Horse Serum Research Articles

INFLUENZA D VIRUSES - PATHOGENS FORMING A NEW GENUS IN THE ORTHOMYXOVIRIDAE FAMILY

Influenza pathogens belong to the Orthomyxoviridae family and are divided into genera: Influenzavirus A, B, C, D, as well as Quaranjavirus, Thogotovirus, and Isavirus. For the first time, the influenza D virus was isolated from swine nasal swabs in 2011 in the United States, and its widespread distribution among cattle in France, China, Italy, Ireland, Japan, and several African countries, as well as its ability to infect ferrets, guinea pigs, is further shown. Antibodies to influenza D virus are found in the blood serum of horses, sheep, goats, and in people who have been in contact with cattle. The RNA genome of the influenza D virus is represented by seven fragments responsible for the synthesis of nine proteins. The longest three segments encode for polymerases PB2, PB1, and P3; the fourth and fifth segments encode for hemagglutinin-esterase fusion protein – HEF and nucleoprotein – NP, respectively. The sixth fragment is involved in the synthesis of membrane polypeptides DM1 and DM2, which, in accordance, lines the viral membrane from the inside and performs the function of proton channels. The seventh segment encodes the non-structural protein NS1 and the nuclear export protein NEP; NS1 helps to neutralize cellular interferon and NEP mediates the nuclear export of ribonucleoprotein. Three phylogenetic lines of the influenza virus D are described – D/OK, D/660, and D/Japan, which must be taken into account when preparing vaccines. It is concluded that from its epidemiological, pathological and biological characteristics, the potential ability to cause disease in humans and be transmitted from person to person, new, more in-depth studies are required using ecological-virological and molecular genetic methods.

The Oxidative Stress Markers of Horses-the Comparison with Other Animals and the Influence of Exercise and Disease.

Simple SummaryAlthough oxidative stress is detrimental in biological systems, direct analysis of the active oxygen species that causes the stress has been difficult at a clinical level. In the current study, we analyzed the levels of diacron-reactive oxygen metabolites (d-ROMs) and biological antioxidant potential (BAP) in the serum of horses. These are easy to measure and they provide information on the level of oxidative stress in an animal. The mean d-ROM level in horses was higher than those in dogs or dairy cattle, and the levels in horses can be used to distinguish those with a disease.Diacron-reactive oxygen metabolite (d-ROM) and biological antioxidant potential (BAP) levels in the serum of horses were measured (ponies, n = 15; thoroughbred, n = 31; other full-sized horses, n = 7). The mean d-ROM levels in horses were significantly higher (p < 0.001) than those in dairy cattle (n = 25) and dogs (n = 31). However, d-ROM levels in horses were lower than the standard levels reported in humans. When d-ROM and BAP levels were plotted graphically, the points for horses with a disease (ringbone in 1 Japanese sports horse, cellulitis in 1 thoroughbred, melanoma in 1 Lipizzaner) fell outside the group of points for other (non-diseased) horses. A similar separation was seen (using data from other authors) for a horse with Rhodococcus equi, a horse following castration surgery, and a mare following delivery. These results, comparing horses, other animals, and humans, are interesting from the standpoint of comparative medicine, and they contribute to the sparse literature available on d-ROM and BAP levels in animals. Because the level of d-ROM and BAP levels were changed depending on the situation of health, those indexes are promising as indices of health in horses.

