CRISPR-Edited Stem Cell Transplantation for HIV-Related Gene Modification In Vivo: A Systematic Review.

BACKGROUND: CRISPR is a novel genomic editing technology which can be useful for the treatment of immune diseases such as HIV. However, the application of CRISPR in stem cells for HIV-related research was not effective, and most of the research was done in vivo. This systematic review is to identify a new research idea about increase CRISPR-editing efficiencies in stem cell transplantation for HIV treatment, as well as its future perspective. METHOD: Four databases were searched for articles published during 1952 to 2020. PRISMA method was used to select appropriate research papers. CAMARADES was used to identify the paper quality. The outcome was engraftment efficiency, gene disruption percentage, differentiation ability, HIV-resistant efficiency. RESULT: Screening method showed 196 papers mentioned the topic. However, only 5 studies were reliable with the research objective. We found that (1) Two research ideas which was double gene knockout and knockout-knockin method to provide HIV-resistant cells, engraftment support and avoid cardiac disease as an HIV disease side effect. (2) Ribonucleoprotein (RNP) delivery was the best way to deliver the CRISPR/Cas9 and Adeno-Associated Virus (AAV) would be effective for knockin purpose. (3) CRISPR/SaCas9 could replace CRISPR/Cas9 role in editing HIV-related gene. CONCLUSION: Potential genes to increase HIV resistance and stem cell engraftment should be explored more in the future. Double knockout and knock-in procedures should be applied to set up a better engraftment for improving HIV treatment or resistance of patients. CRISPR/SaCas9 and RNP delivery should be explored more in the future. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42020203312.

Mycotoxin Fumonisin B1 Interferes Sphingolipid Metabolisms and Neural Tube Closure during Early Embryogenesis in Brown Tsaiya Ducks.

Fumonisin B1 (FB1) is among the most common contaminants produced by Fusarium spp. fungus from corns and animal feeds. Although FB1 has been known to cause physical or functional defects of embryos in humans and several animal species such as Syrian hamsters, rabbits, and rodents, little is known about the precise toxicity to the embryos and the underlying mechanisms have not been fully addressed. The present study aimed to investigate its developmental toxicity and potential mechanisms of action on sphingolipid metabolism in Brown Tsaiya Ducks (BTDs) embryos. We examined the effect of various FB1 dosages (0, 10, 20 and 40 µg/embryo) on BTD embryogenesis 72 h post-incubation. The sphingomyelin content of duck embryos decreased (p < 0.05) in the highest FB1-treated group (40 µg). Failure of neural tube closure was observed in treated embryos and the expression levels of a neurulation-related gene, sonic hedgehog (Shh) was abnormally decreased. The sphingolipid metabolism-related genes including N-acylsphingosine amidohydrolase 1 (ASAH1), and ceramide synthase 6 (CERS6) expressions were altered in the treated embryos compared to those in the control embryos. Apparently, FB1 have interfered sphingolipid metabolisms by inhibiting the functions of ceramide synthase and folate transporters. In conclusion, FB1-caused developmental retardation and abnormalities, such as neural tube defects in Brown Tsaiya Duck embryos, as well as are partly mediated by the disruption of sphingolipid metabolisms.

Effects of electromagnetic waves on oocyte maturation and embryonic development in pigs.

