Lentiviruses inefficiently incorporate human parainfluenza type 3 envelope proteins

We have previously shown that the envelope glycoproteins of human parainfluenza type 3 (HPIV3), F and HN, are able to pseudotype lentiviruses, but the titers of these viruses are too low for use in clinical gene transfer. In this study we investigated the cause of these low titers. We compared the mRNA and protein expression levels of HN and F in transfected cells and in cells infected with wild-type HPIV3. Transfected cells contained similar levels of HN and F cytosolic mRNA, but fewer cell-surface HN and F proteins (3.8- and 1.3-fold less, respectively), than cells infected with wild-type HPIV3. To increase expression of HN in transfected cells, we codon-optimized HN and used it to transfect lentivirus producer cells. Cell surface expression of HN, as well as the amount of HN incorporated into virus particles, increased two- to threefold. Virus titers increased 1.2- to 6.4-fold, and the transduction efficiency of polarized MDCK cells via their apical surfaces increased 1.4-fold. Interestingly, even though codon optimization improved the expression levels of HN and virus titers, we found that HPIV3 pseudotyped viruses contained about 14-fold fewer envelope proteins than lentiviruses pseudotyped with the amphotropic envelope protein. Taken together, our findings suggest that titers are low, not because virus producer cells express levels of HPIV3 envelope proteins that are too low, but because too few of these proteins are incorporated by the lentiviruses for them to be able to efficiently transduce cells.     

An improved culture system for secondary palatal elevation

Summary An organ culture system devised for studying the development of the secondary palate was modified so that it retained high partial pressures of oxygen and supported total anterior and posterior palatal elevation. The cultured tissues appeared healthy as judged by histological examination. Medium was continuously recirculated through the culture system in which Day 13 embryonic mouse heads, with the brain and tongue removed, were totally submerged and suspended. The medium was constantly gassed via hollow fiber devices. A motor-driven stirrer, run at a low rate, agitated the medium so that the boundary layer surrounding the tissue was dispersed. Embryonic mouse heads were cultured in each of four media: Eagle's basal medium, Ham's F-12 medium, Fitton-Jackson's modified BGJb medium, and Waymouth's MB 752/1 medium. Elevation of the palate in both anterior and posterior regions with excellent tissue viability was achieved in all heads grown in BGJb medium. This work was supported by NIH Post-doctoral Fellowship 2F32 DE05038-03 to C. A. L.     

Effects of long-term exercise on the biomechanical properties of the Achilles tendon of guinea fowl

The purpose of this study wasto determine the effect of long-term exercise on tendon compliance andto ascertain whether tendons adapt differently to downhill running vs.running on a level surface. We carried out this investigation on thegastrocnemius tendon of helmeted guinea fowl (Numidameleagris) that were trained for 8-12 wk before commencingexperimental procedures. We used an in situ technique to measure tendonstiffness. The animals were deeply anesthetized with isofluorane duringall in situ procedures. Our results indicate that long-term exerciseincreased tendon stiffness. This finding held true after normalizationfor the cross-sectional area of the free tendon, likely reflecting achange in the material properties of the exercised tendons. Whethertraining consisted of level or downhill running did not appear toinfluence response of the tendon to exercise. We hypothesize that theincreased stiffness observed in tendons after a long-term runningprogram may be a response to repeated stress and may function as amechanism to resist tendon damage due to mechanical fatigue.

     

Analysis of preimplantation golden hamster conceptuses resulting from spermatozoa aged in utero

The effects of aging golden hamster spermatozoa in the female reproductive tract on the percentage of ova fertilized, the stage of development, and the chromosome complement of the resulting zygotes were studied. Females were inseminated artificially with cauda epididymal sperm at 6 h (control), 10, 15,18, or 21 h before the estimated time of ovulation. A decrease in the percentage of ova fertilized was found as the time spermatozoa were aged in utero prior to ovulation increased. The zygotes collected at 56 h postovulation from females inseminated 15 or 18 h prior to ovulation were delayed in development, as judged by the number of blastomeres. Although an increase of chromosomally abnormal zygotes was not found, a possibility exists that mosaicism may have been present, as evidenced by zygotes with unequal sized blastomeres, and went undetected.     

