Serum Components Research Articles

Host type 2 immune response to xenogeneic serum components impairs biomaterial-directed osteo-regenerative therapies

The transformative potential of cells as therapeutic agents is being realized in a wide range of applications, from regenerative medicine to cancer therapy to autoimmune disorders. The majority of these therapies require ex vivo expansion of the cellular product, often utilizing fetal bovine serum (FBS) in the culture media. However, the impact of residual FBS on immune responses to cell therapies and the resulting cell therapy outcomes remains unclear. Here, we show that hydrogel-delivered FBS elicits a robust type 2 immune response characterized by infiltration of eosinophils and CD4+ T cells. Host secretion of cytokines associated with type 2 immunity, including IL-4, IL-5, and IL-13, is also increased in FBS-containing hydrogels. We demonstrate that the immune response to xenogeneic serum components dominates the local environment and masks the immunomodulatory effects of biomaterial-delivered mesenchymal stromal/stem cells. Importantly, delivery of relatively small amounts of FBS (3.2% by volume) within BMP-2-containing biomaterial constructs dramatically reduces the ability of these constructs to promote de novo bone formation in a radial defect model in immunocompetent mice. These results urge caution when interpreting the immunological and tissue repair outcomes in immunocompetent pre-clinical models from cells and biomaterial constructs that have come in contact with xenogeneic serum components.

Uncovering conditional vulnerabilities in human cancer cells

Forward genetic screens based on either RNAi or CRISPR make it possible to systematically identify genes required for the survival and growth of mammalian cells, and thus offer a powerful approach to characterize protein function and identify potential cancer vulnerabilities. Nonetheless, fitness genes can be context‐dependent, and indeed, CRISPR screens across hundreds of human cell lines have revealed that genetic dependencies can vary with natural cell‐intrinsic diversity. However, while the impact of environmental factors on cell physiology has become better appreciated, there has been little investigation into how nutrient availability affects gene essentiality and, further, most forward genetic screens are performed in vitro using culture media with little relevance to human physiology. We previously developed Human Plasma‐Like Medium (HPLM) – the first systematically designed physiologic medium, which contains over 60 components at concentrations that reflect their average values in human blood. To test the hypothesis that medium composition influences gene essentiality, we performed CRISPR‐based loss‐of‐function screens of blood cancer lines HPLM versus traditional media. Analysis of these data revealed that sets of conditionally essential genes are involved in several biological processes and can vary with both cell‐intrinsic diversity and the combination of basal and serum components that comprise typical media. Moreover, our approach allows for the downstream identification of gene‐nutrient interactions that underlie conditional CRISPR phenotypes, which can in turn uncover conditionally essential protein roles.Additionally, most preclinical therapies fail to achieve translation – a major problem attributed in part to the limited physiologic relevance of existing models. Nonetheless, there has been similarly little systematic investigation into how the composition of culture media might affect drug efficacy. Further, virtually all drug screens, initial hit validation efforts, and studies of drug activity are performed in vitro using traditional media as well. Therefore, we with collaborators at NCATS used a library of approved and (pre)‐clinical drugs to screen blood cancer lines in HPLM versus conventional media. Remarkably, we identified many whose killing activity varied with medium composition, including drugs approved for non‐oncology indications. Indeed, neither the drug‐nutrient interaction nor the mechanism of drug activity in cancer cells is immediately apparent in many such cases.

Serum Protein Exposure Activates a Core Regulatory Program Driving Human Proximal Tubule Injury.

The kidneys efficiently filter waste products while retaining serum proteins in the circulation. However, numerous diseases compromise this barrier function, resulting in spillage of serum proteins into the urine (proteinuria). Some studies of glomerular filtration suggest that tubules may be physiologically exposed to nephrotic-range protein levels. Therefore, whether serum components can directly injure the downstream tubular portions of the kidney, which in turn can lead to inflammation and fibrosis, remains controversial. We tested the effects of serum protein exposure in human kidney tubule microphysiologic systems and with orthogonal epigenomic approaches since animal models cannot directly assess the effect of serum components on tubules. Serum, but not its major protein component albumin, induced tubular injury and secretion of proinflammatory cytokines. Epigenomic comparison of serum-injured tubules and intact kidney tissue revealed canonical stress-inducible regulation of injury-induced genes. Concordant transcriptional changes in microdissected tubulointerstitium were also observed in an independent cohort of patients with proteinuric kidney disease. Our results demonstrate a causal role for serum proteins in tubular injury and identify regulatory mechanisms and novel pathways for intervention.

