Percoll Gradient Centrifugation Research Articles

Endonuclease G is dispensable for sperm mitochondrial DNA elimination during spermatogenesis in mice.

Maternal inheritance of mitochondrial DNA (mtDNA) is a widespread phenomenon in eukaryotes. Our earlier research indicated that sperm mtDNA is removed prior to fertilization in mice, and Endonuclease G (ENDOG) orchestrates the degradation of sperm mitochondria in Caenorhabditis elegans. However, the mechanisms underlying sperm mtDNA disposal in mammals remain poorly understood. To investigate the potential role of ENDOG in sperm mtDNA elimination, we created Endog knockout (Endog-/-) mice. Our findings revealed that Endog-/- mice maintained normal spermatogenesis and fertility. Most strikingly, we detected no substantial discrepancy in sperm mtDNA copy number between Endog-/- and control mice. Furthermore, we noted that sperm mtDNA copy numbers were unchanged in both less motile and motile sperm isolated by Percoll gradient centrifugation from Endog-/- and control mice. Taken together, our results indicate that ENDOG is not essential for spermatogenesis or the elimination of sperm mtDNA in mice.

Flow Cytometry Characterization and Analysis of Glial and Immune Cells from the Spinal Cord

Several protocols have been developed with the aim of characterizing glial and immune cells from the central and peripheral nervous systems. However, a small number of these protocols have demonstrated the ability to yield satisfactory results following conventional isolation. Considering this necessity and the difficulties encountered in enzymatic and bead isolation, our work proposes a method for the isolation of glial and immune cells from the spinal cord utilizing a Percoll gradient. For this purpose, C57BL/6J spinal cords were dissected, and the lumbar intumescence was dissociated and subjected to a Percoll gradient centrifugation (70%, 50%, 37%, and 10%). Each layer was then separated and labeled for astrocytes (anti-GFAP, TNF-α, IFN-γ, IL-10, IL-4), microglia (anti-CD45, CD11b, CD206, CD68, TNF-α, IFN-γ), and lymphocytes (anti-CD3, CD4, IFN-γ, IL-4). The gate detections were mathematically performed by computational analysis utilizing the K-means clustering algorithm. The results demonstrated that astrocytes were concentrated at the Percoll 10/37 interface, microglia at the Percoll 37/50 layer, and lymphocytes at the Percoll 50/70 layer. Our findings indicate that astrocytes in healthy animals are putative of the A1 profile, while microglia and lymphocytes are more frequently labeled with M1 and Th1 markers, suggesting a propensity towards inflammatory responses. The computational method enabled the semi-autonomous gate detection of flow cytometry data, which might facilitate and expedite the processing of large amounts of data.

P086 Identification of a Novel Immunometabolic Target and Agonist for PLXDC2 for Amelioration of DSS Colitis Model in Mice

Abstract Background PLXDC2 is expressed on the cell surface of macrophage, dendritic and specific mesenchymal and epithelial cells whose activation shifts cellular metabolism and reduces oxidative stress to rebalance the immune response and decrease inflammation. It’s role in IBD pathogenesis is unknown. PLXDC2 activation improves disease severity in rheumatoid arthritis models1, while loss of PLXDC2 exacerbates the severity of disease in DSS colitis2. PX-04 is a novel, first-in-class, orally active PLXDC2 agonist. Here, we report on the consequences of activating PLXDC2 using PX-04 in an acute DSS colitis model. Methods Mice were given DSS in drinking water for seven days to induce disruption of the epithelial layer. At project initiation, mice were 8 weeks of age and began once daily dosing with 20mg/kg of PX-04 24 hours after being placed on DSS. Mice were weighed and scored daily for 15 symptoms of disease (weight loss, diarrhea, rectal bleeding, rectal inflammation, pain, & overall behavior). Colons were collected and digested, and the resultant cellular suspensions were filtered to produce single cell suspensions. Colonic Lamina Propria Immune cells were isolated by Percoll gradient centrifugation. Cells were labeled with mixtures of extracellular (CD45, CD3, CD4, CD8, CD19, NK1.1, CD25, F4/80, CD11b, Gr1, CX3CR1, CD64) and intracellular (Tbet, RORγT, FOXP3, IFNγ, IL17, IL10, TNFα) antibodies in a sequential live staining in 96-well plates. Data was acquired using a FACS Celesta flow cytometer with FACSDiva software. Results Oral PX-04 treatment decreased the cumulative disease activity of mice challenged with DSS (FIG 1A). Immunologically, PX-04 affected both the adaptive and innate immune responses. First, PX-04 greatly decreased Th17 cells in the colon, while providing a slight increase to regulatory CD4+ T cells (FIG. 1C). A lower proportion of Natural Killer (NK) cells (FIG. 7D) produced IFNγ. Meanwhile, the proportion of TNF producing dendritic cells was decreased by PX-04 treatment. Conclusion Conclusion: PLXDC2 is expressed in cells relevant to IBD pathogenesis. PX-04, which binds to and activates PLXDC2, effectively decreases levels of effector T cells and myeloid cells, as well as overall disease severity in acute DSS colitis, warranting further investigation. 1Tubau-Juni et al. J Immunol 206(Supp) 2Tubau-Juni et al. Sci Rep 10(1)

