Developmental Stage-Specific Imprinting of IPL in Domestic Pigs (Sus scrofa)

Imprinted in placenta and liver (IPL) gene has been identified as an imprinted gene in the mouse and human. Its sequence and imprinting status, however, have not been determined in the domestic pigs. In the present study, a 259 base pair-specific sequence for IPL gene of the domestic pig was obtained and a novel SNP, a T/C transition, was identified in IPL exon 1. The C allele of this polymorphism was found to be the predominant allele in Landrace,Yorkshire, and Duroc. The frequency of CC genotype and C allele are different in Duroc as compared with Yorkshire (P = .038 and P = .005, resp.). Variable imprinting status of this gene was observed in different developmental stages. For example, it is imprinted in 1-dayold newborns (expressed from the maternal allele), but imprinting was lost in 180-day-old adult (expressed from both parental alleles). Real-time PCR analysis showed the porcine IPL gene is expressed in all tested eight organ/tissues. The expression level was significantly higher in spleen, duodenum, lung, and bladder of 180-day-old Lantang adult compared to that in 1-day-old newborns Lantang pigs (P < .05). In conclusion, the imprinting of the porcine IPL gene is developmental stage and tissue specific

Plant-RRBS, a bisulfite and next-generation sequencing-based methylome profiling method enriching for coverage of cytosine positions

Background: Cytosine methylation in plant genomes is important for the regulation of gene transcription and transposon activity. Genome-wide methylomes are studied upon mutation of the DNA methyltransferases, adaptation to environmental stresses or during development. However, from basic biology to breeding programs, there is a need to monitor multiple samples to determine transgenerational methylation inheritance or differential cytosine methylation. Methylome data obtained by sodium hydrogen sulfite (bisulfite)-conversion and next-generation sequencing (NGS) provide genome- wide information on cytosine methylation. However, a profiling method that detects cytosine methylation state dispersed over the genome would allow high-throughput analysis of multiple plant samples with distinct epigenetic signatures. We use specific restriction endonucleases to enrich for cytosine coverage in a bisulfite and NGS-based profiling method, which was compared to whole-genome bisulfite sequencing of the same plant material. Methods: We established an effective methylome profiling method in plants, termed plant-reduced representation bisulfite sequencing (plant-RRBS), using optimized double restriction endonuclease digestion, fragment end repair, adapter ligation, followed by bisulfite conversion, PCR amplification and NGS. We report a performant laboratory protocol and a straightforward bioinformatics data analysis pipeline for plant-RRBS, applicable for any reference-sequenced plant species. Results: As a proof of concept, methylome profiling was performed using an Oryza sativa ssp. indica pure breeding line and a derived epigenetically altered line (epiline). Plant-RRBS detects methylation levels at tens of millions of cytosine positions deduced from bisulfite conversion in multiple samples. To evaluate the method, the coverage of cytosine positions, the intra-line similarity and the differential cytosine methylation levels between the pure breeding line and the epiline were determined. Plant-RRBS reproducibly covers commonly up to one fourth of the cytosine positions in the rice genome when using MspI-DpnII within a group of five biological replicates of a line. The method predominantly detects cytosine methylation in putative promoter regions and not-annotated regions in rice. Conclusions: Plant-RRBS offers high-throughput and broad, genome- dispersed methylation detection by effective read number generation obtained from reproducibly covered genome fractions using optimized endonuclease combinations, facilitating comparative analyses of multi-sample studies for cytosine methylation and transgenerational stability in experimental material and plant breeding populations

Beneficial Effect of Young Oocytes for Rabbit Somatic Cell Nuclear Transfer

Abstract This study was designed to examine the effect of the age of rabbit oocytes on the developmental potential of cloned embryos. The metaphase II oocytes used for nuclear transfer (NT) were collected at 10, 12, 14, and 16h post-hCG injection (hpi). The total number of oocytes collected per donor (21.4-23.7) at 12 to 16 hpi was similar, but significantly higher than that collected at 10 hpi (16.2). Additionally, a significant improvement in blastocyst development was achieved with embryos generated by electrically mediated cell fusion (56.0%), compared to those from nuclear injection (13.1 %) (Experiment 1). Markedly higher blastocyst development (45.8-54.5%) was also achieved with oocytes collected at 10-12 hpi than from those collected 14-16 hpi (8.3-14.3%) (Experiment 2). In Experiment 3, the blastocyst rates of NT embryos derived from oocytes harvested 12 hpi (39.2-42.8 %) were significantly higher than from those collected at 16 hpi (6.8-8.4 %) (p<0.05), regardless of the donor cell age. Kinase activity assays showed variable changes of activity in rabbit oocytes over the period of 10-16 hpi; however, there was no correlation with preimplantational development (blastocyst rate vs. MPF, R=0.326; blastocyst rate vs. MAPK, R=0.131). Embryo transfer of NT embryos utilizing 12 hpi oocytes resulted in one full-term but stillborn, and one live cloned rabbit; thus, an efficiency of 1.7 % (n=117) (Experiment 4). These results demonstrated that NT utilizing relatively young rabbit oocytes, harvested at 10-12h after hCG injection, was beneficial for the development of NT embryos.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78139/1/clo.2008.0042.pd

