Discontinuous Density Gradient Research Articles

Genome-Wide Investigation of Pasteurella multocida Identifies the Stringent Response as a Negative Regulator of Hyaluronic Acid Capsule Production.

ABSTRACTPasteurella multocida is a Gram-negative capsulated bacterium responsible for a range of diseases that cause severe morbidity and mortality in livestock animals. The hyaluronic acid (HA) capsule produced by P. multocida serogroup A strains is a critical virulence factor. In this study, we utilized transposon-directed insertion site sequencing (TraDIS) to identify genes essential for in vitro growth of P. multocida and combined TraDIS with discontinuous density gradients (TraDISort) to identify genes required for HA capsule production and regulation in this pathogen. Analysis of mutants with a high cell density phenotype, indicative of the loss of extracellular capsule, led to the identification of 69 genes important for capsule production. These genes included all previously characterized genes in the capsule biosynthesis locus and fis and hfq, which encode known positive regulators of P. multocida capsule. Many of the other capsule-associated genes identified in this study were involved in regulation or activation of the stringent response, including spoT and relA, which encode proteins that regulate the concentration of guanosine alarmones. Disruption of the autoregulatory domains in the C-terminal half of SpoT using insertional mutagenesis resulted in reduced expression of capsule biosynthesis genes and an acapsular phenotype. Overall, these findings have greatly increased the understanding of hyaluronic acid capsule production and regulation in P. multocida.IMPORTANCE The bacterial pathogen P. multocida can cause serious disease in production animals, including fowl cholera in poultry, hemorrhagic septicemia in cattle and buffalo, atrophic rhinitis in pigs, and respiratory diseases in a range of livestock. P. multocida produces a capsule that is essential for systemic disease, but the complete mechanisms underlying synthesis and regulation of capsule production are not fully elucidated. A whole-genome analysis using TraDIS was undertaken to identify genes essential for growth in rich media and to obtain a comprehensive characterization of capsule production. Many of the capsule-associated genes identified in this study were involved in the stringent response to stress, a novel finding for this important animal pathogen.

Addressing challenges in the removal of unbound dye from passively labelled extracellular vesicles.

Studies of extracellular vesicles (EVs), their trafficking and characterization often employ fluorescent labelling. Unfortunately, little attention has been paid thus far to a thorough evaluation of the purification of EVs after labelling, although the presence of an unbound dye may severely compromise the results or even lead to wrong conclusions on EV functionality. Here, we systematically studied five dyes for passive EV labelling and meticulously compared five typical purification methods: ultracentrifugation (UC), ultracentrifugation with discontinuous density gradient (UCG), ultrafiltration (UF), size exclusion chromatography (SEC), and anion exchange chromatography (AEC). A general methodology for evaluation of EV purification efficiency after the labelling was developed and tested to select the purification methods for the chosen dyes. Firstly, we found that some methods initially lead to high EV losses even in the absence of the dye. Secondly, the suitable purification method needs to be found for each particular dye and depends on the physical and chemical properties of the dye. Thirdly, we demonstrated that the developed parameter Erp (relative purification efficiency) is a useful tool for the pre-screening of the suitable dye-purification method combinations. Additionally, it was also shown that the labelled EVs properly purified from the unbound dye may show significantly reduced contrast and visibility in the target application, e.g. in the live cell fluorescence lifetime imaging.

In-vitro propagation of pre-pubertal bovine Spermatogonial Stem Cell in prepation for transplantation

‌Backgroud: We investigated the expression of stem cell markers in in -vitro cultivated speramatogonial stem cells (SSCs) with the intention of developing a useful system to produce progeny of SSCs. Materials and Mehtods: Testes from 3, 5 and 7 months old calves were utilized to examine isolation, purity and in –vitro propogation of SSCs. Cells were isolated and purified with two-stemp enzymatic digestion combined with centrifugal separation on discontinuous Percoll density gradient layers. Findings: Cell propagation and SSCs marker expression were determined at 5, 10 and 15 days post-culture. Immunostaining in conjunction with transcrioption based marker expression of cultured cells showed that stem cell markers (DBA, UCHL1, Oct4 and Sox2) were expressed in SSCs. Conclusion: The results demonstrated marker expression of bull spermatogonial stem cells and showed that prior to transplantation in-vitro culturing of bull SSCs is implementable.

