Serum Components Research Articles

Abalone Viscera Fermented with Aspergillus oryzae 001 Prevents Pressure Elevation by Inhibiting Angiotensin Converting Enzyme.

Abalone viscera, which accounts for more than 20% of the total weight of abalone, is generally regarded as waste in the food industry, and effective methods are required to utilize it productively. In this study, the viscera were fermented with Aspergillus oryzae 001 to add functionality. Fermented abalone viscera exhibited increased angiotensin I-converting enzyme (ACE) inhibitory activity and enhanced inhibition of blood pressure elevation in spontaneously hypertensive rats (SHRs). Abalone viscera administration had no significant effect on body weight, food intake, liver and kidney weights, or serum components in SHRs. ACE inhibitors specific to fermented abalone viscera were identified through extraction, fractionation, purification, and analysis. The identified substance was L-m-tyrosine, which non-competitively inhibited ACE and, in a single oral administration, significantly reduced blood pressure in SHRs compared to that in the control. This study identified that abalone viscera fermented by A. oryzae 001 has an inhibitory effect on blood pressure elevation, suggesting its potential use as a functional food. In addition, L-m-tyrosine, a unique substance in fermented abalone viscera, was isolated for the first time as a single ACE-inhibitory amino acid.

Effect of PEG Anchor and Serum on Lipid Nanoparticles: Development of a Nanoparticles Tracking Method.

Polyethylene glycol (PEG) is used in Lipid Nanoparticles (LNPs) formulations to confer stealth properties and is traditionally anchored in membranes by a lipid moiety whose length significantly impacts the LNPs fate in vivo. C18 acyl chains are efficiently anchored in the membrane, while shorter C14 lipids are quickly desorbed and replaced by a protein corona responsible for the completely different fate of LNPs. In this context, a method to predict the biological behavior of LNPs depending on the lipid-PEG dissociation was developed using the Nanoparticle Tracking Analysis (NTA) method in serum. Two formulations of siRNA-containing LNPs were prepared including CSL3 or SM-102 lipids and were grafted with different lipids-PEG (C18, C14 lipids-PEG, and Ceramide-PEG). The impact of the lipid-PEG on the interactions between LNPs and serum components was demonstrated by monitoring the mean particle size and the concentration over time. In vitro, these formulations demonstrated low toxicity and efficient gene knockdown on tumor MDA-MB-231 cells, but serum was found to significantly impact the efficiency of C18-PEG-based LNPs, while it did not impact the efficiency of C14-PEG-based LNPs. The NTA method demonstrated the ability to discriminate between the behaviors of LNPs according to serum proteins' interactions. CSL3 lipid and Cer-PEG were confirmed to have promise for LNP formulation.

In vitro culture of bovine fibroblasts using select serum-free media supplemented with Chlorella vulgaris extract

Standard cell culture practices require the addition of animal-derived serum to culture media to achieve adequate cell growth. Typically, 5–10% by volume of fetal bovine serum (FBS) is used, which accounts for a vast majority of the media cost while also imposing environmental and ethical concerns associated with the use of animal serum. Here we tested the efficacy of culturing cells by replacing serum in the media with algae extract and select additives. Using LC–MS, we compared molecular signatures of FBS to Chlorella algae extracts and identified NAD(H)/NADP(H) as common and relatively abundant features in their characteristic profiles. Bovine fibroblasts, cultured in serum-free media supplemented with C. vulgaris extract and just two growth factors plus insulin, showed significant growth with enhanced viability compared to control cells cultured without serum, albeit still lower than that of controls cultured with 10% FBS. Moreover, C. vulgaris extract enhanced cell viability beyond that of cells cultured with the two growth factors and insulin alone. These results suggest that key components in serum which are essential for cell growth may also be present in C. vulgaris extract, demonstrating that it may be used at least as a partial alternative to serum for cell culture applications.

RETRACTED: Effects of the application of a food processing-based classification system in obese women: A randomized controlled pilot study.

