A six-inhibitor culture medium for improving naïve-type pluripotency of porcine pluripotent stem cells.

Understanding essential signaling network requirements and making appropriate adjustments in culture conditions are crucial if porcine pluripotent stem cells (PSC) are to achieve their full potential. Here, we first used two protein factors (LIF and FGF2) and kinase inhibitor combinations in attempts to convert primed type lentiviral-reprogrammed porcine induced PSC (Lv-piPSC) into naïve-like state and developed a medium called FL6i. In addition to FGF2 and LIF, this medium contained inhibitors of MAPK14, MAPK8, TGFB1, MAP2K1, GSK3A and BMP. Crucially, the usual TGFB1 and BMP4 protein components of many stem cell media were replaced in FL6i with inhibitors of TGFB1 and BMP. With this medium, Lv-piPSC were readily transformed from their original primed state into cells that formed colonies with typical features of naïve-state stem cells. The FL6i medium also assisted generation of naïve-type piPSC lines from porcine embryonic fibroblasts with non-integrating episomal plasmids (Epi-piPSC). These lines, despite retaining variable amounts of vector DNA, expressed higher endogenous pPOU5F1 and pSOX2 than Lv-piPSC. They have been cultured without obvious morphological change for >45 passages and retained pluripotent phenotypes in terms of upregulation of genes associated with pluripotency, low expression of genes linked to emergence of somatic cell lineages, and ability to generate well differentiated teratomas in immune-compromised mice. FL6i conditions, therefore, appear to support elevated pluripotent phenotypes. However, FL6i was less able to support the generation of embryonic stem cells from porcine blastocysts. Although colonies with dome-shaped morphologies were evident and the cells had some gene expression features linked to pluripotency, the phenotypes were ultimately not stable. Pathway analysis derived from RNAseq data performed on the various cell lines generated in this study suggest the benefits of employing the FL6i medium on porcine cells reside in its ability to minimize TGFB1 and BMP signaling, which would otherwise de-stabilize the stem cell state.

Early onset preeclampsia in a model for human placental trophoblast.

We describe a model for early onset preeclampsia (EOPE) that uses induced pluripotent stem cells (iPSCs) generated from umbilical cords of EOPE and control (CTL) pregnancies. These iPSCs were then converted to placental trophoblast (TB) representative of early pregnancy. Marker gene analysis indicated that both sets of cells differentiated at comparable rates. The cells were tested for parameters disturbed in EOPE, including invasive potential. Under 5% O2, CTL TB and EOPE TB lines did not differ, but, under hyperoxia (20% O2), invasiveness of EOPE TB was reduced. RNA sequencing analysis disclosed no consistent differences in expression of individual genes between EOPE TB and CTL TB under 20% O2, but, a weighted correlation network analysis revealed two gene modules (CTL4 and CTL9) that, in CTL TB, were significantly linked to extent of TB invasion. CTL9, which was positively correlated with 20% O2 (P = 0.02) and negatively correlated with invasion (P = 0.03), was enriched for gene ontology terms relating to cell adhesion and migration, angiogenesis, preeclampsia, and stress. Two EOPE TB modules, EOPE1 and EOPE2, also correlated positively and negatively, respectively, with 20% O2 conditions, but only weakly with invasion; they largely contained the same sets of genes present in modules CTL4 and CTL9. Our experiments suggest that, in EOPE, the initial step precipitating disease is a reduced capacity of placental TB to invade caused by a dysregulation of O2 response mechanisms and that EOPE is a syndrome, in which unbalanced expression of various combinations of genes affecting TB invasion provoke disease onset.

AAV CRISPR editing rescues cardiac and muscle function for 18 months in dystrophic mice.

