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Cell-based therapies hold tremendous potential to treat cancer, inflammatory conditions, and degenerative diseases. However, despite a few notable successes, cell-based therapies face key clinical barriers, including cell source heterogeneity, inefficient directed migration (homing) to target tissues, cell dosing or controllability, inability to affect tissue microenvironments, and patient safety. Bioengineering may potentially enhance cell capabilities, but raises safety concerns regarding the control of genetically engineered cells in vivo. Therefore, bioengineering methods to improve cell-based therapy efficacy must prioritize predictable behavior and safety. To address these issues, the Klemke Laboratory developed a platform technology that couples cell biongineering with cell enucleation (mass removal of nuclei) to enhance cellular function and controllability. By adapting methods developed in the 1970s, mesenchymal stromal cells (MSCs) were enucleated by ultracentrifugation in discontinuous Ficoll gradients containing cytochalasin B. Combining cell bioengineering with enucleation both decreases the risk of engraftment and oncogenesis, and provides opportunities for customized pre- and/or post-enucleation bioengineering. We named these bioengineered, enucleated cells “CargocytesTM”.Cargocytes were characterized by absent nuclei, viability for 2-3 days, functional protein translational machinery, retention of receptor expression, and directional migration, and then further investigated via bioengineering with therapeutically-relevant properties. For in vitro studies, Cargocytes were bioengineered and then examined for secretion of functional cytokines and expression of functional chemokine receptors. Next, Cargocytes in vivo abilities were examined in two independent mouse models. First, bioengineered Cargocytes injected intratumorally in a mouse breast cancer model produced functional antitumor cytokine that slowed tumor progression and increased animal survival. Second, bioengineered Cargocytes injected intravenously in a mouse ear inflammation model both homed to the site of inflammation and produced functional anti-inflammatory cytokine that reduced inflammation. This suggests that bioengineered, enucleated cells are more efficacious than non-engineered cells, and lacking nuclei uniquely contributes to safety and controllability in vivo.To my knowledge, therapeutic bioengineering of enucleated cells has not been previously described, and Cargocytes represent a unique entity in the cell-based therapy field as either a stand-alone or adjuvant therapy. By retaining biologically important cell-like functions yet with advantages like small size and defined lifespan, Cargocytes potentially resolve key barriers currently limiting cell-based therapies.

Zona-free somatic cell transfer (SCT) and embryo aggregation increase throughput and efficiency of cloned embryo and offspring production, respectively, but both approaches have not been widely adopted. Cloning efficiency is further improved by cell cycle coordination between the interphase donor cell and metaphase-arrested recipient cytoplast. This commonly involves inclusion of caffeine and omission of calcium to maintain high mitotic cyclin-dependent kinase activity and low calcium levels, respectively, in the nonactivated cytoplast. The aim of our study was to integrate these various methodological improvements into a single work stream that increases sheep cloning success. We show that omitting calcium during zona-free SCT improved blastocyst development from 6% to 13%, while caffeine treatment reduced spontaneous oocyte activation from 17% to 8%. In a retrospective analysis, morula aggregation produced high morphological quality blastocysts with better in vivo survival to term than nonaggregated controls (15% vs. 9%), particularly after vitrification (14% vs. 0%). By combining cytoplast cell cycle control with zona-free embryo reconstruction and aggregation, this novel SCT protocol maximizes the benefits of vitrification by producing more cryoresilient blastocysts. The presented cloning methodology is relatively easy to operate and further increases throughput and efficiency of cloned embryo and offspring production. Integration of additional reprogramming steps or alternate donor cells is straightforward, providing a flexible workflow that can be adapted to changing experimental requirements.

Purpose Assisted reproductive technology (ART) is a widely applied fertility treatment. However, the developmental competence of aged oocytes from women of a late reproductive age is seriously reduced and the aged oocytes often fail in fertilization even when ART is used. To resolve this problem, we examined usefulness of a new method “the metaphase II spindle transfer (MESI)” as ART using mouse oocytes. Methods This work was composed of two experiments. First, 24 hours after collection, embryos from oocytes (1‐day‐old oocytes, called postovulatory‐aged oocytes), were observed, after intracytoplasmic sperm injection (ICSI), and it was found that they were not able to reach the blastocyst stage. Next, the metaphase II chromosome‐spindle complexes from 1‐day‐old oocytes were injected into cytoplasts from oocytes just collected, using piezo pulses to generate reconstructed oocytes. This procedure was named metaphase II spindle injection (MESI). Results After ICSI, embryos from the reconstructed oocytes (32/105), which contained the genes of 1‐day‐old oocytes, were able to develop into the blastocyst stage. The fragmentation rate after ICSI was 28.6%. Thus, the developmental competence of 1‐day‐old oocytes was improved by MESI. Conclusions The MESI method has the potential to improve the success rate of infertility treatments for women of a late reproductive age., The metaphase II chromosome‐spindle complexes from 1‐day‐old oocytes (postovulatory‐aged oocytes 24 hours after collection) were injected into cytoplasts from freshly collected oocytes using piezo pulses to generate reconstructed oocytes (MESI). After ICSI, the embryos from reconstructed oocytes (32/105) could develop into the blastocyst stage. However, the developmental competence of 1‐day‐old oocytes was improved by MESI.

The vertical bone increase of alveolar rim is important to obtain good results in rehabilitation with prostheses on implants. This Literature Review sought articles that treat bone increases of alveolar edges using Cytoplast® membranes, seeking to evaluate the resistance to bacterial penetration and capacity to create and maintain space. For this, a literature review was made on the Pubmed and Google scholar search platforms. The researches analyzed found similar results between d-PTFE membrane, titanium meshes and e-PTFE membranes, both in bone gain and bone quality. The space maintenance capacity was evident in the articles in which the titanium reinforced membrane was used. The d-PTFE membrane presented a greater capacity of exposure to the oral environment without compromising the graft material. The authors researched in this study found that titanium-reinforced d-PTFE membranes for bone augamble alveolar rim increases are viable and allow a certain period of exposure to the oral environment without graft contamination. Further studies are needed with the Cytoplast® d-PTFE membrane to explore its characteristics with vertical bone augmentation procedures.

