scholarly journals Stress conditions promote Leishmania hybridization in vitro marked by expression of the ancestral gamete fusogen HAP2 as revealed by single-cell RNAseq

eLife ◽  
2022 ◽  
Vol 11 ◽  
Author(s):  
Isabelle Louradour ◽  
Tiago Rodrigues Ferreira ◽  
Emma Duge ◽  
Nadira D Karunaweeera ◽  
Andrea Paun ◽  
...  
Keyword(s):  
Single Cell ◽  
High Efficiency ◽  
Wide Spectrum ◽  
Sexual Cycle ◽  
Sand Fly ◽  
Large Numbers ◽  
Transcriptomic Signature ◽  
Low Efficiency ◽  
Transformative Approach

Leishmania are protozoan parasites transmitted by the bite of sand fly vectors producing a wide spectrum of diseases in their mammalian hosts. These diverse clinical outcomes are directly associated with parasite strain and species diversity. Although Leishmania reproduction is mainly clonal, a cryptic sexual cycle capable of producing hybrid genotypes has been inferred from population genetic studies, and directly demonstrated by laboratory crosses. Experimentally, mating competence has been largely confined to promastigotes developing in the sand fly midgut. The ability to hybridize culture promastigotes in vitro has been limited so far to low efficiency crosses between two L. tropica strains, L747 and MA37, that mate with high efficiency in flies. Here, we show that exposure of promastigote cultures to DNA damage stress produces a remarkably enhanced efficiency of in vitro hybridization of the L. tropica strains, and extends to other species, including L. donovani, L. infantum, and L. braziliensis, a capacity to generate intra- and interspecific hybrids. Whole genome sequencing and total DNA content analyses indicate that the hybrids are in each case full genome, mostly tetraploid hybrids. Single-cell RNA sequencing of the L747 and MA37 parental lines highlights the transcriptome heterogeneity of culture promastigotes and reveals discrete clusters that emerge post-irradiation in which genes potentially involved in genetic exchange are expressed, including the ancestral gamete fusogen HAP2. By generating reporter constructs for HAP2, we could select for promastigotes that could either hybridize or not in vitro. Overall, this work reveals that there are specific populations involved in Leishmania hybridization associated with a discernible transcriptomic signature, and that stress facilitated in vitro hybridization can be a transformative approach to generate large numbers of hybrid genotypes between diverse species and strains.

scholarly journals Stress conditions promote the mating competency of Leishmania promastigotes in vitro marked by expression of the ancestral gamete fusogen HAP2

2021 ◽  
Author(s):  
Isabelle Louradour ◽  
Tiago Rodrigues Ferreira ◽  
Emma Duge ◽  
Nadira Karunaweera ◽  
Andrea Paun ◽  
...  
Keyword(s):  
High Efficiency ◽  
Wide Spectrum ◽  
Sexual Cycle ◽  
Sand Fly ◽  
Large Numbers ◽  
Transcriptomic Signature ◽  
Total Dna ◽  
Low Efficiency ◽  
Transformative Approach

Leishmania are protozoan parasites transmitted by the bite of sand fly vectors producing a wide spectrum of diseases in their mammalian hosts. These diverse clinical outcomes are directly associated with parasite strain and species diversity. Although Leishmania reproduction is mainly clonal, a cryptic sexual cycle capable of producing hybrid genotypes has been inferred from population genetic studies, and directly demonstrated by laboratory crosses. Experimentally, mating competency has been largely confined to promastigotes developing in the sand fly midgut. The ability to hybridize culture promastigotes in vitro has been limited so far to low efficiency mating between two L. tropica strains, L747 and MA37, that mate with high efficiency in flies. Here, we show that exposure of promastigote cultures to DNA damage stress produces a remarkably enhanced efficiency of in vitro hybridization of the L. tropica strains, and extends to other species, including L. donovani, L. infantum, and L. braziliensis, a capacity to generate intra- and interspecific hybrids. Whole genome sequencing and total DNA content analyses indicate that the hybrids are in each case full genome, polyploid hybrids. Single-cell RNA sequencing of the L747 and MA37 parental lines highlights the transcriptome heterogeneity of culture promastigotes and reveals discrete clusters that emerge post-irradiation in which genes potentially involved in genetic exchange are expressed, including the ancestral gamete fusogen HAP2. By generating reporter constructs for HAP2, we could select for mating-competent and mating-incompetent promastigotes. Overall, this work reveals that there are specific populations involved in Leishmania mating associated with a discernible transcriptomic signature, and that stress facilitated in vitro hybridization can be a transformative approach to generate large numbers of hybrid genotypes between diverse species and strains.

