Fluorescent and Bioluminescent Calcium Indicators with Tuneable Colors and Affinities.

We introduce a family of bright, rhodamine-based calcium indicators with tuneable affinities and colors. The indicators can be specifically localized to different cellular compartments and are compatible with both fluorescence and bioluminescence readouts through conjugation to HaloTag fusion proteins. Importantly, their increase in fluorescence upon localization enables no-wash live-cell imaging, which greatly facilitates their use in biological assays. Applications as fluorescent indicators in rat hippocampal neurons include the detection of single action potentials and of calcium fluxes in the endoplasmic reticulum. Applications as bioluminescent indicators include the recording of the pharmacological modulation of nuclear calcium in high-throughput compatible assays. The versatility and remarkable ease of use of these indicators make them powerful tools for bioimaging and bioassays.

Kinetic and Structural Characterization of the Self-Labeling Protein Tags HaloTag7, SNAP-tag, and CLIP-tag.

The self-labeling protein tags (SLPs) HaloTag7, SNAP-tag, and CLIP-tag allow the covalent labeling of fusion proteins with synthetic molecules for applications in bioimaging and biotechnology. To guide the selection of an SLP-substrate pair and provide guidelines for the design of substrates, we report a systematic and comparative study of the labeling kinetics and substrate specificities of HaloTag7, SNAP-tag, and CLIP-tag. HaloTag7 reaches almost diffusion-limited labeling rate constants with certain rhodamine substrates, which are more than 2 orders of magnitude higher than those of SNAP-tag for the corresponding substrates. SNAP-tag labeling rate constants, however, are less affected by the structure of the label than those of HaloTag7, which vary over 6 orders of magnitude for commonly employed substrates. Determining the crystal structures of HaloTag7 and SNAP-tag labeled with fluorescent substrates allowed us to rationalize their substrate preferences. We also demonstrate how these insights can be exploited to design substrates with improved labeling kinetics.

Site-Specific Conjugation of Native Antibodies Using Engineered Microbial Transglutaminases.

Site-specific bioconjugation technologies are frequently employed to generate homogeneous antibody-drug conjugates (ADCs) and are generally considered superior to stochastic approaches like lysine coupling. However, most of the technologies developed so far require undesired manipulation of the antibody sequence or its glycan structures. Herein, we report the successful engineering of microbial transglutaminase enabling efficient, site-specific conjugation of drug-linker constructs to position HC-Q295 of native, fully glycosylated IgG-type antibodies. ADCs generated via this approach demonstrate excellent stability in vitro as well as strong efficacy in vitro and in vivo. As it employs different drug-linker structures and several native antibodies, our study additionally proves the broad applicability of this approach.

Helicobacter pylori genome evolution during human infection.

High genetic diversity is a hallmark of the gastric pathogen Helicobacter pylori. We used 454 sequencing technology to perform whole-genome comparisons for five sets of H. pylori strains that had been sequentially cultured from four chronically infected Colombians (isolation intervals=3-16 y) and one human volunteer experimentally infected with H. pylori as part of a vaccine trial. The four sets of genomes from Colombian H. pylori differed by 27-232 isolated SNPs and 16-441 imported clusters of polymorphisms resulting from recombination. Imports (mean length=394 bp) were distributed nonrandomly over the chromosome and frequently occurred in groups, suggesting that H. pylori first takes up long DNA fragments, which subsequently become partially integrated in multiple shorter pieces. Imports were present at significantly increased frequency in members of the hop family of outer membrane gene paralogues, some of which are involved in bacterial adhesion, suggesting diversifying selection. No evidence of recombination and few other differences were identified in the strain pair from an infected volunteer, indicating that the H. pylori genome is stable in the absence of mixed infection. Among these few differences was an OFF/ON switch in the phase-variable adhesin gene hopZ, suggesting strong in vivo selection for this putative adhesin during early colonization.

Human Infections with Borna Disease Virus 1 (BoDV-1) Primarily Lead to Severe Encephalitis: Further Evidence from the Seroepidemiological BoSOT Study in an Endemic Region in Southern Germany.