Made available in DSpace on 2020-12-12T01:34:49Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-08-01 The genetic diversity of Neotropical deer is increasingly jeopardized, owing to declining population size. Thus, the formation of cryobanking of somatic cells is important for the preservation of these species using cloning. The transformation of these cells into viable embryos has been hampered by a lack of endangered species oocytes. Accordingly, the aim of this study was to produce brown brocket deer embryos by interspecific somatic cell nuclear transfer (iSCNT), using goat or cattle oocytes as cytoplasts, and to elucidate embryo mitochondrial activity by measuring the expression levels of ATP6, COX3, and ND5. Cattle embryos produced by in vitro fertilization (IVF) were used as a control. There were no differences in the development of embryos produced by traditional SCNT and iSCNT when using either the goat cytoplasts (38.4% vs. 25.0% cleaved and 40.0% vs. 50.0% morula rates, respectively) or cattle cytoplast (72.8% vs. 65.5% cleaved and 11.3% vs. 5.9% blastocyst rates, respectively). Concerning the gene expression, no significant difference was observed when goat oocytes were used as cytoplasts. However, when using cattle oocytes and 16S as a reference gene, the iSCNT upregulated COX3, when compared with SCNT group. In contrast, when GAPDH was used as a reference gene, all the evaluated genes were upregulated in the iSCNT group, when compared with the IVF group. When compared with the SCNT group, only the expression of ATP6 was statistically different. In conclusion, it was demonstrated that interspecific nuclear transfer is a potentially useful tool for conservation programs of endangered similar deer species. Laboratory of Physiology Control of Reproduction Faculty of Veterinary Ceará State University (UECE) Laboratory of Animal Biotechnology National University Toribio Rodriguez de Mendoza Department of Biology University of Saskatchewan Department of Animal Science Deer Research and Conservation Center São Paulo State University (UNESP) Molecular Genetics Research Unit University Center Fametro (UNIFAMETRO) Department of Animal Science Deer Research and Conservation Center São Paulo State University (UNESP)

Guided bone-regeneration techniques use either resorbable or nonresorbable membrane. Ideal membrane material should be biocompatible with tissue integration, be able to create and maintain space, be occlusive with selective permeability, and have good handling properties. Commercially available nonresorbable membranes are Gor-tex (e-PTFE), Cytoplast (d-PTFE), and titanium mesh. Resorbable membranes are available as natural and synthetic. Clinical trials, a systematic review and meta-analysis have shown no statistically significant difference in most clinical indications between both types of membrane. The choice of membrane varies according to the choice of grafting materials and nature of defect.

Along with the rapid appearance of superbacteria with multidrug resistance, it is a challenge to develop new antibacterial materials to address this big issue. Herein, we report a novel amine group-modified fullerene derivative (C70-(ethylenediamine)8 abrr. C70-(EDA)8), which reveals a high performance in killing superbacteria, and most importantly, it shows negligible toxicity to the mammalian cells. The strong antibacterial ability of this material was attributed to its unique molecular structure. On one hand, amino groups on the EDA part make it easy to affix onto the outer membrane of multidrug resistance Escherichia coli by electrostatic interactions. On the other hand, the hydrophobic surface on the C70 part makes it easy to form a strong hydrophobic interaction with the inner membrane of bacteria. Finally, C70-(EDA)8 leads to the cytoplast leakage of superbacteria. In contrast, the C70-(EDA)8 is nontoxic for mammalian cells due to different distributions of the negative charges in the cell membrane. In vivo studies indicated that C70-(EDA)8 mitigated bacterial infection and accelerated wound healing by regulating the immune response and secretion of growth factors. Our amine group-based fullerene derivatives are promising for clinical treatment of wound infection and offer a new way to fight against the superbacteria.

Enucleated cells or cytoplasts (cells whose nucleus is removed in vitro) represent an unexplored biological model for intracellular infection studies due to the abrupt interruption of nuclear processing and new RNA synthesis by the host cell in response to pathogen entry. Using enucleated fibroblasts hosting the protozoan parasite Leishmania amazonensis, we demonstrate that parasite multiplication and biogenesis of large parasitophorous vacuoles in which parasites multiply are independent of the host cell nucleus. Dual RNA sequencing of both host cytoplast and intracellular parasite transcripts identified host transcripts that are more preserved or degraded upon interaction with parasites and also parasite genes that are differentially expressed when hosted by nucleated or enucleated cells. Cytoplasts are suitable host cells, which persist in culture for more than 72 h and display functional enrichment of transcripts related to mitochondrial functions and mRNA translation. Crosstalk between nucleated host de novo gene expression in response to intracellular parasitism and the parasite gene expression to counteract or benefit from these host responses induces a parasite transcriptional profile favoring parasite multiplication and aerobic respiration, and a host-parasite transcriptional landscape enriched in host cell metabolic functions related to NAD, fatty acid, and glycolytic metabolism. Conversely, interruption of host nucleus-parasite cross talk by infection of enucleated cells generates a host-parasite transcriptional landscape in which cytoplast transcripts are enriched in phagolysosome-related pathway, prosurvival, and SerpinB-mediated immunomodulation. In addition, predictive in silico analyses indicated that parasite transcript products secreted within cytoplasts interact with host transcript products conserving the host V-ATPase proton translocation function and glutamine/proline metabolism. The collective evidence indicates parasite-mediated control of host cell transcripts half-life that is beneficial to parasite intracellular multiplication and escape from host immune responses. These findings will contribute to improved drug targeting and serve as database for L. amazonensis-host cell interactions.

