Spatiotemporal distribution of reactive oxygen species production, delivery, and use in Arabidopsis root hairs.

Fluorescent selective probes for reactive oxygen species (ROS) detection in living cells are versatile tools for the documentation of ROS production in plant developmental or stress reactions. We employed high-resolution live-cell imaging and semiquantitative analysis of Arabidopsis (Arabidopsis thaliana) stained with CM-H2DCFDA, CellROX Deep Red, and Amplex Red for functional characterization of the spatiotemporal mode of ROS production, delivery, and utilization during root hair formation. Cell viability marker fluorescein diacetate served as a positive control for dye loading and undisturbed root hair tip growth after staining. Using a colocalization analysis with subcellular molecular markers and two root hair mutants with similar phenotypes of nonelongating root hairs, but with contrasting reasons for this impairment, we found that: (i) CM-H2DCFDA is a sensitive probe for ROS generation in the cytoplasm, (ii) CellROX Deep Red labels ROS in mitochondria, (iii) Amplex Red labels apoplastic ROS and mitochondria and shows high selectivity to root hairs, (iv) the root hair defective 2-1 (rhd2-1) mutant with nonfunctional NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN C/ROOT HAIR-DEFECTIVE 2 (AtRBOHC/RHD2) has a low level of CM-H2DCFDA-reactive ROS in cytoplasm and lacks Amplex Red-reactive ROS in apoplast, and (v) the ACTIN2-deficient deformed root hairs1-3 (der1-3) mutant is not altered in these aspects. The sensitivity of CellROX Deep Red was documented by discrimination between larger ROS-containing mitochondria and small, yet ROS-free premature mitochondria in the growing tip of root hairs. We characterized spatial changes in ROS production and compartmentalization induced by external ROS modulators, ethylene precursor 1-aminocyclopropane-1-carboxylic acid, and ionophore valinomycin. This dynamic and high-resolution study of ROS production and utilization opens opportunities for precise speciation of particular ROS involved in root hair formation.

Methyl viologen-induced changes in the Arabidopsis proteome implicate PATELLIN 4 in oxidative stress responses.

The photosynthesis-induced accumulation of reactive oxygen species in chloroplasts can lead to oxidative stress, triggering changes in protein synthesis, degradation, and the assembly/disassembly of protein complexes. Using shot-gun proteomics, we identified methyl viologen-induced changes in protein abundance in wild-type Arabidopsis and oxidative stress-hypersensitive fsd1-1 and fsd1-2 knockout mutants, which are deficient in IRON SUPEROXIDE DISMUTASE 1 (FSD1). The levels of proteins that are localized in chloroplasts and the cytoplasm were modified in all lines treated with methyl viologen. Compared with the wild-type, fsd1 mutants showed significant changes in metabolic protein and chloroplast chaperone levels, together with increased ratio of cytoplasmic, peroxisomal, and mitochondrial proteins. Different responses in proteins involved in the disassembly of photosystem II-light harvesting chlorophyll a/b binding proteins were observed. Moreover, the abundance of PATELLIN 4, a phospholipid-binding protein enriched in stomatal lineage, was decreased in response to methyl viologen. Reverse genetic studies using patl4 knockout mutants and a PATELLIN 4 complemented line indicate that PATELLIN 4 affects plant responses to oxidative stress by effects on stomatal closure.

Population genomics resolves cryptic species of the ecologically flexible genus Laspinema (Cyanobacteria).

Cyanobacterial taxonomy is entering the genomic era, but only a few taxonomic studies have employed population genomics, which provides a framework and a multitude of tools to understand species boundaries. Phylogenomic and population genomic analyses previously suggested that several cryptic lineages emerged within the genus Laspinema. Here, we apply population genomics to define boundaries between these lineages and propose two new cryptic species, Laspinema olomoucense and L. palackyanum. Moreover, we sampled soil and puddles across Central Europe and sequenced the 16S rRNA gene and 16S-23S ITS region of the isolated Laspinema strains. Together with database mining of 16S rRNA gene sequences, we determined that the genus Laspinema has a cosmopolitan distribution and inhabits a wide variety of habitats, including freshwater, saline water, mangroves, soil crusts, soils, puddles, and the human body.

Zebularine induces enzymatic DNA-protein crosslinks in 45S rDNA heterochromatin of Arabidopsis nuclei.