Follicular Oocytes Better Support Development in Rabbit Cloning Than Oviductal Oocytes

This study was conducted to determine the effect of rabbit oocytes collected from ovaries or oviducts on the developmental potential of nuclear transplant embryos. Donor nuclei were obtained from adult skin fibroblasts, cumulus cells, and embryonic blastomeres. Rabbit oocytes were flushed from the oviducts (oviductal oocytes) or aspirated from the ovaries (follicular oocytes) of superovulated does at 10, 11, or 12-h post-hCG injection. The majority of collected oocytes were still attached to the sites of ovulation on the ovaries. We found that follicular oocytes had a significantly higher rate of fusion with nuclear donor cells than oviductal oocytes. There was no difference in the cleavage rate between follicular and oviductal groups, but morula and blastocyst development was significantly higher in the follicular group than in the oviductal group. Two live clones were produced in follicular group using blastomere and cumulus nuclear donors, whereas one live clone was produced in the oviductal group using a cumulus nuclear donor. These results demonstrate that cloned rabbit embryos derived from follicular oocytes have better developmental competence than those derived from oviductal oocytes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90481/1/cell-2E2011-2E0030.pd

Maternal gut Bifidobacterium breve modifies fetal brain metabolism in germ-free mice

Background: Recent advances have significantly expanded our understanding of the gut microbiome's influence on host physiology and metabolism. However, the specific role of certain microorganisms in gestational health and fetal development remains underexplored. Objective: This study investigates the impact of Bifidobacterium breve UCC2003 on fetal brain metabolism when colonized in the maternal gut during pregnancy. Methods: Germ-free pregnant mice were colonized with or without B. breve UCC2003 during pregnancy. The metabolic profiles of fetal brains were analyzed, focusing on the presence of key metabolites and the expression of critical metabolic and cellular pathways. Results: Maternal colonization with B. breve resulted in significant metabolic changes in the fetal brain. Specifically, ten metabolites, including citrate, 3-hydroxyisobutyrate, and carnitine, were reduced in the fetal brain. These alterations were accompanied by increased abundance of transporters involved in glucose and branched-chain amino acid uptake. Furthermore, supplementation with this bacterium was associated with elevated expression of critical metabolic pathways such as PI3K-AKT, AMPK, STAT5, and Wnt-β-catenin signaling, including its receptor Frizzled-7. Additionally, there was stabilization of HIF-2 protein and modifications in genes and proteins related to cellular growth, axogenesis, and mitochondrial function. Conclusions: The presence of maternal B. breve during pregnancy plays a crucial role in modulating fetal brain metabolism and growth. These findings suggest that Bifidobacterium could modify fetal brain development, potentially offering new avenues for enhancing gestational health and fetal development through microbiota-targeted interventions

Pharmacokinetics and pharmacodynamics of clofazimine for treatment of cryptosporidiosis

