Porcine iGb3s gene silencing provides minimal benefit for clinical xenotransplantation

Background The Galα(1,3)Gal epitope (α-GAL), created by α-1,3-glycosyltransferase-1 (GGTA1), is a major xenoantigen causing hyperacute rejection in pig-to-primate and pig-to-human xenotransplantation. In response, GGTA1 gene-deleted pigs have been generated. However, it is unclear whether there is a residual small amount of α-Gal epitope expressed in GGTA1−/− pigs. Isoglobotrihexosylceramide synthase (iGb3s), another member of the glycosyltransferase family, catalyzes the synthesis of isoglobo-series glycosphingolipids with an α-GAL-terminal disaccharide (iGb3), creating the possibility that iGb3s may be a source of α-GAL epitopes in GGTA1−/− animals. The objective of this study was to examine the impact of silencing the iGb3s gene (A3GalT2) on pig-to-primate and pig-to-human immune cross-reactivity by creating and comparing GGTA1−/− pigs to GGTA1−/−- and A3GalT2−/−-double-knockout pigs. Methods We used the CRISPR/Cas 9 system to target the GGTA1 and A3GalT2 genes in pigs. Both GGTA1 and A3GalT2 genes are functionally inactive in humans and baboons. CRISPR-treated cells used directly for somatic cell nuclear transfer produced single- and double-gene-knockout piglets in a single pregnancy. Once grown to maturity, the glycosphingolipid profile (including iGb3) was assayed in renal tissue by normal-phase liquid chromatography. In addition, peripheral blood mononuclear cells (PBMCs) were subjected to (i) comparative cross-match cytotoxicity analysis against human and baboon serum and (ii) IB4 staining for α-GAL/iGb3. Results Silencing of the iGb3s gene significantly modulated the renal glycosphingolipid profile and iGb3 was not detected. Moreover, the human and baboon serum PBMC cytotoxicity and α-GAL/iGb3 staining were unchanged by iGb3s silencing. Conclusions Our data suggest that iGb3s is not a contributor to antibody-mediated rejection in pig-to-primate or pig-to-human xenotransplantation. Although iGb3s gene silencing significantly changed the renal glycosphingolipid profile, the effect on Galα3Gal levels, antibody binding, and cytotoxic profiles of baboon and human sera on porcine PBMCs was neutral

Silencing porcine CMAH and GGTA1 genes significantly reduces xenogeneic consumption of human platelets by porcine livers

BACKGROUND: A profound thrombocytopenia limits hepatic xenotransplantation in the pig-to-primate model. Porcine livers also have shown the ability to phagocytose human platelets in the absence of immune-mediated injury. Recently, inactivation of the porcine ASGR1 gene has been shown to decrease this phenomenon. Inactivating GGTA1 and CMAH genes has reduced the antibody-mediated barrier to xenotransplantation; herein, we describe the effect that these modifications have on xenogeneic consumption of human platelets in the absence of immune-mediated graft injury. METHODS: Wild type (WT), ASGR1, GGTA1, and GGTA1CMAH knockout pigs were compared for their xenogeneic hepatic consumption of human platelets. An in vitro assay was established to measure the association of human platelets with liver sinusoidal endothelial cells (LSECs) by immunohistochemistry. Perfusion models were used to measure human platelet uptake in livers from WT, ASGR1, GGTA1, and GGTA1 CMAH pigs. RESULTS: GGTA1, CMAH LSECs exhibited reduced levels of human platelet binding in vitro when compared with GGTA1 and WT LSECs. In a continuous perfusion model, GGTA1 CMAH livers consumed fewer human platelets than GGTA1 and WT livers. GGTA1 CMAH livers also consumed fewer human platelets than ASGR1 livers in a single-pass model. CONCLUSIONS: Silencing the porcine carbohydrate genes necessary to avoid antibody-mediated rejection in a pig-to-human model also reduces the xenogeneic consumption of human platelets by the porcine liver. The combination of these genetic modifications may be an effective strategy to limit the thrombocytopenia associated with pig-to-human hepatic xenotransplantation

A search for antiproton decay at the Fermilab Antiproton Accumulator

We report on the search for anti-proton decay at the Fermilab Antiproton Accumulator Ring. Experiment 868 (APEX) was designed to search for two-body p̄ decay modes containing an electron in the final state (→e+X)(p̄→e+X) and to conduct an exploratory search for decays with a muon in the final state (→μ+X).(p̄→μ+X). Data were taken for three months in the Spring of 1995. Preliminary results yield lower limits on /BRτp̄/BR in the range of 105–106105–106 years for selected channels having an electron in the final state, improving on previous results by approximately 3 orders of magnitude. Additionally, we report the first preliminary results for the →μγp̄→μγ and →μπ0p̄→μπ0 decay channels. © 1997 American Institute of Physics.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/87906/2/419_1.pd

Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation

Advances in sequencing technology allow researchers to map genome-wide changes in DNA methylation in development and disease. However, there is a lack of experimental tools to site-specifically manipulate DNA methylation to discern the functional consequences. We developed a CRISPR/Cas9 DNA methyltransferase 3A (DNMT3A) fusion to induce DNA methylation at specific loci in the genome. We induced DNA methylation at up to 50% of alleles for targeted CpG dinucleotides. DNA methylation levels peaked within 50 bp of the short guide RNA (sgRNA) binding site and between pairs of sgRNAs. We used our approach to target methylation across the entire CpG island at the CDKN2A promoter, three CpG dinucleotides at the ARF promoter, and the CpG island within the Cdkn1a promoter to decrease expression of the target gene. These tools permit mechanistic studies of DNA methylation and its role in guiding molecular processes that determine cellular fate

