Direct introduction of cloned DNA into the sea urchin zygote nucleus, and fate of injected DNA

A method is described for microinjection of cloned DNA into the zygote nucleus of Lytechinus variegatus. Eggs of this species are unusually transparent, facilitating visual monitoring of the injection process. The initial fate of injected DNA fragments appears similar to that observed earlier for exogenous DNA injected into unfertilized egg cytoplasm. Thus after end-to-end ligation, it is replicated after a lag of several hours to an extent indicating that it probably participates in most of the later rounds of DNA synthesis undergone by the host cell genomes during cleavage. The different consequences of nuclear versus cytoplasmic injection are evident at advanced larval stages. Larvae descendant from eggs in which exogenous DNA was injected into the nuclei are four times more likely (32% versus 8%) to retain this DNA in cell lineages that replicate very extensively during larval growth, i.e. the lineages contributing to the imaginal rudiment, and thus to display greatly enhanced contents of the exogenous DNA. Similarly, 36% of postmetamorphic juveniles from a nuclear injection sample retained the exogenous DNA sequences, compared to 12% of juveniles from a cytoplasmic injection sample. However, the number of copies of the exogenous DNA sequences retained per average genome in postmetamorphic juveniles was usually less than 0.1 (range 0.05-50), and genome blot hybridizations indicate that these sequences are organized as integrated, randomly oriented, end-to-end molecular concatenates. It follows that only a small fraction of the cells of the average juvenile usually retains the exogenous sequences. Thus, even when introduced by nuclear microinjection, the stable incorporation of exogenous DNA in the embryo occurs in a mosaic fashion, although in many recipients the DNA enters a wider range of cell lineages than is typical after cytoplasmic injection. Nuclear injection would probably be the route of choice for studies of exogenous DNA function in the postembryonic larval rudiment

Negative spatial regulation of the lineage specific CyIIIa actin gene in the sea urchin embryo

The CyIIIa·CAT fusion gene was injected into Strongylocentrotus purpuratus eggs, together with excess ligated competitor sequences representing subregions of the CyIIIa regulatory domain. In this construct, the chloramphenicol acetyltransferase (CAT) reporter gene is placed under the control of the 2300 nucleotide upstream regulatory domain of the lineage-specific CyIIIa cytoskeletal actin gene. CAT mRNA was detected by in situ hybridization in serial sections of pluteus stage embryos derived from the injected eggs. When carrier DNA lacking competitor CyIIIa fragments was coinjected with CyIIIa.CAT, CAT mRNA was observed exclusively in aboral ectoderm cells, i.e. the territory in which the CyIIIa gene itself is normally expressed (as also reported by us previously). The same result was obtained when five of seven different competitor subfragments bearing sites of DNA-protein interaction were coinjected. However, coinjection of excess quantities of either of two widely separated, nonhomologous fragments of the CyIIIa regulatory domain produced a dramatic ectopic expression of CAT mRNA in the recipient embryos. CAT mRNA was observed in gut, mesenchyme cells and oral ectoderm in these embryos. We conclude that these fragments contain regulatory sites that negatively control spatial expression of the CyIIIa gene

Competitive titration in living sea urchin embryos of regulatory factors required for expression of the CyIIIa actin gene

Previous studies have located some twenty distinct sites within the 2.3 kb 5' regulatory domain of the sea urchin CyIIIa cytoskeletal actin gene, where there occur in vitro high-specificity interactions with nuclear DNA-binding proteins of the embryo. This gene is activated in late cleavage, exclusively in cells of the aboral ectoderm cell lineages. In this study, we investigate the functional importance in vivo of these sites of DNA-protein interaction. Sea urchin eggs were coinjected with a fusion gene construct in which the bacterial chloramphenicol acetyltransferase (CAT) reporter gene is under the control of the entire CyIIIa regulatory domain, together with molar excesses of one of ten nonoverlapping competitor subfragments of this domain, each of which contains one or a few specific site(s) of interaction. The exogenous excess binding sites competitively titrate the available regulatory factors away from the respective sites associated with the CyIIIa.CAT reporter gene. This provides a method for detecting in vivo sites within the regulatory domain that are required for normal levels of expression, without disturbing the structure of the regulatory domain. We thus identify five nonoverlapping regions of the regulatory DNA that apparently function as binding sites for positively acting transcriptional regulatory factors. Competition with a subfragment bearing an octamer site results in embryonic lethality. We find that three other sites display no quantitative competitive interference with CyIIIa.CAT expression, though as shown in the accompanying paper, two of these sites are required for control of spatial expression. We conclude that the complex CyIIIa regulatory domain must assess the state of many distinct and individually necessary interactions in order to properly regulate CyIIIa transcriptional activity in development

