Seminiferous tubule transfection in vitro to define post-meiotic gene regulation

Background  

Post-meiotically expressed genes in the testis are essential for the proper progression of spermatogenesis, and yet, aside from the construction of individual transgenic mice using specific promoters to drive reporter plasmids, there are only very limited possibilities for relevant and quantitative analysis of gene promoters. This is due to the special nature of post-meiotic haploid cells, which to date are not represented in any appropriate cell-lines. This article reports the development of novel methodology using isolated and cultured rat seminiferous tubules in a multiwell format, into which promoter-reporter constructs can be introduced by a combination of microinjection and electroporation.     

Experimentelle analyse der infektion und des entwicklungszyklus vonMalamoeba scolyti inDryocoetes autographus [Coleoptera: scolytidae]

Malamoeba scolyti (Purrini), ein Parasit im Verdauungstrakt des BorkenkäfersDryocoetes autographus (Ratz.), schmarotzt intrazellulär in bestimmten Abschnitten des Mitteldarmepithels und im Epithel der Malpighischen Gefä\e. Sie zerstört die befallenen Epithelzellen, wobei sie sich bis zum zystenstadium entwickeln kann. in umfangreichen Infektionsexperimenten wird gezeigt, daß die Infektion in den vordersten Krypten des Mitteldarms beginnt und sich in mehreren Infecktionswellen bis zu den hintersten Darmdivertikeln und den Malpighischen Gefäßen ausbreitet. Bei 20° Dauertemperatur nimmt die Erstinfektion etwa 6 Tage, der gesamte Krankheitsverlauf bis zum Exitus des Wirtes 5 bis 7 Wochen in Anspruch. Im Gegensatz zu anderen Beschreibungen konnte nur eine einzige Form von Trophozoiten beobachtet werden. Auf die mögliche Bedeutung dieses Umstandes für die taxonomische Identität der Amöbe wird hingewiesen.     

The rad16 gene of Schizosaccharomyces pombe: a homolog of the RAD1 gene of Saccharomyces cerevisiae.

The rad10, rad16, rad20, and swi9 mutants of the fission yeast Schizosaccharomyces pombe, isolated by their radiation sensitivity or abnormal mating-type switching, have been shown previously to be allelic. We have cloned DNA correcting the UV sensitivity or mating-type switching phenotype of these mutants and shown that the correcting DNA is encompassed in a single open reading frame. The gene, which we will refer to as rad16, is approximately 3 kb in length, contains seven introns, and encodes a protein of 892 amino acids. It is not essential for viability of S. pombe. The predicted protein is the homolog of the Saccharomyces cerevisiae RAD1 protein, which is involved in an early step in excision-repair of UV damage from DNA. The approximately 30% sequence identity between the predicted proteins from the two yeasts is distributed throughout the protein. Two-hybrid experiments indicate a strong protein-protein interaction between the products of the rad16 and swi10 genes of S. pombe, which mirrors that reported for RAD1 and RAD10 in S. cerevisiae. We have identified the mutations in the four alleles of rad16. They mapped to the N-terminal (rad10), central (rad20), and C-terminal (rad16 and swi9) regions. The rad10 and rad20 mutations are in the splice donor sequences of introns 2 and 4, respectively. The plasmid correcting the UV sensitivity of the rad20 mutation was missing the sequence corresponding to the 335 N-terminal amino acids of the predicted protein. Neither smaller nor larger truncations were, however, able to correct its UV sensitivity.     

Diatom communities of acidic mountain streams in Poland

A comparison has been made of the species composition of diatom communities developing in acidic Polish mountain streams which flow over calcium-poor substrates: sandstones in the Silesian Beskid (section of the Western Carpathians), the witokrzyskie Mts, and over granite in the Karkonosze range (in the Sudetic Mts). The number of taxa and diversity of the diatom assemblages decreased along a decreasing pH gradient. The correlation between pH and the number of taxa was positive and significant (r ² = 0.69, p < 0.005). A small number of species (< 20) and low diversity were found in the communities developing in strongly acidic streams such as in the witokrzyskie Mts with pH 4.1–5.2, and in the Silesian Beskid with pH 3.5–4.0. In the stream of the Karkonosze Mts, with pH 5.2–6.0, the communities were characterized by their greater number of species and higher diversity.Acidobiontic and acidophilous diatoms were generally dominant. The pH-indiferent forms were less abundant, and their proportion increased above pH 5.0. Eunotia exigua, E. paludosa var. trinacria, E. tenella and Pinnularia subcapitata dominated in streams with the lowest pH, while E. exigua, E. sudetica and Achnanthes kryophila predominated in a stream with water pH above 5.2. Eunotia exigua, a common acidobiontic species was present in all the examined communities, and was a strong dominant in waters of pH 5.0. A corresponding decrease in abundance of E. exigua was observed with an increase in pH.     

Tetrazolium [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction by mycoplasmas.

We investigated 22 mycoplasma and acholeplasma species for their ability to reduce tetrazolium salts by using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The test results were evaluated visually, as well as spectrophotometrically, by using an enzyme-linked immunosorbent assay reader. Our results were very similar to the results obtained when the tetrazolium salt reduction assay described by Aluotto et al. was used. However, the MTT reduction assay appeared to be better because it is faster, more objective and sensitive, easier to evaluate, and less expensive; in addition, it allows quantitative determinations. By using regression analysis a linear correlation between formazan production and the number of colony-forming units was demonstrated for all of the species investigated, indicating that the MTT assay can also be used for growth, toxicity, or chemosensitivity tests for the mycoplasma species that are capable of reducing tetrazolium salts.     

In vitro biocompatibility evaluation of a heat‐resistant 3D printing material for use in customized cell culture devices

Additive manufacturing (3D printing) enables the fabrication of highly customized and complex devices and is therefore increasingly used in the field of life sciences and biotechnology. However, the application of 3D‐printed parts in these fields requires not only their biocompatibility but also their sterility. The most common method for sterilizing 3D‐printed parts is heat steam sterilization—but most commercially available 3D printing materials cannot withstand high temperatures. In this study, a novel heat‐resistant polyacrylate material for high‐resolution 3D Multijet printing was evaluated for the first time for its resistance to heat steam sterilization and in vitro biocompatibility with mouse fibroblasts (L929), human embryonic kidney cells (HEK 293E), and yeast (Saccharomyces cerevisiae (S. cerevisiae)). Analysis of the growth and viability of L929 cells and the growth of S. cerevisiae confirmed that the extraction media obtained from 3D‐printed parts had no negative effect on the aforementioned cell types, while, in contrast, viability and growth of HEK 293E cells were affected. No different effects of the material on the cells were found when comparing heat steam sterilization and disinfection with ethanol (70%, v/v). In principle, the investigated material shows great potential for high‐resolution 3D printing of novel cell culture systems that are highly complex in design, customized and easily sterilizable—however, the biocompatibility of the material for other cell types needs to be re‐evaluated.