scholarly journals Systematically improved in vitro culture conditions reveal new insights into the reproductive biology of the human parasite Schistosoma mansoni

2018 ◽  
Author(s):  
Jipeng Wang ◽  
Rui Chen ◽  
James J. Collins
Keyword(s):  
Sexual Development ◽  
Egg Production ◽  
Reproductive Organs ◽  
Culture Conditions ◽  
Physical Contact ◽  
Female Development ◽  
Male Worm ◽  
Female Sexual Development ◽  
Haploid Embryos

ABSTRACTSchistosomes infect over 200 million people. The prodigious egg output of these parasites is the sole driver of pathology due to infection, yet our understanding of their sexual reproduction is limited because egg production is not sustained for more than a few days in vitro. Here, we describe culture conditions that support schistosome sexual development and sustained egg production in vitro. Female schistosomes rely on continuous pairing with male worms to fuel the maturation of their reproductive organs. Exploiting these new culture conditions, we explore the process of male-stimulated female maturation and demonstrate that physical contact with a male worm, and not insemination, is sufficient to induce female development and the production of viable parthenogenetic haploid embryos. We further report the characterization of a novel nuclear receptor, that we call vitellogenic factor 1, that is essential for female sexual development following pairing with a male worm. Taken together, these results provide a platform to study the fascinating sexual biology of these parasites on a molecular level, illuminating new strategies to control schistosome egg production.

In vitro development of haploid mouse embryos produced by bisection of one-cell fertilized eggs

Development ◽  
1977 ◽  
Vol 38 (1) ◽  
pp. 187-202
Author(s):  
Andrzej K. Tarkowski
Keyword(s):  
X Chromosome ◽  
Culture Conditions ◽  
Extrinsic Factors ◽  
Cell Stage ◽  
Glass Needle ◽  
Androgenetic Embryos ◽  
Haploid Embryos ◽  
Vitro Development ◽  
Further Development

F1(CBA × C57BL'10) mouse eggs originating from spontaneous or induced ovulation and fertilized by CBA-T6T6 or PO spermatozoa were bisected with a glass needle into halves each containing a pronucleus. This technique offers a unique opportunity of producing both androgenetic and gynogenetic haploid embryos from one egg. Out of 600 operated eggs, in 406 (67·7%) both halves survived. During 96 h of culture in vitro the fragments were inspected once daily and finally examined in air-dried preparations. Eighty-seven per cent of halves underwent first cleavage but their further development was to a large extent affected by extrinsic factors connected with experimental procedure (mainly by suboptimal and variable culture conditions) and by the origin of eggs (those from spontaneous ovulation being superior). For this reason developmental capabilities of egg halves were assessed in a selected group of pairs in which at least one partner reached the stage of four or more blastomeres. The observed ratio between pairs with both or only one sister embryo developing successfully suggests that androgenetic embryos carrying Y rather than X chromosome can cleave twice but do not survive beyond 4-cell stage. None of the metaphase plates from older embryos contained a Y chromosome. These observations imply that the X chromosome is genetically active during early cleavage and that a full haploid set is required for preimplantation development to be completed. Formation of blastocysts varied from batch to batch, with an average of 12·8% and maximal incidence of 29·5% . In 34 pairs both fragments developed beyond the 4-cell stage but in only one case did both form blastocysts. Haploid blastocysts were composed of 27 cells on average which was about a half of the number of cells in control diploid zona-free whole eggs. Ten out of 51 embryos with metaphase plates proved to be haploid/diploid mosaics.

scholarly journals Optimization of Conditions for In Vitro Culture of the Microphallid DigeneanGynaecotyla adunca

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
Jenna West ◽  
Alexandra Mitchell ◽  
Oscar J. Pung
Keyword(s):  
Egg Production ◽  
Horse Serum ◽  
Calf Serum ◽  
Culture Conditions ◽  
In Vitro Cultivation ◽  
Ilyanassa Obsoleta ◽  
Uca Pugnax ◽  
Newborn Calf Serum ◽  
Chicken Serum

In vitro cultivation of digeneans would aid the development of effective treatments and studies of the biology of the parasites. The goal of this study was to optimize culture conditions for the trematode,Gynaecotyla adunca. Metacercariae of the parasite from fiddler crabs,Uca pugnax, excysted in trypsin, were incubated overnight to permit fertilization, and were cultured in different conditions to find those that resulted in maximum worm longevity and egg production. When cultured in media lacking serum, worms lived longer in Hanks balanced salt solution and Dulbecco’s Modified Eagle medium/F-12 (DME/F-12) than in RPMI-1640 but produced the most eggs in DME/F-12. Worm longevity and egg production increased when worms were grown in DME/F-12 supplemented with 20% chicken, horse, or newborn calf serum but the greatest number of eggs was deposited in cultures containing horse or chicken serum. Horse serum was chosen over chicken serum due to the formation of a precipitate in chicken serum. The optimal concentration of horse serum with respect to egg production ranged from 5 to 20%. Infectivity of eggs deposited by worms in culture was tested by feeding eggs to mud snails,Ilyanassa obsoleta. None of these snails producedG. aduncacercariae.