Antimicrobial activity of serum of cystic fibrosis patients before and after lung transplantation

Cystic fibrosis (CF) is a systemic congenital disease characterized by chronic bronchopulmonary infection of bacterial and fungal origins. Numerous antimicrobial peptides (AMP) play a special role in humoral immunity in CF patients. Each AMP was characterized in detail earlier. A new method has been developed recently in the Russian Federation for estimation of the cumulative antimicrobial activity of all AMPs in different bioliquids.The aim of the study was the application of this method for the estimation of antimicrobial activity of whole sera and the AMP-containing low molecular serum fractions in patients with CF before and after lung transplantation.Methods. Sera from 11 CF patients of 24 – 33 years old obtained before and after lung transplantation were used in the study. Antimicrobial activities of the whole serum and its fractions with the molecular mass under 100 kDa were estimated by the effect of the specimens on Candida albicans test culture cells and by spectrophotometric assay of dye, which was internalized by the killed cells, as compared to the control untreated cells.Results. The total serum activity and AMP fractions activity in patients with CF were lower than in the healthy volunteers (medians): 82.3% vs 87.8 % (p ≤ 0.01) and 17.2% vs 41.9% (p ≤ 0.01). After the lung transplantation, both activities increased to 84.5% vs 33.3%, respectively. The improvement of AMP fractions activity was observed during 12 months after the transplantation. Serum albumin levels were lower in CF patients as compared to the reference values but normalized after lung transplantation.Conclusion. Obviously, the activity of the serum components of humoral immunity, which are responsible for direct antimicrobial defense, is decreased in CF patients compared to the reference values. The lung transplantation increased their activity considerably.

Diagnosis of Parkinson's disease by investigating the inhibitory effect of serum components on P450 inhibition assay

Parkinson’s disease (PD) is the second most common neurodegenerative disease, and diagnostic methods and biomarkers for patients without subjective motor symptoms have not yet been established. Previously, we developed a cytochrome P450 inhibition assay that detects alterations in metabolite levels associated with P450s caused by inflammation and exposure to endogenous or exogenous substances. However, it is unknown whether the P450 inhibition assay can be applied in PD diagnosis. Here, we determined whether the P450 inhibition assay can discriminate sera between patients with PD and healthy individuals. The results of the assay revealed that the P450 inhibition assay can discriminate PD with an area under the receiver operating characteristic curve (AUC) value of 0.814–0.914 in rats and an AUC value of 0.910 in humans. These findings demonstrate that the P450 inhibition assay can aid in the future development of liquid biopsy-based diagnostic methods for PD.

Rolling circle amplification assisted dual signal amplification colorimetric biosensor for ultrasensitive detection of leukemia-derived exosomes

Tumor-derived exosomes as a liquid biopsy marker hold great promising for the accurate tumor diagnosis. However, the visual exosomes detection method with high sensitivity and convenience is still a challenge and highly valuable in the clinical application. Herein, we fabricated a colorimetric biosensor that could visually detect leukemia-derived exosomes, and dual amplified the signal based on the rolling circle amplification (RCA) assisted platform. To avoid the interference of components in serum, trigger-aptamer complex was firstly modified on the magnetic beads (MBs) to recognize exosomes, and then release trigger probe to initiate RCA reaction. Therefore, the trigger probe could produce long repeated sequences and hybridized with numerous signal probe. After that, GNPs-HRP (HRP modified gold nanoparticles) bound with signal probe and induced dramatic color change of TMB to achieve the sensing of exosomes. Benefiting from the RCA assisted dual signal amplification, the presented method showed a limit of detection as low as 100 particles/μL. Moreover, this method exhibited good specificity to distinguish healthy and leukemia patients, suggesting its great potential for clinical diagnosis.