Background: Obesity is one of the major public health problems worldwide and contributes to the onset of many diseases, especially the ones related to the metabolic syndrome. The new Dietary Guidelines for the Brazilian population bring a new food classification based on food processing and prioritizes the consumption of fresh or minimally processed foods. Aim: This study analyzed the effects of an educational intervention on obese women, on their weight loss, quality of life, components of the metabolic syndrome and pain. Methods: Randomized controlled pilot study, including 40 obese women, randomized into 2 groups: control group and intervention group. An educational intervention with 5 biweekly meetings of 90 min based on the Dietary Guidelines for the Brazilian population (2014) was carried out involving the intervention group. Parameters related to weight loss, quality of life (SF-36), pain (McGill), bioelectrical impedance analysis, cardiorespiratory fitness, and serum and clinical components of metabolic syndrome, as well as serum concentrations of cytokines were assessed. Results: Significantly decrease of body mass, waist and hip circumferences, basal metabolic rate, extracellular water, body capacitance, and body cell mass were observed in the intervention group after 3 months. Reduction of pain and improvement in quality of life and cardiorespiratory fitness were also observed in the intervention group. There were reductions in waist circumference and glycemia, components of metabolic syndrome. Conclusions: This study showed that the educational intervention can be associated with weight loss, increase in quality of life, reduction of pain, and better metabolic syndrome parameters in obese women.

Cysteinyl leukotriene receptor 1 is a potent regulator of the endosomal-lysosomal system in the ARPE-19 retinal pigment epithelial cell line.

The endosomal-lysosomal system is central for cell homeostasis and comprises the functions and dynamics of particular organelles including endosomes, lysosomes and autophagosomes. In previous studies, we found that the cysteinyl leukotriene receptor 1 (CysLTR1) regulates autophagy in the retinal pigment epithelial cell line ARPE-19 under basal cellular conditions. However, the underlying mechanism by which CysLTR1 regulates autophagy is unknown. Thus, in the present study, the effects of CysLTR1 inhibition on the endosomal-lysosomal system are analyzed in detail to identify the role of CysLTR1 in cell homeostasis and autophagy regulation. CysLTR1 inhibition in ARPE-19 cells by Zafirlukast, a CysLTR1 antagonist, depleted the lysosomal pool. Furthermore, CysLTR1 antagonization reduced endocytic capacity and internalization of epidermal growth factor and decreased levels of the transferrin receptor, CD71. Serum starvation abolished the effect of Zafirlukast on the autophagic flux, which identifies the endocytic regulation of serum components by CysLTR1 as an important autophagy-modulating mechanism. The role of CysLTR1 in inflammation and cell stress has been exceedingly studied, but its involvement in the endosomal-lysosomal pathway is largely unknown. This current study provides new insights into basal activity of CysLTR1 on cellular endocytosis and the subsequent impact on downstream processes like autophagy.

Quince extract resists atherosclerosis in rats by down-regulating the EGFR/PI3K/Akt/GSK-3β pathway

We identified the effective components and the underlying mechanisms of Quince (Cydonia oblonga Mill, COM) extract against atherosclerosis. The effective components of COM extract were identified with UHPLC-Q-TOF-MS/MS. Network pharmacology was performed. A rat model of atherosclerosis induced by high-fat emulsion combined with vitamin D3 was established. The anti-atherosclerosis effect of COM extract was evaluated from various aspects such as blood lipid regulation, anti-oxidative stress, anti-inflammatory response, and vascular protection function. We identified 14 serum components of COM extract using UHPLC-Q-TOF-MS/MS. Through prediction, 573 targets were obtained, among which 224 targets were atherosclerosis specific targets. The key targets included GSK3β, ESR1, EGFR, and HSP90AA1. The key signaling pathway was PI3K-Akt signaling pathway. Pharmacodynamics analysis showed that COM extract reduced the levels of TC, TG, and LDL-C as well as ALT and AST, while increased the level of HDL-C. Mechanistically, COM extract significantly increased serum SOD and GSH-Px activities, but decreased MDA content in atherosclerosis rats, showing antioxidant effects. Meanwhile, COM extract significantly down-regulated the levels of pro-inflammatory factors IL-1β, IL-6, TNF-α and CRP, but up-regulated anti-inflammatory factor IL-10. Additionally, COM extract increased the levels of NO, eNOS, and 6-keto-PGF1α; whereas, decreased the levels of ET-1 and TXB2. Furthermore, COM extract significantly inhibited the mRNA and protein levels of EGFR, p-PI3K, p-AKT, GSK-3β, Bax, and Caspase-3 as well as the Bax/Bcl-2 ratio. Conclusively, COM extract exerts hypolipidemic, anti-oxidative, anti-inflammatory, anti-thrombotic and vascular endothelium protective effects on atherosclerosis rat model, which may be related to the inhibition of EGFR/PI3K/AKT/GSK-3β signaling pathway.