Adeno-associated virus-mediated (AAV-mediated) CRISPR editing is a revolutionary approach for treating inherited diseases. Sustained, often life-long mutation correction is required for treating these diseases. Unfortunately, this has never been demonstrated with AAV CRISPR therapy. We addressed this question in the mdx model of Duchenne muscular dystrophy (DMD). DMD is caused by dystrophin gene mutation. Dystrophin deficiency leads to ambulation loss and cardiomyopathy. We treated 6-week-old mice intravenously and evaluated disease rescue at 18 months. Surprisingly, nominal dystrophin was restored in skeletal muscle. Cardiac dystrophin was restored, but histology and hemodynamics were not improved. To determine the underlying mechanism, we evaluated components of the CRISPR-editing machinery. Intriguingly, we found disproportional guide RNA (gRNA) vector depletion. To test whether this is responsible for the poor outcome, we increased the gRNA vector dose and repeated the study. This strategy significantly increased dystrophin restoration and reduced fibrosis in all striated muscles at 18 months. Importantly, skeletal muscle function and cardiac hemodynamics were significantly enhanced. Interestingly, we did not see selective depletion of the gRNA vector after intramuscular injection. Our results suggest that gRNA vector loss is a unique barrier for systemic AAV CRISPR therapy. This can be circumvented by vector dose optimization.

Evolution and Expression Divergence of the CYP78A Subfamily Genes in Soybean.

Gene expression divergence is an important evolutionary driving force for the retention of duplicate genes. In this study, we identified three CYP78A subfamily genes in soybean, GmCYP78A70, GmCYP78A57 and GmCYP78A72, which experienced different duplication events. GmCYP78A70 was mainly expressed in leaf tissue and the vegetative phase, whereas GmCYP78A57 was mainly expressed in floral tissue and seed, i.e., the reproductive phase. Expression of GmCYP78A72 could be detected in all the tissues and phases mentioned above. The expression levels of GmCYP78A70 and GmCYP78A57 in different soybean cultivars showed positive correlations with leaf size and 100-seed weight, respectively. The population genetics analysis indicated that the three genes had experienced different selective pressures during domestication and improved breeding of soybean. Deciphering the function of this subfamily of genes may well prove useful to breeders for improving soybean's agronomic traits.

Up-Regulation of TAB3 Is Involved in Neuronal Apoptosis After Intracerebral Hemorrhage.

Human transforming growth factor ß-activated kinase (TAK1)-binding protein 3 (TAB3) is a regulator of NF-κB which has been mainly found in a variety of cancers. While TAB3 is highly expressed in brain tissue, little is known about the function of TAB3 in central nervous system. Our group established an animal ICH model with autologous whole blood injected into brain, and also a cell ICH model with hemin stimulation. Our Western blot result showed up-regulation of TAB3 during neuronal apoptosis in the model of intracerebral hemorrhage (ICH), which was also approved by immunofluorescence and immunohistochemistry result. Besides, increasing TAB3 level was accompanied by the increased expression of active-caspase-3, active-caspase-8, and decreased expression of Bcl-2. Furthermore, in in vitro study, the level of neuronal apoptosis was decreased by applying TAB3- RNA interference in PC12 cells. All the results above suggested that TAB3 probably participates in the process of neuronal apoptosis following ICH.

Efficient long-term cryopreservation of pluripotent stem cells at -80 °C.

Current long term cryopreservation of cell stocks routinely requires the use of liquid nitrogen (LN2), because commonly used cryopreservation media containing cell membrane permeating cryoprotectants are thermally unstable when frozen at higher storage temperatures, e.g. -80 °C. This instability leads to ice recrystallization, causing progressive loss of cell viability over time under the storage conditions provided by most laboratory deep freezers. The dependency on LN2 for cell storage significantly increases operational expense and raises issues related to impaired working efficiency and safety. Here we demonstrate that addition of Ficoll 70 to cryoprotectant solutions significantly improves system thermal stability at the working temperature (~-80 °C) of laboratory deep freezers. Moreover, a medium comprised of Ficoll 70 and dimethyl sulfoxide (DMSO) in presence or absence of fetal bovine serum (FBS) can provide reliable cryopreservation of various kinds of human and porcine pluripotent stem cells at -80 °C for periods that extend up to at least one year, with the post-thaw viability, plating efficiency, and full retention of pluripotent phenotype comparable to that achieved with LN2 storage. These results illustrate the practicability of a promising long-term cryopreservation method that completely eliminates the need for LN2.

Upregulation of PRDM5 Is Associated with Astrocyte Proliferation and Neuronal Apoptosis Caused by Lipopolysaccharide.