Plant diversity patterns in subtropical evergreen broad-leaved forests of Yunnan and Taiwan

The subtropical evergreen broad-leaved forests of Yunnan and Taiwan were compared along environmental and successional gradients with the aim of identifying important taxon and species diversity as well as the drivers of mountain biodiversity patterns. A detrended correspondence analysis of an exhaustive set of data collected from 105 and 223 plots for Yunnan and Taiwan, respectively, was applied to classify natural mature forest types. Additional data from 72 and 68 plots for Yunnan and Taiwan, respectively, were used for analyses of secondary succession. The floristic richness and diversity index were calculated for each type of forest. In Yunnan, the monsoon forests in mesic-humid sites had more taxa and tended to show higher species diversity than the other two forest types. In Taiwan, species diversity values were significantly higher in the Machilus–Castanopsis zone in the middle altitudes (500–1500 m) than for the other three forest zones. For both Yunnan and Taiwan, the forests at the middle successional stage showed significantly higher species diversity than those at the early successional stage. Differences in diversity between the middle and late stages were not significant. These findings highlight the high species diversity of the natural mature evergreen broad-leaved forests of both Yunnan and Taiwan. In the secondary forests, as succession proceeds, species diversity comes to resemble that of the natural mature forests. In both ecosystems, the drivers of species diversity patterns are moisture, altitude, and succession/disturbance.     

Unlimited in vitro expansion of adult bi‐potent pancreas progenitors through the Lgr5/R‐spondin axis

Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt‐agonistic R‐spondins (RSPOs). Intestinal, stomach and liver Lgr5⁺ stem cells grow in 3D cultures to form ever‐expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1‐based cultures, and develop into budding cyst‐like structures (organoids) that expand five‐fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi‐potentiality.     

The ovine newborn and human foetal intervertebral disc contain perlecan and aggrecan variably substituted with native 7D4 CS sulphation motif: spatiotemporal immunolocalisation and co-distribution with Notch-1 in the human foetal disc

Composite agarose (1.2 %) polyacrylamide (0.6 %) gel electrophoresis was used to separate discrete populations of native aggrecan and perlecan in newborn to 10 year old ovine intervertebral discs (IVDs). Semi-dry immunoblotting using core-protein and glycosaminoglycan (GAG) side chain specific monoclonal antibodies in combination with chondroitin ABC lyase demonstrated intra-chain native 7-D-4 chondroitin sulphate (CS) sulphation motifs and variable proportions of non-reducing terminal Δ4,5-unsaturated uronate-N-acetylgalactosamine-4-sulphate [2B6(+)] and Δ4,5-unsaturated glucuronate-N-acetylgalactosamine-6-sulphate [3B3(+)] disaccharides. The relative abundance of 2-B-6(+) aggrecan increased with advancing age of the IVD samples while the converse was true for the 3-B-3(+) aggrecan population. Relative 7D4 levels in aggrecan and perlecan were highest in the newborn IVD and significantly lower in the older IVD and other cartilage samples. Quantitation of 7D4 proteoglycan by enzyme linked immunosorbent inhibition assay confirmed the newborn ovine nucleus pulposus (NP) and inner annulus fibrosus (AF) contained higher levels (1.2-1.32 μg 7-D-4-proteoglycan/mg tissue wet weight) than the 2 (0.35-0.42 μg/mg wet weight tissue) and 10 year old IVD samples (0.16-0.22 μg/mg tissue wet weight) with the outer AF zones consistently containing lower levels of 7-D-4 epitope in all cases (P?<?0.001). Cell populations on the margins of the AF and cartilaginous vertebral rudiments in newborn ovine and human foetal IVD strongly expressed 7-D-4 CS epitope and perlecan, This was co-distributed with Notch-1 expression in human foetal IVDs consistent with the 7-D-4 CS sulphation motif representing a marker of tissue development expressed by disc progenitor cell populations.     

Regionally Specific and Genome-Wide Analyses Conclusively Demonstrate the Absence of CpG Methylation in Human Mitochondrial DNA

Although CpG methylation clearly distributes genome-wide in vertebrate nuclear DNA, the state of methylation in the vertebrate mitochondrial genome has been unclear. Several recent reports using immunoprecipitation, mass spectrometry, and enzyme-linked immunosorbent assay methods concluded that human mitochondrial DNA (mtDNA) has much more than the 2 to 5% CpG methylation previously estimated. However, these methods do not provide information as to the sites or frequency of methylation at each CpG site. Here, we have used the more definitive bisulfite genomic sequencing method to examine CpG methylation in HCT116 human cells and primary human cells to independently answer these two questions. We found no evidence of CpG methylation at a biologically significant level in these regions of the human mitochondrial genome. Furthermore, unbiased next-generation sequencing of sodium bisulfite treated total DNA from HCT116 cells and analysis of genome-wide sodium bisulfite sequencing data sets from several other DNA sources confirmed this absence of CpG methylation in mtDNA. Based on our findings using regionally specific and genome-wide approaches with multiple human cell sources, we can definitively conclude that CpG methylation is absent in mtDNA. It is highly unlikely that CpG methylation plays any role in direct control of mitochondrial function.