Human Serum Mediated Bacteriophage Life Cycle Switch in Aggregatibacter actinomycetemcomitans Is Linked to Pyruvate Dehydrogenase Complex.

Antimicrobial resistance is rising as a major global public health threat and antibiotic resistance genes are widely spread among species, including human oral pathogens, e.g., Aggregatibacter actinomycetemcomitans. This Gram-negative, capnophilic, facultative anaerobe is well recognized as a causative agent leading to periodontal diseases, as well as seriously systemic infections including endocarditis. A. actinomycetemcomitans has also evolved mechanisms against complement-mediated phagocytosis and resiliently survives in serum-rich in vivo environments, i.e., inflamed periodontal pockets and blood circulations. This bacterium, however, demonstrated increasing sensitivity to human serum, when being infected by a pseudolysogenic bacteriophage S1249, which switched to the lytic state as a response to human serum. Concomitantly, the pyruvate dehydrogenase complex (PDHc), which is composed of multiple copies of three enzymes (E1, E2, and E3) and oxidatively decarboxylates pyruvate to acetyl-CoA available for tricarboxylic acid (TCA) cycle, was found up-regulated 10-fold in the bacterial lysogen after human serum exposure. The data clearly indicated that certain human serum components induced phage virion replication and egress, resulting in bacterial lysis. Phage manipulation of bacterial ATP production through regulation of PDHc, a gatekeeper linking glycolysis to TCA cycle through aerobic respiration, suggests that a more efficient energy production and delivery system is required for phage progeny replication and release in this in vivo environment. Insights into bacteriophage regulation of bacterial fitness in a mimic in vivo condition will provide alternative strategies to control bacterial infection, in addition to antibiotics.

Material basis and mechanism of Curcuma longa tuberous roots with and without vinegar processing in treating primary dysmenorrhea

Liquid chromatography-mass spectrometry was employed to analyze the chemical components in Curcuma longa tuberous roots(HSYJ), C. longa tuberous roots processed with vinegar(CHSYJ), and rat serum after the administration. The active components of HSYJ and CHSYJ absorbed in serum were identified based on the secondary spectrum of database and literature. The targets of primary dysmenorrhea was screened out from database. The protein-protein interaction network analysis, gene ontology(GO) functional annotation, and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis were performed for the common targets shared by the drug active components in serum and primary dysmenorrhea, and the component-target-pathway network was constructed. AutoDock was used to conduct molecular docking between the core components and targets. A total of 44 chemical components were identified from HSYJ and CHSYJ, including 18 absorbed in serum. On the basis of network pharmacology, we identified 8 core components(including procurcumenol, isobutyl p-hydroxybenzoate, ferulic acid, and zedoarondiol) and 10 core targets \[including interleukin-6(IL-6), estrogen receptor 1(ESR1), and prostaglandin-endoperoxide synthase 2(PTGS2)\]. The core targets were mainly distributed in the heart, liver, uterus, and smooth muscle. The molecular docking results showed that the core components were well bound to the core targets, indicating that HSYJ and CHSYJ may exert therapeutic effect on primary dysmenorrhea via estrogen, ovarian steroidogenesis, tumor necrosis factor(TNF), hypoxia-inducible factor-1(HIF-1), IL-17 and other signaling pathways. This study clarifies the HSYJ and CHSYJ components absorbed in serum, as well as the corresponding mechanism, providing a reference for further elucidating the therapeutic material basis and clinical application of HSYJ and CHSYJ.

Effect of increasing valine level in the diet on performance, egg quality, and serum components in laying quails.

