The discovery and chemical identification, in the 1920s, of the aliphatic polyester: poly(3-hydroxybutyrate), PHB, as a granular component in bacterial cells proceeded without any of the ...controversies which marked the recognition of macromolecules by Staudinger. Some thirty years after its discovery, PHB was recognized as the prototypical biodegradable thermoplastic to solve the waste disposal challenge. The development effort led by Imperial Chemical Industries Ltd., encouraged interdisciplinary research from genetic engineering and biotechnology to the study of enzymes involved in biosynthesis and biodegradation. From the simple PHB homopolyester discovered by Maurice Lemoigne in the mid-twenties, a family of over 100 different aliphatic polyesters of the same general structure has been discovered. Depending on bacterial species and substrates, these high molecular weight stereoregular polyesters have emerged as a new family of natural polymers ranking with nucleic acids, polyamides, polyisoprenoids, polyphenols, polyphosphates, and polysaccharides. In this historical review, the chemical, biochemical and microbial highlights are linked to personalities and locations involved with the events covering a discovery timespan of 75 years.
Chromatin immunoprecipitation-sequencing (ChIP-seq) is a widely used epigenetic approach for investigating genome-wide protein-DNA interactions in cells and tissues. The approach has been relatively ...well established but several key steps still require further improvement. As a part of the procedure, immnoprecipitated DNA must undergo purification and library preparation for subsequent high-throughput sequencing. Current ChIP protocols typically yield nanogram quantities of immunoprecipitated DNA mainly depending on the target of interest and starting chromatin input amount. However, little information exists on the performance of reagents used for the purification of such minute amounts of immunoprecipitated DNA in ChIP elution buffer and their effects on ChIP-seq data. Here, we compared DNA recovery, library preparation efficiency, and ChIP-seq results obtained with several commercial DNA purification reagents applied to 1 ng ChIP DNA and also investigated the impact of conditions under which ChIP DNA is stored.
We compared DNA recovery of ten commercial DNA purification reagents and phenol/chloroform extraction from 1 to 50 ng of immunopreciptated DNA in ChIP elution buffer. The recovery yield was significantly different with 1 ng of DNA while similar in higher DNA amounts. We also observed that the low nanogram range of purified DNA is prone to loss during storage depending on the type of polypropylene tube used. The immunoprecipitated DNA equivalent to 1 ng of purified DNA was subject to DNA purification and library preparation to evaluate the performance of four better performing purification reagents in ChIP-seq applications. Quantification of library DNAs indicated the selected purification kits have a negligible impact on the efficiency of library preparation. The resulting ChIP-seq data were comparable with the dataset generated by ENCODE consortium and were highly correlated between the data from different purification reagents.
This study provides comparative data on commercial DNA purification reagents applied to nanogram-range immunopreciptated ChIP DNA and evidence for the importance of storage conditions of low nanogram-range purified DNA. We verified consistent high performance of a subset of the tested reagents. These results will facilitate the improvement of ChIP-seq methodology for low-input applications.
Epigenetic dysregulation is involved in the etiology and progression of various human diseases. Formalin-fixed paraffin-embedded (FFPE) samples represent the gold standard for archiving pathology ...samples, and thus FFPE samples are a major resource of samples in clinical research. However, chromatin-based epigenetic assays in the clinical settings are limited to fresh or frozen samples, and are hampered by low chromatin yield in FFPE samples due to the lack of a reliable and efficient chromatin preparation method. Here, we introduce a new chromatin extraction method from FFPE tissues (Chrom-EX PE) for chromatin-based epigenetic assays.
During rehydration of FFPE tissues, applying a tissue-level cross-link reversal into the deparaffinized tissue at 65 °C dramatically increased chromatin yield in the soluble fraction. The resulting chromatin is compatible with targeted ChIP-qPCR and genome-wide ChIP-seq approaches. The chromatin prepared by Chrom-EX PE showed a gradual fragmentation pattern with varying incubation temperature. At temperatures below 37 °C, the majority of soluble chromatin is over 1 kb. The soluble chromatin prepared in the range of 45-60 °C showed a typical nucleosomal pattern. And the majority of chromatin prepared at 65 °C is close to mononucleosomal size. These observations indicate that chromatin preparation from FFPE samples can be controlled for downstream chromatin-based epigenetic assays.