PRDM5 (PR domain containing 5) belongs to PRDM family which consists of transcriptional regulators that modulate cellular processes such as cell growth, differentiation and apoptosis. However, the function of PRDM5 in central nervous system (CNS) inflammatory response is unknown. In recent study, an adult rat neuroinflammation model via lipopolysaccharide (LPS) lateral ventricle injection was constructed. PRDM5 expression was increased in activated astrocytes and apoptotic neurons of the adult rat cerebral cortex after LPS injection. In vitro studies showed that the remarkable upregulation of PRDM5 might be involved in rat primary astrocyte proliferation and rat primary neuronal apoptosis in the cerebral cortex following LPS administration. In addition, using PRDM5 RNA interference both in rat primary asrtocytes and neurons, further indicated that PRDM5 was required for astrocyte proliferation and neuronal apoptosis induced by LPS. Our findings on the cellular signaling pathway may provide a new therapeutic strategy against neuroinflammation in the CNS.

SMAD4 is Involved in the Development of Endotoxin Tolerance in Microglia.

Initial exposure of macrophages to LPS induces hyporesponsiveness to a second challenge with LPS, a phenomenon termed LPS tolerance. Smad4 plays important roles in the induction of LPS tolerance. However, the function of Smad4 in microglia remains unknown. Here we show that expression of Smad4 was highly up-regulated in LPS-tolerized mouse cerebral cortex. Smad4 was mostly colocalized with microglia, rarely with neurons. Using a microglia cell line, BV2, we find that LPS activates endogenous Smad4, inducing its migration into the nucleus and increasing its expression. Smad4 significantly suppressed TLR-triggered production of proinflammatory cytokines (IL-6), increased anti-inflammatory cytokine in LPS-tolerized microglia. Moreover, IL-6 concentrations in culture supernatants after second LPS challenge are higher in SMAD4 small interfering RNA (siRNA) BV2 cells than control siRNA BV2 cells, indicating failure to induce tolerance in absence of Smad4 signaling. In our study, we conclude that both in vivo and in vitro, Smad4 signaling is required for maximal induction of endotoxin tolerance.

Genetic regulation analysis reveals involvement of tumor necrosis factor and alpha-induced protein 3 in stress response in mice.

In order to study whether Tnfaip3 is related to stress response and further to find it's genetic regulation, we use C57BL/6J (B6) and DBA/2 (D2) mice to built the model of chronic unpredictable mild stress. RT-PCR, Western blotting and immunohistochemistry were used for studying the expression variation of Tnfaip3 in hippocampus tissue of B6 and D2 mice after being stressed. We found that the expression of Tnfaip3 was more remarkably increased in chronic unpredictable stress models than that in untreated mice (P<0.05). It is indicated that Tnfaip3 might take part in the process of stress response. The expression of Tnfaip3 is regulated by a cis-acting quantitative trait locus (cis-eQTL). We identified 5 genes are controlled by Tnfaip3 and the expression of 64 genes highly associated with Tnfaip3, 9 of those have formerly been participate in stress related pathways. In order to estimate the relationship between Tnfaip3 and its downstream genes or network members, we transfected SH-SY5Y cells with Tnfaip3 siRNA leading to down-regulation of Tnfaip3 mRNA. We confirmed a significant influence of Tnfaip3 depletion on the expression of Tsc22d3, Pex7, Rap2a, Slc2a3, and Gap43. These validated downstream genes and members of Tnfaip3 gene network provide us new insight into the biological mechanisms of Tnfaip3 in chronic unpredictable stress.

Arabidopsis KLU homologue GmCYP78A72 regulates seed size in soybean.

Soybean (Glycine max) is one of the most important crops in the world, and its yield is largely determined by grain weight and grain size. However, the genes that regulate soybean seed size have not been identified. CYP78A, which is highly conserved within terrestrial plants, regulates organ development. In Arabidopsis, AtCYP78A5/KLU has been shown to determine seed size. In the present study, soybean CYP78A72 (GmCYP78A72), one of the orthologs of KLU, was over-expressed in both Arabidopsis and soybean to examine its function in plant development. GmCYP78A72 heterologous expression in Arabidopsis resulted in enlarged sepals, petals, seeds and carpel. Over-expression of GmCYP78A72 in soybean resulted in increased pea size, which is an extremely desirable trait for enhancing productivity. Moreover, knock-down of GmCYP78A72 does not reduce grain size. However, silencing of GmCYP78A57, GmCYP78A70 and GmCYP78A72 genes in triplet reduces the seed size significantly indicating functional redundancy of these three GmCYP78A genes. In conclusion, we investigated the role of CYP78A in soybean seed regulation, and our strategy can be effectively used to engineer large seed traits in soybean varieties as well as other crops.