Loss of genome stability leads to reduced fitness, fertility and a high mutation rate. Therefore, the genome is guarded by the pathways monitoring its integrity and neutralizing DNA lesions. To analyze the mechanism of DNA damage induction by cytidine analog zebularine, we performed a forward-directed suppressor genetic screen in the background of Arabidopsis thaliana zebularine-hypersensitive structural maintenance of chromosomes 6b (smc6b) mutant. We show that smc6b hypersensitivity was suppressed by the mutations in EQUILIBRATIVE NUCLEOSIDE TRANSPORTER 3 (ENT3), DNA METHYLTRANSFERASE 1 (MET1) and DECREASE IN DNA METHYLATION 1 (DDM1). Superior resistance of ent3 plants to zebularine indicated that ENT3 is likely necessary for the import of the drug to the cells. Identification of MET1 and DDM1 suggested that zebularine induces DNA damage by interference with the maintenance of CG DNA methylation. The same holds for structurally similar compounds 5-azacytidine and 2-deoxy-5-azacytidine. Based on our genetic and biochemical data, we propose that zebularine induces enzymatic DNA-protein crosslinks (DPCs) of MET1 and zebularine-containing DNA in Arabidopsis, which was confirmed by native chromatin immunoprecipitation experiments. Moreover, zebularine-induced DPCs accumulate preferentially in 45S rDNA chromocenters in a DDM1-dependent manner. These findings open a new avenue for studying genome stability and DPC repair in plants.

Possibilities of Interpreting the Night-to-Day Ratio Specified by 24-Hour Blood Pressure Monitoring.

Aim: Specify the risk rate of incorrect patient classification based on the night-to-day ratio specification from singular 24-h ABPM in comparison to the results of 7-day ABPM monitoring. Materials and Methods: 1197 24 h cycles were enrolled in 171 subjects in the study and divided into 4 groups: group 1 (40 healthy men and women without exercise), group 2 (40 healthy exercise-training men and women), group 3 (40 patients with ischemic coronary artery disease without exercise), and group 4 (51 patients with ischemic coronary artery disease following cardiovascular rehabilitation). The subject of the evaluation was the percentage rate of incorrect subject classification (dipper, nondipper, extreme dipper, and riser) based on the mean blood pressure values for 7 days and from seven independent 24-hour cycles (the mean value mode). Results: In the case of the individuals included in the monitored groups, the mean night-to-day ratio-based (mode for the 7 days versus the individual days of 24-hour monitoring) classification accordance ranged between 59% and 62%. Only in singular cases did the accordance reach 0% or 100%. The accordance size was not dependent on the health or cardiovascular disease (p < 0.594; 56% vs. 54%) or physical activity (p < 0.833; 55% vs. 54%) of the monitored individuals. Conclusion: The specification of the night-to-day ratio of each individual for each day of the 7-day ABPM monitoring would be the most convenient option. In many patients, diagnosing could thus be based on the most frequently occurring values (mode specification).

On the relativity of species, or the probabilistic solution to the species problem.

For centuries, both scientists and philosophers have discussed the nature of species resulting in c. 35 species concepts proposed to date. However, in our opinion, none of them incorporated neither recent advances in evolutionary genomics nor dimensionality of species in befitting depth. Our attempt to do so resulted in the following conclusions. Due to the continuous nature of evolution (regardless of its rate and constancy), species are inevitably undefinable as natural discontinuous units (except those originating in saltatory speciation) whenever the time dimension is taken into consideration. Therefore, the very existence of species as a natural discontinuous entity is relative to its dimensionality. A direct consequence of the relativity of species is the duality of speciators (e.g., incipient species) meaning that, in a given time, they may be perceived as both being and not being a species. Finally, the most accurate way to reflect both the relativity of species and the duality of speciators in species delimitation is probabilistic. While the novelty of these ideas may be questionable, they still deserve more extensive attention from the biological community. Here, we hope to draw such attention by outlining one of the possible pathways towards a new kind of probabilistic species delimitation methods based on the probability of irreversible divergence of evolutionary lineages. We anticipate that our probabilistic view of speciation has the potential to facilitate some of the most serious and universal issues of current taxonomy and to ensure unity of the species-level taxonomy across the tree of life.

Single-Shot Three-Dimensional Orientation Imaging of Nanorods Using Spin to Orbital Angular Momentum Conversion.