P-281 Single-cell RNA sequencing analysis of granulosa cells in preovulatory follicles in normal ovarian reserve patients undergoing progestin-primed ovarian stimulation and GnRH-antagonist

Abstract Study question Could the difference in controlled ovarian stimulation protocol between progestin-primed ovarian stimulation and GnRH-antagonist induce change in the expression genes of human granulosa cells? Summary answer The mitochondrial DNA (mtDNA) gene expression of granulosa cells in patients who underwent progestin-primed ovarian stimulation was significantly higher than the GnRH-antagonist. What is known already The progestin-primed ovarian stimulation (PPOS) protocol has attracted attention and many studies have reported similar pregnancy outcomes for both PPOS and the GnRH-antagonist (GnRH-ant). However, our previous study demonstrated that pregnancy rate was significantly lower in PPOS compared to GnRH-ant (38th Hybrid Annual Meeting of the ESHRE, O-130). In this study, we evaluated the gene expression of granulosa cells (GCs) which affects reproductive outcome. Many studies have shown a close association between the mitochondrial status of GCs and embryo’s quality. To date, the change in gene expression of GCs with different controlled ovarian stimulation protocols have not been investigated. Study design, size, duration From November 2021 to January 2022, all samples were obtained from the patients who provided written informed consent before the inclusion and underwent controlled ovarian stimulation cycles at a single-institution. Single cell RNA-seq (scRNA-seq) was performed on 2,224 cells corresponding to GCs of the MII-oocyte follicles from a total of 16 patients with normal ovarian reserve (aged < 40 years, AMH ≧1.1 ng/mL) undergoing PPOS with chlormadinone acetate (n = 8), or GnRH-ant with cetrorelix (n = 8). Participants/materials, setting, methods The GCs were obtained by follicular aspiration from 16 patients undergoing oocyte retrieval. The follicular fluid (FF) from each follicle was collected and all FF were mixed together in MII-oocyte for sequencing. The GCs were isolated from the blood cells and cellular debris using Percoll gradient centrifugation. Samples for sequencing were prepared using Chromium Next GEM Single Cell V(D)J Reagent Kits. Libraries were sequenced in DNBSEQ-G400, and reads processed with CellRanger and visualized with CellxGene. Main results and the role of chance Two population of GCs were used in this investigation: PPOS and GnRH-ant of MII-oocyte. After the quality control, 1,197 cells for PPOS and 1,027 cells for GnRH-ant, respectively, were analyzed. The GCs of MII-oocyte were clustered into 6 groups based on Leiden algorithm and visualized by UMAP analysis. There was no significant difference in the gene expression and cell distribution in each cluster. In the two population of GCs, we identified 12 differentially expressed genes (DEGs). Interestingly, we observed significant increase in expression of mtDNA genes in PPOS of MII-oocyte as compared to GnRH-ant of MII-oocyte. The data were confirmed by qRT-PCR analysis. Limitations, reasons for caution Our conclusions are limited due to the inclusion of Asian patients at a single-institution. The results need to be validated across different centers and other ethnicities. Due to the small sample size, these results should prompt further study to confirm our findings. Wider implications of the findings These findings describe, for the first time, the DEGs of GCs investigated in different COS protocols at single-cell level. This study suggests that progestin for PPOS induce elevation of the mt DNA gene expression of GCs which may be associated with reproductive outcomes. Trial registration number Not applicable