Transferring the cultured anther to a medium without activated charcoal overcomes the recalcitrancy in pepper genotypes

2020 ◽  
Author(s):  
Hasan Pınar ◽  
Nedim Mutlu ◽  
Serhat Yildiz ◽  
Duran Simsek ◽  
Mostafakamal Shams
Keyword(s):  
Activated Charcoal ◽  
Plant Material ◽  
High Efficiency ◽  
In Vitro Growth ◽  
Vegetable Crops ◽  
Crop Breeding ◽  
Breeding Lines ◽  
Low Efficiency ◽  
Solanaceae Family

Anther culture is a relatively easy and high-efficiency technique, however, low efficiency in plant regeneration may restrict its use in crop breeding. Activated charcoal is often used in in-vitro culture, and it may ameliorate or hinder in-vitro growth, depending on genotype and tissue used. Pepper is one of the main vegetable crops of the Solanaceae family, but some pepper genotypes are known to be recalcitrant to androgenesis and formation of haploid regenerants. Therefore, this study was aimed to explore the effect of activated charcoal on response to androgenesis in pepper genotypes. The plant material including 34 Long Green (LG), 13 Bell pepper (BP), 13 Charleston (Ch), 6 California Wonder (CW), and 23 Capia (CP) advanced breeding lines. Initially, anthers were cultured in a medium with activated charcoal (WAC) for 25, 35, or 45 days, and then they were transferred to the same medium without activated charcoal (NAC). In the WAC medium, 15 lines of LG genotype showed the highest recalcitrancy while many lines of CW had the lowest recalcitrancy to androgenesis. However, after transferring the 35-day old anthers to a NAC medium, the androgenesis was observed in recalcitrant LG lines. The results indicated that transferring the cultured anthers from WAC medium, ideally after 35-day, to a NAC medium overcame the recalcitrancy to androgenesis in pepper.

scholarly journals Marine Algae Metabolites as Promising Therapeutics for the Prevention and Treatment of HIV/AIDS

Metabolites ◽  
2019 ◽  
Vol 9 (5) ◽  
pp. 87 ◽  
Author(s):  
Natalya N. Besednova ◽  
Tatyana N. Zvyagintseva ◽  
Tatyana A. Kuznetsova ◽  
Ilona D. Makarenkova ◽  
Tatyana P. Smolina ◽  
...  
Keyword(s):  
Marine Algae ◽  
High Efficiency ◽  
Wide Spectrum ◽  
Antiretroviral Drugs ◽  
Biologically Active ◽  
New Drugs ◽  
Sulfated Polysaccharides ◽  
Natural Immunity

This review presents an analysis of works devoted to the anti-human immunodeficiency virus (HIV) activity of algae metabolites—sulfated polysaccharides (fucoidans, carrageenans), lectins, laminarans, and polyphenols. Despite the presence of a significant number of antiretroviral drugs, the development of new therapeutic and prophylactic agents against this infection remains very urgent problem. This is due to the variability of HIV, the absence of an animal model (except monkeys) and natural immunity to this virus and the toxicity of therapeutic agents and their high cost. In this regard, the need for new therapeutic approaches and broad-spectrum drugs, which in addition to antiviral effects can have anti-inflammatory, antioxidant, and immunomodulatory effects, and to which the minimum resistance of HIV strains would be formed. These requirements meet the biologically active substances of marine algae. The results of experimental and clinical studies conducted in vitro and in vivo are presented, and the issues of the anti-HIV activity of these compounds are considered depending on their structural features. On the whole, the presented data prove the high efficiency of seaweed metabolites and justify the possibility of their use as a potential basis for the development of new drugs with a wide spectrum of activity.