Effect of Electrical Pulse Stimulation on Anabolic Signaling in L6 Myotubes

The mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase with a central role in controlling cellular anabolism. By phosphorylating two downstream targets, p70‐S6 kinase (p70‐S6K) and eIF4E binding protein 1 (4EBP1), mTOR induces an increase in mRNA translation, leading to an upregulation of protein synthesis. In muscle tissue, mTOR activation in response to an exercise stimulus is a key signaling event in the anabolic response to physical activity. As studying the signaling response to exercise at the cellular level is challenging in whole organisms due to difficulty in isolating muscle cell level effects, development of a cell culture model mimicking exercise would provide an avenue for understanding the intracellular cellular signaling response to exercise in skeletal muscle. The aim of this study was to determine whether 12 hours of electrical pulse stimulation (EPS) would alter the anabolic response in L6 myotubes.METHODSMurine L6 myoblasts were cultured on 10 cm plates in standard culture medium supplemented with 1% penicillin/streptomycin, and 10% fetal bovine serum until 60% confluence. At this point, differentiation was induced by switching to medium without sodium pyruvate supplemented with 1% penicillin/streptomycin, 2% horse serum, and 0.5% insulin‐transferrin‐selenium (ITS). After 7 days of differentiation, myotubes were stimulated, in culture, for 12 hours at a low frequency (1 Hz, 2ms, 60V). Differentiation media was changed in all plates immediately before stimulation and immediately after stimulation in half of the plates. Half of the cells were harvested immediately after stimulation to assess the acute effects and the remaining plates were harvested 24 hours post stimulation and re‐feeding. Protein expression of common anabolic markers was measured using western blot techniques.RESULTSOur results indicate an increase in phosphorylated 4EBP1 immediately after stimulation (EPS‐Ea) compared to control (CON‐Ea) (p = 0.05). Additionally, phosphorylation levels return to baseline 24 hours post stimulation. In contrast, phosphorylation levels of p70‐S6K immediately after stimulation (EPS‐Ea) were not different from control (CON‐Ea) but were elevated 24 hours post stimulation (EPS‐La) (p = 0.09). Finally, we determined the ratio of phosphorylated to total levels of 4EBP1 were two‐fold greater immediately after stimulation (EPS‐Ea) compared to 24 hours post stimulation (EPS‐La) (p = 0.09). Conversely, the ratio of phosphorylated to total levels of p70‐S6K were two times greater 24 hours post stimulation (EPS‐La) compared to immediately after stimulation (EPS‐Ea) (p = 0.09).CONCLUSIONWe were able to determine that electrical pulse stimulation of L6 myotubes could be used as a model to better understand anabolic signaling strategies following EPS in culture. This study represents a first step in using electrical pulse stimulation as an in culture exercise mimetic and elucidating the role of mTOR and downstream targets in cell growth.Support or Funding InformationThis work was funded in part by the College of Health and Education and the Huffines Institute for Sports Medicine at Texas A&amp;M University

Restoration of Dystrophin in Duchenne Muscular Dystrophy by Human iPS Cells Derived Skeletal Muscle Progenitor Cells

Background and ObjectiveDuchenne muscular dystrophy (DMD) is caused by mutations of the gene that encodes the protein dystrophin. Loss of dystrophin leads to severe and progressive muscle‐wasting in both skeletal and heart muscles. Human induced pluripotent stem cells (hiPSCs) offer an important opportunity to treat a number of diseases. Here, we investigated whether givinostat, a histone deacetylase inhibitor (HDACi), could reprogram hiPSCs into muscle progenitor cells (MPCs) for DMD treatment.Methods and ResultshiPSCs were treated with CHIR99021(10 μM) for two days and followed by givinostat (100 nM) for 5 days in E6 medium (Fig. 1). After culture in E6 medium for additional seven days, cells were analyzed for myogenic makers (Pax7, desmin) expression. Reprogrammed cells (MPCs) from 4 human iPS cell lines expressed Pax7 and desmin. MPCs were differentiated into myotubes in N2 or DMEM medium with 2% horse serum expressing MF20. Additionally, we also generated MPCs from hiPSCs using CHIR99021 and FGF according to a previous study (Fig. 2) and referred these cells as control‐MPCs. We observed that givinostat induced MPCs exhibited superior proliferation and migration capacity using CCK‐8 assay, colony assay and migration assay compared to control‐MPCs. Moreover, we transplanted human myoblasts (1×105) and MPCs (1×105) labeled with GFP into tibialis anterior (TA) muscles of Mdx/scid mice, after injury with cardiotoxin (CTX) for one day prior to cell transplantation. One month after transplantation, dystrophin and GFP double positive fibers were analyzed. Givinostat induced MPCs showed higher engraftment capacity and restoration of dystrophin than treatment with control‐MPCs and human myoblasts. In addition, givinostat induced MPCs treated muscle contained significantly less inflammatory cells and showed limited muscle necrosis and fibrosis.ConclusionWe successfully generated highly expandable and integration free MPCs from multiple hiPS cell lines using CHIR99021 and givinostat. Givinostat induced MPCs showed marked and impressive regeneration and restored dystrophin in injured tibialis muscle compared to control MPCs. It is concluded that hiPSCs pharmacologically reprogrammed into MPCs with a small molecule, givinostat might be effective cellular source for treatment of muscle injury and restoration of dystrophin in DMD.Support or Funding InformationNational Institutes of Health grants R01 HL134354 &amp; R01 AR070029 (M Ashraf, Y Tang, NL Weintraub)Schematic outline of generation of MPCs from hiPSCs using CHIR99021 and givinostat (Givi) small molecule combo.Figure 1Schematic outline generation of control MPCs from hiPSC.Figure 2

Existence of Circulating Mitochondria in Human and Animal Peripheral Blood.