Dependency of CLL Cell Survival on Direct Physical Interaction with Bone Marrow Stromal Cells In Vitro Is Largely Outweighed by Secretion of Soluble Factors with Time In Culture

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1365-1365 ◽  
Author(s):  
Iris Gehrke ◽  
Simon Jonas Poll-Wolbeck ◽  
Michael Hallek ◽  
Karl-Anton Kreuzer
Keyword(s):  
Bone Marrow ◽  
Cell Survival ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Culture Conditions ◽  
Marrow Stromal Cells ◽  
Physical Contact ◽  
Physical Interaction ◽  
Soluble Factors

Abstract Abstract 1365 The major pathophysiology feature of chronic lymphocytic leukemia (CLL) cells is their extended life span due to a pronounced resistance towards apoptotic stimuli in vivo. Despite this, CLL cells die within a few days when isolated from their natural microenvironment and are placed under cell culture conditions. That is why the bone marrow microenvironment has been ascribed an essential role in maintenance of the apoptotic resistance of the CLL cell. Thereby both, the physical interaction between bone marrow stromal cells and CLL cells and the secretion of soluble factors have been described to be essentially involved. We analysed the survival capacity of CLL cells in monoculture and in coculture with the bone marrow-derived stromal cell line HS5 with and without physical separation using transwells for up to 7 days by flow cytometric determination of Annexin-V/PI status. As expected, in vitro CLL cell survival was significantly reduced when physical contact between CLL cells and bone marrow stromal cells was prevented. Interestingly, this was only the case for short term cultivation for up to three days. With time under culture conditions CLL cell survival became less dependent on direct physical contact with the HS5 feederlayer, suggesting the secretion of soluble factors to compensate for the loss of pro-survival signals obtained from direct cell-cell interactions over time. This was further supported by the fact of reduced survival support for CLL cells when HS5 proliferation, hence production and secretion of soluble factors, was prevented by mitomycin treatment or formaldehyde fixation. The use of an expanded human Cytokine Antibody Array (Affymetrix), which analyses the presence of the most common 36 cytokine proteins, might offer information about the composition of soluble factors present in the supernatant which are essential for CLL cell survival in vitro. In conclusion, while direct cell-cell contact between CLL cells and bone marrow stromal cells provides an immediate protection against in vitro apoptosis of CLL cells, the secretion of soluble factors, most likely by both, CLL and bone marrow stormal cells, leads to the creation of an in vitro environment which can to a certain extent compensate for the loss of prosurvival signals obtained by direct physical interactions. Disclosures: No relevant conflicts of interest to declare.

Neutrophil migration through in vitro interstitial matrix

1992 ◽  
Vol 50 (1) ◽  
pp. 894-895
Author(s):  
J. Roemer ◽  
S.R. Simon
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Cell Migration ◽  
Smooth Muscle Cells ◽  
Inflammatory Cell ◽  
Neutrophil Migration ◽  
Culture Conditions ◽  
Aortic Smooth Muscle ◽  
Specific Culture

We are developing an in vitro interstitial extracellular matrix (ECM) system for study of inflammatory cell migration. Falcon brand Cyclopore membrane inserts of various pore sizes are used as a support substrate for production of ECM by R22 rat aortic smooth muscle cells. Under specific culture conditions these cells produce a highly insoluble matrix consisting of typical interstitial ECM components, i.e.: types I and III collagen, elastin, proteoglycans and fibronectin.

Myofibrillar degeneration with diphtheria toxin

2020 ◽  
Vol 45 (4) ◽  
pp. 351-357
Author(s):  
Bilge Özerman Edis ◽  
Muhammet Bektaş ◽  
Rüstem Nurten
Keyword(s):  
Protein Synthesis ◽  
Actin Filaments ◽  
Diphtheria Toxin ◽  
Cardiac Tissue ◽  
Culture Conditions ◽  
Bacterial Toxin ◽  
Filamentous Actin ◽  
Cardiac Damage ◽  
Tissue Samples