Lineage and fate of each blastomere of the eight-cell sea urchin embryo

A fluoresceinated lineage tracer was injected into individual blastomeres of eight-cell sea urchin (Strongylocentrotus purpuratus) embryos, and the location of the progeny of each blastomere was determined in the fully developed pluteus. Each blastomere gives rise to a unique portion of the advanced embryo. We confirm many of the classical assignments of cell fate along the animal-vegetal axis of the cleavage-stage embryo, and demonstrate that one blastomere of the animal quartet at the eight-cell stage lies nearest the future oral pole and the opposite one nearest the future aboral pole of the embryo. Clones of cells deriving from ectodermal founder cells always remain contiguous, while clones of cells descendant from the vegetal plate (i.e., gut, secondary mesenchyme) do not. The locations of ectodermal clones contributed by specific blastomeres require that the larval plane of bilateral symmetry lie approximately equidistant (i.e., at a 45 degree angle) from each of the first two cleavage planes. These results underscore the conclusion that many of the early spatial patterns of differential gene expression observed at the molecular level are specified in a clonal manner early in embryonic sea urchin development, and are each confined to cell lineages established during cleavage

Spatially deranged though temporally correct expression of a Strongylocentrotus purpuratus actin gene fusion in transgenic embryos of a different sea urchin family

We report the unexpected observation that cis-regulatory sequences of a Strongylocentrotus purpuratus actin gene, which direct a particular, lineage-specific pattern of embryonic expression, confer a completely different spatial pattern of expression when introduced into embryos of another sea urchin species. We utilized a fusion gene construct in which the bacterial chloramphenicol acetyl transferase (CAT) reporter gene is driven by CyIIIa actin regulatory sequences. We previously showed that the regulatory region that is included suffices to promote the accumulation of CAT mRNA in transgenic S. purpuratus embryos, on the same developmental schedule and in the same embryonic region, the aboral ectoderm, in which the CyIIIa actin gene is normally expressed (Flytzanis et al. 1987; Hough-Evans et al. 1987). When injected into zygotes of Lytechinus variegatus, which belongs to a different echinoid family, the expected temporal pattern of expression of CAT enzyme was observed. Thus, in both S. purpuratus and L. variegatus embryos, expression is activated at the early blastula stage, although this stage is attained several hours sooner in L. variegatus embryo cultures. Similar kinetics of CAT enzyme accumulation were obtained whether the gene was introduced directly into the L. variegatus zygote nucleus or into the cytoplasm. However, when examined by in situ hybridization, the transgenic L. variegatus embryos were found to display a totally new pattern of CAT mRNA accumulation. Copious CAT transcripts were detected not only in aboral ectoderm cells, but also in skeletogenic mesenchyme cells, gut cells, and oral ectoderm, all cell types that in the transgenic S. purpuratus controls are invariably devoid of detectable CAT transcripts

Point-of-care versus central testing of hemoglobin during large volume blood transfusion.

BACKGROUND: Point-of-care (POC) hemoglobin testing has the potential to revolutionize massive transfusion strategies. No prior studies have compared POC and central laboratory testing of hemoglobin in patients undergoing massive transfusions. METHODS: We retrospectively compared the results of our point-of-care hemoglobin test (EPOC®) to our core laboratory complete blood count (CBC) hemoglobin test (Sysmex XE-5000™) in patients undergoing massive transfusion protocols (MTP) for hemorrhage. One hundred seventy paired samples from 90 patients for whom MTP was activated were collected at a single, tertiary care hospital between 10/2011 and 10/2017. Patients had both an EPOC® and CBC hemoglobin performed within 30 min of each other during the MTP. We assessed the accuracy of EPOC® hemoglobin testing using two variables: interchangeability and clinically significant differences from the CBC. The Clinical Laboratory Improvement Amendments (CLIA) proficiency testing criteria defined interchangeability for measurements. Clinically significant differences between the tests were defined by an expert panel. We examined whether these relationships changed as a function of the hemoglobin measured by the EPOC® and specific patient characteristics. RESULTS: Fifty one percent (86 of 170) of paired samples\u27 hemoglobin results had an absolute difference of ≤7 and 73% (124 of 170) fell within ±1 g/dL of each other. The mean difference between EPOC® and CBC hemoglobin had a bias of - 0.268 g/dL (p = 0.002). When the EPOC® hemoglobin was \u3c 7 g/dL, 30% of the hemoglobin values were within ±7, and 57% were within ±1 g/dL. When the measured EPOC® hemoglobin was ≥7 g/dL, 55% of the EPOC® and CBC hemoglobin values were within ±7, and 76% were within ±1 g/dL. EPOC® and CBC hemoglobin values that were within ±1 g/dL varied by patient population: 77% for cardiac surgery, 58% for general surgery, and 72% for non-surgical patients. CONCLUSIONS: The EPOC® device had minor negative bias, was not interchangeable with the CBC hemoglobin, and was less reliable when the EPOC® value was \u3c 7 g/dL. Clinicians must consider speed versus accuracy, and should check a CBC within 30 min as confirmation when the EPOC® hemoglobin is \u3c 7 g/dL until further prospective trials are performed in this population