Mitochondria are usually located in the cytoplasm of cells where they generate adenosine triphosphate (ATP) to empower cellular functions. However, we found circulating mitochondria in human and animal blood. Electron microscopy confirmed the presence of mitochondria in adult human blood plasma. Flow cytometry analyses demonstrated that circulating mitochondria from the plasma of human cord blood and adult peripheral blood displayed the immune tolerance-associated membrane molecules such as CD270 and PD-L1 (programmed cell death-ligand 1). Similar data were obtained from fetal bovine serum (FBS) and horse serum of different vendors. Mitochondria remained detectable even after 56 °C heat inactivation. A real-time PCR array revealed purified mitochondria from animal sera expressed several genes that contribute to human T- and B-cell activation. Transwell experiments confirmed the migration capability of mitochondria through their expression of the chemokine receptor CXCR4 in responses to its ligand stromal-derived factor-1α (SDF-1α). Functional analysis established that human plasma mitochondria stimulated the proliferation of anti-CD3/CD28 bead-activated PBMC, up-regulated the percentage of activated CD4+ T and CD8+ T cells, and reduced the production of inflammatory cytokines. These findings suggested that the existence of circulating mitochondria in blood may function as a novel mediator for cell-cell communications and maintenance of homeostasis. Plasma-related products should be cautiously utilized in cell cultures due to the mitochondrial contamination.

Non-specific, agar medium-related peaks can result in false positive Mycoplasma alkalescens and Mycoplasma arginini identification by MALDI-TOF MS

MALDI-TOF MS is a fast and accurate tool to identify Mycoplasma species in liquid media. However, when trying to identify presumptive Mycoplasma bovis (M. bovis) colonies from solid medium (the “direct transfer method”) a surprisingly high occurrence of M. arginini and M. alkalescens identification was observed. It was hypothesized that agar medium components are associated with false positive identification with Mycoplasma spp., as M. bovis colonies are very small and grow into the agar. The objective of this study was to determine whether complete modified pleuropneumonia-like organism (PPLO) agar (supplemented with horse serum, sodium pyruvate, technical yeast extract, ampicillin sodium salt and colistin) and the separate components, result in false identification as Mycoplasma spp. by MALDI-TOF MS. A total of 100 samples were examined, of which 33% of the modified PPLO agar spots were identified as M. alkalescens (16%) and M. arginini (17%)), albeit with relatively low score values (< 1.85). No false identification of M. bovis was obtained. Several medium components (unsupplemented PPLO agar, horse serum and colistin) resulted in spectra with peaks showing close matches with peaks present in the M. alkalescens and M. arginini database spectra. This study shows that the direct transfer method should be interpreted with caution, and one should strive to pick as little as possible agar when sampling Mycoplasma-like colonies from solid medium containing PPLO agar, horse serum and/or colistin.

Usefulness of a Point-of-Care Analyzer to Measure Cardiac Troponin I and D-Dimer Concentrations in Critically Ill Horses With Gastrointestinal Diseases