AbstractObjectivesCardiac damage in patient with diphtheritic myocarditis is reported as the leading cause of mortality. Diphtheria toxin (DTx) is a well-known bacterial toxin inducing various cytotoxic effects. Mainly, catalytic fragment inhibits protein synthesis, induces cytotoxicity, and depolymerizes actin filaments. In this study, we aimed to demonstrate the extent of myofibrillar damage under DTx treatment to porcine cardiac tissue samples.MethodsTissue samples were incubated with DTx for 1–3 h in culture conditions. To analyze whole toxin (both fragments) distribution, conjugation of DTx with FITC was performed. Measurements were carried out with fluorescence spectrophotometer before and after dialysis. Immunofluorescence microscopy was used to show localization of DTx-FITC (15 nM) on cardiac tissue incubated for 2 h. Ultrastructural characterization of cardiac tissue samples treated with DTx (15 or 150 nM) was performed with transmission electron microscopy.ResultsDTx exerts myofibrillar disorganization. Myofilament degeneration, mitochondrial damage, vacuolization, and abundant lipid droplets were determined with 150 nM of DTx treatment.ConclusionsThis finding is an addition to depolymerization of actin filaments as a result of the DTx-actin interactions in in vitro conditions, indicating that myofilament damage can occur with DTx directly besides protein synthesis inhibition. Ultrastructural results support the importance of filamentous actin degeneration at diphtheritic myocarditis.

scholarly journals High-resolution AP-SMALDI MSI as a tool for drug imaging in Schistosoma mansoni

2021 ◽  
Author(s):  
Annika S. Mokosch ◽  
Stefanie Gerbig ◽  
Christoph G. Grevelding ◽  
Simone Haeberlein ◽  
Bernhard Spengler
Keyword(s):  
Schistosoma Mansoni ◽  
Egg Production ◽  
Drug Repurposing ◽  
Cancer Drug ◽  
Ionization Mass ◽  
Parasitic Flatworm ◽  
Paired Female ◽  
First Time ◽  
Organ Tropism

AbstractSchistosoma mansoni is a parasitic flatworm causing schistosomiasis, an infectious disease affecting several hundred million people worldwide. Schistosomes live dioeciously, and upon pairing with the male, the female starts massive egg production, which causes pathology. Praziquantel (PZQ) is the only drug used, but it has an inherent risk of resistance development. Therefore, alternatives are needed. In the context of drug repurposing, the cancer drug imatinib was tested, showing high efficacy against S. mansoni in vitro. Besides the gonads, imatinib mainly affected the integrity of the intestine in males and females. In this study, we investigated the potential uptake and distribution of imatinib in adult schistosomes including its distribution kinetics. To this end, we applied for the first time atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) for drug imaging in paired S. mansoni. Our results indicate that imatinib was present in the esophagus and intestine of the male as early as 20 min after in vitro exposure, suggesting an oral uptake route. After one hour, the drug was also found inside the paired female. The detection of the main metabolite, N-desmethyl imatinib, indicated metabolization of the drug. Additionally, a marker signal for the female ovary was successfully applied to facilitate further conclusions regarding organ tropism of imatinib. Our results demonstrate that AP-SMALDI MSI is a useful method to study the uptake, tissue distribution, and metabolization of imatinib in S. mansoni. The results suggest using AP-SMALDI MSI also for investigating other antiparasitic compounds and their metabolites in schistosomes and other parasites. Graphical abstract

scholarly journals Barley somatic embryogenesis-an attempt to modify variation induced in tissue culture

2021 ◽  
Vol 28 (1) ◽  
Author(s):  
Renata Orłowska
Keyword(s):  
Tissue Culture ◽  
Somatic Embryogenesis ◽  
Taguchi Method ◽  
Culture Conditions ◽  
Dna Demethylation ◽  
Silver Ion ◽  
Donor Plant ◽  
In Vitro Tissue Culture ◽  
Induced Variation

Abstract Background Somatic embryogenesis is a phenomenon carried out in an environment that generates abiotic stress. Thus, regenerants may differ from the source of explants at the morphological, genetic, and epigenetic levels. The DNA changes may be the outcome of induction media ingredients (i.e., copper and silver ions) and their concentrations and time of in vitro cultures. Results This study optimised the level of copper and silver ion concentration in culture media parallel with the induction medium longevity step towards obtaining barley regenerants via somatic embryogenesis with a minimum or maximum level of tissue culture-induced differences between the donor plant and its regenerants. The optimisation process is based on tissue culture-induced variation evaluated via the metAFLP approach for regenerants derived under varying in vitro tissue culture conditions and exploited by the Taguchi method. In the optimisation and verification experiments, various copper and silver ion concentrations and the different number of days differentiated the tested trials concerning the tissue culture-induced variation level, DNA demethylation, and de novo methylation, including symmetric (CG, CHG) and asymmetric (CHH) DNA sequence contexts. Verification of optimised conditions towards obtaining regenerants with minimum and maximum variability compared to donor plants proved useful. The main changes that discriminate optimised conditions belonged to DNA demethylation events with particular stress on CHG context. Conclusions The combination of tissue culture-induced variation evaluated for eight experimental trials and implementation of the Taguchi method allowed the optimisation of the in vitro tissue culture conditions towards the minimum and maximum differences between a source of tissue explants (donor plant) and its regenerants from somatic embryos. The tissue culture-induced variation characteristic is mostly affected by demethylation with preferences towards CHG sequence context.