A New Role Discovered for IGTP: The Protective Effect of IGTP in ICH-Induced Neuronal Apoptosis.

Interferon gamma-induced GTPase (IGTP), which is also named Irgm3, has been widely described in regulating host resistance against intracellular pathogens. Previous researches have demonstrated that IGTP exerts beneficial function during coxsackievirus B3 (CVB3) infection. However, little information is available regarding the role of IGTP in central nervous system. Here, our study revealed that IGTP may have an essential role during ICH-induced neuronal apoptosis. We found the expression level of IGTP adjacent to hematoma was strongly increased after ICH, accompanied with the up-regulation of proliferating cell nuclear antigen (PCNA), active-caspase-3, p-GSK-3ß, and Bax. IGTP was also observed to be co-localized with PCNA in astrocytes and active-caspase-3 in neurons, indicating its association with astrocyte proliferation and neuronal apoptosis after ICH. Finally, in vitro study, knocking down IGTP with IGTP-specific siRNA promoted active-caspase-3, p-GSK-3ß, and Bax expression, and led to more severe neuronal apoptosis after ICH. All these results above suggested that IGTP might play a critical role in protecting neurons from apoptosis after ICH.

Up-Regulation of KPNB1 Involves in Neuronal Apoptosis Following Intracerebral Hemorrhage in Adult Rats.

Kpnb1, also known as Importin ß1, is a member of the Karyopherin protein family which plays a important role in nuclear import and export pathways. Its expression has been shown to be responsive to stress, such as heat shock, ethanol and oxidative stress. Previous studies demonstrated that Kpnb1 had anti-apoptotic in cervical cancer. These together prompted us to explore whether Kpnb1 has some association with neuron apoptosis in the pathophysiology of intracerebral hemorrhage (ICH). In our study, an ICH model was established by injecting into the right basal ganglia of adult rats with their autologous whole blood and assessed by behavioral tests. We found Kpnb1 were significantly up-regulated adjacent to the hematoma following ICH by Western blot and immunohistochemistry. Double immunofluorenscence manifested Kpnb1 was strikingly increased in neurons, not astrocytes or microglia. Furthermore, we also found that kpnb1 had co-localizations with active-caspase-3 which is a neuronal apoptosis marker suggesting its role in neuronal apoptosis. What's more, our in vitro study, using Kpnb1 RNA interference in PC12 cells, further indicated that Kpnb1 might exert its pro-apoptotic function on neuronal apoptosis. Therefore, Kpnb1 may play a role in the neuronal apoptosis following ICH.

Protective Effect of Pyrroloquinoline Quinone (PQQ) in Rat Model of Intracerebral Hemorrhage.

Pyrroloquinoline quinone (PQQ) has invoked considerable interest because of its presence in foods, antioxidant properties, cofactor of dehydrogenase, and amine oxidase. Protective roles of PQQ in central nervous system diseases, such as experimental stroke and spinal cord injury models have been emerged. However, it is unclear whether intracerebral hemorrhage (ICH), as an acute devastating disease, can also benefit from PQQ in experimental conditions. Herein, we examined the possible effect of PQQ on neuronal functions following ICH in the adult rats. The results showed that rats pretreated with PQQ at 10 mg/kg effectively improved the locomotor functions, alleviated the hematoma volumes, and reduced the expansion of brain edema after ICH. Also, pretreated rats with PQQ obviously reduced the production of reactive oxygen species after ICH, probably due to its antioxidant properties. Further, we found that, Bcl-2/Bax, the important indicator of oxidative stress insult in mitochondria after ICH, exhibited increasing ratio in PQQ-pretreated groups. Moreover, activated caspase-3, the apoptotic executor, showed coincident alleviation in PQQ groups after ICH. Collectively, we speculated that PQQ might be an effective and potential neuroprotectant in clinical therapy for ICH.

Up-regulation of Glis2 involves in neuronal apoptosis after intracerebral hemorrhage in adult rats.