This research was conducted to determine the effect of increasing dietary valine level on performance parameters, egg quality criteria, and blood and serum components. In the study, 120 female Japanese quails at the age of 10weeks (241.4 ± 8.6g) did randomly allocate to six experimental groups contained five subgroups. Treatment diets were prepared to contain 0.93% (control), 1.00%, 1.10%, 1.20%, 1.30%, or 1.40% valine. The egg mass decreased linearly with the increasing level of valine in the diet, while egg weight decreased in both linear and quadratic manner (P < 0.05). Increased levels of dietary valine increased eggshell breaking strength in cubic manner (P < 0.05). The administration of the valine to the diet caused a linear decrease in the albumin index and Haugh unit (P < 0.05). Other egg quality parameters were not affected (P > 0.05). Serum glucose and triglyceride concentrations of laying quails were reduced by the increasing dietary valine levels; the effect on serum glucose was linear, while both linear and quadratic effects were significant on triglyceride concentration (P < 0.05). White blood cell, lymphocyte, and neutrophil counts increased with the increasing dietary levels of valine (P < 0.05). Both linear and quadratic effects were significant on white blood cells and lymphocytes, while the effect on neutrophil counts was linear. However, erythrocyte and haematocrit (P < 0.05) decreased quadratically with increment in the dietary levels of valine. In conclusion, the increased valine levels in the diet adversely affected the egg weight, egg mass, and Haugh unit. but it reduced the serum triglyceride and glucose. Results may suggest that dietary valine at 0.93% level is sufficient for egg production and egg quality in laying quails, but positive effects of increased dietary valine levels on white blood cells may worth to investigate further.

Determination of endogenous sphingolipid content in stroke rats and HT22 cells subjected to oxygen-glucose deprivation by LC‒MS/MS

BackgroundStroke is the leading cause of death in humans worldwide, and its incidence increases every year. It is well documented that lipids are closely related to stroke. Analyzing the changes in lipid content in the stroke model after absolute quantification and investigating whether changes in lipid content can predict stroke severity provides a basis for the combination of clinical stroke and quantitative lipid indicators.MethodsThis paper establishes a rapid, sensitive, and reliable LC‒MS/MS analytical method for the detection of endogenous sphingolipids in rat serum and brain tissue and HT22 cells and quantifies the changes in sphingolipid content in the serum and brain tissue of rats from the normal and pMCAO groups and in cells from the normal and OGD/R groups. Using sphingosine (d17:1) as the internal standard, a chloroform: methanol (9:1) mixed system was used for protein precipitation and lipid extraction, followed by analysis by reversed-phase liquid chromatography coupled to triple quadrupole mass spectrometry.ResultsBased on absolute quantitative analysis of lipids in multiple biological samples, our results show that compared with those in the normal group, the contents of sphinganine (d16:0), sphinganine (d18:0), and phytosphingosine were significantly increased in the model group, except sphingosine-1-phosphate, which was decreased in various biological samples. The levels of each sphingolipid component in serum fluctuate with time.ConclusionThis isotope-free and derivatization-free LC‒MS/MS method can achieve absolute quantification of sphingolipids in biological samples, which may also help identify lipid biomarkers of cerebral ischemia.Graphical

Negative interference with antibody-dependent cellular cytotoxicity mediated by rituximab from its interactions with human serum proteins.

Although interactions of small molecular drugs with serum proteins have been widely studied from pharmacokinetic and pharmacodynamic perspectives, there have been few reports on the effects of serum components on therapeutic antibody functions. This study reports the effect of abundant serum proteins on antibody-dependent cellular cytotoxicity (ADCC) mediated by rituximab and Fcγ receptor III (FcγRIII). Human serum albumin (HSA) and the Fab fragment from the pooled serum polyclonal IgG were found to compromise ADCC as non-competitive inhibitors. Our nuclear magnetic resonance data provided direct evidence for the interactions of HSA with both the Fab and Fc regions of rituximab and also with the extracellular region of FcγRIII (sFcγRIII). The degree of involvement in the interaction decreased in the order of rituximab-Fab > rituximab-Fc > sFcγRIII, suggesting preferential binding of HSA to net positively charged proteins. Although much less pronounced than the effect of HSA, polyclonal IgG-Fab specifically interacted with rituximab-Fc. The NMR data also showed that the serum protein interactions cover the Fc surface extensively, suggesting that they can act as pan-inhibitors against various Fc receptor-mediated functions and pharmacokinetics. Our findings highlight the importance of considering serum-protein interactions in the design and application of antibody-based drugs with increased efficacy and safety.

Fertility-associated biochemical components in seminal plasma and serum of buffalo (Bubalus bubalis) bulls.