Synchronization isolation method for multiple types of cells from mouse liver

Objective: To explore a simple and feasible method for the isolation and purification of hepatocytes, hepatic stellate cells (HSC), and lymphocytes from mice. Methods: The cell suspension was obtained from male C57bl/6 mice by hepatic perfusion through the portal vein digestion method and then isolated and purified by discontinuous Percoll gradient centrifugation. Trypan blue exclusion was used to determine cell viability. Glycogen staining, cytokeratin 18, and transmission electron microscopy were used to identify hepatic cells. Immunofluorescence was used to detect α-smooth muscle actin combined with desmin in HSCs. Flow cytometry was used to analyze lymphocyte subsets in the liver. Results: After isolation and purification, about 2.7×10(7) hepatocytes, 5.7×10(5) HSCS, and 4.6×106 hepatic mononuclear cells were obtained from the liver of mice with a body weight of about 22g. The cell survival rate in each group was > 95%. Hepatocytes were apparent in glycogen deposited purple-red granules and cytokeratin 18. Electron microscopy showed that there were abundant organelles in hepatocytes and tight junctions between cells. HSC had expressed α-smooth muscle actin and desmin. Flow cytometry showed hepatic mononuclear cells, including lymphocyte subsets such as CD4, CD8, NKs, and NKTs. Conclusion: The hepatic perfusion through the portal vein digestion method can isolate multiple primary cells from the liver of mice at once and has the features of simplicity and efficiency.

Proteomic changes in human spermatozoa during in vitro capacitation and acrosome reaction in normozoospermia and asthenozoospermia.

The cellular and molecular mechanisms of the events that help spermatozoa acquire their fertilizing capability during capacitation and acrosome reaction are not completely understood. This study was performed with a postulation that the identification of sperm proteins and their changes during in vitro capacitation and acrosome reaction will unravel unknown molecular aspects of fertilization that impact male fertility. Spermatozoa collected from sequential conditions, that is, separation of ejaculated spermatozoa by Percoll gradient centrifugation, in vitro capacitation, and acrosome reaction were processed for tandem mass spectrometric analysis, followed by protein identification, label-free quantitation, and statistical analysis. Collectively, a total of 1088 sperm proteins were identified. In comparison to ejaculated spermatozoa, 44 and 141 proteins were differentially expressed in capacitated and acrosome reacted spermatozoa, respectively. A large number of proteins were found downregulated, including clusterin, pyruvate dehydrogenase E1 component, semenogelin-1 and 2, heat shock protein 90, beta-microseminoprotein, and keratin. It was expected as sperm-membrane-associated proteins are removed during capacitation. There were significant proteomic alterations in asthenozoospermia compared to normozoospermia; however, variation was more noticeable among proteins of acrosome reacted spermatozoa and those released during the acrosome reaction. The processes enriched among downregulated proteins in asthenozoospermia included acrosome assembly, binding of spermatozoa to zona pellucida, nucleosome assembly, flagellated sperm motility, protein folding, oxidative phosphorylation, tricarboxylic acid cycle, chromatin silencing, gluconeogenesis, glycolytic process, and glycolysis. The dynamic information generated about proteomic alterations in spermatozoa during capacitation and acrosome reaction and their variability in asthenozoospermia will contribute not only to enhancing our understanding of processes that prepare spermatozoa to acquire fertilization capability but also help in deciphering novel factors of male infertility.

Enrichment of spermatogonia by density gradient centrifugation for use as a donor of surrogate production of tiger puffer

A method of producing functional gametes from cryopreserved testicular germ cells using surrogate broodstock technology is a powerful tool for preserving the aquacultural genetic resources of the tiger puffer (Takifugu rubripes). Among the various testicular cells, only type A spermatogonia (ASGs) can colonize recipient gonads and shift to gametogenesis; therefore, ASG enrichment is an important factor for successful surrogate production. This study examined the possibility of ASG enrichment by density gradient centrifugation for surrogate production, considering its practical use. Through density gradient centrifugation with Percoll media, the highest distribution of ASGs estimated on the cell morphology and vasa expression was counted at a fraction of 20% Percoll layer. These fractionated testicular cells were transplanted intraperitoneally and successfully incorporated into the gonads of the recipient, grass puffer (T. alboplumbeus); however, no colonization was observed in the control recipients transplanted with unfractionated testicular cells. Moreover, these recipients matured normally and produced functional donor-derived gametes from which tiger puffer offspring originated. Thus, the Percoll gradient centrifugation developed in this study enables the enrichment of ASGs from testicular cells, including many differentiated germ cells, and would be useful for efficiently managing broodstock with desirable genetic traits.