Point-of-care (POC) systems for the joint measurement of Troponin and D-dimers have not been studied in horses. The aim of this study was to perform the validation of a POC system (AQT90 FLEX) for the measurement of cardiac troponin I (cTnI) and D-dimers in the serum of horses with gastrointestinal diseases. The main objective was to evaluate whether or not this system can distinguish healthy animals from diseased animals. A sample of 33 horses was included in the study: control group (n = 10) and horses with gastrointestinal disorders (n = 21), which were classified according to their outcome in survivors (subgroup A = 9) and nonsurvivors (subgroup B = 12). Considering the diagnosis of the process, ill horses were classified into three groups: inflammatory (I = 7), obstructive (O = 9), and strangulating diseases (S = 5). The clinical usefulness of AQT90 FLEX was validated by the study of linearity, coefficient of variation, and detection limits. Later, concentrations of D-dimers and cTnI were measured. A significant increase in both parameters was detected in ill animals (cTnI: control: 0.014 ± 0.01 μg/mL, survivors: 0.27 ± 0.37 μg/mL, nonsurvivors: 0.60 ± 1.21 μg/mL; D-dimers: control: 104.90 ± 30.82 ng/mL, survivors: 1,217.22 ± 1,213.28 ng/mL, nonsurvivors: 1,613.67 ± 1,426.75 ng/mL), although there were no statistically significant differences in concentrations according to diagnosis and outcome. In conclusion, AQT90 FLEX POC analyzer can be used in horses with gastrointestinal diseases to measure cTnI and D-dimer concentrations. It is a quick, practical, and minimally invasive tool that helps in determining the severity of illness.

Lack of a skeletal muscle phenotype in adult human bone marrow stromal cells following xenogeneic-free expansion

BackgroundMany studies have elegantly shown that murine and rat bone marrow-derived mesenchymal stromal cells (bmMSCs) contribute to muscle regeneration and improve muscle function. Yet, the ability of transplanted human bmMSCs to manifest myogenic potential shows conflicting results. While human adipose- and umbilical cord-derived MSCs can be differentiated into a skeletal muscle phenotype using horse serum (HS), bmMSCs have only been shown to differentiate towards the skeletal muscle lineage using a complex mixture of cytokines followed by transfection with notch intracellular domain.MethodsSince xenogeneic-free growth supplements are increasingly being used in the expansion of bmMSCs in clinical trials, we investigated the effects of human plasma and platelet lysate (P/PL) on the expression of neuromuscular markers and whether P/PL-expanded human bmMSCs could be differentiated towards a skeletal myogenic phenotype. Neuromuscular markers were measured using the highly sensitive droplet digital polymerase chain reaction for measuring the expression of Myf5, MyoD, MyoG, ACTA1, Desmin, GAP-43, and Coronin 1b transcripts, by performing immunofluorescence for the expression of Desmin, GAP-43, and MEF2, and flow cytometry for the expression of CD56/neural cell adhesion molecule (NCAM).ResultsDespite that bmMSCs expressed the myogenic regulatory factor (MRF) MEF2 after expansion in P/PL, bmMSCs cultured under such conditions did not express other essential MRFs including Myf5, MyoD, MyoG, or ACTA1 needed for myogenesis. Moreover, HS did not induce myogenesis of bmMSCs and hence did not induce the expression of any of these myogenic markers. P/PL, however, did lead to a significant increase in neurogenic GAP-43, as well as Desmin expression, and resulted in a high baseline expression of the neurogenic gene Coronin 1b which was sustained under further P/PL or HS culture conditions. Fetal bovine serum resulted in equally high levels of GAP-43 and Coronin 1b. Moreover, the proportion of CD56/NCAM-positive bmMSCs cultured in P/PL was 5.9 ± 2.1.ConclusionsThese data suggest that P/PL may prime a small portion of bmMSCs towards an early neural precursor cell type. Collectively, this shows that P/PL partially primes the cells towards a neurogenic phenotype, but does not prime adult human bmMSCs towards the skeletal muscle lineage.

BrightNet: A Deep CNN for OLED-Based Point of Care Immunofluorescent Diagnostic Systems

An automatic tool, targeting low-cost, low-power, point-of-care embedded system, is proposed for fluorescence diagnostic imaging. This allows for a quick and accurate diagnosis even when used by nonexpert operators. To achieve this goal, an embedded system has been equipped with an end-to-end deep-learning algorithm that does not require manual parameter tuning to perform a diagnosis. The proposed deep convolutional model, named BrightNet, is based on a single-shot detector neural network, modified to estimate the brightness of the detected fluorescent spots in a low-density protein or DNA microarray and finalize the diagnosis. Several optimization steps are presented to compress the inference model size, which is required for the deployment into a portable resource-constrained device. The resulting inference time is about 66 [ms] on an i7 3770K desktop CPU and is estimated to be lower than 5 [s] on an ARM-Cortex M7 considering $1.1 \times 10^{9}$ multiply-accumulate operations. BrightNet has been successfully validated for the detection and discrimination of four different serotypes of the dengue virus in a set of human samples as well as for the diagnosis of West Nile virus in horse sera. When evaluated on the considered diagnostic tasks, BrightNet provides better average accuracy than a state-of-the-art variational approach that requires operator intervention, with significant additional advantages of complete automation and quicker diagnosis.

