Profiling SARS-CoV-2 HLA-I peptidome reveals T cell epitopes from out-of-frame ORFs

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Confidence Intervals for the Risk Ratio in Cohort Studies under Inverse Sampling

Three simple interval estimates for the risk ratio in inverse sampling are considered. The first two interval estimates are derived on the basis of Fieller's Theorem and the delta method with the logarithmic transformation, respectively. The third interval estimate is derived on the basis of an F-test statistic proposed by BENNETT (1981) for testing equal probabilities of a disease between two comparison groups when the disease is rare. To evaluate the performance of these three methods, a Monte Carlo simulation is used to compare the actual coverage probability with the nominal confidence level for each method and to estimate the expected length of the corresponding confidence interval in a variety of situations. On the basis of the results found in the simulation, we have concluded that the method with the logarithmic transformation is either equivalent to or better than the other two methods for all situations considered here.     

Cloning and expression of the 3' portion of the T4 denV gene as a lacZ fusion gene

Polyclonal antibodies have been raised against endonuclease V from the bacteriophage T4. This rabbit serum, from which endemic E. coli antibodies have been removed, reacts with a single protein from T4-infected E. coli with a molecular weight of 16078 dalton. It was confirmed that these antibodies were directed against endonuclease V through the inhibition of the pyrimidine dimer specific nicking activity of endonuclease V in an in vitro nicking assay. A phage lambda gt11 T4 dC DNA library was screened for phage which produced a beta-galactosidase-endonuclease V fusion protein. Immunopositive clones were detected at a frequency of 0.25% of the plaques in the library. Restriction enzyme analyses of the DNA from 45 of these phage showed that all contained a 1.8 kb T4 EcoRI fragment which had been inserted within lambda gt11 in a single orientation. Western analysis of proteins which were produced from an induction of lysogens made from these phage reveals a single fusion protein band with a molecular weight slightly larger than native beta-galactosidase.     

Single copy DNA homology in sea stars

Summary The sequence homology in the single copy DNA of sea stars has been measured. Labeled single copy DNA fromPisaster ochraceus was reannealed with excess genomic DNA fromP. brevispinus, Evasterias troschelii, Pycnopodia helianthoides, Solaster stimpsoni, andDermasterias imbricata. Reassociation reactions were performed under two criteria of salt and temperature. The extent of reassociation and thermal denaturation characteristics of hybrid single copy DNA molecules follow classical taxonomic lines.P. brevispinus DNA contains essentially all of the sequences present inP. ochraceus single copy tracer whileEvasterias andPycnopodia DNAs contain 52% and 46% of such sequences respectively. Reciprocal reassociation reactions with labeledEvasterias single copy DNA confirm the amount and fidelity of the sequence homology. There is a small definite reaction of uncertain homology betweenP. ochraceus single copy DNA andSolaster orDermasterias DNA. SimilarlySolaster DNA contains sequences homologous to approximately 18% ofDermasterias unique DNA. The thermal denaturation temperatures of heteroduplexes indicate that the generaPisaster andEvasterias diverged shortly after the divergence of the subfamilies Pycnopodiinae and Asteriinae. The twoPisaster species diverged more recently, probably in the most recent quarter of the interval since the separation of the generaPisaster andEvasterias.     

High-frequency spontaneous deletion of DNA sequences flanked by direct DNA repeats which also contain an internal palindrome

The DNA sequences associated with a very high-frequency, spontaneous deletion event have been determined to be two 11-base direct repeats which also contain an internal 6-base palindrome. A parental M13 replicative form (RF) DNA harboring DNA fragments of the T4 denV gene contained these direct repeats and could only be maintained at 5% of the total RF DNA within an infected cell. The remaining RF DNA was deleted for all intervening sequences between the direct repeats (2.2-kb), but one copy of the direct repeat was retained after the deletion had occurred. This site-specific deletion was highly reproducible in that if parental-sized M13 RF DNA was gel purified and transformed back into cells, the deletion occurred at precisely the same sequence as before. Electron microscopic analyses of DNA extracted from cells transformed with parental-sized DNA revealed the presence of excised 2.2-kb double-stranded circular DNA molecules. This observation thus rules out a copy choice replication/deletion mechanism to account for this high-frequency deletion event.     

Metabolism of copper and zinc in the liver and bone of the perinatal guinea-pig

Hepatic copper concentration in the guinea-pig increased markedly during the second-half of gestation, attaining a maximum shortly after birth; thereafter, concentration declined rapidly during the neonatal period. Changes in perinatal hepatic copper concentrations paralleled the binding of copper to a cytosolic metallothionein-like component, and the loss of hepatic copper in the neonates coincided with increases in serum copper concentrations. Zinc concentrations of the perinatal liver were low and showed no dramatic developmental changes. The humerus showed striking increases in zinc concentration with gestational age, attaining peak concentration before term and a marked depletion of tissue zinc during the neonatal period.     

Eimeria tenella: use of a monoclonal antibody in determining the intracellular fate of the refractile body organelles and the effect on in vitro development

A monoclonal antibody, which recognizes the refractile body of Eimeria sporozoites, was used to study the developmental fate of this organelle during asexual development of E. tenella and to determine the effect of this monoclonal antibody on in vitro development of the parasite. Through use of immunofluorescent antibody and gold-labeling techniques at the light and electron microscopy level, the refractile body at 48 to 96 hr postinoculation was found to separate into 6 to 10 small globules, then diffuse throughout the schizont cytoplasm, and eventually reconcentrate as a small dot of material in each of the mature first-generation merozoites. The schizont did not develop to maturity if diffusion of the refractile body did not occur. The refractile body material was quickly lost as the merozoite left the schizont and invaded new cells and was not detected in any later developmental stages. The in vitro development of first- and second-generation schizonts of E. tenella was greatly inhibited (up to 100%) with exposure to the monoclonal antibody. There was an increase in the number of schizonts with nondispersed refractile body in the monoclonal antibody-treated cells when compared to the untreated controls, and the few mature schizonts seen had up to a 50-fold decrease in the number of merozoites. Immunofluorescent antibody labeling of the refractile body of intracellular sporozoites and schizonts treated in vitro with the monoclonal antibody for 24-96 hr postinoculation indicated that the antibody had crossed the host cell and parasite plasma membrane during incubation.     

Monoclonal antibody produced against partially purified sporozoite antigen inhibits invasion of cells by sporozoites of avian Eimeria species

Previous studies showed that molecules of in vitro-cultured primary turkey kidney cells bound to 23-, 40-, and 60- to 65-kDa antigens of sodium dodecyl sulfate-solubilized sporozoites of Eimeria adenoeides. Similar binding to antigens of three other species of avian Eimeria, E. tenella, E. acervulina, and E. meleagrimitis, is now reported. Strips containing the most avidly bound sporozoite antigen (approximately 40 kDa) were excised from the sodium dodecyl sulfate-polyacrylamide gels on which E. adenoeides antigens had been electrophoretically separated. The strips were homogenized and injected into mice to produce hybridoma cell lines. Twelve cell lines secreting monoclonal antibodies (McAb) that reacted with E. adenoeides sporozoites were detected. One of these McAb, H11C3, reacted with structures in the anterior tip of sporozoites of E. adenoeides and five other species of avian Eimeria. When included in the inoculation medium, this McAb significantly inhibited invasion of cultured kidney cells by sporozoites of E. adenoeides and E. tenella. In contrast, when the sporozoites were pretreated with McAb H11C3 and then washed free of the antibody, no inhibition of invasion was observed.