Continuous Percoll Gradient Centrifugation of Erythrocytes—Explanation of Cellular Bands and Compromised Age Separation

(1) Background: When red blood cells are centrifuged in a continuous Percoll-based density gradient, they form discrete bands. While this is a popular approach for red blood cell age separation, the mechanisms involved in banding were unknown. (2) Methods: Percoll centrifugations of red blood cells were performed under various experimental conditions and the resulting distributions analyzed. The age of the red blood cells was measured by determining the protein band 4.1a to 4.1b ratio based on western blots. Red blood cell aggregates, so-called rouleaux, were monitored microscopically. A mathematical model for the centrifugation process was developed. (3) Results: The red blood cell band pattern is reproducible but re-centrifugation of sub-bands reveals a new set of bands. This is caused by red blood cell aggregation. Based on the aggregation, our mathematical model predicts the band formation. Suppression of red blood cell aggregation reduces the band formation. (4) Conclusions: The red blood cell band formation in continuous Percoll density gradients could be explained physically by red blood cell aggregate formation. This aggregate formation distorts the density-based red blood cell age separation. Suppressing aggregation by osmotic swelling has a more severe effect on compromising the RBC age separation to a higher degree.

A novel Ush transcription factor involving in hematopoiesis of Eriocheir sinensis.

The FOG transcriptional factor is a co-regulator that recognizes and binds to the GATA N-terminal zinc-finger domain and participates in hemocyte production and differentiation. In this study, an FOG-like gene, Ush, was characterized from Eriocheir sinensis, which consists of an 897bp full-length open reading frame, encoding a polypeptide of 298 amino acids with four ZnF_C2H2 domains. The EsUsh mRNA transcripts were mainly expressed in the hematopoietic tissue (HPT) and hemocytes, and were significantly higher in hyalinocytes than semi-granulocytes and granulocytes, which were separated by Percoll gradient centrifugation. The transcription levels of EsUsh were found to be significantly upregulated in HPT, but downregulated in hemocytes after exsanguination. By using flow cytometry to determine the percentage of hemocyte sub-population after exsanguination, the percentage of hyalinocytes was found to significantly downregulated, while the percentage of granulocytes was significantly upregulated. Silencing EsUsh by dsRNA interference significantly decreased the percentage of hyalinocytes and small granulocytes, and increased the percentage of medium granulocytes and large granulocytes. Such findings suggest that EsUsh might be involved in hemocyte production and differentiation, especially in promoting hyalinocyte formation and limiting granulocyte generation and differentiation.

Growth and division mechanisms by which genetic resisters emerge from the rifampicin-surviving population of differentially antibiotic-susceptible mycobacterial subpopulations.

We recently reported the de novo emergence of unusually high numbers of antibiotic resisters from the in vitro cultures of Mycobacterium tuberculosis and Mycobacterium smegmatis surviving in the presence of minimum bactericidal concentration (MBC) of antituberculosis antibiotics. The resisters emerged due to multiple asymmetric divisions of elongated mother cells containing multiple nucleoids and multiple septae. We had earlier found a minor subpopulation of short-sized cells (SCs) and a major subpopulation of normal-sized cells (NCs) (10% and 90%, respectively, of the whole population), with significant difference in antibiotic susceptibility and resister generation frequency, in the in vitro cultures of M. tuberculosis, M. smegmatis, and Mycobacterium xenopi, as well as in pulmonary tuberculosis patients' sputum. However, the mechanisms of growth and division promoting the emergence of antibiotic resisters from these subpopulations remained unknown. Therefore, here, we took up the first-time study to find out the mechanism of growth and division by which antibiotic resisters emerge from the antibiotic-surviving population of the two subpopulations of M. smegmatis. M. smegmatis SCs and NCs were fractionated from mid-log phase cultures using Percoll gradient centrifugation; their purity was checked and exposed to 10×, 2×, and 0.4× MBC of rifampicin for 120 h. The colony-forming units (CFUs) were determined on rifampicin-free plates for the total population and on rifampicin-containing plates for scoring rifampicin resisters. The phenotype and the morphology of the cells at various stages of the exposure were determined using transmission electron microscopy. The dynamic growth and division mechanisms of the cells to emerge as rifampicin resisters were monitored using live-cell time-lapse imaging. The rifampicin resisters were sequenced for mutations in the rifampicin resistance determining region of rpoB gene. Statistical significance was calculated using two-tailed paired t-test, with *P ≤ 0.05 and **P ≤ 0.01. Multinucleated and multiseptated elongated cells emerged from their respective antibiotic-surviving populations. They produced a large number of sibling-daughter cells through multiple asymmetric divisions in short durations, showing abnormally high spurts in CFUs of antibiotic resisters. The CFUs were several-fold higher than that expected from the mass-doubling time of the subpopulations. Despite this commonality, the subpopulations showed specific differences in their response to different multiples of their respective MBC of rifampicin. Mycobacterial subpopulations come out of rifampicin stress by undergoing multiple nucleoid replications, multiple septation for nucleoid segregation, and acquisition of antibiotic target-specific mutations, followed by multiple asymmetric divisions to generate unusually a large number of rifampicin resisters. Because we had earlier shown that SCs and NCs are present in the pulmonary tuberculosis patients' sputum, the present findings have clinical relevance on the mechanism of emergence of antibiotic-resistant strains from mycobacterial subpopulations.