Our living environment has been full of electromagnetic radiation (EMR) due to the prevailing electronic devices and equipment. Intermediate frequency electromagnetic field (IF-EMF) or waves constitute a significant part of EMR; therefore, an increasing number of household electrical appliances have become a source of IF-EMF, and concerns about IF-EMF on health are gaining more attention. However, little information is available about its impact on female reproductive traits, such as germ cell viability and early embryonic development, particularly at the cellular and molecular levels. In this study, we used porcine oocytes as a model system to explore the effect of IF-EMF at various intensities on the in vitro maturation (IVM) of oocytes and their subsequent embryonic development. Our results showed that no difference in oocyte maturation rates was detected among groups, but the cleavage and blastocyst rates of parthenotes derived from EMF-treated oocytes decreased with the weaker IF-EMF intensity (25 and 50 Gauss) groups compared to the control group (P < 0.05). For cytoplasmic maturation, the weaker IF-EMF intensity groups also showed a peripheral pattern of mitochondrial distribution resembling that of immature oocytes and increased autophagy activity. No obvious differences in cytoskeletal distribution and total cell numbers of blastocysts were investigated in the four IF-EMF treatments compared to those in the control group. Although the underlying mechanism associated with EMF effects on oocytes and embryos is still elusive, we have demonstrated that low intensity IF-EMF exerts harmful effects on porcine oocytes during the maturation stage, carrying over such effects to their subsequent embryonic development.

Differential Effects of Green Tea Powders on the Protection of Brown Tsaiya and Kaiya Ducklings against Trichothecene T-2 Toxin Toxicity.

A 3-week feeding trial in a 3 × 2 × 2 factorial design was conducted with three concentrations (0, 0.5, and 5 mg/kg) of T-2 toxin (T-2) and two levels (0% and 0.5%) of green tea powder (GTP) supplements used in the diets of female brown Tsaiya ducklings (BTDs) and Kaiya ducklings (KDs), respectively. Breed had a significant effect on the growth performances and the relative weights of organs and carcass. In general, the growth performances of KDs were better than BTDs. The relative weights of organs and carcass of BTDs were typically heavier than those of KDs; however, the breast of KDs was heavier than those of BTDs. Both ducklings received 5 mg/kg of T-2 blended in the diet showed lower feed intake and body weight gain (BWG) in the second and the third week. The diet containing 5 mg/kg of T-2 and 0.5% GTP improved the BWG compared to those fed the diet supplemented with 5 mg/kg of T-2 without GTP in BTDs. Ducklings fed the diet containing 5 mg/kg of T-2 induced hypocalcemia and hypomagnesemia, as well as decreased concentrations of creatine phosphokinase and alkaline phosphatase. The concentrations of blood urea nitrogen (BUN) and glutamate oxaloacetate transaminase (GOT) were increased in KDs and BTDs fed the diet containing 5 mg/kg of T-2 without GTP, respectively. However, duckling diets containing 5 mg/kg of T-2 with 0.5% GTP lowered concentrations of BUN and GOT in the blood plasma of KDs and BTDs, respectively. The diet containing 5 mg/kg of T-2 increased the relative kidney weight but decreased the relative breast weight of ducklings. Enlarged gizzards and reduced relative leg weights were observed in BTDs fed the diets containing 5 mg/kg of T-2. In summary, BTDs are more sensitive than KDs in responding to T-2 toxicity and GTP detoxification. Green tea powder has detoxification ability and could potentially mitigate T-2 toxicity on BWG, BUN, and GOT in ducklings.

The Potential of Peroxidases Extracted from the Spent Mushroom (Flammulina velutipes) Substrate Significantly Degrade Mycotoxin Deoxynivalenol.

Little is known about the degradability of mycotoxin deoxynivalenol (DON) by the spent mushroom substrate (SMS)-derived manganese peroxidase (MnP) and lignin peroxidase (LiP) and its potential. The present study investigated the growth inhibition of Fusarium graminearum KR1 and the degradation of DON by MnP and LiP extracted from SMS. The results from the 7-day treatment period showed that mycelium inhibition of F. graminearum KR1 by MnP and LiP were 23.7% and 74.7%, respectively. Deoxynivalenol production in the mycelium of F. graminearum KR1 was undetectable after treatment with 50 U/mL of MnP or LiP for 7 days. N-acetyl-D-glucosamine (GlcNAc) content and chitinase activity both increased in the hyphae of F. graminearum KR1 after treatment with MnP and LiP for 1, 3, and 6 h, respectively. At 12 h, only the LiP-treated group had higher chitinase activity and GlcNAc content than those of the control group (p < 0.05). However, more than 60% of DON degradabilities (0.5 mg/kg, 1 h) were observed under various pH values (2.5, 4.5, and 6.5) in both MnP (50 U/g) and LiP (50 U/g) groups, while DON degradability at 1 mg/kg was 85.5% after 50 U/g of LiP treatment for 7 h in simulated pig gastrointestinal tracts. Similarly, DON degradability at 5 mg/kg was 67.1% after LiP treatment for 4.5 h in simulated poultry gastrointestinal tracts. The present study demonstrated that SMS-extracted peroxidases, particularly LiP, could effectively degrade DON and inhibit the mycelium growth of F. graminearum KR1.