The present study looks for components in seminal plasma (SP) and/or serum that are closely related to in vivo fertility of buffalo bulls. Fourteen healthy mature buffalo bulls were classified according to their in vivo fertility into fertile (n = 10) and subfertile (n = 4) groups. Semen and serum samples were collected from all animals for 12 replicates. The collected ejaculates were examined for sperm characteristics before being centrifuged to collect SP for hormonal (FSH, LH, testosterone, and IGF-1), biochemical [total antioxidant capacity (TAC), catalase (CAT), glutathione peroxidase (GPx), nitric oxide (NO), malondialdehyde (MDA), fructose, total protein, albumin, triglycerides, cholesterol, and high-density lipoprotein (HDL)] and proteomic (SDS-PAGE) analyses. Likewise, serum levels of FSH, LH, testosterone, IGF-1, glucose, total protein, albumin, triglycerides, cholesterol, and HDL were determined. All sperm characteristics and the majority of sperm kinematics were (P < 0.01) different between fertile and subfertile groups. Seminal and serum levels of FSH, LH, testosterone, and IGF-1 were higher (P < 0.01) in the fertile group, but only seminal fructose, total protein, albumin, triglycerides, cholesterol, and HDL were higher (P < 0.01) in the fertile group. Moreover, the fertile group had greater TAC, CAT, GPx, and NO, but the subfertile group had greater MDA. Protein bands of 14, 15, 26, 30, and 55 kDa were larger and denser in the SP of the fertile group but were smaller and faint to absent in that of the subfertile group. Also, the protein fractions of detected protein bands demonstrated a substantial influence of fertility on those of 16, 26, 30, and 55 kDa. In conclusion, sperm characteristics and kinematics with serum, and/or seminal hormonal and biochemical components, should be evaluated for reliable prediction of buffalo bull fertility. Furthermore, protein bands of 26, 30, and 55 kDa may represent fertility-associated proteins in buffalo bull SP.

Single-Particle Functionality Imaging of Antibody-Conjugated Nanoparticles in Complex Media.

The properties of nanoparticles (NPs) can change upon contact with serum components, occluding the NP surface by forming a biomolecular corona. It is believed that targeted NPs can lose their functionality due to this biological coating, thus losing specificity and selectivity toward target cells and leading to poor therapeutic efficiency. A better understanding of how the biomolecular corona affects NP ligand functionality is needed to maintain NP targeting capabilities. However, techniques that can quantify the functionality of NPs at a single-particle level in a complex medium are limited and often laborious in sample preparation, measurement, and analysis. In this work, the influence of serum exposure on the functionality of antibody-functionalized NPs was quantified using a straightforward total internal reflection fluorescence (TIRF) microscopy method and evaluated in cell uptake studies. The single-particle resolution of TIRF reveals the interparticle functionality heterogeneity and the substantial differences between NPs conjugated with covalent and noncovalent methods. Notably, only NPs covalently conjugated with a relatively high amount of antibodies maintain their functionality to a certain extent and still showed cell specificity and selectivity toward high receptor density cells after incubation in full serum. The presented study emphasizes the importance of single-particle functional characterization of NPs in complex media, contributing to the understanding and design of targeted NPs that retain their cell specificity and selectivity in biologically relevant conditions.

Assessment of anti-inflammatory bioactivity of extracellular vesicles is susceptible to error via media component contamination

Extracellular vesicles (EVs) are widely implicated as novel diagnostic and therapeutic modalities for a wide range of diseases. Thus, optimization of EV biomanufacturing is of high interest. In the course of developing parameters for a human embryonic kidney cells (HEK293T) EV production platform, we examined the combinatorial effects of cell culture conditions (i.e., static versus dynamic) and isolation techniques (i.e., ultracentrifugation versus tangential flow filtration versus size-exclusion chromatography) on functional characteristics of HEK293T EVs, including anti-inflammatory bioactivity using a well-established lipopolysaccharide-stimulated mouse macrophage model. We unexpectedly found that, depending on culture condition and isolation strategy, HEK293T EVs appeared to significantly suppress the secretion of pro-inflammatory cytokines (i.e., interleukin-6, RANTES [regulated upon activation, normal T cell expressed and secreted]) in the stimulated mouse macrophages. Further examination revealed that these results were most likely due to non-EV fetal bovine serum components in HEK293T EV preparations. Thus, future research assessing the anti-inflammatory effects of EVs should be designed to account for this phenomenon.

Removal of hemolysis interference in serum Raman spectroscopy by multivariate curve resolution analysis for accurate classification of oral cancers

Removing hemolysis components in serum by MCR-ALS assisted Raman spectroscopy. Disease specific information in serum preserved after MCR-ALS analysis. Digital removal of undesirable components in Raman spectra improves oral cancer diagnosis.