A novel method for the isolation of CD45-positive and CD45-negative cells from malignant pleural effusion

Background: Patient-derived malignant pleural effusion (MPE) samples can be used to identify a patient-specific drug combination since MPE samples are readily available and cost-effective tumor cell sources. However, the isolation of target cancer cells from MPE has been inefficient because MPE samples contain a complex mixture of immune cells, non-cancerous cells, and cancer cells. Hence, new methods need to be developed to effectively isolate target cancer cells from MPE samples that can be used for 3-dimensional (3D) cell culture. Patient-derived in vitro 3D tumor models are expected to facilitate more precise drug treatment.Methods: MPE samples were obtained from Seoul St. Mary’s Hospital, The Catholic University of Korea with consent from patients previously diagnosed with lung adenocarcinoma. We isolated target cells from MPE samples using 2 different Percoll-gradient centrifugation methods.Results: The use of 40% and 75% Percoll-gradient centrifugation led to a clearer separation of CD45-positive (CD45pos) and CD45-negative (CD45neg) cells than the traditional 44% and 67% Percoll-gradient centrifugation method.Conclusion: Our findings strongly suggest that the 40% and 75% Percoll-gradient centrifugation method is more useful for the isolation of CD45pos or CD45neg cells than the previously described Percoll-gradient centrifugation method. Furthermore, our novel method was useful for the isolation of MPE-derived target cancer cells that can be used to construct in vitro patient-specific 3D tumor models.

Isolation and Measurement of Respiration and Structural Studies of Purified Mitochondria from Heterotrophic Plant Tissues.

Mitochondria are the power houses of eukaryotic cells. These organelles contain various oxidoreductase complexes. Electron transfer from different reducing equivalents channeled via these complexes drives proton translocation across the inner mitochondrial membrane, leading to ATP generation. Plant mitochondria contain alternative NAD(P)H dehydrogenases, alternative oxidase, and uncoupling protein, and TCA cycle enzymes are located in their matrix. Apart from ATP production, mitochondria are also involved in synthesis of vitamins and cofactors and participate in fatty acid, nucleotide, photorespiratory, and antioxidant metabolism. Recent emerging evidence suggests that mitochondria play a role in redox signaling and generation of reactive oxygen and nitrogen species. For mitochondrial studies, it is essential to isolate physiologically active mitochondria with good structural integrity. In this article, we explain a detailed procedure for isolation of mitochondria from various heterotrophic tissues, such as germinating chickpea seeds, potato tubers, and cauliflower florets. This procedure requires discontinuous Percoll gradient centrifugation and can give a good yield of mitochondria, in the range of 4 to 8 mg per 50 g tissue with active respiratory capacity. After MitoTracker staining, isolated mitochondria can be visualized by using a confocal microscope. The structure of mitochondria can be monitored by scanning electron microscopy. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Isolation of mitochondria from germinating chickpea seeds, potato tubers, and cauliflower florets Basic Protocol 2: Quantification of mitochondrial protein concentration by Bradford assay Basic Protocol 3: Quantification of mitochondrial respiration using single-channel free-radical analyzer Basic Protocol 4: Staining of mitochondria and confocal imaging Basic Protocol 5: Visualization of isolated mitochondria under scanning electron microscope.

Proteomic Analysis of Trichomonas vaginalis Phagolysosome, Lysosomal Targeting, and Unconventional Secretion of Cysteine Peptidases

The lysosome represents a central degradative compartment of eukaryote cells, yet little is known about the biogenesis and function of this organelle in parasitic protists. Whereas the mannose 6-phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide-independent sorting has been reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting and the involvement of lysosomes in hydrolase secretion. The organelles were purified using Percoll and OptiPrep gradient centrifugation and a novel purification protocol based on the phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in a lysosomal proteome of 462 proteins, which were sorted into 21 classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate a dynamic phagolysosomal compartment. Several cysteine proteases such as TvCP2 were previously shown to be secreted. Our experiments showed that secretion of TvCP2 was strongly inhibited by chloroquine, which increases intralysosomal pH, thus indicating that TvCP2 secretion occurs through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified divergent homologues of the M6P receptor TvMPR in the phagolysosomal proteome, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP, which possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, the introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as a recognition marker for lysosomal hydrolases. Our findings provide the first insight into the complexity of T. vaginalis phagolysosomes, their biogenesis, and role in the unconventional secretion of cysteine peptidases.