Background The nucleus, besides its functions in the gene maintenance and regulation, plays a significant role in the cell mechanosensitivity and mechanotransduction. It is the largest cellular organelle that is often considered as the stiffest cell part as well. Interestingly, the previous studies have revealed that the nucleus might be dispensable for some of the cell properties, like polarization and 1D and 2D migration. Here, we studied how the nanomechanical properties of cells, as measured using nanomechanical mapping by atomic force microscopy (AFM), were affected by the removal of the nucleus. Methods The mass enucleation procedure was employed to obtain cytoplasts (enucleated cells) and nucleoplasts (nuclei surrounded by plasma membrane) of two cell lines, REF52 fibroblasts and HT1080 fibrosarcoma cells. High-resolution viscoelastic mapping by AFM was performed to compare the mechanical properties of normal cells, cytoplasts, and nucleoplast. The absence or presence of the nucleus was confirmed with fluorescence microscopy, and the actin cytoskeleton structure was assessed with confocal microscopy. Results Surprisingly, we did not find the softening of cytoplasts relative to normal cells, and even some degree of stiffening was discovered. Nucleoplasts, as well as the nuclei isolated from cells using a detergent, were substantially softer than both the cytoplasts and normal cells. Conclusions The cell can maintain its mechanical properties without the nucleus. Together, the obtained data indicate the dominating role of the actomyosin cytoskeleton over the nucleus in the cell mechanics at small deformations inflicted by AFM.

The kodkod (Leopardus guigna) is a small felid endemic of Chile and is considered a vulnerable species. Domestic cat oocytes have been successfully used as recipient cytoplast to reprogram somatic cells from different felids by interspecific somatic cell nuclear transfer (iSCNT). The developmental competence of felid embryos generated by iSCNT can be improved by the aggregation method using a zona-free culture system. The objective of this research was to evaluate the developmental competence of kodkod embryos generated by iSCNT using domestic cat oocytes and the aggregation method. For this purpose, five experimental group were done: (1) cat embryos generated by IVF, (2) cat embryos generated by SCNT (Ca1x), (3) aggregated cat embryos generated by SCNT (Ca2x), (4) kodkod embryos generated by iSCNT (K1x) and (5) aggregated kodkod embryos generated by iSCNT (K2x). Cleavage, morulae and blastocyst rates were estimated. The blastocyst diameter was evaluated. The gene expression level of pluripotency (OCT4, SOX2 and NANOG) and differentiation markers (CDX2 and GATA6) was analyzed in blastocysts. Morulae rate was higher in the IVF group and when cloned embryos were cultured in aggregates (IVF: 68.2%, Ca2x: 58.0% and K2x: 62.4%) compared to individually cultured kodkod embryos (K1x: 37.0%) (P 0.05). Embryo aggregation increased blastocysts formation in the Ca2x group (30.9%) to a similar rate compared to the IVF group (44.5%) (P 0.05). No blastocysts were generated in the K1x group, whereas blastocysts formation was obtained in K2x group (5.9%). The diameter of blastocysts from the K2x group (172.8 μm) was significantly lower than blastocysts from the Ca2x group (P 0.05). The relative expression of OCT4 was lower in blastocysts from Ca1x than in blastocysts from IVF (P 0.05). Furthermore, CDX2 expression was lower in blastocysts from Ca2x than in blastocysts from Ca1x and IVF groups (P 0.05). In kodkod embryos, only one blastocyst from the K2x group expressed OCT4. No expression of SOX2, NANOG, CDX2 and GATA6 was detected in kodkod blastocysts. In conclusion, after iSCNT, domestic cat oocytes support the development of kodkod embryos until the morula stage. The aggregation method increases the morulae rate of kodkod cloned embryos and allows blastocysts formation. However, kodkod blastocysts have a poor morphological quality and a lacking expression of pluripotency and differentiation markers, probably caused by an incomplete nuclear reprogramming.

The current study was conducted for the period (10/4/2019 to 1/6/2019) on the white mouse of Swiss origin for the purpose of demonstrating the effect of methionine on the appearance of tissue lesions of some members and the possibility of treatment with extract grape seeds, And used 16 animals of white mice for this purpose, was used oral dosage of methionine dosage for 10 days and treatment with grape extract for 30 days. The results of the study of the histopathological effects in the liver, kidney and heart tissues of the groups treated with methionine showed changes in the tissues of the heart, liver and kidneys, which were characterized by the presence of hemorrhage, lymphocyte infiltration, necrosis of cell cytoplasts, and nucleus. The fourth group, which included the best grape seed extract group among the three concentration groups of the three aquatic extracts, showed a very good improvement compared to the first and the second group and the third group where the closest natural form of the tissue was found in the control group, This is due to the different susceptibility of the body of the animal used in the study to the similarity of treatment with the extract of the seeds of aqueous grapes. And that the level of enzymes decreased when treated with methionine and that the levels approached the control group when treatment with the extract of water grapes with increasing concentrations.

In regular IVF, a portion of oocytes exhibit abnormal numbers of pronuclei (PN) that is considered as abnormal fertilization, and they are routinely discarded. However, it is known that abnormal ploidy still does not completely abandon embryo development and implantation. To explore the potential of cytoplasm from those abnormally fertilized oocytes, we developed a novel technique for the transfer of large cytoplasm between pronuclear-stage mouse embryos, and assessed its impact. A large volume of cytoplast could be efficiently transferred in the PN stage using a novel two-step method of pronuclear-stage cytoplasmic transfer (PNCT). PNCT revealed the difference in the cytoplasmic function among abnormally fertilized embryos where the cytoplasm of 3PN was developmentally more competent than 1PN, and the supplementing of fresh 3PN cytoplasm restored the impaired developmental potential of postovulatory “aged” oocytes. PNCT-derived embryos harbored significantly higher mitochondrial DNA copies, ATP content, oxygen consumption rate, and total cells. The difference in cytoplasmic function between 3PN and 1PN mouse oocytes probably attributed to the proper activation via sperm and may impact subsequent epigenetic events. These results imply that PNCT may serve as a potential alternative treatment to whole egg donation for patients with age-related recurrent IVF failure.