Effective substances and mechanism of Yishen Guluo Mixture in treatment of chronic glomerulonephritis based on metabolomics and serum pharmacochemistry

This study aimed to investigate the effective substances and mechanism of Yishen Guluo Mixture in the treatment of chronic glomerulonephritis(CGN) based on metabolomics and serum pharmacochemistry. The rat model of CGN was induced by cationic bovine serum albumin(C-BSA). After intragastric administration of Yishen Guluo Mixture, the biochemical indexes related to renal function(24-hour urinary protein, serum urea nitrogen, and creatinine) were determined, and the efficacy evaluations such as histopathological observation were carried out. The serum biomarkers of Yishen Guluo Mixture in the treatment of CGN were screened out by ultra-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry(UPLC-Q-TOF-MS) combined with multivariate statistical analysis, and the metabolic pathways were analyzed. According to the mass spectrum ion fragment information and metabolic pathway, the components absorbed into the blood(prototypes and metabolites) from Yishen Guluo Mixture were identified and analyzed by using PeakView 1.2 and MetabolitePilot 2.0.4. By integrating metabolomics and serum pharmacochemistry data, a mathematical model of correlation analysis between serum biomarkers and components absorbed into blood was constructed to screen out the potential effective substances of Yishen Guluo Mixture in the treatment of CGN. Yishen Guluo mixture significantly decreased the levels of 24-hour urinary protein, serum urea nitrogen, and creatinine in rats with CGN, and improved the pathological damage of the kidney tissue. Twenty serum biomarkers of Yishen Guluo Mixture in the treatment of CGN, such as arachidonic acid and lysophosphatidylcholine, were screened out, involving arachidonic acid metabolism, glycerol phosphatide metabolism, and other pathways. Based on the serum pharmacochemistry, 8 prototype components and 20 metabolites in the serum-containing Yishen Guluo Mixture were identified. According to the metabolomics and correlation analysis of serum pharmacochemistry, 12 compounds such as genistein absorbed into the blood from Yishen Guluo Mixture were selected as the potential effective substances for the treatment of CGN. Based on metabolomics and serum pharmacochemistry, the effective substances and mechanism of Yishen Guluo Mixture in the treatment of CGN are analyzed and explained in this study, which provides a new idea for the development of innovative traditional Chinese medicine for the treatment of CGN.

Evaluation of Raman spectroscopy combined with the gated recurrent unit serum detection method in early screening of gastrointestinal cancer.

Gastric and colorectal cancers are significant causes of human mortality. Conventionally, the diagnosis of gastrointestinal tumors has been accomplished through image-based techniques, including endoscopic and biopsy procedures coupled with tissue staining. Most of these methods are invasive. In contrast, Raman spectroscopy has the advantages of being non-invasive and label-free and requiring no additional reagents, making it a potential tool for the detection of serum components. In this study, we collected Raman spectra of serum samples from patients with gastric cancer (n = 93) and colorectal cancer (n = 92) and from healthy individuals (n = 100). Analysis of Raman peak areas revealed that cancer patients had significantly higher peak areas at around 2923 cm-1 compared to normal individuals, which corresponded to the presence of lipids and proteins. We successfully achieved the early screening of gastrointestinal tumors using the improved gated recurrent unit (GRU) algorithm and traditional machine learning methods. The accuracy of identifying digestive tract tumors using different recognition models exceeds 84.72%, with support vector machine (SVM) and GRU achieving 100% accuracy. The use of GRU further demonstrated its ability to differentiate subtypes of gastric and colorectal cancers based on the degree of differentiation and stage, with a recognition accuracy exceeding 95%, which is challenging using traditional machine learning methods. Furthermore, our study revealed that principal component analysis (PCA) dimensionality reduction has a limited impact on the recognition results obtained using different recognition models.

Analysis of Human and Mouse Neutrophil Phagocytosis by Flow Cytometry.

Neutrophils are primary phagocytes that recognize their targets through surface chemistry, either through pattern recognition receptor (PPR) interaction with pathogen-associated molecular patterns (PAMPs) or through immunoglobulin (Ig) or complement mediated recognition. Opsonization can be important for target recognition and phagocytosis by neutrophils. Therefore, phagocytosis assays performed using neutrophils in whole blood, compared to isolated cells, will differ due to the presence of opsonizing blood serum components, as well as other blood components like platelets. Powerful and sensitive flow cytometry-based methods are presented to measure phagocytosis by human blood neutrophils and mouse peritoneal neutrophils.