Characterization of Biochars Produced From Peanut Hulls and Pine Wood with Different Pyrolysis Conditions

Background Application of modern biomass pyrolysis methods for production of biofuels and biochar is potentially a significant approach to enable global carbon capture and sequestration. To realize this potential, it is essential to develop methods that produce biochar with the characteristics needed for effective soil amendment. Methods Biochar materials were produced from peanut hulls and pine wood with different pyrolysis conditions, then characterized by cation exchange (CEC) capacity assays, nitrogen adsorption–desorption isotherm measurements, micro/nanostructural imaging, infrared spectra and elemental analyses. Results Under a standard assay condition of pH 8.5, the CEC values of the peanut hull-derived biochar materials, ranging from 6.22 to 66.56 cmol kg−1, are significantly higher than those of the southern yellow pine-derived biochar, which are near zero or negative. The biochar produced from peanut hulls with a steam activation process yielded the highest CEC value of 66.56 cmol kg−1, which is about 5 times higher than the cation exchange capacity (12.51 cmol kg−1) of a reference soil sample. Notably, biochar produced from peanut hulls with batch barrel retort pyrolysis also has a much higher CEC value (60.12 cmol kg−1) than that (12.45 cmol kg−1) from Eprida’s H2-producing continuous steam injection process. The CEC values were shown to correlate well with the ratios of oxygen atoms to carbon atoms (O:C ratios) in the biochar materials. The higher O:C ratio in a biochar material may indicate the presence of more hydroxyl, carboxylate, and carbonyl groups that contribute to a higher CEC value for the biochar product. In addition, the increase in surface area can also play a role in increasing the CEC value of biochar, as in the case of the steam activation char. Conclusion Comparison of characterization results indicated that CEC value is determined not only by the type of the source biomass materials but also by the pyrolysis conditions. Biochar with the desirable characteristics of extremely high surface area (700 m2/g) and cation exchange capacity (\u3e 60 cmol kg) was created through steam activation

The Lantern Vol. 15, No. 1, Fall 1946

• Public, Speaking • Concept • The Storm • Yes Sir! • Messengers of Death • The Anonymous Letter • Best Trust the Happy Moments • Disillusionment • The Man With the Water-Brown Eyes • Poetry • Who Knows?https://digitalcommons.ursinus.edu/lantern/1040/thumbnail.jp

Ontogenic expression of a CyI actin fusion gene injected into sea urchin eggs

The 5' terminus of the CyI actin gene transcription unit of Strongylocentrotus purpuratus was located by primer extension and other procedures, and the flanking upstream region was partially sequenced and mapped. A fusion gene was constructed containing about 2.5 kb of 5' flanking sequence, the transcribed leader sequence, and the first few codons of the CyI gene ligated to the bacterial gene coding for chloramphenicol acetyl transferase (CAT). This was micro-injected into the cytoplasm of S. purpuratus eggs, and CAT enzyme activity was measured at various stages of embryonic development. CAT synthesis was activated between 10 and 14 h postfertilization, the same time at which newly synthesized transcripts of the endogenous CyI gene first appear. The exogenous CyI.CAT fusion DNA replicated actively during cleavage, as observed previously for other DNAs injected into sea urchin egg cytoplasm. Thus the absence of CAT activity prior to 10 h postfertilization could not be due to insufficient CyI.CAT genes. The amounts of CAT enzyme produced by embryos bearing CyI.CAT deletions that lack various regions of the CyI sequence were measured. As little as 254 nucleotides of upstream CyI sequence suffice for correct temporal activation of the fusion construct, although the level of CAT enzyme produced in embryos bearing any deletion retaining less than 850 nucleotides of upstream sequence was significantly lowered compared to controls bearing the complete CyI.CAT fusion construct