SARS Unique Domain (SUD) of Severe Acute Respiratory Syndrome Coronavirus Induces NLRP3 Inflammasome-Dependent CXCL10-Mediated Pulmonary Inflammation.

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) initiates the cytokine/chemokine storm-mediated lung injury. The SARS-CoV unique domain (SUD) with three macrodomains (N, M, and C), showing the G-quadruplex binding activity, was examined the possible role in SARS pathogenesis in this study. The chemokine profile analysis indicated that SARS-CoV SUD significantly up-regulated the expression of CXCL10, CCL5 and interleukin (IL)-1ß in human lung epithelial cells and in the lung tissues of the mice intratracheally instilled with the recombinant plasmids. Among the SUD subdomains, SUD-MC substantially activated AP-1-mediated CXCL10 expression in vitro. In the wild type mice, SARS-CoV SUD-MC triggered the pulmonary infiltration of macrophages and monocytes, inducing CXCL10-mediated inflammatory responses and severe diffuse alveolar damage symptoms. Moreover, SUD-MC actuated NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome-dependent pulmonary inflammation, as confirmed by the NLRP3 inflammasome inhibitor and the NLRP3-/- mouse model. This study demonstrated that SARS-CoV SUD modulated NLRP3 inflammasome-dependent CXCL10-mediated pulmonary inflammation, providing the potential therapeutic targets for developing the antiviral agents.

Ultrastructural Characterization of Porcine Growing and In Vitro Matured Oocytes.

This study aimed to investigate ultrastructural changes of growing porcine oocytes and in vitro maturated oocytes. Light microscopy was used to characterize and localize the primordial, primary, secondary, and tertiary follicles. During oocyte growth and maturation, the morphology of mitochondria was roundish or ovoid in shape depending on the differentiation state, whereas their mean diameters oscillated between 0.5 and 0.7 µm, respectively, from primary and secondary follicles. Hooded mitochondria were found in the growing oocytes of the tertiary follicles. In addition to the pleomorphism of mitochondria, changes in the appearance of lipid droplets were also observed, along with the alignment of a single layer of cortical granules beneath the oolemma. In conclusion, our study is apparently the first report to portray morphological alterations of mitochondria that possess the hooded structure during the growth phase of porcine oocytes. The spatiotemporal and intrinsic changes during oogenesis/folliculogenesis are phenomena at the ultrastructural or subcellular level of porcine oocytes, highlighting an in-depth understanding of oocyte biology and impetus for future studies on practical mitochondrion replacement therapies for oocytes.

Enzyme Degradation Reagents Effectively Remove Mycotoxins Deoxynivalenol and Zearalenone from Pig and Poultry Artificial Digestive Juices.

