell repair process. We found that acute acidification induced more differentially expressed genes than shell damage in the mantle, and the biomineralization-related Gene Ontology terms and KEGG pathways were significantly enriched by these DEGs. Most DEGs were upregulated in enriched pathways, indicating the activation of biomineralization-related processes in the mussel mantle under acute acidification. The expression levels of some shell matrix proteins and antimicrobial peptides increased under acute acidification and/or shell damage, suggesting the molecular modulation of the mantle for the preparation and activation of the shell repairing and anti-infection under adverse environmental conditions. In addition, morphological and microstructural analyses were performed for the mantle edge and shell cross-section, and changes in the mantle secretory capacity and shell inner film system induced by the two stressors were observed. Our findings highlight the adaptation of M. coruscus in estuarine areas with dramatic fluctuations in pH and may prove instrumental in its ability to survive ocean acidification.

Optimization and validation of flow cytometry method for quantification of SARS-CoV-2 antigen-reactive human memory T cells

A proper and representative monitoring of SARS-CoV-2 herd immunity including a long-term health impact on recovered patients and vaccinated individuals is of great importance. For this, a monitoring campaign should assesses both humoral and T-cell immune arms. Upon that, analyzing antigen specific-cell activation and cellular phenotype are informative. We developed a flow cytometry method for detection of intracellular IFN-producing antigen-reactive T cells after exposure of human peripheral blood mononuclear cells (PBMC) to SARS-CoV-2 virus antigens. The method was validated according to the following characteristics: sensitivity, specificity, precision, and robustness. We used positive samples from donors recovered from COVID-19 and negative samples from donors who had no contact with COVID-19 patients and lacking antibodies to SARS-CoV-2. All samples were tested by laboratory methods. Peripheral blood mononuclear cells were isolated from donor blood by centrifugation in a Ficoll density gradient. Specific T cells were stimulated with S-protein as well as N, M, ORF3a, and ORF7a protein peptides to count IFN-producing T cells by flow cytometer. The data were statistically analyzed. The area limited by ROC-curve and false positive rate (AUC) for CD4 and CD8 cells was from 0.97 to 1.00. Precision was considered acceptable because the coefficient of variation for all PBCM did not exceed 20%. Robustness was confirmed for frozen and freshly prepared PBMC samples. The thresholds levels to recognize immune and non-immune samples were defined for CD4-positive T-cells (0.029%) and CD8-positive T-cells (0.0640.068%). Also, acceptance criteria for positive and negative controls were defined. Based on the validation, the suitability of the method Evaluation of antigen-reactive T cells that produce intracellular IFN in response to SARS-CoV-2 virus antigens by flow cytometry was confirmed. The method allows for reliable data that was used to characterize standard control samples for internal quality control of TigraTest SARS-CoV-2 kits.

Silkworm pupa protein peptide improved DSS-induced colitis in C57BL/6 mice through the MAPK/NF-κB signaling pathway

Numerous studies have revealed the multifunctional activities of silkworm pupa peptides (SPP). In this study, we investigated the protective effects of SPP on dextran sulfate sodium (DSS)-induced colitis in mice. The results showed that SPP improved the clinical symptoms of colitis in mice, including increased colon length, slowed weight loss, and reduced disease activity index (DAI) scores. Furthermore, SPP significantly alleviated DSS-induced colonic injury by reducing the expression of myeloperoxidase (MPO) in colonic tissues, enhancing mucin secretion in the colon, and decreasing colonic cell apoptosis. SPP also mitigated the severity of colitis by modulating serum inflammatory factors and improving colonic oxidative stress levels. Western blot analysis revealed that SPP suppressed the activation of the MAPK and NF-κB signaling pathways. In conclusion, SPP protected against DSS-induced colitis in mice by reducing cell apoptosis, attenuating inflammatory responses, and improving oxidative stress levels, potentially through the inhibition of MAPK and NF-κB signaling pathways.

Exploration of the mechanism of reinfusion of fresh autologous blood in type 2 diabetes mice to induce macrophage polarization and inhibit erythrocyte damage.

Type 2 diabetes triggers an inflammatory response that can damage red blood cells. M2 macrophages have inhibitory effects on inflammation, and play an important role in tissue damage repair and fibrosis. Autologous blood transfusion has the potential to inhibit red blood cell damage by mediating macrophage polarization. Swiss mice were used to establish a suitable type 2 diabetes model, and autologous blood transfusion was carried out. The mice were killed, the blood of the mice was collected and CD14+ monocytes were sorted. The expression levels of phenotypic molecules CD16, CD32 and CD206 in CD14+ monocytes were analyzed by flow cytometry. The proportion of M1 and M2 macrophages were analyzed by flow cytometry. The Q value, P50 , 2,3-diphosphoglycerate and Na+ -K+ -ATPase of red blood cells were detected. The red blood cell osmotic fragility test analyzed the red blood cell osmotic fragility. Western blot analysis was used to analyze the expression changes of erythrocyte surface membrane proteins or transporters erythrocyte membrane protein band 4.1, sphingosine-1-phosphate, glycolipid transfer protein and signal peptide peptidase-like 2A. Autologous blood transfusion induced a significant increase in the number of macrophages. The state and capacity of blood cells improved with autologous blood transfusion. Reinfusion of fresh autologous blood in type 2 diabetes mice made erythrocytes shrink. The expression of erythrocyte-related proteins proved that the erythrocyte injury in the reinfusion of fresh autologous blood + type 2 diabetes group was significantly reduced. The reinfusion of fresh autologous blood into the body of patients with type 2 diabetes can induce macrophage polarization to M2, thereby inhibiting red blood cell damage.

