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Here we present a formal description of Biremis panamae Barka, Witkowski et Weisenborn sp. nov., which was isolated from the marine littoral environment of the Pacific Ocean coast of Panama. The description is based on morphology (light and electron microscopy) and the rbcL, psbC and SSU sequences of one clone of this species. The new species is included in Biremis due to its morphological features; i.e. two marginal rows of foramina, chambered striae, and girdle composed of numerous punctate copulae. The new species also possesses a striated valve face which is not seen in most known representatives of marine littoral Biremis species. In this study we also present the relationship of Biremis to other taxa using morphology, DNA sequence data and observations of auxosporulation. Our results based on these three sources point to an evolutionary relationship between Biremis, Neidium and Scoliopleura. The unusual silicified incunabular caps present in them are known otherwise only in Muelleria, which is probably related to the Neidiaceae and Scoliotropidaceae. We also discuss the relationship between Biremis and the recently described Labellicula and Olifantiella

Device-Controlled Microcondensation for Spatially Confined On-Tissue Digests in MALDI Imaging of <i>N</i>-Glycans

On-tissue enzymatic digestion is a prerequisite for MALDI mass spectrometry imaging (MSI) and spatialomic analysis of tissue proteins and their N-glycan conjugates. Despite the more widely accepted importance of N-glycans as diagnostic and prognostic biomarkers of many diseases and their potential as pharmacodynamic markers, the crucial sample preparation step, namely on-tissue digestion with enzymes like PNGaseF, is currently mainly carried out by specialized laboratories using home-built incubation arrangements, e.g., petri dishes placed in an incubator. Standardized spatially confined enzyme digests, however, require precise control and possible regulation of humidity and temperature, as high humidity increases the risk of analyte dislocation and low humidity compromises enzyme function. Here, a digestion device that controls humidity by cyclic ventilation and heating of the slide holder and the chamber lid was designed to enable controlled micro-condensation on the slide and to stabilize and monitor the digestion process. The device presented here may help with standardization in MSI. Using sagittal mouse brain sections and xenografted human U87 glioblastoma cells in CD1 nu/nu mouse brain, a device-controlled workflow for MALDI MSI of N-glycans was developed

Identification and Affinity Determination of Protein-Antibody and Protein-Aptamer Epitopes by Biosensor-Mass Spectrometry Combination

Analytical methods for molecular characterization of diagnostic or therapeutic targets have recently gained high interest. This review summarizes the combination of mass spectrometry and surface plasmon resonance (SPR) biosensor analysis for identification and affinity determination of protein interactions with antibodies and DNA-aptamers. The binding constant (KD) of a protein–antibody complex is first determined by immobilizing an antibody or DNA-aptamer on an SPR chip. A proteolytic peptide mixture is then applied to the chip, and following removal of unbound material by washing, the epitope(s) peptide(s) are eluted and identified by MALDI-MS. The SPR-MS combination was applied to a wide range of affinity pairs. Distinct epitope peptides were identified for the cardiac biomarker myoglobin (MG) both from monoclonal and polyclonal antibodies, and binding constants determined for equine and human MG provided molecular assessment of cross immunoreactivities. Mass spectrometric epitope identifications were obtained for linear, as well as for assembled (“conformational”) antibody epitopes, e.g., for the polypeptide chemokine Interleukin-8. Immobilization using protein G substantially improved surface fixation and antibody stabilities for epitope identification and affinity determination. Moreover, epitopes were successfully determined for polyclonal antibodies from biological material, such as from patient antisera upon enzyme replacement therapy of lysosomal diseases. The SPR-MS combination was also successfully applied to identify linear and assembled epitopes for DNA–aptamer interaction complexes of the tumor diagnostic protein C-Met. In summary, the SPR-MS combination has been established as a powerful molecular tool for identification of protein interaction epitopes

Location of the sampling site on the coast of Panama.

<p>Location of the sampling site on the coast of Panama.</p

<i>Biremis panamae</i> sp. nov., SEM: external valve views.