More than 40 human cases of severe encephalitis caused by Borna disease virus 1 (BoDV-1) have been reported to German health authorities. In an endemic region in southern Germany, we conducted the seroepidemiological BoSOT study ("BoDV-1 after solid-organ transplantation") to assess whether there are undetected oligo- or asymptomatic courses of infection. A total of 216 healthy blood donors and 280 outpatients after solid organ transplantation were screened by a recombinant BoDV-1 ELISA followed by an indirect immunofluorescence assay (iIFA) as confirmatory test. For comparison, 288 serum and 258 cerebrospinal fluid (CSF) samples with a request for tick-borne encephalitis (TBE) diagnostics were analyzed for BoDV-1 infections. ELISA screening reactivity rates ranged from 3.5% to 18.6% depending on the cohort and the used ELISA antigen, but only one sample of a patient from the cohort with requested TBE diagnostics was confirmed to be positive for anti-BoDV-1-IgG by iIFA. In addition, the corresponding CSF sample of this patient with a three-week history of severe neurological disease tested positive for BoDV-1 RNA. Due to the iIFA results, all other results were interpreted as false-reactive in the ELISA screening. By linear serological epitope mapping, cross-reactions with human and bacterial proteins were identified as possible underlying mechanism for the false-reactive ELISA screening results. In conclusion, no oligo- or asymptomatic infections were detected in the studied cohorts. Serological tests based on a single recombinant BoDV-1 antigen should be interpreted with caution, and an iIFA should always be performed in addition.

Prevalence of pSmeSM11a-like plasmids in indigenous Sinorhizobium meliloti strains isolated in the course of a field release experiment with genetically modified S. meliloti strains.

Plasmid pSmeSM11a, residing in the indigenous Sinorhizobium meliloti strain SM11 originating from a field in Strassmoos (Bavaria, Germany), was analysed previously at the genomic level. Thirty-seven indigenous S. meliloti strains, originating from two different locations in Germany, were screened for genes identified previously on pSmeSM11a. Seven of these strains harbour accessory plasmids that are very similar to pSmeSM11a. The identified pSmeSM11a-like plasmids are c. 130-150 kb in size and possess nearly identical restriction profiles. Up to 30 genes identified previously on pSmeSM11a could be detected on these plasmids by hybridisation experiments, e.g., the nodulation genes nodP and nodQ, the ethylene level modulation gene acdS and the taurine metabolism gene tauD. A few pSmeSM11a genes were also detected on other plasmids. The reference plasmid pSmeSM11a contains a region that is similar to a segment of S. meliloti strain Rm1021 pSymA. Regions with similarity to pSymA were also detected on the aforementioned seven pSmeSM11a-like plasmids. The specifications of these regions are nearly identical to the one on pSmeSM11a and differ from Rm1021 pSymA as determined by nucleotide sequence analysis. Two further plasmids similar to pSmeSM11a completely lack the pSymA-region. Those strains carrying accessory plasmids that contain the acdS gene encoding 1-aminocyclopropane-1-carboxylate deaminase are able to grow on 1-aminocyclopropane-1-carboxylate as the sole source of nitrogen, demonstrating functionality of the acdS gene product. About 36% of the analysed plasmids, including three pSmeSM11a-like plasmids, could be transferred to another S. meliloti recipient strain, allowing for their dissemination in S. meliloti populations.

The systematic position of the parasitoid marine dinoflagellate Paulsenella vonstoschii (Dinophyceae) inferred from nuclear-encoded small subunit ribosomal DNA.

Members of the genus Paulsenella Chatton are marine phagotrophic dinoflagellates that specifically attack marine diatoms. In this phylogenetic study, we show that Paulsenella groups with Amyloodinium ocellatum (Brown) Brown et Hovasse, Pfiesteria piscicida Steidinger et Burkholder (Dinophyceae), Pfiesteria shumwayae Glasgow et Burkholder, and the cryptoperidiniopsoids, all members of the order Peridiniales. In the phylogenetic tree, Paulsenella diverged after Amyloodinium ocellatum but prior to Pfiesteria and the cryptoperidiniopsoids. This suggests that Paulsenella also belongs to the order Peridiniales and its earlier description as gymnodinioid and athecate has to be revised.

Phylogenetic position of the parasitoid nanoflagellate Pirsonia inferred from nuclear-encoded small subunit ribosomal DNA and a description of Pseudopirsonia n. gen. and Pseudopirsonia mucosa (Drebes) comb. nov.