The key information about any nanoscale system relates to the orientations and conformations of its parts. Unfortunately, these details are often hidden below the diffraction limit, and elaborate techniques must be used to optically probe them. Here we present imaging of the 3D rotation motion of metal nanorods, restoring the distinct nanorod orientations in the full extent of azimuthal and polar angles. The nanorods imprint their 3D orientation onto the geometric phase and space-variant polarization of the light they scatter. We manipulate the light angular momentum and generate optical vortices that create self-interference images providing the nanorods' angles via digital processing. After calibration by scanning electron microscopy, we demonstrated time-resolved 3D orientation imaging of sub-100 nm nanorods under Brownian motion (frame rate up to 500 fps). We also succeeded in imaging nanorods as nanoprobes in live-cell imaging and reconstructed their 3D rotational movement during interaction with the cell membrane (100 fps).

Overexpression of alfalfa SIMK promotes root hair growth, nodule clustering and shoot biomass production.

Nitrogen-fixing rhizobia and legumes have developed complex mutualistic mechanism that allows to convert atmospheric nitrogen into ammonia. Signalling by mitogen-activated protein kinases (MAPKs) seems to be involved in this symbiotic interaction. Previously, we reported that stress-induced MAPK (SIMK) shows predominantly nuclear localization in alfalfa root epidermal cells. Nevertheless, SIMK is activated and relocalized to the tips of growing root hairs during their development. SIMK kinase (SIMKK) is a well-known upstream activator of SIMK. Here, we characterized production parameters of transgenic alfalfa plants with genetically manipulated SIMK after infection with Sinorhizobium meliloti. SIMKK RNAi lines, causing strong downregulation of both SIMKK and SIMK, showed reduced root hair growth and lower capacity to form infection threads and nodules. In contrast, constitutive overexpression of GFP-tagged SIMK promoted root hair growth as well as infection thread and nodule clustering. Moreover, SIMKK and SIMK downregulation led to decrease, while overexpression of GFP-tagged SIMK led to increase of biomass in above-ground part of plants. These data suggest that genetic manipulations causing downregulation or overexpression of SIMK affect root hair, nodule and shoot formation patterns in alfalfa, and point to the new biotechnological potential of this MAPK.

In vivo light-sheet microscopy resolves localisation patterns of FSD1, a superoxide dismutase with function in root development and osmoprotection.

Superoxide dismutases (SODs) are enzymes detoxifying superoxide to hydrogen peroxide while temporal developmental expression and subcellular localisation are linked to their functions. Therefore, we aimed here to reveal in vivo developmental expression, subcellular, tissue- and organ-specific localisation of iron superoxide dismutase 1 (FSD1) in Arabidopsis using light-sheet and Airyscan confocal microscopy. FSD1-GFP temporarily accumulated at the site of endosperm rupture during seed germination. In emerged roots, it showed the highest abundance in cells of the lateral root cap, columella, and endodermis/cortex initials. The largest subcellular pool of FSD1-GFP was localised in the plastid stroma, while it was also located in the nuclei and cytosol. The majority of the nuclear FSD1-GFP is immobile as revealed by fluorescence recovery after photobleaching. We found that fsd1 knockout mutants exhibit reduced lateral root number and this phenotype was reverted by genetic complementation. Mutant analysis also revealed a requirement for FSD1 in seed germination during salt stress. Salt stress tolerance was coupled with the accumulation of FSD1-GFP in Hechtian strands and superoxide removal. It is likely that the plastidic pool is required for acquiring oxidative stress tolerance in Arabidopsis. This study suggests new developmental and osmoprotective functions of SODs in plants.

Oxygen Is an Ambivalent Factor for the Differentiation of Human Pluripotent Stem Cells in Cardiac 2D Monolayer and 3D Cardiac Spheroids.

