Monoclonal antibodies to antigens abnormally expressed in breast cancer

We report the production, screening, and characterization of ten murine monoclonal antibodies directed at antigens that are expressed abnormally in human breast tumors. Immunoperoxidase staining of frozen and fixed tissues shows the antigens to be present at low levels on the luminal membrane of normal breast cells and at high levels in the cytoplasm and surface membrane of breast tumor cells. The ten antibodies appear to recognize six different epitopes on the basis of their quantitative differences in reactivity against four antigen preparations, as measured by ELISA. Immunoblots show that eight of the ten antibodies recognize a 300,000 MW molecule from breast tumor preparations; six of these antibodies also react with a second molecule from the same tumor preparations of 280,000 MW. Seven antibodies react with an antigen from milk fat globule membrane of 330,000 MW. It therefore appears that the two molecules from tumor tissue and the one molecule from normal tissue share common epitopes. Selected antibodies were tested for reactivity against 25 primary breast tumors and 14 pairs of primary and metastatic breast tumors. Three antibodies have broad reactivity and stain more than 80% of primary tumors; the three other antibodies identify subsets of those tumors. Results of staining pairs of primary and metastatic lesions show that metastases continue to express antigens of the primary lesion in a high percentage of cells.     

The molecular basis of the requirement for antigen processing of pigeon cytochrome c prior to T cell activation

Antigen-induced activation of T lymphocytes that co-recognize Ia molecules has been shown to require an antigen-processing step by the presenting cell before T cell stimulation can occur. In this report, we demonstrate that antigen presentation of pigeon cytochrome c to an E kappa beta:E kappa alpha-restricted T cell hybridoma, 2C2, is inhibited by pretreatment of the antigen-presenting cells (APC) either with chloroquine or with fixation by paraformaldehyde. The chloroquine effect was partially reversible after 22 hr; the paraformaldehyde effect was not. In contrast, these treatments had little or no effect on the presentation of the carboxy-terminal cyanogen bromide cleavage fragment of pigeon cytochrome c, residues 81 to 104. There was at least a 50-fold greater potency of the fragment, as compared to that of the intact molecule, when paraformaldehyde-fixed APC were used. In addition, the fixed cells did not present synthetic fragments of the cytochrome c that were nonstimulatory when presented by unfixed cells. This observation showed that the loss of potency, demonstrated previously for analogs of pigeon cytochrome c with single amino acid substitutions at positions such as 99, was not a consequence of an alteration in the rate of antigen-processing. This result is consistent with our earlier hypothesis that these residues are contact amino acids with the antigen-specific T cell receptor or the Ia molecule. The major goal of these experiments was to define the molecular transition that occurred as a result of antigen processing. To achieve this end, we tested a variety of pigeon cytochrome c molecules and fragments for their ability to be presented by paraformaldehyde-fixed APC. Apocytochrome c, the denatured form of the molecule with the heme removed, could not be presented by the fixed cells, nor could the fragment 60-104, derived by acid cleavage of the tryptophan at position 59. Both molecules stimulated an IL 2 response from the T cell hybridoma when unfixed APC were utilized, demonstrating that the conditions used to prepare these two molecules did not destroy their antigenic determinant. In contrast, carboxy-terminal fragments, both native and synthetic, ranging in size from 16 to 39 amino acids, were capable of stimulating in the presence of paraformaldehyde-fixed APC. In particular, the partial-digest cyanogen bromide fragment, residues 66 to 104, was only twofold less potent than the pigeon fragment 81-104.(ABSTRACT TRUNCATED AT 400 WORDS)     

The incorporation of macromolecules into the germ cells of male mice via electroporation and dimethyl sulfoxide

Electroporation (incorporation of macromolecules into the living cells by means of electric pulses) provides inclusion of plasmid 14C-DNA into immature cells of spermatogenic epithelium. The highest level of foreign DNA incorporation into spermatocytes and spermatids has been induced by 8kV electric pulses applied 3 times with 20 sec intervals. Meanwhile, mature sperms are found to be exclusively resistant to exogenous DNA irrespective of the voltage level, the number of pulses and Ca++ uptake (contents). Incubation of mature sperms for two hours in the medium with Ca++ (10 mM) and dimethylsulfoxide--(DMSO, 33%) provides highly reliable incorporation of plasmid 14C-DNA into sperm heads. The sperm cells with foreign DNA incorporated by means of Ca++ and DMSO treatment still remain alive and mobile. The possibilities of mature sperms loaded with foreign DNA for the creation of transgenic mammals are discussed.     