Infection with Cryptosporidium spp. can cause severe diarrhea leading to long-term adverse impacts and even death in malnourished children and immunocompromised patients. The only FDA-approved drug for treating cryptosporidiosis, nitazoxanide, has limited efficacy in the populations impacted the most by the diarrheal disease, and safe, effective treatment options are urgently needed. Initially identified by a large-scale phenotypic screening campaign, the antimycobacterial therapeutic clofazimine demonstrated great promise in both in vitro and in vivo preclinical models of Cryptosporidium infection. Unfortunately, a Phase 2a clinical trial in HIV infected adults with cryptosporidiosis did not identify any clofazimine treatment effect on Cryptosporidium infection burden or clinical outcomes. To explore whether clofazimine's lack of efficacy in the Phase 2a trial may have been due to subtherapeutic clofazimine concentrations, a pharmacokinetic/pharmacodynamic modeling approach was undertaken to determine the relationship between clofazimine in vivo concentrations and treatment effects in multiple preclinical infection models. Exposure-response relationships were characterized using Emax and logistic models which allowed predictions of efficacious clofazimine concentrations for the control and reduction of disease burden. After establishing exposure-response relationships for clofazimine treatment of Cryptosporidium infection in our preclinical model studies, it was unmistakable that the clofazimine levels observed in the Phase 2a study participants were well below concentrations associated with anti-Cryptosporidium efficacy. Thus, despite a dosing regimen above the highest doses recommended for mycobacterial therapy, it is very likely the lack of treatment effect in the Phase 2a trial was at least partially due to clofazimine concentrations below those required for efficacy against cryptosporidiosis. It is unlikely that clofazimine will provide a remedy for the large number of cryptosporidiosis patients currently without a viable treatment option unless alternative, safe clofazimine formulations with improved oral absorption are developed

Envelope Deglycosylation Enhances Antigenicity of HIV-1 gp41 Epitopes for Both Broad Neutralizing Antibodies and Their Unmutated Ancestor Antibodies

The HIV-1 gp41 envelope (Env) membrane proximal external region (MPER) is an important vaccine target that in rare subjects can elicit neutralizing antibodies. One mechanism proposed for rarity of MPER neutralizing antibody generation is lack of reverted unmutated ancestor (putative naive B cell receptor) antibody reactivity with HIV-1 envelope. We have studied the effect of partial deglycosylation under non-denaturing (native) conditions on gp140 Env antigenicity for MPER neutralizing antibodies and their reverted unmutated ancestor antibodies. We found that native deglycosylation of clade B JRFL gp140 as well as group M consensus gp140 Env CON-S selectively increased the reactivity of Env with the broad neutralizing human mAbs, 2F5 and 4E10. Whereas fully glycosylated gp140 Env either did not bind (JRFL), or weakly bound (CON-S), 2F5 and 4E10 reverted unmutated ancestors, natively deglycosylated JRFL and CON-S gp140 Envs did bind well to these putative mimics of naive B cell receptors. These data predict that partially deglycoslated Env would bind better than fully glycosylated Env to gp41-specific naïve B cells with improved immunogenicity. In this regard, immunization of rhesus macaques demonstrated enhanced immunogenicity of the 2F5 MPER epitope on deglyosylated JRFL gp140 compared to glycosylated JRFL gp140. Thus, the lack of 2F5 and 4E10 reverted unmutated ancestor binding to gp140 Env may not always be due to lack of unmutated ancestor antibody reactivity with gp41 peptide epitopes, but rather, may be due to glycan interference of binding of unmutated ancestor antibodies of broad neutralizing mAb to Env gp41

Demonstration of quantum volume 64 on a superconducting quantum computing system

We improve the quality of quantum circuits on superconducting quantum computing systems, as measured by the quantum volume, with a combination of dynamical decoupling, compiler optimizations, shorter two-qubit gates, and excited state promoted readout. This result shows that the path to larger quantum volume systems requires the simultaneous increase of coherence, control gate fidelities, measurement fidelities, and smarter software which takes into account hardware details, thereby demonstrating the need to continue to co-design the software and hardware stack for the foreseeable future.Comment: Fixed typo in author list. Added references [38], [49] and [52

Induction of Antibodies in Rhesus Macaques That Recognize a Fusion-Intermediate Conformation of HIV-1 gp41

A component to the problem of inducing broad neutralizing HIV-1 gp41 membrane proximal external region (MPER) antibodies is the need to focus the antibody response to the transiently exposed MPER pre-hairpin intermediate neutralization epitope. Here we describe a HIV-1 envelope (Env) gp140 oligomer prime followed by MPER peptide-liposomes boost strategy for eliciting serum antibody responses in rhesus macaques that bind to a gp41 fusion intermediate protein. This Env-liposome immunization strategy induced antibodies to the 2F5 neutralizing epitope 664DKW residues, and these antibodies preferentially bound to a gp41 fusion intermediate construct as well as to MPER scaffolds stabilized in the 2F5-bound conformation. However, no serum lipid binding activity was observed nor was serum neutralizing activity for HIV-1 pseudoviruses present. Nonetheless, the Env-liposome prime-boost immunization strategy induced antibodies that recognized a gp41 fusion intermediate protein and was successful in focusing the antibody response to the desired epitope