En este estudio se demuestra el uso de la transferencia nuclear de células somáticas para producir los primeros bovinos clonados en el Perú. Se obtuvieron fibroblastos de piel y células de cúmulos de donantes adultos para ser usados como carioplastos; asimismo, ovocitos obtenidos a partir de ovarios de camal fueron madurados in vitro por 24 h. Los ovocitos madurados se incubaron 2 h en demecolcina (2.5 ìg/ml) para promover la formación del cono con el plato metafásico y para orientar la enucleación manual. Se eliminó la zona pelúcida en pronasa (2 mg/ml) por 3 min. La enucleación fue manual con una microcuchilla dividiendo el óvulo en dos mitades, donde las mitades carentes de núcleo fueron fusionadas por el método «sandwich» (citoplasto–fibroblasto–citoplasto) por electrofusión. Las estructuras reconstruidas se activaron químicamente mediante incubación por 5 min en 7% de etanol absoluto, seguido por 5 h de citocalacina B (5 ìg/ml) y cicloheximida (10 ìg/ml). Las estructuras se cultivaron durante 7 d hasta la fase de incubación/eclosión de blastocisto. Siete blastocistos fueron transferidos a seis vacas receptoras sincronizadas siete días después de la ovulación. Se logró la permanencia de cuatro y tres vesículas embrionarias hasta los días 28 y 60, respectivamente. Dos terneras llegaron a nacer a partir de embriones reconstruidos con células de piel y con células de cúmulos. Mediante el análisis de genotipos, utilizando 15 marcadores (SSR) para bovinos, se confirmó que los terneros clonados fueron derivados de las líneas celulares de las donantes., This study demonstrates the use of nuclear somatic cell transfer to produce the first cloned cattle in Peru. Skin fibroblasts and cumulus cells from adult donors were obtained for use as carioplasts; likewise, oocytes obtained from ovaries in the slaughterhouse were matured in vitro for 24 h. The mature oocytes were incubated 2 h in demecolcin (2.5 μg/ml) to promote cone formation with the metaphase plate and to guide manual enucleation. The zona pellucida in pronase (2 mg/ml) was removed for 3 min. The enucleation was manual with a microblade dividing the ova into two halves, where the nucleus-lacking halves were fused by the «sandwich» method (cytoplast–fibroblast– cytoplast). The reconstructed structures were chemically activated by incubation for 5 min in 7% absolute ethanol, followed by 5 h of cytochalacin B (5 μg/ml) and cycloheximide (10 μg/ml). The structures were cultured for 7 d until the blastocyst incubation/hatching phase. Seven blastocysts were transferred to six synchronized recipient cows seven days after ovulation. The permanence of four and three embryonic vesicles was achieved until days 28 and 60, respectively. Two calves were born from embryos reconstructed with skin cells and cumulus cells. By the genotype analysis using 15 markers (SSR) for cattle, it was confirmed that cloned calves were derived from donor cell lines.

We previously demonstrated that the cellular thermotolerance of cloned cattle produced by combination of ooplasm (o) derived from Taiwan native yellow cattle (Y) and the donor (d) nucleus derived from Holstein (H) cattle (Yo-Hd) transmits to their offspring (Yo-Hd-F1). In the present study, the responses of mitochondria in these cloned cattle and their offspring after heat shock were investigated to elucidate influence of cytoplasmic input (i.e., ooplasm) during the course of heat stress. After heat shock, oxidative phosphorylation proteins (Complex III and IV) of ear fibroblast cells with Y-originated cytoplasm (including Y, Yo-Hd, and Yo-Hd-F1 cattle) were significantly greater (P 0.05) than those of ear fibroblast cells with H-originated cytoplasm (including H, Ho-Hd, and Ho-Hd-F1 cattle). However, the expressions of Complex I and Complex II protein in heat shocked cells derived from Yo-Hd-F1 cattle were significantly (P 0.05) higher than those of cell derived from cattle with the H-cytoplasm. The catalase (CAT) expression in heat shocked ear fibroblast cells derived from Yo-Hd and Yo-Hd-F1 cattle were significantly (P 0.05) higher than that of cells derived from Ho-Hd-F1 cattle. However, the level of glutathione peroxidase (GPx) expression was higher (P 0.05) in ear fibroblast cells with Y-originated cytoplasm compared to cells with H-originated cytoplasm. In conclusion, the expression of proteins involved in mitochondrial oxidative phosphorylation and antioxidant enzymes after heat shock was increased in ear fibroblast cells from cattle with Y-originated cytoplasm, which can be transmitted to their offspring.