<p><b>A.</b> Frustule with detached valves and a part of a copula with several rows of pores (arrowhead). <b>B.</b> Valve face showing occlusions of the marginal row of small areolae (arrowhead). <b>C, D.</b> External valve view showing variation in the valve face morphology. Note the depressed surface of the valve face areola occlusions (arrowhead).</p

<i>Biremis panamae</i> sp. nov., TEM. A.

<p>A whole valve in valve view. <b>B.</b> A whole specimen observed from the valve interior. <b>C, D.</b> Close ups of a specimen illustrated in Fig. 5A; note the finely porous areolae occlusions, arrowhead in Fig. 5D.</p

Vegetative cells and auxosporulation in <i>Biremis</i> sp. A, B.

<p>Two focuses of a vegetative cell in girdle view. Each cell contains two chloroplasts either side of the centre, each of which comprises two plates (one is shown for each chloroplast in Fig. 7A, the other being out of focus beneath, on the opposite side of the cell) connected by a narrow bridge containing the pyrenoid (e.g. p). <b>C, D.</b> Two paired gametangia, each containing two rounded, rearranged gametes. The gametangia were paired with their girdles adjacent, the cell shown in Fig. 7D lying immediately below that in Fig. 7C. <b>E</b>. Two paired gametangia, unusual in being in contact only via their valves. Each gametangium contains a single subspherical zygote. Two nuclei are visible in the left-hand cell (arrows) and two of the four chloroplasts in the right-hand cell. <b>F, G.</b> Two focuses of a gametangium containing a zygote on the point of transformation into an auxospore. Note the slight central inflection of the zygote's outline, marking the deposition of the primary transverse perizonial band (cf. Fig. 7H, arrowhead). The two rows of foramina on the valves can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114508#pone-0114508-g002" target="_blank">Fig. 2G</a> (arrowheads). <b>H</b>. Expanded auxospore containing the initial epivalve (in section at arrow). The auxospore is encased in a well developed perizonium, containing a primary transverse band flanked by several secondary bands (see in section: see also Figs 7I, J). <b>I.</b> Peripheral focus of an expanded auxospore containing the initial epivalve. The two rows of foramina on one of the gametangium valves can be seen (arrowheads). The end of the auxospore is covered by a siliceous cap (arrow). <b>J.</b> Expanded auxospore containing a completed initial cell. The initial hypovalve (in section at h) lies at a distance from the perizonium, as a result of a strong contraction of the protoplast immediately before its formation; the initial epivalve lies opposite, directly moulded by the interior of the perizonium. The auxospore casing can be seen to consist of a perizonium of transverse bands (e.g. at white arrows) and two silicified hemispherical caps (e.g. at black arrow). [Scale bar 10 µm].</p

Maximum likelihood phylogeny (with bootstrap values at nodes) inferred from a concatenated alignment of <i>rbc</i>L, <i>psb</i>C and SSU markers.

<p><i>Neidium</i> sp. NEI323TM, <i>Neidium</i> sp. NEI 44, <i>Neidium</i> sp. NEI428T and <i>Neidium</i> sp. NEI Balk482 represent previously unpublished <i>rbc</i>L gene sequences from different <i>Neidium</i> species. <i>Biremis</i> sp. represents a <i>rbc</i>L gene sequence from an unpublished <i>Biremis</i> sp. The tree is rooted with the pennate araphid taxa <i>Ctenophora pulchella</i> and <i>Tabularia</i> cf. <i>tabulata</i>. Support values lower than 50% were not included in the tree. The GenBank <i>Achnanthidium coarctatum</i> name has been changed to <i>Achnanthes coarctata</i>.</p

Bayesian Inference phylogeny inferred from a concatenated alignment of <i>rbc</i>L, <i>psb</i>C and SSU markers.

<p>Posterior probabilities are shown at the nodes. The tree is rooted with the araphid pennate taxa <i>Ctenophora pulchella</i> and <i>Tabularia</i> cf. <i>tabulata</i>.</p