A modified density gradient proteomic-based method to analyze endolysosomal proteins in cardiac tissue

SummaryThe importance of lysosomes in cardiac physiology and pathology is well established, and evidence for roles in calcium signaling is emerging. We describe a label-free proteomics method suitable for small cardiac tissue biopsies based on density-separated fractionation, which allows study of endolysosomal (EL) proteins. Density gradient fractions corresponding to tissue lysate; sarcoplasmic reticulum (SR), mitochondria (Mito) (1.3 g/mL); and EL with negligible contamination from SR or Mito (1.04 g/mL) were analyzed using Western blot, enzyme activity assay, and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis (adapted discontinuous Percoll and sucrose differential density gradient). Kyoto Encyclopedia of Genes and Genomes, Reactome, Panther, and Gene Ontology pathway analysis showed good coverage of RAB proteins and lysosomal cathepsins (including cardiac-specific cathepsin D) in the purified EL fraction. Significant EL proteins recovered included catalytic activity proteins. We thus present a comprehensive protocol and data set of guinea pig atrial EL organelle proteomics using techniques also applicable for non-cardiac tissue.

Effect of High-Density Lipoprotein from Healthy Subjects and Chronic Kidney Disease Patients on the CD14 Expression on Polymorphonuclear Leukocytes.

In uremic patients, high-density lipoprotein (HDL) loses its anti-inflammatory features and can even become pro-inflammatory due to an altered protein composition. In chronic kidney disease (CKD), impaired functions of polymorphonuclear leukocytes (PMNLs) contribute to inflammation and an increased risk of cardiovascular disease. This study investigated the effect of HDL from CKD and hemodialysis (HD) patients on the CD14 expression on PMNLs. HDL was isolated using a one-step density gradient centrifugation. Isolation of PMNLs was carried out by discontinuous Ficoll-Hypaque density gradient centrifugation. CD14 surface expression was quantified by flow cytometry. The activity of the small GTPase Rac1 was determined by means of an activation pull-down assay. HDL increased the CD14 surface expression on PMNLs. This effect was more pronounced for HDL isolated from uremic patients. The acute phase protein serum amyloid A (SAA) caused higher CD14 expression, while SAA as part of an HDL particle did not. Lipid raft disruption with methyl-β-cyclodextrin led to a reduced CD14 expression in the absence and presence of HDL. HDL from healthy subjects but not from HD patients decreased the activity of Rac1. Considering the known anti-inflammatory effects of HDL, the finding that even HDL from healthy subjects increased the CD14 expression was unexpected. The pathophysiological relevance of this result needs further investigation.

Dual-process brain mitochondria isolation preserves function and clarifies protein composition

The brain requires continuously high energy production to maintain ion gradients and normal function. Mitochondria critically undergird brain energetics, and mitochondrial abnormalities feature prominently in neuropsychiatric disease. However, many unique aspects of brain mitochondria composition and function are poorly understood. Developing improved neuroprotective therapeutics thus requires more comprehensively understanding brain mitochondria, including accurately delineating protein composition and channel-transporter functional networks. However, obtaining pure mitochondria from the brain is especially challenging due to its distinctive lipid and cell structure properties. As a result, conflicting reports on protein localization to brain mitochondria abound. Here we illustrate this problem with the neuropsychiatric disease-associated L-type calcium channel Cav1.2α1 subunit previously observed in crude mitochondria. We applied a dual-process approach to obtain functionally intact versus compositionally pure brain mitochondria. One branch utilizes discontinuous density gradient centrifugation to isolate semipure mitochondria suitable for functional assays but unsuitable for protein localization because of endoplasmic reticulum (ER) contamination. The other branch utilizes self-forming density gradient ultracentrifugation to remove ER and yield ultrapure mitochondria that are suitable for investigating protein localization but functionally compromised. Through this process, we evaluated brain mitochondria protein content and observed the absence of Cav1.2α1 and other previously reported mitochondrial proteins, including the NMDA receptor, ryanodine receptor 1, monocarboxylate transporter 1, excitatory amino acid transporter 1, and glyceraldehyde 3-phosphate dehydrogenase. Conversely, we confirmed mitochondrial localization of several plasma membrane proteins previously reported to also localize to mitochondria. We expect this dual-process isolation procedure will enhance understanding of brain mitochondria in both health and disease.