Arachidonoylcholine and Other Unsaturated Long-Chain Acylcholines Are Endogenous Modulators of the Acetylcholine Signaling System.

Cholines acylated with unsaturated fatty acids are a recently discovered family of endogenous lipids. However, the data on the biological activity of acylcholines remain very limited. We hypothesized that acylcholines containing residues of arachidonic (AA-CHOL), oleic (Ol-CHOL), linoleic (Ln-CHOL), and docosahexaenoic (DHA-CHOL) acids act as modulators of the acetylcholine signaling system. In the radioligand binding assay, acylcholines showed inhibition in the micromolar range of both α7 neuronal nAChR overexpressed in GH4C1 cells and muscle type nAChR from Torpedo californica, as well as Lymnaea stagnalis acetylcholine binding protein. Functional response was checked in two cell lines endogenously expressing α7 nAChR. In SH-SY5Y cells, these compounds did not induce Ca2+ rise, but inhibited the acetylcholine-evoked Ca2+ rise with IC50 9 to 12 μM. In the A549 lung cancer cells, where α7 nAChR activation stimulates proliferation, Ol-CHOL, Ln-CHOL, and AA-CHOL dose-dependently decreased cell viability by up to 45%. AA-CHOL inhibited human erythrocyte acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BChE) by a mixed type mechanism with Ki= 16.7 ± 1.5 μM and αKi= 51.4 ± 4.1 μM for AChE and Ki= 70.5 ± 6.3 μM and αKi= 214 ± 17 μM for BChE, being a weak substrate of the last enzyme only, agrees with molecular docking results. Thus, long-chain unsaturated acylcholines could be viewed as endogenous modulators of the acetylcholine signaling system.

Human antibodies neutralizing diphtheria toxin in vitro and in vivo

Diphtheria is an infectious disease caused by Corynebacterium diphtheriae. The bacterium primarily infects the throat and upper airways and the produced diphtheria toxin (DT), which binds to the elongation factor 2 and blocks protein synthesis, can spread through the bloodstream and affect organs, such as the heart and kidneys. For more than 125 years, the therapy against diphtheria has been based on polyclonal horse sera directed against DT (diphtheria antitoxin; DAT). Animal sera have many disadvantages including serum sickness, batch-to-batch variation in quality and the use of animals for production. In this work, 400 human recombinant antibodies were generated against DT from two different phage display panning strategies using a human immune library. A panning in microtiter plates resulted in 22 unique in vitro neutralizing antibodies and a panning in solution combined with a functional neutralization screening resulted in 268 in vitro neutralizing antibodies. 61 unique antibodies were further characterized as scFv-Fc with 35 produced as fully human IgG1. The best in vitro neutralizing antibody showed an estimated relative potency of 454 IU/mg and minimal effective dose 50% (MED50%) of 3.0 pM at a constant amount of DT (4x minimal cytopathic dose) in the IgG format. The targeted domains of the 35 antibodies were analyzed by immunoblot and by epitope mapping using phage display. All three DT domains (enzymatic domain, translocation domain and receptor binding domain) are targets for neutralizing antibodies. When toxin neutralization assays were performed at higher toxin dose levels, the neutralizing capacity of individual antibodies was markedly reduced but this was largely compensated for by using two or more antibodies in combination, resulting in a potency of 79.4 IU/mg in the in vivo intradermal challenge assay. These recombinant antibody combinations are candidates for further clinical and regulatory development to replace equine DAT.