Prediction of the mechanism of Dachengqi Decoction treating colorectal cancer based on the analysis method of " into serum components -action target-key pathway"

Ethnopharmacological relevanceColorectal cancer (CRC) is a common digestive tract malignant tumor that its morbidity and mortality seriously affect human health. At present, Dachengqi Decoction (DCQ), a traditional Chinese medicine formula, has been clinically used as an adjuvant therapy for CRC. However, pharmacodynamic substance basis and therapeutic mechanism are still unclear. Aim of the studyThe main constituents absorbed in the blood and possible active targets after DCQ administration were explored based on the analysis method of “into serum components, action target and key pathway”, which may provide reference for the study of the pharmacodynamic material basis and action mechanism of Dachengqi Decoction in the treatment of CRC. Material and methodsBased on the serum pharmacochemistry of traditional Chinese medicine (TCM), the prescription prototype ingredients of DCQ in mice serum samples were identified by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry technology (UPLC-Q-TOF-MSE). Taking the prototype ingredients absorbed into serum as the research object, the possible targets and key pathways of DCQ in vivo were demonstrated by network pharmacology. Finally, using molecular docking verified the binding activity of prototype components and potential action targets. ResultsA total of 46 prototype components of DCQ were identified in mice serum, most of which were derived from flavonoids and anthraquinones in Citrus aurantium L. and Rheum palmatum L. Network pharmacology prediction results indicated that the drug prototype components entering the serum may mainly regulate targets including mitogen-activated protein kinase (MAPK), interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), etc. and main pathways such as (phosphatidylinositol 3-kinase/protein kinase B) PI3K-AKT signaling pathway, advanced glycation end products-receptor for AGE (AGE-RAGE) signaling pathway and IL-17 signaling pathway, etc. Molecular docking showed that the prototype active components had strong binding activity to VEGF, Harvey rat sarcoma viral oncogene homolog (HRAS) and MAPK1. ConclusionsThis study elucidated that most of the direct acting substances of DCQ in vivo were flavonoids and anthraquinones, which may play a role in regulating cell reproduction and apoptosis and inhibiting inflammation, providing a reference for the research of pharmacodynamic material basis and mechanism of DCQ in the treatment of CRC.

Increasing productivity of arylsulfatase B-producing cell line by coexpression of formylglycine-generating enzyme

Mucopolysaccharidosis type VI (Maroteaux–Lamy syndrome) is an orphan genetic disease caused by deficiency of the lysosomal enzyme arylsulfatase B (ASB). The need to develop a highly productive cell line for the production of recombinant ASB, is behind the concept and relevance of this study. The most promising approach seems to be the development of CHO producer cell lines coexpressing the target ASB enzyme and an auxiliary formylglycine-generating enzyme (FGE). At the same time, it is important from a practical perspective to have the possibility of cultivating producer cell lines as suspensions free of serum or other components of animal origin. The aim of the study was to develop highly productive cell lines for the production of recombinant ASB by coexpression of the auxiliary FGE. Materials and methods: a suspension CHO cell line was used in the study. CHO cells were transfected by electroporation using the MaxCyte STX system. Monoclonal cell lines were obtained with the help of the Cell Metric system. Enzyme-linked immunosorbent assay was used for determination of ASB concentration in the culture fluid. Culture fluid samples were analysed using polyacrylamide gel electrophoresis and Western blotting. The mRNA level was measured by real-time polymerase chain reaction. Results: producer cell lines coexpressing the target ASB enzyme and auxiliary FGE were obtained. An increase in the yield of the active target ASB enzyme from 2 to 100 mg/L was achieved by selecting the optimal ratio of plasmids during transfection. The highest yield of the target ASB enzyme was achieved at the 90:10 ratio (%) of plasmids encoding the ASB and FGE genes, respectively. Conclusions: the authors developed highly productive cell lines for the production of recombinant ASB, which coexpress the target and auxiliary enzymes. The coexpression of ASB and FGE improves the growth and production characteristics of the cell line, probably due to the modification of the ASB active site. The obtained results will help resolve the problem of low enzyme yield, which is typical of this class of medicines.