The novel Krüppel-like zinc finger protein Gli-similar 2 (Glis2), one member of the transcription factors, is involved in controlling the flow of genetic information and the modulation of diverse cellular activities. Accumulating evidence has demonstrated its important roles in adult development and several diseases. However, information regarding the regulation and possible function of Glis2 in the central nervous system is still limited. In this study, we explored the roles of Glis2 during the pathophysiological process of intracerebral hemorrhage (ICH). An ICH rat model was established and assessed by behavioral tests. Expression of Glis2 was significantly up-regulated in brain areas surrounding the hematoma following ICH. Immunofluorescence showed that Glis2 was strikingly increased in neurons, but not astrocytes or microglia. Up-regulation of Glis2 was found to be accompanied by the increased expression of active caspase-3 and Bax and decreased expression of Bcl-2 in vivo and vitro studies. Moreover, knocking down Glis2 by RNA-interference in PC12 cells reduced active caspase-3 and Bax expression while increased Bcl-2. Collectively, we speculated that Glis2 might exert pro-apoptotic function in neurons following ICH.

Abnormal oxidative stress responses in fibroblasts from preeclampsia infants.

BACKGROUND: Signs of severe oxidative stress are evident in term placentae of infants born to mothers with preeclampsia (PE), but it is unclear whether this is a cause or consequence of the disease. Here fibroblast lines were established from umbilical cords (UC) delivered by mothers who had experienced early onset PE and from controls with the goal of converting these primary cells to induced pluripotent stem cells and ultimately trophoblast. Contrary to expectations, the oxidative stress responses of these non-placental cells from PE infants were more severe than those from controls. METHODS AND FINDINGS: Three features suggested that UC-derived fibroblasts from PE infants responded less well to oxidative stressors than controls: 1) While all UC provided outgrowths in 4% O2, success was significantly lower for PE cords in 20% O2; 2) PE lines established in 4% O2 proliferated more slowly than controls when switched to 20% O2; 3) PE lines were more susceptible to the pro-oxidants diethylmaleate and tert-butylhydroquinone than control lines, but, unlike controls, were not protected by glutathione. Transcriptome profiling revealed only a few genes differentially regulated between PE lines and controls in 4% O2 conditions. However, a more severely stressed phenotype than controls, particularly in the unfolded protein response, was evident when PE lines were switched suddenly to 20% O2, thus confirming the greater sensitivity of the PE fibroblasts to acute changes in oxidative stress. CONCLUSIONS: UC fibroblasts derived from PE infants are intrinsically less able to respond to acute oxidative stress than controls, and this phenotype is retained over many cell doublings. Whether the basis of this vulnerability is genetic or epigenetic and how it pertains to trophoblast development remains unclear, but this finding may provide a clue to the basis of the early onset, usually severe, form of PE.

Neuromodulation independently determines correlated channel expression and conductance levels in motor neurons of the stomatogastric ganglion.

Neuronal identity depends on the regulated expression of numerous molecular components, especially ionic channels, which determine the electrical signature of a neuron. Such regulation depends on at least two key factors, activity itself and neuromodulatory input. Neuronal electrical activity can modify the expression of ionic currents in homeostatic or nonhomeostatic fashion. Neuromodulators typically modify activity by regulating the properties or expression levels of subsets of ionic channels. In the stomatogastric system of crustaceans, both types of regulation have been demonstrated. Furthermore, the regulation of the coordinated expression of ionic currents and the channels that carry these currents has been recently reported in diverse neuronal systems, with neuromodulators not only controlling the absolute levels of ionic current expression but also, over long periods of time, appearing to modify their correlated expression. We hypothesize that neuromodulators may regulate the correlated expression of ion channels at multiple levels and in a cell-type-dependent fashion. We report that in two identified neuronal types, three ionic currents are linearly correlated in a pairwise manner, suggesting their coexpression or direct interactions, under normal neuromodulatory conditions. In each cell, some currents remain correlated after neuromodulatory input is removed, whereas the correlations between the other pairs are either lost or altered. Interestingly, in each cell, a different suite of currents change their correlation. At the transcript level we observe distinct alterations in correlations between channel mRNA amounts, including one of the cell types lacking a correlation under normal neuromodulatory conditions and then gaining the correlation when neuromodulators are removed. Synaptic activity does not appear to contribute, with one possible exception, to the correlated expression of either ionic currents or of the transcripts that code for the respective channels. We conclude that neuromodulators regulate the correlated expression of ion channels at both the transcript and the protein levels.