​Spermatozoa Sorting Techniques for the Sex Pre-selection: A Review

Furtherance of sex pre-selection techniques is extremely beneficial for the farm productivity and economic growth of the country. Several techniques such as flow cytometry, swim-up, percoll gradient centrifugation, lumisort and immunogenic spermatozoa sexing developed so far are reviewed with their principles, advantages, disadvantages and possible ways for developing a highly reliable and efficient technique to achieve success in obtaining offspring of the desired sex. Out of all these techniques available till now, sorting X- and Y- spermatozoa using fluorescence-activated cell sorter (FACS) is the most successful and commercially available technique, which employs sorting of spermatozoa based on DNA content. Despite its effectiveness, there are disadvantages concerning cost, sperm damage, trained technical person, low conception rate, etc. An alternative approach that might have potential significance could be the identification of sex-specific membrane marker proteins for the immunological method of spermatozoa sorting.

A method for the enrichment, isolation and validation of Mycobacterium smegmatis population surviving in the presence of bactericidal concentrations of rifampicin and moxifloxacin.

The bacterial populations surviving in the presence of antibiotics contain cells that have gained genetic resistance, phenotypic resistance and tolerance to antibiotics. Isolation of live bacterial population, surviving against antibiotics, from the milieu of high proportions of dead/damaged cells will facilitate the study of the cellular/molecular processes used by them for survival. Here we present a Percoll gradient centrifugation based method for the isolation of enriched population of Mycobacterium smegmatis surviving in the presence of bactericidal concentrations of rifampicin and moxifloxacin. From the time of harvest, throughout the enrichment and isolation processes, and up to the lysis of the cells for total RNA preparation, we maintained the cells in the presence of the antibiotic to avoid changes in their metabolic status. The total RNA extracted from the enriched population of live antibiotic-surviving population showed structural integrity and purity. We analysed the transcriptome profile of the antibiotic-surviving population and compared it with the orthologue genes of Mycobacterium tuberculosis that conferred antibiotic tolerance on tubercle bacilli isolated from the tuberculosis patients under treatment with four antitubercular antibiotics. Statistically significant comparability between the gene expression profiles of the antibiotic tolerance associated genes of M. smegmatis and M. tuberculosis validated the reliability/utility of the method.

Evaluation of in vitro production rates of bovine embryos using melatonin-supplemented culture medium

In this study, we aimed to evaluate the rate of bovine embryo production by using 50 ng/mL melatonin supplementation in in vitro culture medium. For this, oocytes from slaughterhouse ovaries were matured in vitro in TCM-199 medium with Earle’s balanced salt solution + 10% SFB, FSH, and LH in an atmosphere of 5% CO2. Twenty-four hours after IVM, the oocytes underwent in vitro fertilization in human tubal fluid under the same conditions as above, for 18 h. Semen was fractionated by Percoll gradient centrifugation and the concentration of sperm was adjusted to 1 × 106/mL. Probable zygotes were then divided into two groups: the control group grown in drops of 90 μL SOFaa medium + 0.6% BSA + 2.5% SFB, in an atmosphere of 5% CO2, 90% N2, and a melatonin group (Mel), similarly cultured in 90 μL drops of SOFaa medium + 0.6% BSA + 2.5% SFB + 50 ng/mL melatonin. Cleavage rates were assessed on day 3 (D3). On D7, blastocyst formation rates were evaluated. Eight routines were performed (320 oocytes per routine). Data were analyzed with ANOVA, followed by Tukey’s range test using a general linear model. The level of statistical significance was set at 5%. There were no differences in the rates of cleavage or blastocyst formation between the control and melatonin groups (P > 0.05). Thus, under the conditions used in this study, supplementation with melatonin did not yield benefits in increasing the rate of in vitro bovine embryo production.