The Prevalence of Atypical Pneumonia Caused by Mycoplasma pneumoniae (P1 gene) in Patients with Respiratory Infections by Culture and Molecular PCR Methods in Tehran, Iran

Background: Atypical pneumonia is an upper and lower respiratory tract infection. Mycoplasma pneumoniae is a major cause of community-acquired pneumonia (CAP). Objectives: The present study aimed to determine the prevalence of atypical pneumonia caused by M. pneumonia by culture and molecular PCR methods in Tehran. Methods: In the present study, 102 samples of throat swab were collected from patients with respiratory infections. All samples were cultured in liquid PPLO Broth And solid PPLO agar media (1% glucose and 20% horse serum). The PCR technique with specific primers was implemented after culture and genome extraction through phenol-chloroform technique. Results: In this study, 27 (26.47%) colonies of Mycoplasma were isolated on PPLO agar medium. Using specific primers, it was found that 33 samples (32.4%) were positive in terms of Mycoplasma genus and 14 samples (13.7%) were positive for the presence of M. pneumonia. Conclusions: Mycoplasma pneumonia is a pathogen that causes respiratory tract infections in humans. Molecular PCR method is a quick and sensitive technique that has higher sensitivity and specificity than other methods. The obtained results may contribute to the specific treatment of some patients with symptoms of respiratory infections.

Vigilancia epidemiológica al virus del Oeste del Nilo en municipios del Departamento del Meta

Objetivo. Identificar la presencia del virus del Oeste del Nilo en equinos y mosquitos en ocho municipios del departamento del Meta. Materiales y métodos. La investigación contó con el aval del Comité de bioética de la Universidad de los Llanos. Se analizaron mediante pruebas serológicas y moleculares 613 muestras de equinos criollos y de raza cuarto de milla, destinados a actividades deportivas y de trabajo, con un rango de edad de 2 a 15 años, en los transectos: Villavicencio-Restrepo-Cumaral, San Martín-Castilla la Nueva-Granada y Puerto López-Puerto Gaitán, analizados en 62 pool y 213 mosquitos. Los pool de sueros de equinos y mosquitos fueron analizados por ELISA y PCR. Resultados. No se encontraron animales seropositivos mediante la prueba de ELISA y las pruebas moleculares también fueron negativas. Conclusiones. Aunque en este estudio no se evidenció la presencia de anticuerpos IgM por la técnica de Elisa y las pruebas moleculares (RT-PCR) también fueron negativas para circulación viral, en los municipios objeto de estudio, es importante indicar que la detección molecular en sueros, requiere unos niveles de viremia representativos y que el animal se encuentre en la fase aguda de la enfermedad. Aunque es posible que la población equina se mantenga libre de contacto con el virus, se debe mantener la vigilancia epidemiológica frente a este importante patógeno para la salud humana, especialmente por la presentación de brotes de otros virus zoonóticos como la Encefalitis Equina del Este y Encefalitis Equina Venezolana en los departamentos del Meta y Casanare, contiguo a este.

Role of Vascular Endothelial Growth Factor and Human Umbilical Vein Endothelial Cells in Designing An In Vitro Vascular-Muscle Cellular Model Using Adipose-Derived Stem Cells.

Objective Researchers have been interested in the creation of a favorable cellular model for use in vascular-muscle tissue engineering. The main objective of this study is to determine the myogenic effects of vascular endothelial growth factor (VEGF) and human umbilical vein endothelial cells (HUVECs) on adipose-derived stem cells (ADSCs) to achieve an in vitro vascular-muscle cellular model. Materials and Methods The present experimental research was conducted on two primary groups, namely ADSCs monoculture and ADSCs/HUVECs co-culture that were divided into control, horse serum (HS), and HS/VEGF differentiation subgroups. HUVECs were co-cultured by ADSC in a ratio of 1:1. The myogenic differentiation was evaluated using the reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence in different experimental groups. The interaction between ADSCs and HUVECs, as well as the role of ADSCs conditional medium, was investigated for endothelial tube formation assay. Results Immunofluorescence staining indicated that Tropomyosin was positive in ADSCs and ADSCs and HUVECs co-culture groups on HS and HS/VEGF culture medium. Furthermore, the MyHC2 gene expression significantly increased in HS and HS/VEGF groups in comparison with the control group (P<0.001). More importantly, there was a significant difference in the mRNA expression of this gene between ADSCs and ADSCs and HUVECs co-culture groups on HS/VEGF culture medium (P<0.05). Current data revealed that the co-culture of ADSCs and HUVECs could develop endothelial network formation in the VEGF-loaded group. Also, the ADSCs-conditioned medium improved the viability and formation of the endothelial tube in the HS and VEGF groups, respectively. ConclusionIt was concluded that ADSCs/HUVECs co-culture and dual effects of VEGF can lead to the formation of differentiated myoblasts in proximity to endothelial network formations. These in vitro cellular models could be potentially used in vascular-muscle tissue engineering implanted into organ defects where muscle tissue and vascular regeneration were required.