Functional Genomic Analysis of the let-7 Regulatory Network in Caenorhabditis elegans

The let-7 microRNA (miRNA) regulates cellular differentiation across many animal species. Loss of let-7 activity causes abnormal development in Caenorhabditis elegans and unchecked cellular proliferation in human cells, which contributes to tumorigenesis. These defects are due to improper expression of protein-coding genes normally under let-7 regulation. While some direct targets of let-7 have been identified, the genome-wide effect of let-7 insufficiency in a developing animal has not been fully investigated. Here we report the results of molecular and genetic assays aimed at determining the global network of genes regulated by let-7 in C. elegans. By screening for mis-regulated genes that also contribute to let-7 mutant phenotypes, we derived a list of physiologically relevant potential targets of let-7 regulation. Twenty new suppressors of the rupturing vulva or extra seam cell division phenotypes characteristic of let-7 mutants emerged. Three of these genes, opt-2, prmt-1, and T27D12.1, were found to associate with Argonaute in a let-7–dependent manner and are likely novel direct targets of this miRNA. Overall, a complex network of genes with various activities is subject to let-7 regulation to coordinate developmental timing across tissues during worm development.     

Aerobic transformation of zinc into metal sulfide by photosynthetic microorganisms

Industrial activity over the last two centuries has increased heavy metal contamination worldwide, leading to greater human exposure. Zinc is particularly common in industrial effluents and although an essential nutrient, it is highly toxic at elevated concentrations. Photoautotrophic microbes hold promise for heavy metal bioremediation applications because of their ease of culture and their ability to produce sulfide through metabolic processes that in turn are known to complex with the metal ion, Hg(II). The green alga Chlamydomonas reinhardtii, the red alga Cyanidioschyzon merolae, and the cyanobacterium Synechococcus leopoliensis were all able to synthesize sulfide and form zinc sulfide when exposed to Zn(II). Supplementation of their respective media with sulfite and cysteine had deleterious effects on growth, although ZnS still formed in Cyanidioschyzon cells to the same extent as in unsupplemented cells. The simultaneous addition of sulfate and Zn(II) had similar effects to that of Zn(II) alone in all three species, whereas supplying sulfate prior to exposure to Zn(II) enhanced metal sulfide production. The coupled activities of serine acetyltransferase and O-acetylserine(thiol)lyase (SAT/OASTL) did not increase significantly in response to conditions in which enhanced ZnS formation occurred; sulfate added prior to and simultaneously with Zn(II). However, even low activity could provide sufficient sulfate assimilation over this relatively long-term study. Because the extractable activity of cysteine desulfhydrase was elevated in cells that produced higher amounts of zinc sulfide, cysteine is the probable source of the sulfide in this aerobic process.     

The Dynamics of Genetic Draft in Rapidly Adapting Populations

The accumulation of beneficial mutations on competing genetic backgrounds in rapidly adapting populations has a striking impact on evolutionary dynamics. This effect, known as clonal interference, causes erratic fluctuations in the frequencies of observed mutations, randomizes the fixation times of successful mutations, and leaves distinct signatures on patterns of genetic variation. Here, we show how this form of “genetic draft” affects the forward-time dynamics of site frequencies in rapidly adapting asexual populations. We calculate the probability that mutations at individual sites shift in frequency over a characteristic timescale, extending Gillespie’s original model of draft to the case where many strongly selected beneficial mutations segregate simultaneously. We then derive the sojourn time of mutant alleles, the expected fixation time of successful mutants, and the site frequency spectrum of beneficial and neutral mutations. Finally, we show how this form of draft affects inferences in the McDonald–Kreitman test and how it relates to recent observations that some aspects of genetic diversity are described by the Bolthausen–Sznitman coalescent in the limit of very rapid adaptation.     

Spatiotemporal Evolution of Ebola Virus Disease at Sub-National Level during the 2014 West Africa Epidemic: Model Scrutiny and Data Meagreness

Background

The Ebola outbreak in West Africa has infected at least 27,443 individuals and killed 11,207, based on data until 24 June, 2015, released by the World Health Organization (WHO). This outbreak has been characterised by extensive geographic spread across the affected countries Guinea, Liberia and Sierra Leone, and by localized hotspots within these countries. The rapid recognition and quantitative assessment of localised areas of higher transmission can inform the optimal deployment of public health resources.

Methods

A variety of mathematical models have been used to estimate the evolution of this epidemic, and some have pointed out the importance of the spatial heterogeneity apparent from incidence maps. However, little is known about the district-level transmission. Given that many response decisions are taken at sub-national level, the current study aimed to investigate the spatial heterogeneity by using a different modelling framework, built on publicly available data at district level. Furthermore, we assessed whether this model could quantify the effect of intervention measures and provide predictions at a local level to guide public health action. We used a two-stage modelling approach: a) a flexible spatiotemporal growth model across all affected districts and b) a deterministic SEIR compartmental model per district whenever deemed appropriate.

Findings

Our estimates show substantial differences in the evolution of the outbreak in the various regions of Guinea, Liberia and Sierra Leone, illustrating the importance of monitoring the outbreak at district level. We also provide an estimate of the time-dependent district-specific effective reproduction number, as a quantitative measure to compare transmission between different districts and give input for informed decisions on control measures and resource allocation. Prediction and assessing the impact of control measures proved to be difficult without more accurate data. In conclusion, this study provides us a useful tool at district level for public health, and illustrates the importance of collecting and sharing data.