Mycotoxin removers include enzymes and adsorbents that may be used in animal feeds to eliminate the toxic effects of mycotoxins. This study aimed to determine the removability of two different types of mycotoxin removers, adsorbents and enzyme degradation reagents (EDRs), in the simulated gastrointestinal conditions of pigs and poultry. Seven commercial mycotoxin removers, including five EDRs and two adsorbents, were tested in vitro. In this study, the supplemented dosages of mycotoxin removers used in pig and poultry feeds were the commercial recommendation ranging from 0.05% to 0.2%. For pigs, the in vitro gastric and small intestinal simulations were performed by immersing the mycotoxin-tainted feed in artificial gastric juice (AGJ) at pH 2.5 for 5 h or in artificial intestinal juice (AIJ) at pH 6.5 for 2 h to mimick in vivo conditions. For poultry, mycotoxin-tainted feeds were immersed in AGJ for 2 h at pH 4.5 and 0.5 h at pH of 2.5, respectively, to simulate crop/glandular stomach and gizzard conditions; the small intestinal simulation was in AIJ for 2 h at pH 6.5. For the pig, EDRs and adsorbents had deoxynivalenol (DON) removability (1 mg/kg) of 56% to 100% and 15% to 19%, respectively. Under the concentration of 0.5 mg/kg, the zearalenone (ZEN) removability by EDRs and adsorbents was 65% to 100% and 0% to 36%, respectively. For the simulation in poultry, the removability of DON by EDRs and adsorbents (5 mg/kg) was 56% to 79% and 1% to 36%, respectively; for the concentration of 0.5 mg/kg, the removability of ZEN by EDRs and adsorbents was 38% to 69% and 7% to 9%, respectively. These results suggest that EDRs are more effective in reducing DON and ZEN contamination compared to the adsorbent methods in the simulated gastrointestinal tracts of pig and poultry. The recoveries of DON and ZEN of pig in vitro gastrointestinal simulations were higher than 86.4% and 84.7%, respectively, with 88.8% and 85.9%, respectively, in poultry. These results demonstrated the stability and accuracy of our mycotoxin extraction process and in vitro simulation efficiency.

Nucleus, Cytoskeleton, and Mitogen-Activated Protein Kinase p38 Dynamics during In Vitro Maturation of Porcine Oocytes.

The mitogen-activated kinase (MAPK) p38, a member of the MAPK subfamily, is conserved in all mammalian cells and plays important roles in response to various physiologic cues, including mitogens and heat shock. In the present study, MAPK p38 protein expression in porcine oocytes was analyzed during in vitro maturation (IVM) by Western blotting and immunocytochemistry. The levels of p-p38 or activated p38 and p38 expression were at the lowest in the germinal vesicle (GV) stage oocyte, gradually rising at the germinal vesicle breakdown (GVBD) and then reaching a plateau throughout the IVM culture (p < 0.05). Similarly, the expression level of total p38 was also lower in the GV oocyte than in the oocyte of other meiotic stages and uprising after GVBD and remained high until the metaphase III (MII) stage (p < 0.05). In the GV stage, phosphorylated p38 (p-p38) was initially detectable in the ooplasm and subsequently became clear around the nucleus and localized in the ooplasm at GVBD (18 h post-culture). During the metaphase I (MI) and metaphase II (MII) stages, p-p38 was evenly distributed throughout the ooplasm after IVM for 30 or 42 h. We found that the subcellular localization increased in p-p38 expression throughout oocyte maturation (p < 0.05) and that dynamic reorganization of the cytoskeleton, including microfilaments and microtubules, was progressively changed during the course of meiotic maturation which was likely to be associated with the activation or networking of p38 with other proteins in supporting oocyte development. In conclusion, the alteration of p38 activation is essential for the regulation of porcine oocyte maturation, accompanied by the progressive reorganization and redistribution of the cytoskeleton and MAPK p38, respectively, in the ooplasm.

Developmental Toxicity of Mycotoxin Fumonisin B1 in Animal Embryogenesis: An Overview.