Propionic acid regulates immune tolerant properties in B Cells.

Interleukin 10 (IL‐10)‐producing B cells (B10 cells) are a canonical cell fraction for regulating other activities of immune cells. Posttranscriptional modification of IL‐10 in B10 cells is not yet fully understood. Short‐chain fatty acids play an important role to regulate the functions of immune cells. This study aims to clarify the role of propionic acid (PA), a short‐chain fatty acid, in regulating the expression of IL‐10 in B10 cells. Blood samples were collected from patients with food allergy (FA) and healthy subjects. Serum and cellular components were prepared with the samples, and analysed by enzyme‐linked immunosorbent assay and flow cytometry, respectively. The results showed that serum PA levels were lower in FA patients. PA concentrations were negatively correlated with serum cytokine Th2 concentrations, specific IgE concentrations in serum and skin prick test results. The peripheral frequency of B10 cells and the production of IL‐10 in B cells were also associated with serum PA concentrations. Activation of B cells by CpG induced the production of IL‐10 and tristetretrprolin (TTP), in which TTP caused the spontaneous decay of IL‐10 mRNA. PA was necessary to stabilize the IL‐10 mRNA in B cells by inducing the production of granzyme B, which resulted in the degradation of the IL‐10 mRNA. Administration of PA attenuated FA response in mice by maintaining homeostasis of B10 cells. In conclusion, PA is needed to stabilize the expression of IL‐10 in B10 cells. PA administration can mitigate experimental FA by maintaining B10 cell functions.

Detection of Circulating Peanut Components in Serum after Ingestion

Introduction: Detection of specific food allergens brought into circulation following ingestion is complicated by the minute amounts present in serum. We therefore aimed to assess the utility of selective removal of IgE against peanut components using ImmunoCAP combined with the basophil histamine release (HR) assay (BHRA) to identify the absorbed allergen components after ingestion of peanut. Methods: Serum from six healthy individuals was drawn before and 1 h after ingestion of 100 g peanut. Serum from two peanut allergic patients was depleted for IgE against single allergen components by incubation with ImmunoCAPs coupled with either Ara h 1; 2; 3 or 6 before donor basophils were passively sensitized with the serum preparations. The sensitized cells were challenged with serum obtained from the six individuals before and after ingestion of peanut, and HR was measured after serum provocation. Results: Ara h 2 and Ara h 6 were detected in serum 1 h after ingestion in 6/6 individuals by negative selection. Depletion of specific IgE against Ara h 2 or Ara h 6 almost completely abolished the response to serum provocation, indicating a sequence homology between the two allergen components in serum. Ara h 1 was demonstrated in 5/6 sera and Ara h 3 in 1/6 sera. Conclusion: This study is a proof of concept showing that passive sensitization of basophils with sera depleted of component-specific IgE can be used to identify food allergen components present in serum after ingestion.

Identification of Resolvin D1 and Protectin D1 as Potential Therapeutic Agents for Treating Kidney Stones.