Blastocystis incidence, spontaneous clearance, persistence and risk factors in a rural community in Thailand: A prospective cohort study

Objective: To investigate the incidence, spontaneous clearance, persistence and risk factors of Blastocystis infection in a rural community in Thailand. Methods: In February 2016, a cohort study was conducted in a rural community of Thailand. Baseline information of intestinal parasitic infections and follow-up visits were conducted. Blastocystis infection was detected in stool samples examined with short-term in vitro cultivation using Jones’ medium supplemented with 10% horse serum. Participants were tested for Blastocystis infection at every study visit. Cox proportional hazard regression analysis was used to evaluate co-variates to determine the incidence of Blastocystis infection. Results: Of 733 enrolled participants, 57 (7.78%) were positive and 676 (92.22%) were negative for Blastocystis infection. All 676 participants were followed-up for Blastocystis infection between December 2016 and January 2018. In all, 52 of 482 cases (10.79%) comprised individual Blastocystis infection. The incidence density rates of Blastocystis infection, of spontaneous clearance of the disease and of persistent infection were 8.13, 63.14 and 38.70 per 1 000 person-months of follow-up, respectively. Our multivariate analysis revealed that opened defecation in fields or bushes (HR=4.8; 95% CI =2.1-10.4, P Conclusions: Our study indicates that the best way to prevent Blastocystis infection can be by maintaining good personal hygiene, hand washing after animal contact and improving sanitary facilities.

Extracellular xenogeneic hemoglobin suppresses the capacity for C2C12 myoblast myogenic differentiation

Functional characteristics of satellite cells (SCs) that act as myogenesis initiators and have emerged as a promising target for cell therapy, are dependent on their microenvironment. The aim of this study was to investigate the effect of cell-free hemoglobin, as a part of the microenvironment of SCs, on their functional characteristics. The C2C12 cell line served as the experimental model of SCs; hemoglobin isolated from porcine (PHb) and bovine (BHb) slaughterhouse blood served as the experimental model for extracellular hemoglobin. The proliferation rate of C2C12 cells was assessed by the MTT test, migration capacity by the scratch assay, and myogenic differentiation capacity by histochemical staining and RT-PCR analysis of the expression of genes specific for myogenic lineage. The effect of hemoglobin on the proliferation and migration of C2C12 cells was dependent on its concentration and the animal species it was isolated from, but the effect of BHb was more prominent. Both PHb and BHb decreased the expression levels of myogenin and muscle specific creatine kinase at a 10 ?M concentration. While PHb had no effect on the morphometric parameters of C2C12 myotubes, BHb modified the area and length of C2C12 myotubes cultivated in DMEM/2% horse serum and DMEM/10% fetal calf serum. While PHb and BHb had no effect on heme oxygenase 1 (Hmox1) expression, they stimulated the expression of hypoxia-inducible factor 1-alpha (Hif1?) at a concentration of 10 ?M. The mainly inhibitory effect of cell-free hemoglobin on myogenic differentiation suggests that it could be a relevant factor in the outcome of cell therapy of muscle injury.

Role of histone methylation enzyme EZH2 and VEGF165 in momymoya disease

Objective To study on the role of histone methylation enzyme enhancer of zeste homolog 2 (EHZ2) and vascular endothelial growth factor 165 (VEGF165) in momymoya disease. Methods The animal model of moyamoya disease was established by ear vein injection of horse serum in New Zealand rabbits. VEGF165 was over-expressed in situ by packaging lentivirus. Real-time quantitative PCR and Western Blot were used to detect the expression of VEGF165, EZH2 and H3K27me3 in the brain tissues of the animal models. Results Compared with the normal control group, the expression levels of mRNA and protein of EZH2 in the moyamoya disease model group were increased (EZH2 mRNA: P<0.01), and the level of histone H3K27me3 was increased. After overexpression of VEGF165 in the moyamoya disease model group, the expression levels of mRNA and protein of EZH2 was further increased (EZH2 mRNA: P<0.01), and the level of histone H3K27me3 was also increased. Conclusions EZH2 plays a certain role in the pathogenesis of moyamoya disease, and the expression of EZH2 is regulated by VEGF 165, which provides a theoretical basis for the study of the pathogenesis of moyamoya disease. Key words: Moyamoya disease; Enhancer of zeste homolog 2; Vascular endothelial growth factor 165