Influence of maggot excretions/secretions on the anti-Pseudomonas aeruginosa effect of neutrophils in patients with diabetic foot ulcer

Objective: To investigate the effects of medical maggot excretions/secretions (ES) on neutrophils phagocytosis and bactericidal effect in patients with diabetic foot ulcer (DFU). Methods: The experimental research method was used. Thirty DFU patients (16 males and 14 females, aged (64±7) years)who were admitted to the Diabetes Foot Center, the Department of Endocrinology of Air Force Hospital of Eastern Theater Command from June to December 2020 and met the inclusion criteria were recruited. Discontinuous percoll gradient centrifugation method was used to separate the neutrophils. Cells from each patient were enrolled into normal saline group and maggot ES group (30 wells in each group), respectively; sterile normal saline and ES with a final mass concentration of 357 μg/mL (the same as below) were added, respectively. After 1 and 2 hour(s) of culture, the phagocytosis rate and phagocytic index of cells were observed and counted under Wright's staining. Ten patients were selected, then the cells of each patient were enrolled into Pseudomonas aeruginosa+neutrophils group and Pseudomonas aeruginosa+neutrophils+maggot ES group (10 wells in each group) and were treated corresponding, respectively. Pseudomonas aeruginosa alone group and Pseudomonas aeruginosa+maggot ES group (10 wells in each group) were set up respectively; Pseudomonas aeruginosa+RPMI 1640 culture medium+sterile normal saline and Pseudomonas aeruginosa+RPMI 1640 culture medium+maggot ES were added, respectively. After 2 hours of culture, the number of viable bacteria colony was counted by plate colony number method. Six, six, and three patients were selected respectively, and the cells of each patient were respectively enrolled into maggot ES group and normal saline group (6, 6, and 3 wells in each group, respectively) and treated accordingly. After 6 hours of culture, real-time fluorescent quantitative reverse transcription polymerase chain reaction was used to detect the mRNA expressions of interleukin 1β (IL-1β), IL-6, and lysozyme in cells, the content of IL-1β and IL-6 in cell culture supernatant were determined by enzyme-linked immunosorbent assay, and the positive cells expressing lysozyme were observed with immunofluorescence method. Data were statistically analyzed with one-way analysis of variance, paired sample t test, least significant difference test, and Wilcoxon rank sum test. Results: After 1 hour of culture, the phagocytosis rate and phagocytic index of cells in maggot ES group (53.5% (49.7%, 58.0%) and 3.18 (2.96, 3.32)) were similar to 52.0% (47.5%, 55.2%) and 3.15 (2.96, 3.25) of normal saline group (Z=-1.701, -1.092, P>0.05). After 2 hours of culture, the phagocytosis rate and phagocytic index of cells in maggot ES group (70.0% (66.7%, 72.0%) and 4.47 (4.22, 4.96)) were significantly higher than 58.0% (55.0%, 60.0%) and 4.11 (3.52, 4.24) in normal saline group (Z=-4.786, -4.279, P<0.01). After 2 hours of culture, the number of viable bacteria colony in Pseudomonas aeruginosa+neutrophils group was significantly lower than that in Pseudomonas aeruginosa alone group (P<0.01), and the number of viable bacteria colony in Pseudomonas aeruginosa+neutrophils+maggot ES group was significantly lower than that in Pseudomonas aeruginosa+maggot ES group and Pseudomonas aeruginosa+neutrophils group (P<0.01). After 6 hours of culture, the mRNA expressions of IL-1β, IL-6, and lysozyme of cells in maggot ES group were significantly higher those in normal saline group (t=-3.279, -4.273, -4.763, P<0.05 or P<0.01); the concent of IL-1β and IL-6 in cell culture supernatant of maggot ES group were significantly higher than those of normal saline group (t=-9.526, -6.447, P<0.01); there were significantly more positive cells expressing lysozyme in maggot ES group than in normal saline group. Conclusions: Maggot ES can enhance the phagocytosis and bactericidal effect of neutrophils on Pseudomonas aeruginosa by promoting the production of neutrophils immune defense related cytokines and lysozyme in DFU patients.