A teratogenic agent or teratogen can disturb the development of an embryo or a fetus. Fumonisin B1 (FB1), produced by Fusarium verticillioides and F. proliferatum, is among the most commonly seen mycotoxins and contaminants from stale maize and other farm products. It may cause physical or functional defects in embryos or fetuses, if the pregnant animal is exposed to mycotoxin FB1. Due to its high similarity in chemical structure with lipid sphinganine (Sa) and sphingosine (So), the primary component of sphingolipids, FB1 plays a role in competitively inhibiting Sa and So, which are key enzymes in de novo ceramide synthase in the sphingolipid biosynthetic pathway. Therefore, it causes growth retardation and developmental abnormalities to the embryos of hamsters, rats, mice, and chickens. Moreover, maternal FB1 toxicity can be passed onto the embryo or fetus, leading to mortality. FB1 also disrupts folate metabolism via the high-affinity folate transporter that can then result in folate insufficiency. The deficiencies are closely linked to incidences of neural tube defects (NTDs) in mice or humans. The purpose of this review is to understand the toxicity and mechanisms of mycotoxin FB1 on the development of embryos or fetuses.

Novel histone deacetylase inhibitors and embryo aggregation enhance cloned embryo development and ES cell derivation in pigs.

The histone deacetylase inhibitor (HDACi) has been investigated for treating cancers and many other diseases as well as enhancing the reprogramming efficiency in cloned embryos for decades. In the present study, we investigated the effects of two novel HDAC inhibitors, i.e., HDACi-14 and -79, at the concentrations of 0, 1, 2, or 4 µM on the development of embryos cloned by the oocyte bisection cloning technique (OBCT). Blastocyst rates for the reconstructed embryos reached 60% in the 2 µM HDACi-14-treated groups, which was higher (P < 0.05) compared to the untreated group (36.9%). Similarly, HDACi-79 treatment at 2 and 4 µM also conferred higher (P < 0.05) blastocyst rates than that of the untreated group (79.4, 74.2, and 50.0%, respectively). Both HDACi-14 and -79 treatments had no beneficial effect on total cell numbers and apoptotic indices of cloned embryos (P > 0.05). Histone acetylation profile by both HDACi-14 (2 µM) and -79 (2 µM) treatments demonstrated a drastic increase (P < 0.05) mainly in two-cell stage embryos when compared to the control group. After seeding on the feeder cells, the aggregated cloned blastocysts produced by the HDACi-79 treatment showed a significant increase of primary outgrowths compared to the control group (60.0% vs. 42.9%; P < 0.05). Finally, the cloned embryo-derived ES cell lines from aggregated cloned embryos produced from the HDACi-79-treated, HDACi-14-treated and control groups were established (5, 3, and 2 lines, respectively). In conclusion, the novel histone deacetylation inhibitors improve blastocyst formation and potentially increase the derivation efficiency of ES cell lines from the cloned porcine embryos produced in vitro. Depending on the purposes, some fine-tuning may be required to maximize its beneficial effects of these newly synthesized chemicals.

Characterizing early embryonic development of Brown Tsaiya Ducks (Anas platyrhynchos) in comparison with Taiwan Country Chicken (Gallus gallus domestics).

Avian embryos are among the most convenient and the primary representatives for the study of classical embryology. It is well-known that the hatching time of duck embryos is approximately one week longer than that of chicken embryos. However, the key features associated with the slower embryonic development in ducks have not been adequately described. This study aimed to characterize the pattern and the speed of early embryogenesis in Brown Tsaiya Ducks (BTD) compared with those in Taiwan Country Chicken (TCC) by using growth parameters including embryonic crown-tail length (ECTL), primitive streak formation, somitogenesis, and other development-related parameters, during the first 72 h of incubation. Three hundred and sixty eggs from BTD and TCC, respectively, were incubated at 37.2°C, and were then dissected hourly to evaluate their developmental stages. We found that morphological changes of TCC embryos shared a major similarity with that of the Hamburger and Hamilton staging system during early chick embryogenesis. The initial primitive streak in TCC emerged between 6 and 7 h post-incubation, but its emergence was delayed until 10 to 13 h post-incubation in BTD. Similarly, the limb primordia (wing and limb buds) were observed at 51 h post-incubation in TCC embryos compared to 64 h post-incubation in BTD embryos. The allantois first appeared around 65 to 68 h in TCC embryos, but it was not observed in BTD embryos. At the 72 h post-incubation, 40 somites were clearly formed in TCC embryos while only 32 somites in BTD embryos. Overall, the BTD embryos developed approximately 16 h slower than the chicken embryo during the first 72 h of development. To our best knowledge, this is the first study to describe two distinct developmental time courses between TCC and BTD, which would facilitate future embryogenesis-related studies of the two important avian species in Taiwan.