Metalloproteinase Regulation Determines Integrin αIIbβ3 Function and Enhances the Capacity of ES Cell-Derived Platelets.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 140-140
Author(s):  
Hidekazu Nishikii ◽  
Koji Eto ◽  
Taisuke Kanaji ◽  
Jerry Ware ◽  
Hiromitsu Nakauchi
Keyword(s):  
High Efficiency ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Egfp Expression ◽  
Transfusion Therapy ◽  
Integrin Αiibβ3 ◽  
Differentiated Cells ◽  
Low Efficiency

Abstract To resolve the lack of sources for products used in blood transfusion therapy, the utilization of embryonic stem cells (ESCs) has been proposed. However, the use of ESC-derived cells has potential drawbacks including, immunological rejection and teratoma formation. Being fully differentiated cells lacking a nucleus, platelets can be irradiated to eliminate passenger ESCs and other lineages before transfusion. We previously established in vitro differentiation system whereby proplatelet-producing megakaryocytes can be derived from murine ESCs on OP9 stroma, but the platelets were generated with a low efficiency and exhibited weak agonist-induced integrin αIIbβ3 activation and spreading on immobilized fibrinogen. Here, we describe an improved method to obtain ESC-derived platelets of potential use for hemostasis and thrombosis in vivo. We generated a novel murine ESC line wherein the GPIbα promoter regulates EGFP expression as a marker (GP-G ESCs). The combination of flow cytometry with GP-G-derived embryoid bodies and OP9 co-culture enabled us to enrich integrin αIIb + c-kit + CD9 + Sca-1 − progenitors on day 5–6 of culture as a major fraction capable of differentiating into GFP+ mature megakaryocytes on day 8–10 in the presence of TPO. This strategy increased platelet production by 10-fold (P<0.01) compared to our previous report (PNAS, 2002, 99:12819). Interestingly, αIIb+platelets included 35–40% GPIbα-negative population besides GPIbα-positive on day 13–15, which was caused by an increased activity of matrix metalloproteinase (MMP) and TACE (ADAM17) to shed GPIbα in culture. Inhibition of MMP and TACE with GM6001 or TAP1 delayed the clearance rate on post transfusion recovery in vivo as transfused into a platelet-depleted mouse model. Maintaining GPIbα expression in the presence of MMP/TACE inhibitors restored the specific fibrinogen-bound to integrin αIIbβ3 and platelet spreading on fibrinogen as evidenced by the generation of filopodia and lamellipodia. Collectively, these results suggest that 1) ESC-derived αIIb+ c-kit + CD9+ Sca-1 − progenitors may contribute to enrich megakaryocytopoiesis at high efficiency; 2) inhibition of metalloproteinase activity to retain GPIbα expression is required for αIIbβ 3 integrin inside-out and outside-in signaling in ESC-derived platelets; and 3) these platelets may be useful alternatives for future clinical application.

scholarly journals Efficient and Rapid generation of neural stem cells by direct conversion Fibroblasts with A Single microRNA

2021 ◽  
Author(s):  
yuesi wang ◽  
Yuanyuan Li ◽  
Jing Sun ◽  
Tingting Xu ◽  
Xiaobin Weng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
High Efficiency ◽  
Disease Modeling ◽  
System Development ◽  
Direct Conversion ◽  
Tumor Formation ◽  
Low Efficiency