Testing Models of Accretion-driven Coronal Heating and Stellar Wind Acceleration for T Tauri Stars

Classical T Tauri stars are pre-main-sequence objects that undergo simultaneous accretion, wind outflow, and coronal X-ray emission. The impact of plasma on the stellar surface from magnetospheric accretion streams is likely to be a dominant source of energy and momentum in the upper atmospheres of these stars. This paper presents a set of models for the dynamics and heating of three distinct regions on T Tauri stars that are affected by accretion: (1) the shocked plasmas directly beneath the magnetospheric accretion streams, (2) stellar winds that are accelerated along open magnetic flux tubes, and (3) closed magnetic loops that resemble the Sun's coronal active regions. For the loops, a self-consistent model of coronal heating was derived from numerical simulations of solar field-line tangling and turbulent dissipation. Individual models are constructed for the properties of 14 well-observed stars in the Taurus-Auriga star-forming region. Predictions for the wind mass loss rates are, on average, slightly lower than the observations, which suggests that disk winds or X-winds may also contribute to the measured outflows. For some of the stars, however, the modeled stellar winds do appear to contribute significantly to the measured mass fluxes. Predictions for X-ray luminosities from the shocks and loops are in general agreement with existing observations. The stars with the highest accretion rates tend to have X-ray luminosities dominated by the high-temperature (5-10 MK) loops. The X-ray luminosities for the stars having lower accretion rates are dominated by the cooler accretion shocks.Comment: 20 pages (emulateapj style), 13 figures, ApJ, in press (v. 706, December 1, 2009

Evaluation of regression models in metabolic physiology: predicting fluxes from isotopic data without knowledge of the pathway

This study explores the ability of regression models, with no knowledge of the underlying physiology, to estimate physiological parameters relevant for metabolism and endocrinology. Four regression models were compared: multiple linear regression (MLR), principal component regression (PCR), partial least-squares regression (PLS) and regression using artificial neural networks (ANN). The pathway of mammalian gluconeogenesis was analyzed using [U−(13)C]glucose as tracer. A set of data was simulated by randomly selecting physiologically appropriate metabolic fluxes for the 9 steps of this pathway as independent variables. The isotope labeling patterns of key intermediates in the pathway were then calculated for each set of fluxes, yielding 29 dependent variables. Two thousand sets were created, allowing independent training and test data. Regression models were asked to predict the nine fluxes, given only the 29 isotopomers. For large training sets (>50) the artificial neural network model was superior, capturing 95% of the variability in the gluconeogenic flux, whereas the three linear models captured only 75%. This reflects the ability of neural networks to capture the inherent non-linearities of the metabolic system. The effect of error in the variables and the addition of random variables to the data set was considered. Model sensitivities were used to find the isotopomers that most influenced the predicted flux values. These studies provide the first test of multivariate regression models for the analysis of isotopomer flux data. They provide insight for metabolomics and the future of isotopic tracers in metabolic research where the underlying physiology is complex or unknown

The state of the Martian climate

60°N was +2.0°C, relative to the 1981–2010 average value (Fig. 5.1). This marks a new high for the record. The average annual surface air temperature (SAT) anomaly for 2016 for land stations north of starting in 1900, and is a significant increase over the previous highest value of +1.2°C, which was observed in 2007, 2011, and 2015. Average global annual temperatures also showed record values in 2015 and 2016. Currently, the Arctic is warming at more than twice the rate of lower latitudes

Method for Quantitative Study of Airway Functional Microanatomy Using Micro-Optical Coherence Tomography

We demonstrate the use of a high resolution form of optical coherence tomography, termed micro-OCT (μOCT), for investigating the functional microanatomy of airway epithelia. μOCT captures several key parameters governing the function of the airway surface (airway surface liquid depth, periciliary liquid depth, ciliary function including beat frequency, and mucociliary transport rate) from the same series of images and without exogenous particles or labels, enabling non-invasive study of dynamic phenomena. Additionally, the high resolution of μOCT reveals distinguishable phases of the ciliary stroke pattern and glandular extrusion. Images and functional measurements from primary human bronchial epithelial cell cultures and excised tissue are presented and compared with measurements using existing gold standard methods. Active secretion from mucus glands in tissue, a key parameter of epithelial function, was also observed and quantified

The Role of Climate Variability in the Spread of Malaria in Bangladeshi Highlands

Malaria is a major public health problem in Bangladesh, frequently occurring as epidemics since the 1990s. Many factors affect increases in malaria cases, including changes in land use, drug resistance, malaria control programs, socioeconomic issues, and climatic factors. No study has examined the relationship between malaria epidemics and climatic factors in Bangladesh. Here, we investigate the relationship between climatic parameters [rainfall, temperature, humidity, sea surface temperature (SST), El Niño-Southern Oscillation (ENSO), the normalized difference vegetation index (NDVI)], and malaria cases over the last 20 years in the malaria endemic district of Chittagong Hill Tracts (CHT)