Hematopoietic prostaglandin D synthase (HPGDS): a high stability, Val187Ile isoenzyme common among African Americans and its relationship to risk for colorectal cancer.

Intestinal tumors in Apc(Min/+) mice are suppressed by over-production of HPGDS, which is a glutathione transferase that forms prostaglandin D(2) (PGD(2)). We characterized naturally occurring HPGDS isoenzymes, to see if HPGDS variation is associated with human colorectal cancer risk. We used DNA heteroduplex analysis and sequencing to identify HPGDS variants among healthy individuals. HPGDS isoenzymes were produced in bacteria, and their catalytic activities were tested. To determine in vivo effects, we conducted pooled case-control analyses to assess whether there is an association of the isoenzyme with colorectal cancer. Roughly 8% of African Americans and 2% of Caucasians had a highly stable Val187lle isoenzyme (with isoleucine instead of valine at position 187). At 37°C, the wild-type enzyme lost 15% of its activity in 1h, whereas the Val187Ile form remained >95% active. At 50°C, the half life of native HPGDS was 9min, compared to 42 min for Val187Ile. The odds ratio for colorectal cancer among African Americans with Val187Ile was 1.10 (95% CI, 0.75-1.62; 533 cases, 795 controls). Thus, the Val187Ile HPGDS isoenzyme common among African Americans is not associated with colorectal cancer risk. Other approaches will be needed to establish a role for HPGDS in occurrence of human intestinal tumors, as indicated by a mouse model.

Cinnamic acid pretreatment mitigates chilling stress of cucumber leaves through altering antioxidant enzyme activity.

To elucidate the physiological mechanism of chilling stress mitigated by cinnamic acid (CA) pretreatment, a cucumber variety (Cucumis sativus cv. Jinchun no. 4) was pretreated with 50 µM CA for 2d and was then cultivated at two temperatures (15/8 and 25/18 °C) for 1d. We investigated whether exogenous CA could protect cucumber plantlets from chilling stress (15/8 °C) and examined whether the protective effect was associated with the regulation of antioxidant enzymes and lipid peroxidation. At 2d, exogenous CA did not influence plant growth, but induced the activities of some antioxidant enzymes, including superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), guaiacol peroxidase (GPX, EC 1.11.1.7), glutathione peroxidase (GSH-Px, EC 1.6.4.2) and ascorbate peroxidase (APX, EC 1.11.1.11) in cucumber leaves, and it also elevated the contents of reduced glutathione (GSH) and ascorbate (AsA). When CA was rinsed and the CA-pretreated seedlings were exposed to different temperatures, the antioxidant activities in leaves at 3d had undergone additional change. Chilling increased the activities of CAT, GSH-PX, APX, GSH and AsA in leaves, but the combination of CA pretreatment and chilling enhanced the antioxidant activities even more. Moreover, chilling inhibited plant growth and increased the contents of malonaldehyde (MDA), superoxide radical (O2⁻) and hydrogen peroxide (H2O2) in cucumber leaves, and the stress resulted in 87.5% of the second leaves being withered. When CA pretreatment was combined with the chilling stress, we observed alleviated growth inhibition and decreased contents of MDA, H2O2 and O2⁻ in comparison to non-pretreated stressed plants, and found that the withered leaves occurred at a rate of 25.0%. We propose that CA pretreatment increases antioxidant enzyme activities in chilling-stressed leaves and decreases lipid peroxidation to some extent, enhancing the tolerance of cucumber leaves to chilling stress.

Long-acting erythropoietin: clinical studies and potential uses in neonates.

Aranesp (darbepoietin alfa) is a biologically modified form of recombinant human erythropoietin (rHuEpo). Two additional carbohydrate-binding sites give Aranesp a half-life about three times that of rHuEpo. Extensive studies in adults and early studies in children indicate that Aranesp can be administered far less frequently than rHuEpo with an equivalent erythropoietic effect. This article reviews these studies and reports on the in vitro effects of Aranesp on human fetal and neonatal erythroid progenitors.