Identifying therapeutic antibacterial peptides against Vibrio cholerae to inhibit the function of Na(+)-translocating NADH-quinone reductase

Vibrio cholerae is the bacteria responsible for cholera, which is a significant threat to many nations. Curing and treating this infection requires identification of the critical protein and development of a drug to inhibit its function. In this context, Na(+)-translocating NADH-quinone reductase was considered a potential therapeutic target. A library of antibacterial peptides with residue lengths of 50 was screened using a docking method, and the five most potent peptides were selected on the basis of a weighted score derived from solvent accessible surface area and docking score. To investigate the stability of the protein–peptide complex, a 100-ns molecular dynamics simulation was performed. These peptides targeted the native dimeric binding interface of Na(+)-transporting NADH-quinone reductase. This study evaluated the binding affinity and conformational stability of these peptides with the protein using different post-simulation metrics. A peptide, CCL28, exhibited steady RMSD characteristics; nonetheless, it modified the docked conformation but stabilized in the new conformation. This peptide also demonstrated the best performance in addressing the protein’s native binding interface. It demonstrated a binding free energy of −120 kcal/mol with the protein. Principal component analysis (PCA) revealed that the first PC had the lowest conformational variation and the greatest coverage. Eventually, these peptides were also evaluated using steered molecular dynamics, and it was discovered that CCL28 had a greater maximum force than the other five peptides, at 1139.08 kJ/mol/nm. Targeting the native binding interface, we present a CCL28 peptide with a strong potential to block the biological activity of Vibrio cholerae’s Na(+)-translocating NADH-quinone reductase. Communicated by Ramaswamy H. Sarma

Walnut Protein Peptides Ameliorate DSS-Induced Ulcerative Colitis Damage in Mice: An in Silico Analysis and in Vivo Investigation.

Walnut (Juglans regia L.) is a food with food-medicine homology, whose derived protein peptides have been shown to have anti-inflammatory activity in vitro. However, the effects and mechanisms of walnut protein peptides on ulcerative colitis (UC) in vivo have not been systematically and thoroughly investigated. In this study, we applied virtual screening and network pharmacology screening of bioactive peptides to obtain three novel WPPs (SHTLP, HYNLN, and LGTYP) that may alleviate UC through TLR4-MAPK signaling. In vivo studies have shown that WPPs improve intestinal mucosal barrier dysfunction and reduce inflammation by inhibiting activation of the TLR4-MAPK pathway. In addition, WPPs restore intestinal microbial homeostasis by reducing harmful bacteria (Helicobacter and Bacteroides) and increasing the relative abundance of beneficial bacteria (Candidatus_Saccharimonas). Our study showed that the WPPs obtained by virtual screening were effective in ameliorating colitis, which has important implications for future screening of bioactive peptides from medicinal food homologues as drugs or dietary supplements.

Identification of a functional peptide of a probiotic bacterium-derived protein for the sustained effect on preventing colitis

ABSTRACT Several probiotic-derived factors have been identified as effectors of probiotics for exerting beneficial effects on the host. However, there is a paucity of studies to elucidate mechanisms of their functions. p40, a secretory protein, is originally isolated from a probiotic bacterium, Lactobacillus rhamnosus GG. Thus, this study aimed to apply structure-functional analysis to define the functional peptide of p40 that modulates the epigenetic program in intestinal epithelial cells for sustained prevention of colitis. In silico analysis revealed that p40 is composed of a signal peptide (1–28 residues) followed by a coiled-coil domain with uncharacterized function on the N-terminus, a linker region, and a β-sheet domain with high homology to CHAP on the C-terminus. Based on the p40 three-dimensional structure model, two recombinant p40 peptides were generated, p40N120 (28–120 residues) and p40N180 (28–180 residues) that contain first two and first three coiled coils, respectively. Compared to full-length p40 (p40F) and p40N180, p40N120 showed similar or higher effects on up-regulating expression of Setd1b (encoding a methyltransferase), promoting mono- and trimethylation of histone 3 on lysine 4 (H3K4me1/3), and enhancing Tgfb gene expression and protein production that leads to SMAD2 phosphorylation in human colonoids and a mouse colonic epithelial cell line. Furthermore, supplementation with p40F and p40N120 in early life increased H3K4me1, Tgfb expression and differentiation of regulatory T cells (Tregs) in the colon, and mitigated disruption of epithelial barrier and inflammation induced by DSS in adult mice. This study reveals the structural feature of p40 and identifies a functional peptide of p40 that could maintain intestinal homeostasis.

Proteomics analysis of an individual formalin-fixed paraffin-embedded tissue section using isobaric-tag amplification.