// Lu Li 1, 4 , Lu Wang 4 , Kevin M. Prise 2 , K.N. Yu 3 , Guodong Chen 1 , Lianyun Chen 6 , Yide Mei 7 , Wei Han 1, 5 1 Anhui Province Key Laboratory of Medical Physics and Technology/Center of Medical Physics and Technology, Hefei Institutes of Physical Sciences, Chinese Academy of Sciences, Hefei, Anhui, China 2 Centre for Cancer Research & Cell Biology, Queen’s University, Belfast, UK 3 Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong, Hong Kong 4 Clinical College of Integrated Traditional Chinese and Western Medicine, Anhui University of Chinese Medicine, Hefei, Anhui, China 5 Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions and School for Radiological and Interdisciplinary Sciences (RAD-X), Soochow University, Suzhou, Jiangsu, China 6 Institute of Technical Biological & Agriculture Engineering, Hefei Institutes of Physical Sciences, Chinese Academy of Sciences, Hefei, Anhui, China 7 School of Life Science, University of Science and Technology of China, Hefei, Anhui, China Correspondence to: Wei Han, email: hanw@hfcas.ac.cn Keywords: radiation-induced bystander effect, cytoplasm, Akt, mTOR, cytoplast Received: September 20, 2016 Accepted: December 27, 2016 Published: February 01, 2017 ABSTRACT Cytoplasm is an important target for the radiation-induced bystander effect (RIBE). In the present work, the critical role of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway in the generation of RIBE signaling after X-ray irradiation and the rapid phosphorylation of Akt and mTOR was observed in the cytoplasm of irradiated human lung adenocarcinoma epithelial (A549) cells. Targeting A549 cytoplasts with individual protons from a microbeam showed that RIBE signal(s) mediated by the Akt/mTOR pathway were generated even in the absence of a cell nucleus. These results provide a new insight into the mechanisms driving the cytoplasmic response to irradiation and their impact on the production of RIBE signal(s).

Maturation conditions and oocytes quality have substantial roles on developmental competence of unreconstructed or reconstructed oocytes. Cloning has been reported successfully with low efficiency through embryonic or somatic nuclear transfer into enucleated metaphase II oocytes. It has been suggested that introducing embryonic or somatic nucleus to cytoplast at earlier stage might improve reprogramming of the introduced nucleus. In addition, the synchronization between the donor nucleus and recipient cytoplasts might effect on reprogramming and further embryonic development. Therefore, the question arises; does the cytoplast of germinal vesicle (GV) oocyte containing nuclear sap improve developmental competence upon reconstruction with embryonic/somatic nucleus compared with MII cytoplast. It has been indicated that GV material is essential for remodeling of sperm or embryonic or somatic nucleus in GV oocyte cytoplast and their further embryonic developmental competence. GV cytoplasts could be obtained through micromanipulation. Different micromanipulation techniques of immature oocytes at different stages were adapted in addition to introducing donor nuclei at G0/G1, S and G2/M phase, and enucleolation technique as well. Upon micromanipulation, it could obtain GV cytoplasts; cumulus-free and without GV material, cumulus-complexes and without GV material, cumulus-free and with GV material, and cumulus-complexes and with GV material in addition to enucleolated GV oocytes. Therefore, this short review will address briefly the importance of maturation conditions, cumulus cells, oocyte quality, the techniques of enucleation GV oocyte, the cell cycle stage of the introduced donor cell, or nucleus for oocyte maturation and embryo development.

SummaryChemical oocyte enucleation holds the potential to ease somatic cell nuclear transfer (SCNT), although high enucleation rates remain limited to micromanipulation-based approaches. Therefore, this study aimed to test mitomycin C (MMC) for use in bovine functional chemical oocyte enucleation. Incubation of denuded eggs in 10 µg ml−1MMC for different periods did not affect most maturation rates (0.5 h: 85.78%A, 1.0 h: 72.77%B, 1.5 h: 83.87%A, and 2.0 h: 82.05%A) in comparison with non-treated controls (CTL; 85.77%A). Parthenogenetic development arrest by MMC was efficient at cleavage (CTL: 72.93%A, 0.5 h: 64.92%A,B, 1.0 h: 60.39%B,C, 1.5 h: 66.35%A,B, and 2.0 h: 53.84%C) and blastocyst stages (CTL: 33.94%A, 0.5 h: 7.58%B, 1.0 h: 2.47%C, 1.5 h: 0.46%C, and 2.0 h: 0.51%C). Blastocysts were obtained after nuclear transfer (NT) using MMC enucleation [NT(MMC): 4.54%B] but at lower rates than for the SCNT control [NT(CTL): 26.31%A]. The removal of the meiotic spindle after MMC incubation fully restored SCNT blastocyst development [NT(MMC+SR): 24.74%A]. Early pregnancies were obtained by the transfer of NT(MMC) and NT(MMC+SR) blastocysts to synchronized recipients. In conclusion, MMC leads to functional chemical oocyte enucleation during SCNT and further suggests its potential for application towards technical improvements.

Enucleated mammalian cells (cytoplasts) have been widely used for studying differential roles of the cytoplasm and nucleus in various cellular processes. Here, we reported an improved enucleation protocol, in which cells were seeded in extracellular matrix (ECM)-coated 24-wells and spun at 4600 g and 35 °C for 60 min in the presence of cytochalasin B and colchicine. When glass-bottom wells were used, cellular structures and organelles in cytoplasts could be examined directly by confocal microscopy. Nuclear envelope rupture did not occur probably due to mild centrifugation conditions used in this study. Addition of paclitaxel or doxorubicin completely blocked proliferation of residual nucleated cells; however, to our surprise, paclitaxel dramatically prolonged the survival of cytoplasts. Results from Annexin V and Propidium Iodide staining showed that cytoplasts died predominantly by apoptosis, which was partially inhibited by ECM and further by paclitaxel. Mitochondria were mostly rod-shaped and formed a connected network in paclitaxel-treated cytoplasts, indicating lack of fusion and fission dynamics. Moreover, paclitaxel increased mitochondrial membrane potential, suggesting that perturbation of mitochondria might be critical to the survival of cytoplasts. In conclusion, we had established an efficient and fast procedure for enucleation of adherent animal cells, which could facilitate the investigation of nucleocytoplasmic interaction.