Isolation and comparative proteomic analysis of mitochondria from the pulp of ripening citrus fruit

Mitochondria are crucial for the production of primary and secondary metabolites, which largely determine the quality of fruit. However, a method for isolating high-quality mitochondria is currently not available in citrus fruit, preventing high-throughput characterization of mitochondrial functions. Here, based on differential and discontinuous Percoll density gradient centrifugation, we devised a universal protocol for isolating mitochondria from the pulp of four major citrus species, including satsuma mandarin, ponkan mandarin, sweet orange, and pummelo. Western blot analysis and microscopy confirmed the high purity and intactness of the isolated mitochondria. By using this protocol coupled with a label-free proteomic approach, a total of 3353 nonredundant proteins were identified. Comparison of the four mitochondrial proteomes revealed that the proteins commonly detected in all proteomes participate in several typical metabolic pathways (such as tricarboxylic acid cycle, pyruvate metabolism, and oxidative phosphorylation) and pathways closely related to fruit quality (such as γ-aminobutyric acid (GABA) shunt, ascorbate metabolism, and biosynthesis of secondary metabolites). In addition, differentially abundant proteins (DAPs) between different types of species were also identified; these were found to be mainly involved in fatty acid and amino acid metabolism and were further confirmed to be localized to the mitochondria by subcellular localization analysis. In summary, the proposed protocol for the isolation of highly pure mitochondria from different citrus fruits may be used to obtain high-coverage mitochondrial proteomes, which can help to establish the association between mitochondrial metabolism and fruit storability or quality characteristics of different species and lay the foundation for discovering novel functions of mitochondria in plants.

Time intervals between semen production, initiation of analysis, and IUI significantly influence clinical pregnancies and live births.

Does IDEF mapping help monitor the technical process of IUI and explore the potential improvements which might contribute to increased pregnancy and live birth rates? Retrospective analysis of 1729 homologous IUI cycles of couples attending a fertility clinic in a university hospital setting. Standardized conventional semen parameters were analyzed and the semen samples prepared via discontinuous density gradient centrifugation. There was no significant association between sperm concentration, motility and morphology (analysis phase), and pregnancy outcome. Only female and male ages were significantly associated with the pregnancy outcome. There was a significant difference in the odds on clinical pregnancies and live births when analysis was ≤ 21min initiated, and < 107min between sample production and IUI, adjusted for male and female age. Adjusting for the couple's age, we could show that time intervals between semen production and analysis and IUI when kept low significantly influenced clinical pregnancies and live births.

Regulation of the plasminogen activator activity and inflammatory environment via transforming growth factor-beta regulation of sperm in porcine uterine epithelial cells

The aims of the present study were to confirm that regulation of the PA and environment via TGF-β regulation of sperm by Percoll-separated in porcine uterine epithelial cells. And, it was performed to identify the cytokines (TGF-β1, 2 and 3, TGF-β receptor1 and 2; interleukin, IL-6, IL-8) and PA-related genes (urokinase-PA, uPA; tissue- PA, tPA; PA inhibitor, PAI; uPA-receptor, uPAR) by spermatozoa. The experiment used porcine uterus epithelial cells (pUECs) and uterine tissue epithelial cells, Boar sperm were separated by discontinuous Percoll density gradient (45/90%), and tissues were co-incubated with spermatozoa, followed by real-time PCR. PA activity was measured of sperm by discontinuous Percoll density gradient (45/90%) for 24 hours. To measure viability and acrosome damage of sperm double stained propidium iodide (PI) and SYBR- 14 or FITC-PNA were used. In results, binding ratio of Percoll-separated sperm was found no differences, but sperms isolated from 90% Percoll layer reduced PA activity (p < 0.05). when co-cultured sperm selected Percoll in porcine uterus tissues epithelial cells, 90% layer sperm increased TGF-β R1, contrastively tPA and PAI-1 in comparison with control (p < 0.05). 45% sperm was decreased the expression of uPA (p < 0.05). TGF-β decreased PA activity in the supernatant collected from pUECs (p < 0.05). Especially, The group including uPA, PAI-1 were induce sperm intact, while it was reduced in sperm damage when compared to control (p < 0.05). Also, there was no significant difference group of tPA and tPA+I in the dead sperm and acrosome damage compared to control. The expression of tPA and PAI showed a common response. Percoll-separated spermatozoa in 90% layer reduced tPA and IL-related gene mRNA expression. Thus, Percoll-sparated sperm in 90% layer show that it can suppress inflammation through increased expression of TGF-β and downregulation of PA and IL in epithelial cells compared to 45% layer Percoll.