Intrarenal calcium oxalate (CaOx) crystals induce renal tubular epithelial cell (TEC) inflammatory and oxidative injury. This study is aimed at exploring potential therapeutic lipid components in kidney stones because lipids are involved in the development of several diseases and indicate the risk of kidney stones. Serum specimens were collected from 35 kidney stone patients and 35 normal controls. The lipid components in serum were measured, and differences were analyzed. The documented biological importance was comprehensively reviewed to identify lipids that differed significantly between the two groups to find potential agents associated with kidney stones. CaOx nephrocalcinosis mouse model was established to examine the therapeutic effects of specific lipids on CaOx deposition and CaOx-induced oxidative renal injury. Several lipids with significantly different levels were present in the serum of patients with stones and normal controls. Resolvin D1 (RvD1) (4.93-fold change, P < 0.001) and protectin D1 (PD1) (5.06-fold change, P < 0.001) were significantly decreased in the serum of patients with kidney stones, and an integrative review suggested that these factors might be associated with inflammatory responses, which is a crucial mechanism associated with stone damage. The administration of RvD1 and PD1 significantly inhibited kidney CaOx deposition and suppressed CaOx-induced renal tubular cell inflammatory injury and necrosis in a CaOx nephrocalcinosis mouse model. Furthermore, RvD1 and PD1 facilitated the expression of the oxidative indicator superoxide dismutase 2 (SOD2), inhibited NADPH oxidase 2 (NOX2) expression, and diminished intracellular reactive oxygen species (ROS) levels. This study preliminarily elucidated the role of lipids in kidney stones. The inhibitory effects of RvD1 and PD1 on oxidative damage induced by CaOx deposition provide a promising perspective for kidney stone treatment strategies.

Characterization of Serum Metabolome and Proteome Profiles Identifies SNX5 Specific for Pregnancy Failure in Holstein Heifers.

Pregnancy loss predominantly occurs during the first 3–4 weeks due to fertilization failure or early embryonic losses in cattle. Insufficient biochemical communication between conceptus (embryo plus extraembryonic membranes) and endometrium has been suspected as the primary cause for early embryonic losses. If molecules regulating this communication were identified, molecular mechanisms associated with early pregnancy losses could be better understood. To identify candidate molecules as detection markers of non-pregnant or females undergoing embryonic loss, peripheral blood from embryo-transferred heifers on day 7 (day 0 = day of estrus) were collected on days 17 (pre-attachment), 20 (during attachment), and 22 (post-attachment), which were subjected to metabolome and global proteome iTRAQ analyses. The metabolome analysis partly divided serum components into pregnant or not. In the iTRAQ analysis, heatmap analysis with top 25 proteins was separated into pregnant or not on day 20 or 22. Furthermore, receiver operating characteristic curve (ROC) analysis identified five candidate proteins detecting non-pregnant heifers, of which SNX5 in day 22 serum had the highest area under the curve (AUC): 0.983. We also detected SNX5 in day 22 serum from non-pregnant heifers using western blotting. These results suggest that high SNX5 in day 22 serum could predict early pregnancy loss in heifers.

Diagnostic and Prognostic Value of Neutrophil Extracellular Trap Levels in Patients With Acute Aortic Dissection.

BackgroundAcute aortic dissection (AAD) is a fatal disease demanding prompt diagnosis and proper treatment. There is a lack of serum markers that can effectively assist diagnosis and predict prognosis of AAD patients.MethodsNinety-six AAD patients were enrolled in this study, and 249 patients with chest pain due to acute myocardial infarction, pulmonary embolism, intramural hematoma, angina or other causes and 80 healthy controls were included as control group and healthy control group. Demographics, biochemical and hematological data and risk factors were recorded as baseline characteristics. The 1-year follow-up data were collected and analyzed. The diagnostic performance and ability to predict disease severity and prognosis of NET components in serum and aortic tissue were evaluated.ResultsCirculating NET markers, citH3 (citrullination of histone 3), cell-free DNA (cfDNA) and nucleosomes, had good diagnostic value for AAD, with superior diagnostic performance to D-dimer in discriminating patients with chest pain due to other reasons in the emergency department. Circulating NET marker levels (i.e., citH3, cfDNA and nucleosomes) of AAD patients were significantly higher than that of control group and healthy control group. In addition, circulating NET markers levels were closely associated with the disease severity, in-hospital death and 1-year survival of AAD patients. Systolic blood pressure < 90 mmHg and serum citH3 levels were identified as independent risk factors for 1-year survival of AAD patients. Excessive NET components (i.e., neutrophil elastase and citH3) in the aortic tissue of AAD patient were significantly higher than that of healthy donor aortic tissue. The expression levels of granules and nuclear NET components were significantly higher in aortic tissue from AAD patients than controls.ConclusionsCirculating NET markers, citH3, cfDNA and nucleosomes, have significant diagnostic value and predictive value of disease severity and prognosis of AAD patients. The NETs components may constitute a useful diagnostic and prognostic marker in AAD patients.