Neural stem cells (NSCs) have great potential in the application of neurodegenerative disease therapy, drug screening and disease modeling. NSC can be generated by reprogramming from terminally differentiated cells with transcription factors or small molecules. However, current methods for producing NSCs involve the danger of integrating foreign genes into the genome and the problem of low efficiency. Here, we report an efficient method to generate NSCs from human skin-derived fibroblasts with microRNA (mir-302a) in 2-3 days. The induced NSCs (iNSCs) have more than 90% of purity. Their morphology is similar to regular NSCs, expressing key markers including Nestin, Pax6 and Sox2, and can be expanded for more than 20 passages in vitro. They can also differentiate into functional neuron progeny, astrocytes and oligodendrocytes as well. Those cells can elicit action potential, can be xeno-transplanted into the brain of immune-deficient mice, and can survive and differentiate in vivo without tumor formation. This study shows that a single part of pluripotency-inducing mir-302 cluster can drive fibroblasts reprogramming, providing a general platform for high-efficiency generation of individual-specific human NSCs for studies of neuron system development and regenerative cell therapy.

scholarly journals Differential regulation of OCT4 targets facilitates reacquisition of pluripotency

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Sudhir Thakurela ◽  
Camille Sindhu ◽  
Evgeny Yurkovsky ◽  
Christina Riemenschneider ◽  
Zachary D. Smith ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
High Efficiency ◽  
Experimental System ◽  
Regulatory Elements ◽  
Somatic Cell Reprogramming ◽  
Transient Period ◽  
Low Efficiency ◽  
Transcription Factor Expression

Abstract Ectopic transcription factor expression enables reprogramming of somatic cells to pluripotency, albeit with generally low efficiency. Despite steady progress in the field, the exact molecular mechanisms that coordinate this remarkable transition still remain largely elusive. To better characterize the final steps of pluripotency induction, we optimized an experimental system where pluripotent stem cells are differentiated for set intervals before being reintroduced to pluripotency-supporting conditions. Using this approach, we identify a transient period of high-efficiency reprogramming where ectopic transcription factors, but not serum/LIF alone, rapidly revert cells to pluripotency with near 100% efficiency. After this period, cells reprogram with somatic-like kinetics and efficiencies. We identify a set of OCT4 bound cis-regulatory elements that are dynamically regulated during this transient phase and appear central to facilitating reprogramming. Interestingly, these regions remain hypomethylated during in vitro and in vivo differentiation, which may allow them to act as primary targets of ectopically induced factors during somatic cell reprogramming.

scholarly journals CLONAL GROWTH IN VITRO OF HUMAN CELLS WITH FIBROBLASTIC MORPHOLOGY

1957 ◽  
Vol 106 (1) ◽  
pp. 145-158 ◽  
Author(s):  
Theodore T. Puck ◽  
Steven J. Cieciura ◽  
Harold W. Fisher
Keyword(s):  
Single Cell ◽  
Cell Lines ◽  
High Efficiency ◽  
Single Cells ◽  
Previous History ◽  
Good Growth ◽  
Epithelioid Cells ◽  
Cultivation In Vitro ◽  
Fibroblastic Cells

A methodology has been described for reliable cultivation in vitro of dispersed fibroblastic cells obtained from normal human organs. The procedure has permitted establishment of stable cell lines from almost every sample taken, among which the following organs were represented: skin, spleen, amnion, lung, liver, bone marrow, brain, muscle, and heart. Equally good growth has been achieved with cells from embryonic or adult tissues. The methods previously developed whereby single cells plated in Petri dishes grow into isolated macroscopic colonies can successfully be applied to the plating of human fibroblastic stocks. Plating efficiencies in the neighborhood of 50 to 60 per cent are readily achieved with such strains. The resulting colonies can be picked and clonal stocks established. Fibroblastic morphology is maintained in the colonies arising from every single cell of such clonal stocks. All of the single cells from epithelioid clonal strains also maintain their integrity throughout repeated subculture. Since the difference between clonal stocks of these two types is always maintained whenever the respective single cells are plated in the same medium, regardless of the previous history of these stocks, it may be concluded that a true genetic difference exists in these cell lines. In addition to the morphological differences between epithelioid and fibroblastic cell strains, the latter have more demanding nutritional requirements for single cell growth. Thus, single cells of fibroblastic lines almost never produce colonies with high efficiency unless the growth medium which is sufficient for epithelioid cells is supplemented with embryo extract, or a cell feeder layer. Fibroblastic cells are also more resistant to tryptic digestion of the bond uniting the cells to glass surfaces. By use of differential media, growth of both fibroblastic and epithelioid cells, respectively, has been obtained, from dispersed single cells obtained by trypsinization of a specimen of human embryonic lung.