International audience; The centrosome is the main organizer of microtubules and as such, its position is a key determinant of polarized cell functions. As the name says, the default position of the centrosome is considered to be the cell geometrical center. However, the mechanism regulating centrosome positioning is still unclear and often confused with the mechanism regulating the position of the nucleus to which it is linked. Here, we used enucleated cells plated on adhesive micropatterns to impose regular and precise geometrical conditions to centrosome-microtubule networks. Although frequently observed there, the equilibrium position of the centrosome is not systematically at the cell geometrical center and can be close to cell edge. Centrosome positioning appears to respond accurately to the architecture and anisotropy of the actin network, which constitutes, rather than cell shape, the actual spatial boundary conditions the microtubule network is sensitive to. We found that the contraction of the actin network defines a peripheral margin in which microtubules appear bent by compressive forces. The progressive disassembly of the actin network at distance from the cell edges defines an inner zone where actin bundles were absent, where microtubules were more radially organized and where dynein concentration was higher. We further showed that the production of dynein-based forces on microtubules places the centrosome at the center of this zone. In conclusion, the spatial distribution of cell adhesion and the production of contractile forces define the architecture of the actin network with respect to which the centrosome-microtubule network is centered.

Reduced developmental competence after IVF has been reported using oocyte derived from small follicles in several species including cattle, sheep, and goats. No information is currently available about the effect of follicle size of the cytoplast donor on in vivo development after somatic cell nuclear transfer (SCNT) in goats. Oocytes collected from large (≥3 mm) and small follicles (

Confocal laser scanning microscopy for the first time is used to examine the structure of the tobacco microsporocytes involved in the intercellular migration of nuclei (cytomixis). As is observed, the cytomictic channels are distributed over the surface of tobacco microsporocytes in a non-random manner and their number depends on the meiotic stage. Analysis of non-squash cells demonstrates the differences in cytological patterns of cytomixis in a normal meiosis of control tobacco plants (SR1 line) and the abnormal meiosis of polyploids. As a rule, two to three adjacent cells are involved in cytomixis during meiosis of control tobacco plants; after cytomixis, several micronuclei are formed in recipient cells; cytoplasts (enucleated cells) are rare; and polyads are undetectable. In the meiosis of polyploids, cytomixis is massive, with a larger number of cells (sometimes, over ten) involved in nuclear migration simultaneously; recipient cells on completion of cytomixis develop tens of micronuclei; cytoplasts and polyads are frequently detectable.

Interspecies somatic cell nuclear transfer (iSCNT) can be a solution for preservation of endangered species that have limited oocytes. It has been reported that blastocyst production by iSCNT is successful even if the genetic distances between donors and recipients are large. In particular, domestic pig oocytes can support the development of canine to porcine iSCNT embryos. Therefore, we examined whether porcine oocytes may be suitable recipient oocytes for Korean raccoon dog iSCNT. We investigated the effects of trichostatin A (TSA) treatment on iSCNT embryo developmental patterns and nucleolus formation. Enucleated porcine oocytes were fused with raccoon dog fibroblasts by electrofusion and cleavage, and blastocyst development and nucleolus formation were evaluated. To our knowledge, this study is the first in which raccoon dog iSCNT was performed using porcine oocytes; we found that 68.5% of 158 iSCNT embryos had the ability to cleave. However, these iSCNT embryos did not develop past the 4-cell stage. Treatment with TSA did not affect iSCNT embryonic development; moreover, the nuclei failed to form nucleoli at 48 and 72 h post-activation (hpa). In contrast, pig SCNT embryos of the control group showed 18.8% and 87.9% nucleolus formation at 48 and 72 hpa, respectively. Our results demonstrated that porcine cytoplasts efficiently supported the development of raccoon dog iSCNT embryos to the 4-cell stage, the stage of porcine embryonic genome activation (EGA); however, these embryos failed to reach the blastocyst stage and showed defects in nucleolus formation.

The objective of this study was to compare the thermotolerance of ear fibroblasts derived from various SCNT cattle. Specimens were produced from cloned embryos that had been reconstructed using donor cells (d) from the same Holstein cow (Hd) and the ooplasm (o) from Holstein cattle (Ho) or Taiwan yellow cattle (Yo). Polymorphism in the D-loop region of mitochondrial DNA in ear fibroblasts derived from SCNT cattle reconstructed with the Y ooplasm and H donor cells (SCNT-Yo-Hd) indicates that the cytoplasm originated from Bos indicus. The rates of apoptosis in heat-shocked ear fibroblasts derived from SCNT-Yo-Hd cattle (1.9%) and purebred Y cattle (1.5%) were significantly (P < 0.05) lower than those of cells derived from SCNT cattle reconstructed with the H ooplasm (SCNT-Ho-Hd: 3.4%), donor cells (4.0%), and purebred Holstein (4.1%) cattle. At the protein level, the relative abundances of apoptosis-inducing factor, B cell lymphoma 2-associated X protein, endonuclease G, cytochrome c, cysteinyl aspartate-specific proteinases 3, 8 and 9 in ear fibroblasts derived from SCNT-Yo-Hd cattle were significantly (P < 0.05) lower than those of cells derived from SCNT-Ho-Hd cattle after heat shock. In contrast, the relative abundances of heat shock proteins 27, 70 and B cell lymphoma 2 in ear fibroblasts derived from SCNT-Yo-Hd cattle were higher (P < 0.05) than those of fibroblasts derived from SCNT-Ho-Hd cattle. Moreover, heat-shocked ear fibroblasts derived from SCNT-Yo-Hd cattle have a significantly (P < 0.05) lower percentage of apoptosis-inducing factor-positive nuclei than do heat-shocked ear fibroblasts derived from SCNT-Ho-Hd cattle (11.1% vs. 18.5%). Taken together, these results report that ear fibroblasts derived from SCNT cattle reconstructed using the Y ooplasm are more thermotolerant than ear fibroblasts derived from SCNT cattle reconstructed using the H ooplasm. This is an indication that the cytoplasm may be a major determinant of thermal sensitivity in bovine ear fibroblasts.