scholarly journals Transcriptome comparisons of in vitro intestinal epithelia grown under static and microfluidic gut-on-chip conditions with in vivo human epithelia

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kornphimol Kulthong ◽  
Guido J. E. J. Hooiveld ◽  
Loes Duivenvoorde ◽  
Ignacio Miro Estruch ◽  
Victor Marin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Culture Conditions ◽  
Gene Set Enrichment Analysis ◽  
Shear Stresses ◽  
Static Culture ◽  
Dynamic Culture ◽  
Intestinal Segments ◽  
On Chip

AbstractGut-on-chip devices enable exposure of cells to a continuous flow of culture medium, inducing shear stresses and could thus better recapitulate the in vivo human intestinal environment in an in vitro epithelial model compared to static culture methods. We aimed to study if dynamic culture conditions affect the gene expression of Caco-2 cells cultured statically or dynamically in a gut-on-chip device and how these gene expression patterns compared to that of intestinal segments in vivo. For this we applied whole genome transcriptomics. Dynamic culture conditions led to a total of 5927 differentially expressed genes (3280 upregulated and 2647 downregulated genes) compared to static culture conditions. Gene set enrichment analysis revealed upregulated pathways associated with the immune system, signal transduction and cell growth and death, and downregulated pathways associated with drug metabolism, compound digestion and absorption under dynamic culture conditions. Comparison of the in vitro gene expression data with transcriptome profiles of human in vivo duodenum, jejunum, ileum and colon tissue samples showed similarities in gene expression profiles with intestinal segments. It is concluded that both the static and the dynamic gut-on-chip model are suitable to study human intestinal epithelial responses as an alternative for animal models.

scholarly journals TMOD-15. EFFICACY OF THE MDM2 INHIBITOR KRT-232 IN GLIOBLASTOMA PATIENT-DERIVED XENOGRAFT MODELS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii231-ii231
Author(s):  
Rachael Vaubel ◽  
Ann Mladek ◽  
Yu Zhao ◽  
Shiv K Gupta ◽  
Minjee Kim ◽  
...  
Keyword(s):  
Target Genes ◽  
Xenograft Model ◽  
Culture Conditions ◽  
Patient Derived Xenograft ◽  
Fold Induction ◽  
Extended Survival ◽  
Mdm2 Amplification ◽  
Mdm2 Inhibitor

Abstract Non-genotoxic reactivation of p53 by MDM2 inhibitors represents a promising therapeutic strategy for tumors with wild-type TP53, particularly tumors harboring MDM2 amplification. MDM2 controls p53 levels by targeting it for degradation, while disruption of the MDM2-p53 interaction causes rapid accumulation of p53 and activation of the p53 pathway. We examined the efficacy of the small molecule MDM2 inhibitor KRT-232, alone and in combination with radiation therapy (RT), in MDM2-amplified and/or p53 wildtype patient-derived xenograft (PDX) models of glioblastoma in vitro and in vivo. In vitro, glioblastoma PDX explant cultures showed sensitivity to KRT-232, both tumors with MDM2 amplification (GBM108 and G148) and non-amplified but TP53-wildtype lines (GBM10, GBM14, and GBM39), with IC50s ranging from 300-800 nM in FBS culture conditions. A TP53 p.F270C mutant PDX (GBM43) was inherently resistant, with IC50 >3000 nM. In the MDM2-amplified GBM108 line, KRT-232 led to a robust (5-6 fold) induction of p53-target genes p21, PUMA, and NOXA, with initiation of both apoptosis and senescence. Expression of p21 and PUMA was greater with KRT-232 in combination with RT (25-35 fold induction), while stable knock-down of p53 in GBM108 led to complete resistance to KRT-232. In contrast, GBM10 showed lower induction of p21 and PUMA (2-3 fold) and was more resistant to KRT-232. In an orthotopic GBM108 xenograft model, treatment with KRT-232 +/- RT for one week extended survival from 22 days (placebo) to 46 days (KRT-232 alone); combination KRT-232 + RT further extended survival (77 days) over RT alone (31 days). KRT-232 is an effective treatment in a subset of glioblastoma pre-clinical models alone and in combination with RT. Further studies are underway to understand the mechanisms conferring innate sensitivity or resistance to KRT-232.

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