Sonic Hedgehog promotes in vitro oocyte maturation and term development of embryos in Taiwan native goats.

The aim of this study was to investigate the effects of Shh (Sonic Hedgehog) protein on caprine oocyte maturation, early embryo development, and developmental competence after embryo transfer of vitrified-thawed in vitro-produced embryos. Cumulus-oocyte complexes (COCs) derived from abattoir were randomly allocated to the in vitro maturation (IVM) medium supplemented with 0 (Control), 0.125, 0.25, 0.5, or 1.0 µg mL-1 recombinant mouse Shh protein. After IVM, COCs were fertilized with frozen-thawed semen and the presumptive zygotes were cultured on goat oviduct epithelial monolayers in M199 medium for 9 days. Our results showed that supplementation of Shh (0.25 or 0.5 µg mL-1) enhanced oocyte maturation as compared with the control group (92.4% and 95.0% vs. 86.2%, P < 0.05), yet the effect could be reversed by the simultaneous addition of cyclopamine (an inhibitor of Shh signaling by direct binding to the essential signal transducer Smo). Subsequently, an improved blastocyst rate (66.3 ± 10.9, P < 0.05) was observed for the embryos derived from the oocytes matured in the presence of 0.5 µg mL-1 Shh compared with the control group (41.4 ± 12.9). Expressions of Shh, SMO and Gli1 were observed in the ovaries, granulosa cells, COCs, cumulus cells, oocytes and oviduct epithelia. Notably, Ptch1 was expressed in nearly all of the aforementioned tissues and cells except cumulus cells. The embryos exhibited a higher survival rates in the Shh-supplemented group (37.5%) compared to those without Shh supplementation (14.8%; P < 0.05) after embryo transfer. This study demonstrated the beneficial effects of Shh supplementation on oocyte maturation and subsequent embryo development both in vitro and in vivo, suggesting a functional existence of Shh signaling during the final stage of folliculogenesis and early embryogenesis in caprine.

Effects of cytoplasts from Taiwan native yellow cattle on the cellular antioxidant ability of cloned Holstein cattle and their offspring.

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.

Ear fibroblasts derived from Taiwan yellow cattle are more heat resistant than those from Holstein cattle.

The objective of this study was to compare the thermotolerances of ear fibroblasts derived from Holstein (H) and Taiwan yellow cattle (Y) and their apoptosis-related protein expressions with (1, 3, 6, 12, and 24h) or without heat shock treatment. The results showed that the vaginal temperatures of Y (38.4-38.5°C) were (P<0.05) lower than that of H (38.8°C) during the hot season. The apoptotic rates of ear fibroblasts derived from Y (6h: 1.1%; 12h: 1.6%; 24h: 2.6%) were lower (P<0.05) than those of cells derived from H (6h: 1.8%; 12h: 4.0%; 24h: 6.9%), respectively, after heat shock (42°C). The expression level of apoptosis inducing factor (AIF) in ear fibroblasts derived from H was higher (P<0.05) than those derived from Y after the heat shock treatment for 6h and 12h, respectively. The level of cytochrome c of ear fibroblasts derived from H was higher (P<0.05) than those derived from Y after the heat shock treatment for 1-12h, respectively. The abundances of Caspase-3, Caspase-8 and Caspase-9 of ear fibroblasts derived from H were higher (P<0.05) than those of cells derived from Y after 12h and 24h of heat shock, respectively; the Bcl-2/Bax ratios of ear fibroblasts derived from H were lower (P<0.05) than those from Y-derived fibroblasts after heated for 1-24h. The expression level of HSP-70 of Y-derived ear fibroblasts was also higher (P<0.05) than that from H after the same duration of heat shock treatments. Taken together, the thermotolerance of ear fibroblasts derived from Taiwan yellow cattle was better than that of cells derived from Holstein cattle.