Early responses of in vitro cultured fish‐growing glochidia: The effects of taurine, lipids and sera onMargaritifera margaritifera

In vitro culture has great potential for the propagation of freshwater mussels in both commercial and conservation aquaculture. The use of in vitro techniques precludes the need for host fish, thus decreasing costs and increasing efficiency. However, protocols are still lacking for many species that grow substantially during the parasitic stage. In this study, we tested the effects of taurine addition, serum type and source of lipids on the survival rate and increase in length of Margaritifera margaritifera larvae during the initial stage of culture (first 11 days of exposure to media). Our results show that taurine has no significant effect on the early survival rate of glochidia; however, the possible importance of this amino acid in subsequent stages is discussed. The use of an emulsified lipid mixture instead of traditional fish oil showed significantly higher rates of survival. Finally, the addition of serum showed variable effects, with both horse serum and newborn calf serum having higher survival in trials using mussel populations from different sources. These findings can contribute to the invention or improvement of in vitro protocols for species growing during infection and at the same time show the potential that early survival assessments could have for the development of in vitro methods in species with long parasitic stages.

Establishment of an enzyme-linked immunosorbent assay for Getah virus infection in horses using a 20-mer synthetic peptide for the E2 glycoprotein as an antigen.

An enzyme-linked immunosorbent assay (ELISA) using a synthetic peptide for the E2 glycoprotein was developed for the serodiagnosis of Getah virus infection in horses. To identify an immunogenic epitope, a series of 20-mer peptides (n = 22) for the E2 protein was screened with pooled sera from horses infected with Getah virus. Peptide P11 (PTEEEIDMHTPPDIPDITLL) showed the strongest reaction. ELISA using P11 (E2-P11-ELISA) detected increased antibody levels in all seven experimentally infected horses and in five out of nine vaccinated horses. Out of 28 naturally infected horses, 25 were seronegative in their acute sera but turned seropositive in their convalescent sera. For the remaining three horses whose acute sera were seropositive, an endpoint method with serial dilutions detected a ≥ 4-fold increase in titer between paired sera. The concordance between E2-P11-ELISA and a virus-neutralization test in terms of seropositivity was assessed using a series of 220 horse sera, resulting in almost perfect agreement, with a kappa coefficient value of 0.865. E2-P11-ELISA had a sensitivity of 93.3% (95% CI 86.6-97.1%) and a specificity of 95.0% (95% CI 92.5-96.4%). This highly sensitive and specific E2-P11-ELISA should be useful for serodiagnosis of Getah virus infection in horses.

ASSESSMENT OF NITRITE LEVEL - NITRIC OXIDE DERIVATIVE - IN HORSES WITH INTESTINAL COLIC BY ESR SPECTROSCOPY

The pathogenesis of diseases of the digestive tract of horses, accompanied by the development of inflammation and oxidative stress, can be associated with a lack of nitrogen monoxide, which controls a variety of signaling pathways in the body. Nitrogen monoxide (NO) takes part in the work of the immune and nervous systems, its level directly determines the tone of all blood vessels and the course of many pathological processes. NO should be considered as one of the most important factors in the protection of the gastric and intestinal mucosa. The article describes in detail the method for determining the level of nitrite-nitric oxide metabolite - in diseases of horses with the symptom complex of “true” colic, and also presents the results of measurements of nitrite in the serum of healthy animals of two age groups: 1-5 years and 6-25 years and animals with pathology. The concentration of nitrite in blood serum in horses of the age group of 6-25 years was 3.42 ± 4.22 μM, while in young animals (1-5 years) the level of this indicator was 8.24 ± 5.42 μM, which is 2,4 times higher. A sharp decrease of nitrite was noted in all horses with intestinal diseases of 2.07 ± 0.9 μM, especially with meteorism 0.6 ± 0.4 μM and spastic colic 1.78 ± 0.5 μM. These facts determine the diagnostic and prognostic value of nitric oxide as a biomarker for the regulation of intestinal motility in normal and pathological conditions.