The comprehensive analysis of formalin-fixed paraffin-embedded (FFPE) tissues is essential for retrospective clinical studies. However, detecting low-abundance proteins and obtaining proteome-scale data from FFPE samples pose analytical challenges in mass spectrometry-based proteomics. To overcome this challenge, our study focuses on implementing an isobaric labeling approach to improve the detection of low-abundance target proteins in FFPE tissues, thereby enhancing the qualitative and quantitative analysis. We employed an isobaric labeling approach utilizing synthetic peptides or proteins to enable the qualitative and quantitative measurement of target proteins in FFPE tissue samples. To achieve this, we incorporated tandem mass tag (TMT)-labeled recombinant proteins or synthetic peptides into TMT-labeled metastatic breast cancer FFPE tissues. Through this strategy, we successfully detect coexisting CD276 (B7-H3) and CD147 proteins while identifying over 6000 proteins using targeted analysis of individual FFPE tissue sections. Our findings provide compelling evidence that the incorporation of isobaric labeling, along with the inclusion of TMT-labeled peptides or proteins, greatly enhances the detection of target proteins in FFPE tissue samples. By employing this approach, we were able to obtain robust qualitative measurements of CD276 and CD147 proteins, showcasing its effectiveness in identifying more than 6000 proteins in FFPE samples. The integration of an isobaric labeling approach, in conjunction with synthetic peptides or proteins, presents a valuable strategy for enhancing the detection and validation of target proteins in FFPE tissue analysis. This technique holds immense potential in retrospective clinical studies, as it enables comprehensive analysis of low-abundance proteins and facilitating proteome-scale investigations in FFPE samples. By leveraging this methodology, researchers can unlock new insights into disease mechanisms and advance our understanding of complex biological processes.

THU396 Digital Spatial Profiling Of Human Parathyroid Tumors Reveals Distinct Cellular And Molecular Alterations Linked To Vitamin D Deficiency

Abstract Disclosure: C. Tu: None. W. Chang: None. J.A. Sosa: Advisory Board Member; Self; Novo Nordisk, AstraZeneca, Eli Lilly & Company. Grant Recipient; Self; Exelixis, Inc., Eli Lilly & Company. J. Koh: None. Primary hyperparathyroidism (PHPT) is a common endocrine neoplastic disorder characterized by disrupted calcium homeostasis secondary to inappropriately elevated parathyroid hormone (PTH) secretion. Low levels of serum 25-hydroxyvitamin D (25OHD) are significantly more prevalent in PHPT patients than in the general population, but the pathophysiological basis for this association remains unclear. We employed a spatially defined in situ whole-transcriptomics and selective proteomics profiling approach to compare gene expression patterns and cellular composition in parathyroid adenomas from 9 vitamin D-deficient or 34 vitamin D-replete PHPT patients with 12 eucalcemic cadaveric donor parathyroid glands as normal tissue controls. Parathyroid oxyphil cell content is markedly higher in tumors from vitamin D-deficient PHPT patients (Def-Ts) relative to tumors from vitamin D-replete PHPT patients (Rep-Ts) (47.8% vs 17.8%, respectively; p <0.0001) and normal donor parathyroid glands (7.7%). Def-Ts display increased expression of genes encoding electron transport chain (corr. coefficient=0.546), proteasomal catabolism (corr. coefficient=0.587), and oxidative phosphorylation (corr. coefficient=0.982) pathway components. In contrast, Rep-Ts are characterized by upregulation of the ras (corr. coefficient=0.632) and myc (corr. coefficient=0.811) signaling pathways, suggestive of an oncogene-driven etiology. Parathyroid oxyphil cells are comparable to chief cells at the transcriptional level, and vitamin D-status is reflected in the transcriptional profiles of both cell types in a similar manner. Among PHPT patients who did not present as vitamin D deficient, three distinct tumor transcriptional profile clusters showed a dose-dependent relationship to pre-operative 25OHD levels (p <0.0001 by ANOVA). Relative to normal tissue, the abundance of calcium sensing receptor (CaSR) protein is reduced in Def-Ts (mean protein count 253.6 vs 492.3, respectively; p=0.039 for difference), while the type B γ-aminobutyric acid receptor 1 (GABAB1R) is increased (mean protein count 114.5 vs 64.65, respectively; p=0.0099 for difference). These expression changes resulted in a vitamin D dose-dependent reduction in the ratio of CaSR to GABAB1R receptors in Def-T tumor cells (r2 = 0.218, p<0.0001), favoring the formation of calcium sensing-attenuated CaSR:GABAB1R heteromers. The abundance of the amyloid precursor protein (APP) peptide derivative Aβ1-42, a putative GABAB1R ligand, is preferentially increased in Def-Ts compared to normal tissue (mean protein count 5797 vs 4500, respectively; p=0.0318). Collectively, these findings suggest a novel mechanism where chronic vitamin D deficiency can contribute to tonic PTH hypersecretion in PHPT patients by increasing GABAB1R-mediated antagonism of CaSR activity in parathyroid adenomas. Presentation: Thursday, June 15, 2023