Discontinuous density gradient sperm processing concentrates normal morphological spermatozoa better in the yield: An observational study

Aim: To evaluate the efficiency of Discontinuous Double Density Gradient Sperm Preparation over Swim up preparation in concentrating normomorphic spermatozoa in the yield. Materials and Methods: 50 semen samples obtained from patients visiting the NIMS Fertility and Research Center were analyzed for normal morphology prior to processing, then the sample was aliquoted in to two equal halves, each of it was processed by Swim up and Discontinuous Density Gradient preparation simultaneously. Yields obtained from the both methods were tested for normal morphology of sperms using Diff quick staining method and micrometry according to WHO Manual 2010. Results: Analyzed neat semen samples showed mean Sperm concentration of 38.2 m/ml and mean sperm morphology of 7.54%. Post processing yield from Swim up preparation had mean concentration of 30.08 m/ml and normal morphology of 8.66% (p-value 0.05) where as the yield from discontinuous density Gradient preparation showed up a mean concentration of 32.2 m/ml and normal morphology of 9.8% (p-value 0.001) Conclusion: From the study it was evident that the Discontinuous Density Gradient Sperm Preparation method has a clear edge over Swim up preparation in concentrating normal morphological spermatozoa in the yield. Keywords: Sperm Processing, Normal morphology, Swim up technique, Density gradient technique.

Diagnostic performance of discontinuous density gradient centrifugation for estimating human semen quality.

Semen analysis plays an important role in the diagnosis of male infertility. However, many studies have demonstrated that the current methods of semen analysis are inefficient for assessing male fertility. To test whether prior discontinuous density gradient centrifugation (DDCG) improves the performance of semen analysis in diagnosing male infertility. Infertile men and fertile men were recruited from the clinic. Pre- and post-DDGC values for the semen parameters of sperm concentration, total sperm number, percent total motility, percent progressive motility, percent normal sperm morphology, and sperm DNA fragmentation rate were compared. A total of 528 men (252 infertile men and 276 fertile men) were enrolled in the present study. After DDGC, sensitivity was significantly increased for sperm concentration, total sperm number, and sperm morphology (P<.01); specificity was significantly increased for progressive motility and sperm morphology (P<.01); and diagnostic accuracy was significantly improved for all of these parameters (area under the curve (AUC): P<.01). Total motility and sperm DNA fragmentation rate exhibited no obvious change in sensitivity, specificity or accuracy after DDGC (all P>.01). For the combination of all these semen parameters, diagnostic accuracy improved significantly after DDGC (AUC: P<.01). In a multiple regression analysis, only sperm morphology and sperm DNA fragmentation rate had P values less than 0.05 before DDGC, whereas all parameters except total sperm number contributed to the equation after DDGC. DDGC is a mature, standardized procedure for clinical commonly used to optimize spermatozoa. The diagnostic accuracy of semen analysis was significantly improved after DDGC, which indicated that assessing "functional spermatozoa" might be a more suitable method for semen analysis than the WHO 2010 criteria. Assessing semen parameters after DDGC might improve their diagnostic accuracy for male infertility.

SIGNIFICANT IMPROVEMENT IN SPERM MOTILITY IN SEMEN SAMPLES FROM ASTHENOZOOSPERMIC MEN BY USING CONTROLLED INCUBATION PROTOCOLS WITH SINGLE CONTINUOUS CULTURE MEDIUM (CSCM®)