The aim of this study was to evaluate if the positive effects of inhibiting histone deacetylase enzymes on cell reprogramming and development of somatic cell nuclear transfer (SCNT) embryos is affected by the cell cycle stage of nuclear donor cells and host oocytes at the time of embryo reconstruction. SCNT embryos were produced with metaphase II (MII) or telophase II (TII) cytoplasts and nuclear donor cells that were either at the G1-0 or G2/M stages. Embryos reconstructed with the different cell cycle combinations were treated or not with the histone deacetylase inhibitor (HDACi) Scriptaid for 15 h and then cultured in vitro for 7 days. Embryos reconstructed with MII-G1-0 and TII-G2/M developed to the blastocyst stage with a higher frequency compared to the other groups, confirming the importance of cell cycle interactions on cell reprogramming and SCNT embryo development. Treatment with HDACi improved development of SCNT embryos produced with MII but not TII cytoplasts, independently of the cell cycle stage of nuclear donor cells. These findings provide evidence that the positive effect of HDACi treatment on development of SCNT embryos depends upon cell cycle interactions between the host cytoplast and the nuclear donor cells.

Purpose The present study compared the in vivo efficacy of a novel synthesized polycaprolactone (PCL)/polyethylene glycol (PEG)/bioactive glass (BG) nanocomposite membrane versus a cytoplast (Cy) membrane in terms of the average percentage of new bone formation and inflammation levels. Materials and Methods In the present interventional animal study, 12 male New Zealand rabbits were tested. In the parietal bone of the rabbits, 24 defects were prepared (2 defects for each rabbit), which were divided into 3 equal groups (Cy, PCL, and control). Each rabbit's calvarial bone was prepared for the histologic and histomorphometric survey. The amount of regenerated bone (ie, length, area, percentage), necrosis rate, fibrosis (fibrosis plus and percentage), and inflammation in the standard defects of parietal bone in the rabbits were examined and compared after 10 weeks. Results A significant difference was found between the Cy and PCL groups regarding the mean area and thickness of the bone. We also found a significant difference in the bone length, area, and percentage formed between PCL and control groups. Also, the rate of fibrous tissue formation was significantly different statistically between the PCL and control groups. The results showed the influence of the PCL membrane in generating more bone and less fibrous tissue. In all 3 groups, negligible inflammation and no necrosis was observed. Conclusions The results of the present study have shown that combining PCL, PEG, and BGs could be promising for bone regeneration in jaw defects, around dental implants, and in oral and maxillofacial defects.

Neutrophil extracellular trap (NET) formation can generate short-term, functional anucleate cytoplasts and trigger loss of cell viability. We demonstrated that the necroptotic cell death effector mixed lineage kinase domain–like (MLKL) translocated from the cytoplasm to the plasma membrane and stimulated downstream NADPH oxidase– independent ROS production, loss of cytoplasmic granules, breakdown of the nuclear membrane, chromatin decondensation, histone hypercitrullination, and extrusion of bacteriostatic NETs. This process was coordinated by receptor-interacting protein kinase-1 (RIPK1), which activated the caspase-8–dependent apoptotic or RIPK3/ MLKL-dependent necroptotic death of mouse and human neutrophils. Genetic deficiency of RIPK3 and MLKL prevented NET formation but did not prevent cell death, which was because of residual caspase-8–dependent activity. Peptidylarginine deiminase 4 (PAD4) was activated downstream of RIPK1/RIPK3/MLKL and was required for maximal histone hypercitrullination and NET extrusion. This work defines a distinct signaling network that activates PAD4-dependent NET release for the control of methicillin-resistant Staphylococcus aureus (MRSA) infection.

Significance Trypanosoma brucei is a highly invasive pathogen capable of penetrating deeply into host tissues. To understand how flagellar motility facilitates cell penetration, we used cryo-electron tomography (cryo-ET) to visualize two genetically anucleate mutants with different flagellar motility behaviors. We found that the T. brucei cell body is highly deformable as defined by changes in cytoskeletal twist and spacing, in response to flagellar beating and environmental conditions. Based on the cryo-ET models, we proposed a mechanism of how flagellum motility is coupled to cell shape changes, which may facilitate penetration through size-limiting barriers., In the unicellular parasite Trypanosoma brucei, the causative agent of human African sleeping sickness, complex swimming behavior is driven by a flagellum laterally attached to the long and slender cell body. Using microfluidic assays, we demonstrated that T. brucei can penetrate through an orifice smaller than its maximum diameter. Efficient motility and penetration depend on active flagellar beating. To understand how active beating of the flagellum affects the cell body, we genetically engineered T. brucei to produce anucleate cytoplasts (zoids and minis) with different flagellar attachment configurations and different swimming behaviors. We used cryo-electron tomography (cryo-ET) to visualize zoids and minis vitrified in different motility states. We showed that flagellar wave patterns reflective of their motility states are coupled to cytoskeleton deformation. Based on these observations, we propose a mechanism for how flagellum beating can deform the cell body via a flexible connection between the flagellar axoneme and the cell body. This mechanism may be critical for T. brucei to disseminate in its host through size-limiting barriers.

The cell wall provides external support of the plant cells, while the cytoskeletons including the microtubules and the actin filaments constitute an internal framework. The cytoskeletons contribute to the cell wall biosynthesis by spatially and temporarily regulating the transportation and deposition of cell wall components. This tight control is achieved by the dynamic behavior of the cytoskeletons, but also through the tethering of these structures to the plasma membrane. This tethering may also extend beyond the plasma membrane and impact on the cell wall, possibly in the form of a feedback loop. In this review, we discuss the linking components between the cytoskeletons and the plasma membrane, and/or the cell wall. We also discuss the prospective roles of these components in cell wall biosynthesis and modifications, and aim to provide a platform for further studies in this field.