Tceal5 and Tceal7 Function in C2C12 Myogenic Differentiation via Exosomes in Fetal Bovine Serum.

The proliferation and differentiation of skeletal muscle cells are usually controlled by serum components. Myogenic differentiation is induced by a reduction of serum components in vitro. It has been recently reported that serum contains not only various growth factors with specific actions on the proliferation and differentiation of myogenic cells, but also exogenous exosomes, the function of which is poorly understood in myogenesis. We have found that exosomes in fetal bovine serum are capable of exerting an inhibitive effect on the differentiation of C2C12 myogenic cells in vitro. In this process of inhibition, the downregulation of Tceal5 and Tceal7 genes was observed. Expression of these genes is specifically increased in direct proportion to myogenic differentiation. Loss- or gain- of function studies with Tceal5 and Tceal7 indicated that they have the potential to regulate myogenic differentiation via exosomes in fetal bovine serum.

Study on serum pharmacodynamic material basis of Gegen Qinlian Decoction in treatment of ulcerative colitis based on UHPLC-Q-Orbitrap-MS

This study established a mouse model of ulcerative colitis and explored the serum transitional components of Gegen Qinlian Decoction by UHPLC-Q-Orbitrap-MS. Based on the exact relative molecular weight and MS/MS spectrum, 55 prototype components and 59 metabolites were identified from the model group, while 18 prototype components and 35 metabolites from the control group. The prototype components in serum were mainly flavonoids and the characteristic components of the model group were alkaloids. Glucuronidation, sulfonation, and glycosylation have been confirmed to be the main metabolic types in vivo. The results of comparative analysis of differences indicated that puerarin, baicalin, wogonoside, wogonin, chrysin, oroxylin A, berberine, berberrubine, and palmatine were the characteristic components in model state, which at the same time, were confirmed by pharmacological studies to be the serum pharmacodynamic material basis of Gegen Qinlian Decoction in the treatment of ulcerative colitis. This study has provided reference for explaining the metabolic transformation pattern and mechanism of action of Gegen Qinlian Decoction in vivo.

POS-103 Serum and Glomerular Complement Components as Biomarkers in the First South-Asian Prospective Longitudinal Observational IgA Nephropathy cohort (GRACE-IgANI)

The Glomerular Research And Clinical Experiments- IgA Nephropathy in Indians is a prospective longitudinal cohort . The study protocol has been published and is registered with WHO trial id: ISRCTN36834159. The role of serum and glomerular complement components in South Asian IgAN is unknown.

Analysis of serum total bilirubin content based on dual-position joint spectrum of "M plus N" theory and the logarithmic method.

At present, in the chemical quantitative analysis of complex solutions based on spectroscopy, the accuracy of the content analysis of complex solution is difficult to meet the requirements due to the overlapping spectral lines, low signal-to-noise ratio, and scattering characteristics of various components of complex solution. In this paper, the logarithmic method is used to preprocess the spectral data in the spectral preprocessing stage, and the two-position and multi-mode joint spectral strategy of "M plus N" theory is applied to the quantitative analysis of serum components. The serum samples are illuminated by dual-position ultraviolet LED light source, and the two spectra of the vertical position and the coaxial facing position of the light source and the optical fiber are collected respectively. Then the partial least square (PLS) method was used to establish models to analyze the concentration of total bilirubin in serum by the spectrum of vertical position, the spectrum of coaxial facing position, and the spectrum of the combination of the former two. Among the experimental results, the model established by the combination of the two spectra collected by two positions has a good result. The correlation coefficient of all samples predicted by this model is 0.971223, and the root mean square error is 1.96645μmol/L. This study shows that the method of the logarithmic, collecting spectra and analyzing the composition content of complex solutions by using the multi-location and multi-mode strategy of "M + N" theory can effectively improve the prediction accuracy of the model and has practical significance for the chemical quantitative analysis of complex solutions based on spectroscopy.