To test different incubation periods and to assess an appropriate commercially available medium to improve the performance of semen samples from asthenozoospermic men. Prospective study Semen samples (progressive motility <32%) from 54 asthenozoospermic patients (men aged 21-45 years) were used in the study after signed the informed consent form. Fresh semen samples separated by a discontinuous density gradient or by simple washing, were incubated in different culture media and then analyzed. Four different culture media were studied: a) Single Continuous Culture Medium (CSCM®) containing 10% Human Serum Albumin (HSA®); b) Sperm Rinse medium (SR®) supplemented with fructose and carnitine; c) In Vitro Fertilization Medium (G-IVF®) with fructose and 15% Synthetic Serum Substitute (SSS®) and; d) a control group in which Human Tubal Fluid (HTF®) supplemented with 15% SSS® was used. The last one represents the most commonly used medium by labs worldwide. Samples were then incubated at 37°C in a 5% CO2 atmosphere for 2-hour (defined as the optimal incubation time in previous study). Sperm parameters were then analyzed both in fresh samples and after incubation in different media: manual sperm analysis; motility via Sperm Computer Analysis (SCA®); sperm chromatin integrity via Sperm Chromatin Structural Assay (SCSA®); Reactive Oxygen Species level (ROS) by chemiluminescent detection and mitochondrial activity by staining with 3.3′-diaminobenzidine (DAB). Data were analyzed using the IBM SPSS software. Analysis of variance (ANOVA) and independent student t-tests were used to evaluating statistical significance (P<0.05). CSCM® medium demonstrated a significant increase in post-incubation motility (progressive motility = 24.55±13.97%; total motility = 41.17±19.06%) compared to pre-incubation parameters (progressive motility = 8.33±6.82; total motility = 26.61±18.10%; P<0.025). For all other culture media, no significant differences were observed in seminal parameters. Asthenozoospermic men may have significant improvement in their semen samples in the andrology lab after a 2-hours incubation in CSCM®+HSA® medium. This technique has proven to be very attractive because of its efficiency, low cost and ease of reproduction.

Mass-Added Density Modulation for Sorting Cells Based on Differential Surface Protein Levels.

Cell sorting is a powerful tool in basic research and therapeutic enrichment. However, common cell sorting methods, such as fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) have significant limitations, such as generally low cell yields or restriction to binary separation, respectively. To address these limitations, we developed a two-step cell sorting method called mass-added density centrifugation (MADC) to enable nonbinary separation of large cell numbers based on surface protein levels. In the first MADC step (mass-adding), antibody-directed massive microparticles bind target surface proteins to modulate single-cell density proportionally to target protein level. Second, microparticle-laden cells are subjected to discontinuous density gradient centrifugation, whereby they separate into discrete density bands which can be isolated for downstream use. MADC will prove especially advantageous for obtaining sufficient cell numbers for protein analyses from large source populations, and it is a fast process that can facilitate live cell enrichment for therapies that require tens of millions of cells. Here, we demonstrate MADC's utility for both live and fixed cell sorts of multiple cell types based on abundance of an example target protein, CD44. CD44 quantity in separated cell groups was assayed with western blots and correlated with modulated cell density. This novel sorting method enables rapid, nonbinary isolation of large quantities of cells based on surface protein levels and should prove useful in both basic science and therapeutic applications. © 2020 International Society for Advancement of Cytometry.

X-sperm Enrichment of Bovine Semen by Percoll Density Gradient Method and Its Effect on Semen Quality, Sex Ratio and Conception Rate

In cattle, sex selection has a significant economic impact when it improves herd capacity of milk or meat production and is commonly used for the production of calves of the desired gender. New separation techniques which present both better accuracy and low costs are necessary. Density gradient centrifugation might be an approach to sexing spermatozoa because of the additional DNA content and volume of X-bearing sperm head. The present study was designed to apply the modified percoll discontinuous density gradient for X-sperm enrichment in cross bred bovine semen. We attempted to amplify the level of X-sperm enrichment by using double centrifugation in percoll gradient. In addition, effect of percoll density gradient on fertility of semen was also evaluated at three stages; before centrifugation (stage I), after 1st centrifugation (stage II) and after 2nd centrifugation (stage III) respectively and ultrasonography was carried out to confirm the sex of fetus at 60-90 days post AI. Semen was collected from two cross bred bovine bulls and 34 cows were inseminated with liquid X-sperm enriched semen after double centrifugation in percoll gradient. 22 cows were inseminated with non-sexed fresh liquid semen (control). Results showed that the volume, progressive motility, live sperms, concentration and HOST reactive sperms significantly (P is less than 0.05) decreased at stage II and stage III compared to stage I. However, no significant (P is greater than 0.05) change was observed in pH and mass motility of semen at all three stages. There was non-significant effect on per cent spermatozoa with intact acrosomal membrane and fully damaged acrosomal membrane at all three stages however, partially damaged acrosomal membrane sperms were significantly (P is less than 0.05) decreased at Stage II and Stage III compared to Stage I. The conception rate significantly (P is less than 0.05) decreased with X-enriched semen (41.17%) compared to non-sexed liquid semen (59.09%). The female sex ratio of calves significantly (P is less than 0.05) increased to 66.66% with X- sperm enriched semen compared to the male percentage of 33.33% with sexed semen and female percentage of 46.15% with non-sexed semen.