Exploring the role of Tibetan medicinal formula Qishiwei Zhenzhu Pills (Ranasampel) against diabetes mellitus-linked cognitive impairment of db/db mice through serum pharmacochemistry and microarray data analysis.

Diabetes cognitive impairment (DCI) is a common diabetic central nervous system disorder that severely affects the quality of life of patients. Qishiwei Zhenzhu Pills (Ranasampel) is a valuable Tibetan medicine formula with the ability to improve cerebral blood vessels, protect nerves and improve learning and memory, which has also been widely verified in clinical and basic research. Currently, the prevention and treatment of DCI are still in the exploratory research stage, and the use of Ranasampel will provide new ideas and insights for its treatment. This study is to explore the absorbed components in serum derived from Ranasampel using serum pharmacochemistry, then identify the potential mechanism of Ranasampel for the treatment of DCI through bioinformatics and microarray data validation. The UPLC-Q-Exactive MS/MS-based serum pharmacochemistry method was conducted to identify the main active components in serum containing Ranasampel. Then, these components were used to predict the possible biological targets of Ranasampel and explore the potential targets in treating DCI by overlapping with differentially expressed genes (DEGs) screened from Gene Expression Omnibus datasets. Afterward, the protein-protein interaction network, enrichment analyses, hub gene identification, and co-expression analysis were used to study the potential mechanism of Ranasampel. Particularly, the hub genes and co-expression transcription factors were further validated using hippocampal expression profiles of db/db mice treated with Ranasampel, while the Morris water-maze test and H&E staining were used to assess the spatial learning and memory behaviors and histopathological changes. Totally, 40 compounds derived from Ranasampel had been identified by serum sample analysis, and 477 genes related to these identified compounds in Ranasampel, 110 overlapping genes were collected by the intersection of Ranasampel target genes and DEGs. Further comprehensive analysis and verification emphasized that the mechanism of Ranasampel treatment of DCI may be related to the improvement of learning and memory function as well as insulin resistance, hyperglycemia-induced neuronal damage, and neuroinflammation. This study provided useful strategies to explore the potential material basis for compound prescriptions such as Ranasampel. These hub genes and common pathways also provided new ideas for further study of therapeutic targets of DCI and the pharmacological mechanism of Ranasampel.

Low molecular weight cytotoxic components (DAMPs) form the post-COVID-19 syndrome

We studied the role of cytotoxic components (DAMPs) formed in the body of patients with COVID-19 in ensuring the long-term preservation of post-COVID-19 manifestations and the possibility of creating an experimental model by transferring DAMPs to rats. In patients with post-COVID-19 syndrome (PCS) 2 months after SARS-CoV-2 infection we determined the presence of cytotoxic components in the blood serum (Terasaki test, Dunaliella viridis test and content of DAMPs). In post-COVID-19 syndrome patients with a high content of serum cytotoxic oligopeptide fraction (selective group, n = 16) we determined the number of leukocytes, lymphocytes, neutrophil granulocytes and monocytes in the blood, the content of C-reactive protein (CRP), the concentration of C3 and C4 complement components and circulating immune complexes, the serum content of IL-6, IL −10, IL-18, TNF-α, phagocytic activity of neutrophils, presence of neutrophil traps and autoantibodies ANA.It has been shown that in patients with PCS, there are components with cytotoxicity in the blood serum, form specific immunopathological patterns, which are characterized by: an increased content of CRP, complement system components C3 and C4 and cytokines (TNF-α, IL-6, IL-10, IL-18) activation, the formation of a wide range of autoantibodies ANA, the low efficiency of endocytosis in oxygen-independent phagocytosis; their phagocytic activity reaches its functional limit, and against this background, activation of neutrophil traps occurs, which can contribute to further induction of DAMPs. This self-sustaining cell-killing activation provided long-term preservation of PCS symptoms.The transfer of blood serum components from selective group patients with PCS to rats was accompanied by the appearance of cytotoxic components in them which induced sensitization and immunopathological reactions. Preventive administration of a biologically active substance with polyfunctional properties MF to experimental animals “corrected” the initial functional state of the body's immune-metabolic system and eliminated or facilitated immuno-inflammatory reactions.