Bisphenol A and S, but Not F, Alter Oxidative Stress Levels in Spermatozoa

Exposures to endocrine disrupting compounds (EDCs) have been linked to male reproductive dysfunction. The increase in prevalence of male infertility to date has been correlated with the widespread use of bisphenols, particularly bisphenol A and its analogs. Current literature suggests that exposure to EDCs is associated with abnormalities in spermatogenesis, as well as reduced sperm motility and morphology. At this time, the mechanism for bisphenols’ action in spermatozoa is still unknown, though it has been proposed that bisphenols alter sperm parameters by affecting their oxidative stress pathways. The aim of this project is to elucidate whether BPA and its analogs affect oxidative stress levels inducing decreased sperm quality and fertilization capability. An in vitro bovine model was used as the most appropriate translational model to humans. Motile spermatozoa were separated using Percoll gradient centrifugation of frozen-thawed sperm and a pilot experiment was conducted in a time-dependent manner to establish the ideal incubation time of 4 hours. Sperm was then incubated in five treatment groups: control, vehicle (0.01% ethanol), BPA, BPS, and BPF, at the physiological relevant dose of 0.05 mg/ml in 0.01% ethanol. Key reactive oxygen species (ROS) scavenger genes (superoxide mutase 1 [SOD1], superoxide mutase 2 [SOD2], catalase [CAT], glutathione peroxidase 1 [GPX1], glutathione peroxidase 4 [GPX4]) were quantified at the RNA level using qPCR. Of the five genes examined: BPA, BPS, and BPF exposure significantly decreased GPX4 expression (p<0.05) compared to control and vehicle groups. BPA and BPS exposure significantly increased SOD1 expression compared to BPF, control, and vehicle (p<0.05). No significant differences were found in GPX1 after bisphenols’ exposure. SOD2 and CAT expression was not detectable in all five groups suggesting a lack of expression in bovine sperm. Future investigations will involve experiments at the protein level and quantifying total ROS levels. Additionally, sperm morphology was evaluated using Spermac stain. Preliminary results indicate an increased number of abnormal spermatozoa with notably tail defects after 4 hours of bisphenols exposure. These initial findings support BPA and BPS’s ability to alter oxidative stress pathways in sperm via changes in antioxidant enzymes but suggest that BPF likely acts through different mechanisms. Overall, this research aims to understand the effects of bisphenols on male fertility to improve male factors during assisted reproductive biotechnologies.

Visualizing the Integrity of Chloroplast Envelope by Rhodamine and Nile Red Staining

Chloroplasts are essential organelles in plant cells with many important functions. Chloroplasts isolated by Percoll density gradient centrifugation are widely used in the study of chloroplasts. The intactness of isolated chloroplasts is necessary for many of the experiments. In the past, those isolated chloroplasts were either simply believed to be intact or had to be analyzed by indirect biochemical methods. Here we show a new method to check the intactness of isolated chloroplasts by staining their envelope with fluorescent dyes, Rhodamine or Nile red, and then observing them with a fluorescence microscope. With this method, broken chloroplasts and intact chloroplasts can be distinguished easily and their integrity can be checked in a few minutes. Results of this method agreed well with those of biochemical methods. Moreover, we have also found that sometimes the middle layer chloroplasts from the Percoll gradient centrifugation could be mostly broken, which could cause mistakes in the experiment. With our method, this problem can be easily found. This chloroplast envelope staining method can be used in the preparation of isolated chloroplasts to ensure the intactness.

Safety and feasibility of apheresis to harvest and concentrate parasites from subjects with induced blood stage Plasmodium vivax infection

BackgroundIn the absence of a method to culture Plasmodium vivax, the only way to source parasites is ex vivo. This hampers many aspects of P. vivax research. This study aimed to assess the safety of apheresis, a method for selective removal of specific components of blood as a means of extracting and concentrating P. vivax parasites.MethodsAn iterative approach was employed across four non-immune healthy human subjects in single subject cohorts. All four subjects were inoculated with ~ 564 blood stage P. vivax (HMP013-Pv) and subjected to apheresis 10 to 11 days later. Blood samples collected during apheresis (haematocrit layers 0.5% to 11%) were tested for the presence and concentration of P. vivax by microscopy, flow cytometry, 18S rDNA qPCR for total parasites, and pvs25 qRT-PCR for female gametocyte transcripts. Safety was determined by monitoring adverse events. Malaria transmission to mosquitoes was assessed by membrane feeding assays.ResultsThere were no serious adverse events and no significant safety concerns. Apheresis concentrated asexual parasites by up to 4.9-fold (range: 0.9–4.9-fold) and gametocytes by up to 1.45-fold (range: 0.38–1.45-fold) compared to pre-apheresis densities. No single haematocrit layer contained > 40% of all the recovered P. vivax asexual parasites. Ex vivo concentration of parasites by Percoll gradient centrifugation of whole blood achieved greater concentration of gametocytes than apheresis. Mosquito transmission was enhanced by up to fivefold in a single apheresis sample compared to pre-apheresis.ConclusionThe modest level of parasite concentration suggests that the use of apheresis may not be an ideal method for harvesting P. vivax.Trial Registration Australia New Zealand Clinical Trials Registry (ANZCTR) Trial ID: ACTRN12617001502325 registered on 19th October 2017. https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=373812.