Hematological and serum biochemical reference intervals of rainbow trout, Oncorhynchus mykiss cultured in Himalayan aquaculture: Morphology, morphometrics and quantification of peripheral blood cells

De novo reference intervals (RIs) for a total of thirty two hematological and serum biochemical attributes were established for rainbow trout (Oncorhynchus mykiss) cultured in Himalayan aquaculture system. For this purpose, long term assessment of hemato-biochemical parameters was carried over a period of one year from March 2019 to February 2020 and a total of 444 blood samples were analysed. Blood examination results were recorded systematically and reference intervals were established, notably for erythrocyte parameters: hematocrit (Hct) 29–40%, hemoglobin 8.32–12.28 g/dL, red blood cell (RBC) count 1.01–2.04 (×106/mm3); leukocyte parameters (x 103/mm3): total leukocytes 31.32–90.60, neutrophils 4.21–18.85, total lymphocytes 20.55–63.63, small lymphocytes 14.86–46.50, large lymphocytes 6.35–22.34 and monocytes 1.22–7.56; thrombocyte count 23.00–68.00 (×106/mm3). RIs were also established for red blood cell indices, vital serum constituents involved in carbohydrate, protein, lipid and nitrogen metabolism including the less known, diagnostically important, serum enzymes and electrolyte concentrations. Principal component analysis revealed that certain serum components were more efficient at distinguishing between the life stages (juvenile, adult) of fish by explaining about 92.7% of variation in the whole dataset compared to the principal hematological components which explained only about 80% of the variation. Significant (P < 0.05) differences were noted for RBC count, total leukocyte count (TLC), total protein, total cholesterol and uric acid with respect to the sex of fish. Moreover, clearly differentiable morphometric and morphological attributes were also noticed among erythrocytes, leukocytes (lymphocytes, neutrophils and monocytes) and thrombocytes. To our knowledge, the present study is the first of its kind that elucidates blood chemistry of cultured rainbow trout, O. mykiss in accordance to the guidelines framed by the American society of veterinary clinical pathologists (ASVCP). RIs reported here can help monitor the fish health status by improving the use of non-lethal diagnostics in piscine medicine.

Establishment of serum-free adapted Chinese hamster ovary cells with double knockout of GDP-mannose-4,6-dehydratase and GDP-fucose transporter.

The online version contains supplementary material available at 10.1007/s10616-021-00501-3.

Changing lanes: seasonal differences in cellular metabolism of adipocytes in grizzly bears (Ursus arctos horribilis).

Obesity is among the most prevalent of health conditions in humans leading to a multitude of metabolic pathologies such as type 2 diabetes and hyperglycemia. However, there are many wild animals that have large seasonal cycles of fat accumulation and loss that do not result in the health consequences observed in obese humans. One example is the grizzly bear (Ursus arctos horribilis) that can have body fat content > 40% that is then used as the energy source for hibernation. Previous in vitro studies found that hibernation season adipocytes exhibit insulin resistance and increased lipolysis. Yet, other aspects of cellular metabolism were not addressed, leaving this in vitro model incomplete. Thus, the current studies were performed to determine if the cellular energetic phenotype-measured via metabolic flux-of hibernating bears was retained in cultured adipocytes and to what extent that was due to serum or intrinsic cellular factors. Extracellular acidification rate and oxygen consumption rate were used to calculate proton efflux rate and total ATP defined as both ATP from glycolysis and from mitochondrial respiration. Hibernation adipocytes treated with hibernation serum produced less ATP and exhibited lower maximal respiration and glycolysis ratesthan active season adipocytes. These effects were reversed with serum from the opposite season. Insulin had little influence on total ATP production and lipolysis in both hibernation and active serum-treated adipocytes. Together, these results suggest that the metabolic suppression occurring in hibernation adipocytes are downstream of insulin signaling and likely due to a combined reduction in mitochondria number and/or function and glycolytic processes. Future elucidation of the serum components and the cellular mechanisms that enable alterations in mitochondrial function could provide a novel avenue for the development of treatments for human metabolic diseases.