This study provided a new method that achieves efficient extraction of high-quality chromatin suitable for chromatin-based epigenetic assays with less damage on chromatin. This approach may provide a way to circumvent the over-fixed nature of FFPE tissues for future technology development.
sPHENIX is a new experiment under construction for the Relativistic Heavy Ion Collider at Brookhaven National Laboratory which will study the quark-gluon plasma to further the understanding of ...quantum chromodynamics (QCP) matter and interactions. A prototype of the sPHENIX electromagnetic calorimeter (EMCal) was tested at the Fermilab Test Beam Facility in Spring 2018 as experiment T-1044. The EMCal prototype corresponds to a solid angle of <inline-formula> <tex-math notation="LaTeX">\Delta \eta \times \Delta \phi = 0.2 \times 0.2 </tex-math></inline-formula> centered at pseudo-rapidity <inline-formula> <tex-math notation="LaTeX">\eta = 1 </tex-math></inline-formula>. The prototype consists of scintillating fibers embedded in a mix of tungsten powder and epoxy. The fibers project back approximately to the center of the sPHENIX detector, giving 2-D projectivity. The energy response of the EMCal prototype was studied as a function of position and input energy. The energy resolution of the EMCal prototype was obtained after applying a position-dependent energy correction and a beam profile correction. Two separate position-dependent corrections were considered. The EMCal energy resolution was found to be <inline-formula> <tex-math notation="LaTeX">\sigma (E)/\langle E\rangle = 3.5(0.1) \oplus 13.3(0.2)/\sqrt {E} </tex-math></inline-formula> based on the hodoscope position-dependent correction, and <inline-formula> <tex-math notation="LaTeX">\sigma (E)/\langle E\rangle = 3.0(0.1) \oplus 15.4(0.3)/\sqrt {E} </tex-math></inline-formula> based on the cluster position-dependent correction. These energy resolution results meet the requirements of the sPHENIX physics program.
Ejaculates were collected by artificial vagina from 3 Holstein sires and sorted to 90% purity for X-chromosome-bearing spermatozoa (range 88 to 93%) using flow cytometry. Sorted sperm were diluted to ...2.1, 3.5, or 5.0×106 sperm per dose in an egg yolk (20%), Tris, glycerol (7%) extender. Collections were repeated until >600 straws per sperm dose per sire were obtained. Each sperm dose was loaded into color-coded 0.25-mL French straws, with alternate colors used to define treatments across sires. Within sires, straws were packaged at 9 per cane (3 of each color) and strategically allocated to 75 Holstein herds with targets for 50% use in heifers and 50% in lactating cows. Straw color was recorded in the on-farm record-keeping system at the time of insemination. Data were analyzed separately for cows and heifers. Among heifers, a total of 2,125 usable records were retrieved from 51 herds (238±5.5 services/ sperm dose per sire, range: 218 to 263). Conception rates in heifers were influenced by the sire×sperm dosage interaction. Within sire A, conception rates of heifers were greater for the 5×106 (59.5%) than for the 2.1×106 (46.4%) sperm dose and intermediate for the 3.5×106 sperm dose (52.2%). However, across sires, sperm dosage had no effect on heifer conception rates (46.7, 51.2, and 52.5% for the 2.1, 3.5, and 5.0×106 sperm dosages, respectively). Among cows, a total of 2,369 services were retrieved from 56 herds (263±8.8 services/sperm dose per sire, range: 233 to 303). Conception rates of cows (29.4%) were not affected by sire or sperm dosage (27.0, 29.1, and 30.3% for the 2.1, 3.5, and 5.0×106 sperm dosages, respectively). In conclusion, these data indicate that an increased sperm dosage may enhance virgin heifer conception rates for some (but not all) sires, whereas neither sire nor sexed-sperm dosage affected conception rates of lactating cows. Additional studies of sexed-sperm dosage across a larger sampling of bulls are warranted to determine whether and how such a practice can be implemented cost effectively for the benefit of the dairy industry.