Cybridization should be a way to induce phenotypical variations in highly heterozygous secondary citrus species or genotypes, without breaking their very complex multilocus structure by sexual recombination. It should have particular interest to manipulate the nucleo-cytoplasmic male sterility, to transfer tolerance traits with cytoplasmic determinisms and eventually to improve tolerance to oxidative stress. The present work is focused on the creation of new cybrids through three different approaches of protoplast fusion: (i) symmetric hybridization, (ii) donor-recipient protoplast fusion by UV irradiation of donor protoplasts, and (iii) cytoplasts + protoplast fusion. Sixteen combinations of symmetric protoplast fusion have produced cybrids. No cybrid was obtained after UV irradiation of donor protoplasts while three combinations of cytoplasts + protoplast fusion produced cybrids. Therefore, cytoplast fusion appears much more promising than the UV approach for targeted cybridization. The innovative plant material is currently propagated for further cultivar and rootstock selection and for basic researches on the impact of nucleo-cytoplasmic interaction on desirable traits. (Resume d'auteur)

Somatic cell nuclear transfer is a technique to create an embryo using an enucleated oocyte and a donor nucleus. Nucleus of somatic cells must be reprogrammed in order to participate in normal development within an enucleated egg. Reprogramming refers to the erasing and remodeling of cellular epigenetic marks to a lower differentiation state. Somatic nuclei must be reprogrammed by factors in the oocyte cytoplasm to a rather totipotent state since the reconstructed embryo must initiate embryo development from the one cell stage to term. In embryos reconstructed by nuclear transfer, the donor genetic material must respond to the cytoplasmic environment of the cytoplast and recapitulate this normal developmental process. Enucleation is critically important for cloning efficiency because may affect the ultrastructure of the remaining cytoplast, thus resulting in a decline or destruction of its cellular compartments. Nonetheless, the effects of in vitro culturing are yet to be fully understood. In vitro oocyte maturation can affect the abundance of specific transcripts and are likely to deplete the developmental competence. The epigenetic modifications established during cellular differentiation are a major factor determining this low efficiency as they act as epigenetic barriers restricting reprogramming of somatic nuclei. In this review we discuss some factors that could impact cell differentiation in embryo generated by nuclear transfer.

To compare the expression profile of developmentally important genes between hand-made cloned buffalo embryos produced from reprogramming of donor cell with oocyte extracts and selection of recipient cytoplast through brilliant cresyl blue staining and in vitro fertilized (IVF) embryos.Hand-made cloned embryos were produced using oocyte extracts treated donor cells and brilliant cresyl blue (BCB) stained recipient cytoplasts. IVF embryos were produced by culturing 15-20 COCs in BO capacitated sperms from frozen thawed buffalo semen and the mRNA expression patterns of genes implicated in metabolism (GLUT1), pluripotency (OCT4), DNA methylation (DNMT1), pro- apoptosis (BAX) and anti-apoptosis (BCL2) were evaluated at 8- to16- cell stage embryos.A significantly (P 0.05) higher number of 8- to16- cell and blastocyst stages (73.9 %, 32.8 %, respectively) were reported in hand-made cloning (HMC) as compared to in vitro fertilization (49.2 %, 24.2 %, respectively). The amount of RNA recovered from 8- to 16- cell embryos of HMC and in vitro fertilization did not appear to be influenced by the method of embryo generation (3.76 ± 0.61 and 3.82 ± 0.62 ng/μl for HMC and in vitro fertilization embryos, respectively). There were no differences in the expression of the mRNA transcripts of genes (GLUT1, OCT4, DNMT1, BAX and BCL2) were analysed by real-time PCR between hand-made cloned and IVF embryos.Pre-treatment of donor cells with oocyte extracts and selection of developmentally competent oocytes through BCB staining for recipient cytoplast preparations may enhance expression of developmentally important genes GLUT1, OCT4, DNMT1, BAX, and BCL2 in hand-made cloned embryos at levels similar to IVF counterparts. These results also support the notion that if developmental differences observed in HMC and in vitro fertilization produced foetuses and neonates are the results of aberrant gene expression during the pre-implantation stage, those differences in expression are subtle or appear after the maternal to zygotic transition stage of development.

The gradual decline in the genetic diversity of farm animals has threatened their survival and risk of their extinction has increased many fold in the recent past. Endangered species could be rescued using interspecies embryo production. The objective of this study was to investigate the effect of three different culture media on the development of Handmade cloned intraspecies (goat-goat) and interspecies (goat-sheep) embryo reconstructs. Research vitro cleave media (RVCL) yielded higher cleavage and morula-blastocyst development in intraspecies and interspecies nuclear transfer groups compared with G1.G2 and modified synthetic oviductal fluid (mSOFaaci). Cleavage frequency of intraspecies cloned embryos in RVCL, mSOFaaci, and G1.G2 did not differ significantly (87.12%, 82.45%, and 92.52%, respectively). However, the morula/blastocyst frequency in RVCL was greater in mSOFaaci and G1.G2 (51.18% vs. 38.28% vs. 36.50%, respectively). Cleavage and morula/blastocyst frequency in interspecies cloned embryos was greater in RVCL than in mSOFaaci and G1.G2 (76.14% and 42.3% vs. 65.9% and 38.3% vs. 58.56% and 33.1%, respectively). Goat oocytes were parthenogenetically activated and cultured in RVCL, mSOFaaci, and G1.G2 and kept as control. Cleavage and morula/blastocyst frequency in this group was greater in RVCL than in mSOFaaci and G1.G2 (89.66% and 65.26% vs. 85.44% and 48.05% vs. 86.58% and 42.06%, respectively). Conclusively, the results suggest that not only can the interspecies embryos of goat be produced using sheep oocytes as donor cytoplast but also the percentages can be improved by using RVCL media for culturing of the embryos.

Neutrophil cytoplasts promote T helper 17 cell responses that propagate neutrophilic inflammation in severe asthma.