Fabrication of a reticular poly(lactide-co-glycolide) cylindrical scaffold for the in vitro development of microvascular networks.

The microvascular network is a simple but critical system that is responsible for a range of important biological mechanisms in the bodies of all animals. The ability to generate a functional microvessel not only makes it possible to engineer vital tissue of considerable size but also serves as a platform for biomedical studies. However, most of the current methods for generating microvessel networks in vitro use rectangular channels which cannot represent real vessels in vivo and have dead zones at their corners, hence hindering the circulation of culture medium. We propose a scaffold-wrapping method which enables fabrication of a customized microvascular network in vitro in a more biomimetic way. By integrating microelectromechanical techniques with thermal reflow, we designed and fabricated a microscale hemi-cylindrical photoresist template. A replica mold of polydimethylsiloxane, produced by casting, was then used to generate cylindrical scaffolds with biodegradable poly(lactide-co-glycolide) (PLGA). Human umbilical vein endothelial cells were seeded on both sides of the PLGA scaffold and cultured using a traditional approach. The expression of endothelial cell marker CD31 and intercellular junction vascular endothelial cadherin on the cultured cell demonstrated the potential of generating a microvascular network with a degradable cylindrical scaffold. Our method allows cells to be cultured on a scaffold using a conventional culture approach and monitors cell conditions continuously. We hope our cell-covered scaffold can serve as a framework for building large tissues or can be used as the core of a vascular chip for in vitro circulation studies.

Cellular thermotolerance is inheritable from Holstein cattle cloned with ooplasts of Taiwan native yellow cattle.

We have previously demonstrated that the somatic cells from cattle cloned with Holstein (H) donor cells and Taiwan native yellow cattle (Y) ooplasm (Yo-Hd) had better thermotolerance than those from cattle cloned with both Holstein donor cells and ooplasm (Ho-Hd). The present study aimed to investigate whether the cellular thermotolerance of these cloned cattle is transmissible to their offspring (Ho-Hd-F1 and Yo-Hd-F1). Thermotolerance of ear fibroblasts derived from these cloned cattle and their offspring were analyzed. Polymorphisms in mitochondrial DNA (mtDNA) D-loop of ear fibroblasts derived from Yo-Hd and Yo-Hd-F1 indicated that the cytoplasm is originated from Bos indicus (Y). After heat shock, the apoptotic rates, B-cell lymphoma 2-associated X protein/B-cell lymphoma 2 ratios, and relative expression levels of cysteine-aspartic proteases (caspases)-3, -8, and -9 of ear fibroblasts with Y-originated cytoplasm (including Y, Yo-Hd, and Yo-Hd-F1) were lower (P < 0.05) than those of ear fibroblasts with H-originated cytoplasm (including H, Ho-Hd, and Ho-Hd-F1). In contrast, the relative level of HSP-70 was higher (P < 0.05) in ear fibroblasts with Y-originated cytoplasm than that of with H-originated cytoplasm. Based on our results, thermotolerance of ear fibroblasts derived from Yo-Hd and Yo-Hd-F1 cattle is better and can be transmitted, at least at the cellular level, to their offspring.

Nonylphenol reduces sperm viability and fertility of mature male breeders in Brown Tsaiya ducks (Anas platyrhynchos).

