The conjugative plasmid SLP2 of Streptomyces lividans is a 50 kb linear molecule

The SLP2 plasmid had previously been demonstrated genetically to exist In Streptomyces lividans by its ability to promote conjugation and to elicit‘pocks’on recipient (SLP2^?) cultures, but it had not been physically detected. Using pulsed-field gel electrophoresis, a 50kb linear DNA was isolated from SLP2⁺ but not SLP2^? strains of S. lividans, and from Streptomyces coelicolor and Streptomyces parvulus strains to which SLP2 had been transferred by conjugation or transformation. We conclude that this linear DNA is SLP2. The terminal fragments of SLP2 were cloned. The determined sequences revealed a 44 bp imperfect terminal inverted repeat. The terminal 12 bp sequence of SLP2 was identical to those of two other Streptomyces linear plasmids, pSLA2 and pSCL, and similar to the terminal sequences of another Streptomyces linear plasmid, SCP1. The termini of SLP2 DNA were resistant to digestion by λ exonuclease and ExoIII. A truncated (probably crippled) copy of Tn4811 is present on the plasmid. While the SLP2 plasmid exists as a tree form in the host, a 15.7 kb sequence corresponding to the segment of SLP2 from Tn4811 to the right terminus is also present (at a copy number similar to the free form) elsewhere in the genome of S. lividans. Furthermore, SLP2 is partially homologous to a newly discovered 650 kb linear plasmid in S. parvulus.     

Using real-world data to predict health outcomes—The prediction design: Application and sample size planning

The gold standard for investigating the efficacy of a new therapy is a (pragmatic) randomized controlled trial (RCT). This approach is costly, time-consuming, and not always practicable. At the same time, huge quantities of available patient-level control condition data in analyzable format of (former) RCTs or real-world data (RWD) are neglected. Therefore, alternative study designs are desirable. The design presented here consists of setting up a prediction model for determining treatment effects under the control condition for future patients. When a new treatment is intended to be tested against a control treatment, a single-arm trial for the new therapy is conducted. The treatment effect is then evaluated by comparing the outcomes of the single-arm trial against the predicted outcomes under the control condition. While there are obvious advantages of this design compared to classical RCTs (increased efficiency, lower cost, alleviating participants’ fear of being on control treatment), there are several sources of bias. Our aim is to investigate whether and how such a design—the prediction design—may be used to provide information on treatment effects by leveraging external data sources. For this purpose, we investigated under what assumptions linear prediction models could be used to predict the counterfactual of patients precisely enough to construct a test and an appropriate sample size formula for evaluating the average treatment effect in the population of a new study. A user-friendly R Shiny application (available at: https://web.imbi.uni-heidelberg.de/PredictionDesignR/ ) facilitates the application of the proposed methods, while a real-world application example illustrates them.     

Statistical shape modelling of the thoracic spine for the development of pedicle screw insertion guides

Spinal fixation and fusion are surgical procedures undertaken to restore stability in the spine and restrict painful or degenerative motion. Malpositioning of pedicle screws during these procedures can result in major neurological and vascular damage. Patient-specific surgical guides offer clear benefits, reducing malposition rates by up to 25%. However, they suffer from long lead times and the manufacturing process is dependent on third-party specialists. The development of a standard set of surgical guides may eliminate the issues with the manufacturing process. To evaluate the feasibility of this option, a statistical shape model (SSM) was created and used to analyse the morphological variations of the T4–T6 vertebrae in a population of 90 specimens from the Visible Korean Human dataset (50 females and 40 males). The first three principal components, representing 39.7% of the variance within the population, were analysed. The model showed high variability in the transverse process (~ 4 mm) and spinous process (~ 4 mm) and relatively low variation (< 1 mm) in the vertebral lamina. For a Korean population, a standardised set of surgical guides would likely need to align with the lamina where the variance in the population is lower. It is recommended that this standard set of surgical guides should accommodate pedicle screw diameters of 3.5–6 mm and transverse pedicle screw angles of 3.5°–12.4°.

     

The Role of the Cytoskeleton and Dictyosome Activity in the Pulsatory Growth of Nicotiana tabacum and Petunia hybrida Pollen Tubes

Pollen tubes of Nicotiana tabacum and Petunia hybrida show pulsatory growth. Phases of slow growth lasting minutes are interrupted by pulse-like elongations lasting 10–20 seconds involving an increase of growth rate by up to 24-fold. Inhibition of dictyosome activity with brefeldin A or monensin did not result in an inhibition of pulsatory growth but eventually stopped pollen tube elongation. In contrast to this the inhibition of the cytoskeletal elements with cytochalasin D and colchicine caused the pollen tubes to abandon the pulse-like elongations. It was concluded that the activity of the dictyosomes does not have a controlling function in the mechanism of pulsatory growth, even though it is necessary for pollen tube elongation, since cell wall material is provided by secretory vesicles deriving from the Golgi apparatus. In contrast the cytoskeletal elements, actin and microtubules, seem to play an important regulatory role in the pulse-like elongations. In addition, it was observed that during the experiments several pollen tubes burst upon the completion of a pulse-like expansion, indicating on the one hand that the internal turgor is the driving force of the pulse-like expansions. On the other hand, the bursting shows that the pollen tube cell wall is rather weak at the end of a pulse, indicating that at this point of time it is either thinner or less stable than during the slow growth phase or at the beginning of a pulse.     