Assessment of peripheral blood neutrophil respiratory burst, phagocytosis and apoptosis in obese non-insulin dysregulated horses

Obesity is a highly prevalent condition in horses. Dysfunctional neutrophil activity has been reported in metabolically healthy obese humans, but minimal data exist regarding horses. The present study evaluated the effect of obesity on apoptosis, phagocytosis and oxidative burst activity of peripheral blood neutrophils from lean and obese non-insulin dysregulated horses. Seven lean (BCS, body condition score 4–6/9) and five obese (BCS 8–9) horses were enrolled in the study. All animals underwent two metabolic tests (OGT, oral glucose test; IRT, insulin response test) before their selection to ensure their metabolic status (non-insulin dysregulated). A single blood sample was obtained from each horse, and a discontinuous density gradient was carried out to isolate neutrophils. Phagocytosis, apoptosis and reactive oxygen species (ROS) production assays were performed for each animal. All statistical analyses were performed with unpaired two-tailed t-tests. Results indicate that neutrophils from obese non-insulin dysregulated horses have a significantly increased ROS production (P < .0001), with no changes observed on phagocytosis (P > .05) or apoptosis (P > .05) when compared to the control group. In conclusion, our results demonstrate that obesity per se, in absence of other endocrine disorders, alters neutrophil reactive oxygen species production. More research is needed to understand the role of obesity on the equine immune system of horses, and its role in the development of endocrine disorders.

Variability in the cleavage of exosome-associated transferrin receptor questions the utility of clinically useful soluble transferrin receptor assays for dogs, cats, and horses

Whole transferrin receptor (TfR) is present in reticulocyte exosomes. Soluble transferrin receptor (sTfR) is cleaved from whole TfR in human plasma, with the remnant cytoplasmic domain (cTfR) remaining membrane associated. In humans, sTfR is a biomarker that can detect iron deficiency in the presence of inflammatory disease. This condition is still a diagnostic dilemma in veterinary species. We aimed to (1) confirm the presence of exosomes and exosome-associated TfR in the serum of dogs, cats, and horses; and (2) to assess and compare the proportion of cTfR to total (cTfR + whole) in exosomal membranes of healthy and diseased dogs and cats and in healthy horses to indirectly predict their anticipated levels of circulating sTfR. We used discarded serum and whole blood samples from canine and feline patients, separated into healthy and diseased groups based on the health status of each patient, and healthy equine participants from a previous study. Ultracentrifugation, followed in some experiments by OptiPrep discontinuous density gradient fractionation, was used to isolate exosomes. Exosomes and associated TfR were identified using TEM and Western blot for TfR, respectively. Densitometry tracings of Western blots of serum exosomes were used to measure the proportion of cTfR to total TfR. Extracellular vesicles compatible with exosomes were successfully isolated and expressed TfR. The proportion of cTfR in dogs was greater than 50%, indicating that a majority of the whole TfR was cleaved to produce sTfR (and remnant cTfR). There was significant interindividual variation and no significant difference between healthy and diseased animals. The proportion of cTfR in cats was very low at 11%, indicating that very little sTfR was likely produced. There was a small yet significant difference between healthy and diseased cats. Healthy horses do not appear to cleave exosome-associated TfR. Diagnosis of iron deficiency in the presence of inflammatory disease remains a challenge in veterinary medicine. Our results indicate that TfR is poorly or unpredictably cleaved in veterinary species, revealing that there are species differences in exosomal TfR handling. These data suggest that development of an assay for the detection and quantification of sTfR in the species investigated may not be warranted.

Comparative Efficacy of PercollTM Discontinuous Density Gradient Centrifugation and Glass Wool Filtration Techniques for Spermatozoa Selection in Buffalo (Bubalus bubalis)

Comparative Efficacy of PercollTM Discontinuous Density Gradient Centrifugation and Glass Wool Filtration Techniques for Spermatozoa Selection in Buffalo (Bubalus bubalis)

Isolation, culture, and downstream characterization of primary microglia and astrocytes from adult rodent brain and spinal cord