Sequences of the nuclear encoded small subunit (SSU) rRNA were determined for Pirsonia diadema, P. guinardiae, P. punctigerae, P. verrucosa, P. mucosa and three newly isolated strains 99-1, 99-2, 99-S. Based on phylogenetic analysis all Pirsonia strains, except P. mucosa, clustered together in one clade, most closely related to Hyphochytrium catenoides within the group of stramenopiles. However, P. mucosa was most closely related to Cercomonas sp. SIC 7235 and Heteromita globosa and belongs to the heterogenic group of Cercozoa. In addition to the SSU rDNA sequences, P. mucosa differs from the stramenopile Pirsonia species in some characteristics and was therefore redescribed in this paper as Pseudopirsonia mucosa. The three newly isolated strains 99-1, 99-2, and 99-S differed by 28 bp in their SSU rDNA sequences from their closest neighbour P. diadema and only 1 to 3 bp among themselves. These base differences and a host range similar to P. formosa were sufficient to assign them as new strains of P. formosa.

Laser printing of skin cells and human stem cells.

Laser printing based on laser-induced forward transfer (LIFT) is a new biofabrication technique for the arrangement of biological materials or living cells in well-defined patterns. In the current study, skin cell lines (fibroblasts/keratinocytes) and human mesenchymal stem cells (hMSC) were chosen for laser printing experiments due to their high potential in regeneration of human skin and new application possibilities of stem cell therapy. To evaluate the influence of LIFT on the cells, their survival rate, their proliferation and apoptotic activity, and the DNA damages and modifications of their cell surface markers were assessed and statistically evaluated over several days. The cells survived the transfer procedure with a rate of 98% +/- 1% standard error of the mean (skin cells) and 90% +/- 10% (hMSC), respectively. All used cell types maintain their ability to proliferate after LIFT. Further, skin cells and hMSC did not show an increase of apoptosis or DNA fragmentation. In addition, the hMSC keep their phenotype as proven by fluorescence activated cell sorting (FACS) analysis. This study demonstrates LIFT as a suitable technique for unharmed computer-controlled positioning of different cell types and a promising tool for future applications in the ex vivo generation of tissue replacements.

pH effect on the susceptibility to parasitoid infection in the marine diatom Coscinodiscus spp. (Bacillariophyceae).

The pH on the frustule of individual cells of the marine centric diatoms Coscinodiscus granii and Coscinodiscus wailesii (Bacillariophyceae) was measured with pH microsensors in culture media with increasing pH values of 8.04, 8.14, and 8.22, respectively. In 85-96% of the C.granii cells the pH on the frustule was up to 0.4 units higher than that of the medium, reaching a maximum pH 8.95. Only in 2-3% the surface pH exceeded that of the medium by up to 0.7 pH units. These results strongly suggest that diatoms in batch cultures differ, at least temporarily, in their individual photosynthetic activities. Infection experiments with the parasitoid nanoflagellate Pirsonia diadema (Stramenopile) showed that flagellates failed to infect when the culture pH was 8.8 and above. pH measurements on freshly infected C. granii showed that the prevalence of infection was higher in tendency on diatoms with low surface pH. Application of these results to parasitoid-diatom interactions in natural waters suggests that within phytoplankton populations a strong photosynthetic activity might prevent diatom cells temporarily from infection by pH-sensitive parasitoids.

Photosynthetic oscillation in individual cells of the marine diatom Coscinodiscus wailesii (Bacillariophyceae) revealed by microsensor measurements.

Oscillations with a period of 1-2 min in the rate of photosynthesis have been found in leaves of C(3 )and C(4 )land plants under invariant, saturating, light and carbon dioxide. This article reports the occurrence of similar oscillations with a period of 2-2.5 min in individual cells of the marine diatom Coscinodiscus wailesii. These oscillations were determined by measurements of both oxygen (oxygen microelectrode) and carbon dioxide (pH microelectrode) just outside the plasmalemma. These oscillations were found in less than 1% of the cells examined. The occurrence of oscillations in unicelluar diatoms rules out for these organisms hypotheses as to the origin of oscillations in land plant leaves that are based on cell-cell interactions.