scholarly journals Advantages of the composition and actyvity of a new combined ointment with ethony for treatment of the wound process

2019 ◽  
pp. 126-133
Author(s):  
L. B. Ivantsyk ◽  
S. M. Drogovoz ◽  
N. A. Gerbina ◽  
К. А. Каlко ◽  
V. V. Shtroblia
Keyword(s):  
High Efficiency ◽  
Wide Spectrum ◽  
Experimental Studies ◽  
Antimicrobial Effect ◽  
E Coli ◽  
Anti Inflammatory ◽  
Treatment Of Wounds ◽  
Osmotic Activity ◽  
Active Substances

           An experimental study of a new combined ointment with ethony for treatment of wounds was carried out and its advantages were established compared with the similar drugs Inflarax (LLC FC "Health"), Levomekol (ZAO SPC "Borshchagovsky HFZ") and Oflokain-Darnitsa® (ZAO FF "Darnitsa"), having the same indications for use as a new ointment. The osmotic activity of ointment with ethony was studied by the method of kinetics of water absorption in in vitro experiments. The antimicrobial effect of ointment with ethony relative to standard and hospital strains of microorganisms by diffusion in agar in the modification of wells was determined: S. aureus ATCC 25923, E. coli ATCC 25922, P. aeruginosa ATCC 27853, B. subtilis ATCC 6633, P. vulgaris ATCC 4636, C. albicans ATCC 885/653, S. aureus 23, E. coli 15, P. aeruginosa 39, P. vulgaris 59, K. pneumoniae 6. The anti-inflammatory activity of ointment with ethony was established in a model of non-allergic contact dermatitis caused by turpentine. The results of experimental studies indicate the high efficiency of the proposed combined composition of the ointment with ethony due to the optimal combination of the components of the ointment base and active substances. It was established that the ointment with ethony showed a pronounced and prolonged osmotic activity, which contributes to the complete penetration and release of the active substances of the ointment in the tissue. An ointment with ethony revealed a wide spectrum of antimicrobial activity with respect to standard and hospital strains: with respect to C. albicans ATCC 885/653 and K. pneumoniae 6, this ointment was superior in activity to all comparison drugs. The ointment with ethony showed a pronounced anti-inflammatory effect, superior to the comparison drugs in effectiveness. Thus, due to the presence of a wide spectrum of pharmacological activity, ethony ointment can be recommended for the treatment of wounds with severe exudation in the first phase of the wound process, for wounds infected with mixed bacterial and fungal microflora, and for the prevention of their complications, as well as in complex therapy of the skin inflammatory processes.

scholarly journals Functional profiling of single CRISPR/Cas9-edited human long-term hematopoietic stem cells

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Elvin Wagenblast ◽  
Maria Azkanaz ◽  
Sabrina A. Smith ◽  
Lorien Shakib ◽  
Jessica L. McLeod ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Single Cell ◽  
High Efficiency ◽  
Hematopoietic Stem ◽  
Functional Profiling ◽  
Differentiation And Proliferation ◽  
Gata1 Transcription Factor

Abstract In the human hematopoietic system, rare self-renewing multipotent long-term hematopoietic stem cells (LT-HSCs) are responsible for the lifelong production of mature blood cells and are the rational target for clinical regenerative therapies. However, the heterogeneity in the hematopoietic stem cell compartment and variable outcomes of CRISPR/Cas9 editing make functional interrogation of rare LT-HSCs challenging. Here, we report high efficiency LT-HSC editing at single-cell resolution using electroporation of modified synthetic gRNAs and Cas9 protein. Targeted short isoform expression of the GATA1 transcription factor elicit distinct differentiation and proliferation effects in single highly purified LT-HSC when analyzed with functional in vitro differentiation and long-term repopulation xenotransplantation assays. Our method represents a blueprint for systematic genetic analysis of complex tissue hierarchies at single-cell resolution.

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