BackgroundNeuroimmunologists aspire to understand the interactions between neurons, microglia, and astrocytes in the CNS. To study these cells, researchers work with either immortalized cell lines or primary cells acquired from animal tissue. Primary cells reflect in vivo characteristics and functionality compared to immortalized cells; however, they are challenging to acquire and maintain. New MethodEstablished protocols to harvest primary glia use neonatal rodents, here we provide a method for simultaneously isolating microglia and astrocytes from brain and/or spinal cord from adult rodents. We utilized a discontinuous percoll density gradient enabling easy discrimination of these cell populations without enzymatic digestion or complex sorting techniques. ResultsWe found cells isolated from the percoll interface between 70 %−50 % were microglia, as they express ionizing calcium-binding adaptor molecule 1 (Iba1) in immunocytochemistry and CD11bhi and CD45lo using flow cytometry. Isolated cells from the 50 %−30 % interface were astrocytes as they express glial fibrillary acidic protein (GFAP) in immunocytochemistry and Glutamate aspartate transporter (GLAST)-1 using flow cytometry. Cultured microglia and astrocytes showed a functional increase in IL-6 production after treatment of lipopolysaccharide (LPS). Comparison with Existing MethodsOur method allows for rapid isolation of both microglia and astrocytes in one protocol with relatively few resources, preserves cellular phenotype, and yields high cell numbers without magnetic or antibody sorting. ConclusionHere we show a novel, single protocol to isolate microglia and astrocytes from brain and spinal cord tissue, allowing for culturing and other downstream applications from the cells of animals of various ages, which will be useful for researchers investigating these two major glial cell types from the brain or spinal cord of the same rodent.

A2E Distribution in RPE Granules in Human Eyes.

A2E (N-retinylidene-N-retinylethanolamine) is a major fluorophore in the RPE (retinal pigment epithelium). To identify and characterize A2E-rich RPE lipofuscin, we fractionated RPE granules from human donor eyes into five fractions (F1–F5 in ascending order of density) by discontinuous sucrose density gradient centrifugation. The dry weight of each fraction was measured and A2E was quantified by liquid chromatography/mass spectrometry (LC/MS) using a synthetic A2E homolog as a standard. Autofluorescence emission was characterized by a customer-built spectro-fluorometer system. A significant A2E level was detected in every fraction, and the highest level was found in F1, a low-density fraction that makes up half of the total weight of all RPE granules, contains 67% of all A2E, and emits 75% of projected autofluorescence by all RPE granules. This group of RPE granules, not described previously, is therefore the most abundant RPE lipofuscin granule population. A progressive decrease in autofluorescence was observed from F2 to F4, whereas no autofluorescence emission was detected from the heavily pigmented F5. The identification of a novel and major RPE lipofuscin population could have significant implications in our understanding of A2E and lipofuscin in human RPE.

In vitro Antisickling and Sub Chronic Toxicity Studies of the Ethanol Extract of Cnidoscolus aconitifolius in Wistar Rats

Objective: The current study investigated the mechanism(s) of action of the antisickling property of Cnidiscolus aconitifolius ethanol extract and determined its histopathological effects on the liver, kidney and spleen of Wistar rats. Methods: The antisickling properties of C. aconitifolius (CA) ethanol extract was tested at different concentrations, using the inhibitory and reversal models. Its effect on the density and mean cell volume of Hb S red blood cells was evaluated using the discontinuous density gradient assay. Ciklavit®, an antisickling nutritional supplement was employed as positive while phosphate buffered saline (pH 7.0) was the negative control. Changes in percentage Red Cell Count (RBC) and Mean Cell Volume (MCV) in each density fraction were determined. Quantification of extracellular potassium leakage of RBC before and after incubation with CA extract was also conducted. CA was evaluated for its acute toxicity (LD50) while sub-acute toxicity was evaluated by oral administration to Wistar rats for 28 days at 125, 250 and 500 mg/kg body weight. Rats were anaesthetized on the 29th day and blood collected through cardiac puncture for hematological analysis. The liver, kidney and spleen of the rats were harvested for histopathological examinations. Results: The results showed that the percentage of dense cells in blood treated with CA was reduced significantly (p<0.05) compared to positive control. CA increased MCV of cells and inhibited basal potassium efflux with a rightward shift in the dose-response curve. The median lethal dose (LD50) of CA was 3808 mg/kg; neither mortality nor structural or histological alterations were observed in the liver, kidney and spleen at the administered dose. The extract of CA enhanced erythropoiesis. Conclusion: C. aconitifolius extract prevented potassium efflux and reduced red cell density in vitro. The CA crude extract was safe for consumption and increased the production of red blood cells in vivo.