The aim of this study was to investigate the effect of nonylphenol (NP), a widely used surfactant, on the reproductive performance of male Brown Tsaiya ducks (Anas platyrhynchos) (MBBTDs). Mature MBBTDs (n=100) were treated with NP by daily gavaging of 0, 1 (NP1), 10 (NP10) and 250 (NP250) mg/kg-BW/d for 14 wk. Semen quality, fertilization rate and specific factors in blood plasma were measured. Weights of organs were also measured at 14 wk after NP administration. Ducks from each treatment (n=4) were continually treated with NP thereafter for 12 mo to observe changes of tissue ultrastructure by microscopic examination. The results showed that ducks treated with amounts of NP of greater than 1mg NP/kg BW/d (NP1) for 14 wk had decreased sperm viability (32.3%) compared to those in the control group (74.1%, P<0.05). The fertilization rate of ducks treated with 250mg NP/kg-BW/d (NP250) for 14 wk was reduced (21.0%) compared to the control group (74.5%, P<0.05). Plasma aminotransferase (AST) and alanine aminotransferase (ALT) activities were also greater in NP250 group at the 14th wk post-treatment. Plasma testosterone concentrations were increased by NP1 treatment at the 14th wk post-treatment. Administration at dosage 250mg NP/kg-BW/d for 12 mo resulted in reduced sperm counts (P<0.05) and histopathological changes, such as dilated seminiferous tubules (P<0.05) and degenerated spermatocytes (P<0.05). These findings strongly suggest that NP adversely affects the reproductive performance of MBBTDs.

Derivation and characterization of putative embryonic stem cells from cloned rabbit embryos.

The present study aimed to establish embryonic stem (ES) cell lines, i.e., ntES cells, using rabbit blastocyst stage embryos cloned by somatic cell nuclear transfer. First, we investigated the development of cloned rabbit embryos reconstructed with normal fibroblasts and fibroblasts transfected with enhanced green fluorescence protein (eGFP). Blastocyst rates were 27.4% and 23.9%, respectively, for the embryos reconstructed with normal fibroblasts and fibroblasts transfected with eGFP compared with that from the parthenogenetic group (43.1%). One ntES cell line was established from embryos reconstructed with eGFP-transfected fibroblasts (1 of 17, 5.9%), and three ntES cell lines were derived from those with normal fibroblasts (3 of 17, 17.6%). All the ntES cell lines retained alkaline phosphatase activity and expressed ES cell-specific markers SSEA-4, Oct-4, TRA-1-60, and TRA-1-81. The pluripotency was further confirmed by reverse transcription-polymerase chain reaction analyses of Oct-4, Nanog, and Sox-2 expressions in ntES cell lines. The differentiation capacity of ntES cells was also examined in vitro and in vivo, by which these ntES cell lines were able to differentiate into all three germ layers through embryoid bodies and teratomas. In conclusion, it is apparent that the efficiency of ntES cells derived using eGFP-transfected donor cells is lower than that with nontransfected, normal fibroblasts donor cells. Similar to those from parthenogenetic embryos, all ntES cell lines derived from cloned rabbit embryos are able to express pluripotency markers and retain their capability to differentiate into various cell lineages both in vitro and in vivo.

An endothelial cultured condition medium embedded porous PLGA scaffold for the enhancement of mouse embryonic stem cell differentiation.

In this study, we have developed a microporous poly(lactic-co-glycolic acid) (PLGA) scaffold that combines a continuous release property and a three-dimensional (3D) scaffolding technique for the precise and efficient formation of endothelial cell lineage from embryonic stem cells (ESCs). Eight PLGA scaffolds (14.29%, 16.67%, 20% and 25% concentrations of PLGA solutions) mixed with two crystal sizes of sodium chloride (NaCl) were fabricated by leaching. Then, vascular endothelial cell conditioned medium (ECCM) mixed with gelatin was embedded into the scaffold for culturing of mouse embryonic stem cells (mESCs). The 14.29% PLGA scaffolds fabricated using non-ground NaCl particles (NG-PLGA) and the 25% PLGA containing scaffolds fabricated using ground NaCl particles (G-PLGA) possessed minimum and maximum moisture content and bovine serum albumin (BSA) content properties, respectively. These two groups of scaffolds were used for future experiments in this study. Cell culture results demonstrated that the proposed porous scaffolds without growth factors were sufficient to induce mouse ESCs to differentiate into endothelial-like cells in the early culture stages, and combined with embedded ECCM could provide a long-term inducing system for ESC differentiation.