Ultrastructural immunolocalization of periodic pectin depositions in the cell wall ofNicotiana tabacum pollen tubes

Summary The monoclonal antibody (MAb) JIM5, marking acidic pectins, was used to localize ultrastructurally pectin molecules in the pollen tube wall ofNicotiana tabacum. Longitudinal sections of LR-White embedded pollen tubes were exposed to antibody treatment; accumulations of pectins were identified by counting the density of the gold particles representing the pectin epitopes along the pollen tube wall. Significant accumulations of gold grains were marked and the distances between them were measured. In many pollen tubes a more or less regular distribution of the accumulations was observed along the tube indicating a periodical deposition of pectin. The distances between the accumulations were 4–6 m. Most of the label was found in the inner part of the outer layer of the bilayered cell wall. These findings correspond to and confirm the earlier observation by our group reporting ring-shaped periodical deposits in pollen tubes after immunofluorescence labelling with the MAb JIM5 under the confocal laser scanning microscope.Abbreviations Ab antibody - MAb monoclonal antibody     

Mapping of genes involved in macromolecular synthesis on the chromosome of Streptomyces coelicolor A3(2).

The genes for the beta, beta', and seven sigma factor subunits of RNA polymerase, for elongation factors EF-Tu1 and EF-Tu3, and for six rRNA operons were mapped on the combined genetic and physical map of the Streptomyces coelicolor chromosome. Like the previously mapped tRNA genes, the RNA polymerase and rRNA genes map to scattered positions. The lack of rRNA operons in the immediate vicinity of the origin of replication (oriC) and the absence of tRNA genes in any of the rRNA operons are novel features of the Streptomyces chromosome.     

Integrated DNA sequences in three streptomycetes form related autonomous plasmids after transfer to Streptomyces lividans

When Streptomyces parvulus ATCC 12434 was crossed with a plasmid-free S. lividans 66 derivative, some S. lividans exconjugants contained plasmid DNA, pIJ110 (13.6 kb). In a similar way, pIJ408 (15.05 kb) was found after mating S. glaucescens ETH 22794 with S. lividans. CCC DNA was not visualized in the donor strains. pIJ110 and pIJ408 each originates from a larger replicon, probably the chromosome, of S. parvulus or S. glaucescens. Restriction maps of pIJ110 and pIJ408, each for 10 enzymes, were derived. Derivatives of each plasmid were constructed carrying antibiotic-resistance markers (thiostrepton or viomycin) in a nonessential region and each plasmid was cloned into an Escherichia coli plasmid vector (pBR327 or pBR325). pIJ110 and pIJ408 resemble, in their origin, the previously known SLP1 plasmids (such as SLP1.2) which come from integrated sequences in the chromosome of S. coelicolor A3(2). pIJ110 and pIJ408, like SLP1.2, are self-transmissible, elicit the so-called lethal zygosis reaction (pock formation) and mobilize chromosomal markers. The three plasmids, in spite of their very different restriction maps, were found to be related: SLP1.2 and pIJ110 were strongly incompatible, showed complete resistance to each other's lethal zygosis reaction, and shared a segment of DNA with a considerable degree of cross-hybridization; pIJ110 and pIJ408 were weakly incompatible and showed partial resistance to lethal zygosis and a weak DNA cross-hybridization; pIJ408 and SLP1.2 were only distantly related on these criteria. pIJ110, pIJ408, and SLP1.2 hybridized with varying degrees of homology in Southern transfer experiments to DNA from 7 out of 13 of an arbitrary collection of wild-type streptomycetes. Integrated sequences capable of forming plasmids after transfer to S. lividans may therefore be widespread in the genus Streptomyces.     

Restriction analysis of the Streptomyces glaucescens genome by agarose gel electrophoresis

A method for the analysis of total DNA of Streptomyces glaucescens is described. The relevant steps are (a) extraction and purification of DNA, (b) restriction of DNA samples with type II restriction enzymes, (c) one dimensional separation of restriction fragments by agarose gel electrophoresis. A typical banding pattern was obtained for each wild type strain, independant of growth conditions or age of the culture. Mutant strains exhibited in most cases the same banding pattern as the parent wild type strain. Only in one specific mutant class a fragment of about 9 megadalton was missing.