Computer-assisted sperm analysis of fresh and frozen-thawed bovine sperm requires proper handling and preparation, and the type of slide used in the assessment is critical if the resultant data are ...to be useful quality control measurements. In the present study, 4 different slide viewing chambers, a Makler chamber, a clean slide-coverslip, or a 2- or 4-cell chamber Leja slide, were compared with assess their utility in providing reliable measurements of sperm motility variables. A Hamilton-Thorne IVOS Computer-Assisted Semen Analyzer (CASA) was the instrument used to determine sperm measurements utilizing the 4 different chambers. Fifty-eight different freeze batches of bovine semen that had been collected from 47 bulls at 7 sites that sex-sort sperm using Sexing Technologies sorting criteria were incorporated into the trial. Neither the percentage of motile sperm nor the percentage of progressively motile sperm differed for the Makler chamber vs. slide-coverslip comparisons. Similarly, total and progressively motile sperm did not differ between the 2- and 4-cell chambered Leja slides. However, total and progressive motility of sperm determined with the Makler chamber and slide-coverslip were greater (P < 0.0001) than motilities recorded by the 2- or 4-cell chambered Leja slides. Based on the results, the type of viewing chamber can affect the range of sperm motility values when CASA is used for quality control evaluations of thawed, cryopreserved sex-sorted sperm samples.
Purified Ralstonia eutropha polyhydroxybutyrate (PHB) synthase from recombinant cells can exist as monomer and dimer. The polymerization reaction catalyzed by this enzyme displays a lag phase, which ...causes difficulties for kinetic and mechanistic characterization of the enzymatic polymerization reaction. In this study, we developed a method to eliminate the lag phase of PHB synthase by physical means, i.e., adding multihydroxyl compounds to the enzyme solution. This method allows us to recognize the nature of the lag phase as a physical rather than a chemical process. With such lag-phase-free-enzyme, the kinetic properties of the enzyme were investigated. The results indicate that 3-hydroxybutyryl-CoA (3HBCoA) is the optimal substrate for the enzyme. A slower catalytic rate and lower binding ability account for a lower reactivity of 3-hydroxyvaleryl-CoA (3HVCoA) compared to that of 3HBCoA. The change of hydroxyl group from the beta to the gamma position causes dramatic decreases in the binding ability of 4-hydroxybutyryl-CoA (4HBCoA). By using a dilution strategy and size exclusion chromatographic technique, the active form of the enzyme was identified to be the dimeric form. The number of catalytic sites in the dimeric form of the enzyme was examined by comparing the molecular weight of polyhydroxybutyrate as a function of substrate-to-enzyme ratio. The results suggest that the dimeric enzyme has only one catalytic site. A revised model of polymerization reaction catalyzed by R. eutropha PHB synthase is described.
This study examined DNA damage and postthaw motility of white-tailed deer sperm (n = 28) before and after sex selection and conventional sorting using MoFlo XDP SX flow cytometry. Semen samples from ...the same individuals were treated in 4 different ways: 1) chilled-extended sperm samples (without glycerol); 2) cryopreserved conventional samples, samples directly cryopreserved after the addition of extenders; 3) cryopreserved conventionally sorted samples, sorted samples to remove the dead sperm subpopulation; and 4) cryopreserved sex-sorted samples; sorted samples to remove the dead sperm subpopulation and separation of X- and Y-chromosome-bearing sperm. In all the cases (n = 6), conventional samples showed decreased postthaw motilities (43 ± 26%) when compared with X-sorted samples (59 ± 20%; P < 0.05) and Y-sorted samples (54 ± 20%; P > 0.05). The DNA fragmentation baseline was <5% for frozen-thawed conventional samples, but even less after sex sorting and conventional sorting: 2.4 and 1.7%, respectively. On the other hand, conventional samples showed greater (P < 0.05) DNA fragmentation than the sex-sorted sperm (n = 6) at 96 h (average of 4.8 ± 4.5% and 5.3 ± 4%, respectively). Conventionally sorted samples (n = 8) did not have greater (P > 0.05) DNA fragmentation when compared with the sex-sorted samples. Fragmentation of DNA on X-chromosome and Y-chromosome-bearing sorted sperm were not significantly different (n = 10, P > 0.05) after 96 h (2.6 ± 3.6% and 2.2 ± 0.5%, respectively). Future research should be implemented for examining the fertilizing potential of sex-sorted white-tailed deer sperm (e.g., AI fertility trials).
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