Numerous protocols of cardiac differentiation have been established by essentially focusing on specific growth factors on human pluripotent stem cell (hPSC) differentiation efficiency. However, the optimal environmental factors to obtain cardiac myocytes in network are still unclear. The mesoderm germ layer differentiation is known to be enhanced by low oxygen exposure. Here, we hypothesized that low oxygen exposure enhances the molecular and functional maturity of the cardiomyocytes. We aimed at comparing the molecular and functional consequences of low (5% O2 or LOE) and high oxygen exposure (21% O2 or HOE) on cardiac differentiation of hPSCs in 2D- and 3D-based protocols. hPSC-CMs were differentiated through both the 2D (monolayer) and 3D (embryoid body) protocols using several lines. Cardiac marker expression and cell morphology were assessed. The mitochondrial localization and metabolic properties were evaluated. The intracellular Ca2+ handling and contractile properties were also monitored. The 2D cardiac monolayer can only be differentiated in HOE. The 3D cardiac spheroids containing hPSC-CMs in LOE further exhibited cardiac markers, hypertrophy, steadier SR Ca2+ release properties revealing a better SR Ca2+ handling, and enhanced contractile force. Preserved distribution of mitochondria and similar oxygen consumption by the mitochondrial respiratory chain complexes were also observed. Our results brought evidences that LOE is moderately beneficial for the 3D cardiac spheroids with hPSC-CMs exhibiting further maturity. In contrast, the 2D cardiac monolayers strictly require HOE.

Dystrophin Deficiency Causes Progressive Depletion of Cardiovascular Progenitor Cells in the Heart.

Duchenne muscular dystrophy (DMD) is a devastating condition shortening the lifespan of young men. DMD patients suffer from age-related dilated cardiomyopathy (DCM) that leads to heart failure. Several molecular mechanisms leading to cardiomyocyte death in DMD have been described. However, the pathological progression of DMD-associated DCM remains unclear. In skeletal muscle, a dramatic decrease in stem cells, so-called satellite cells, has been shown in DMD patients. Whether similar dysfunction occurs with cardiac muscle cardiovascular progenitor cells (CVPCs) in DMD remains to be explored. We hypothesized that the number of CVPCs decreases in the dystrophin-deficient heart with age and disease state, contributing to DCM progression. We used the dystrophin-deficient mouse model (mdx) to investigate age-dependent CVPC properties. Using quantitative PCR, flow cytometry, speckle tracking echocardiography, and immunofluorescence, we revealed that young mdx mice exhibit elevated CVPCs. We observed a rapid age-related CVPC depletion, coinciding with the progressive onset of cardiac dysfunction. Moreover, mdx CVPCs displayed increased DNA damage, suggesting impaired cardiac muscle homeostasis. Overall, our results identify the early recruitment of CVPCs in dystrophic hearts and their fast depletion with ageing. This latter depletion may participate in the fibrosis development and the acceleration onset of the cardiomyopathy.

Comparison of Pulsed Radiofrequency, Oxygen-Ozone Therapy and Epidural Steroid Injections for the Treatment of Chronic Unilateral Radicular Syndrome.

Background and objectives: For the treatment of chronic unilateral radicular syndrome, there are various methods including three minimally invasive computed tomography (CT)-guided methods, namely, pulsed radiofrequency (PRF), transforaminal oxygen ozone therapy (TFOOT), and transforaminal epidural steroid injection (TFESI). Despite this, it is still unclear which of these methods is the best in terms of pain reduction and disability improvement. Therefore, the purpose of this study was to evaluate the short and long-term effectiveness of these methods by measuring pain relief using the visual analogue scale (VAS) and improvement in disability (per the Oswestry disability index (ODI)) in patients with chronic unilateral radicular syndrome at L5 or S1 that do not respond to conservative treatment. Materials and Methods: After screening 692 patients, we enrolled 178 subjects, each of whom underwent one of the above CT-guided procedures. The PRF settings were as follows: pulse width = 20 ms, f = 2 Hz, U = 45 V, Z ˂ 500 Ω, and interval = 2 × 120 s. For TFOOT, an injection of 4-5 mL of an O2-O3 mixture (24 µg/mL) was administered. For the TFESI, 1 mL of a corticosteroid (betamethasone dipropionate), 3 mL of an anaesthetic (bupivacaine hydrochloride), and a 0.5 mL mixture of a non-ionic contrast agent (Iomeron 300) were administered. Pain intensity was assessed with a questionnaire. Results: The data from 178 patients (PRF, n = 57; TFOOT, n = 69; TFESI, n = 52) who submitted correctly completed questionnaires in the third month of the follow-up period were used for statistical analysis. The median pre-treatment visual analogue scale (VAS) score in all groups was six points. Immediately after treatment, the largest decrease in the median VAS score was observed in the TFESI group, with a score of 3.5 points (a decrease of 41.7%). In the PRF and TFOOT groups, the median VAS score decreased to 4 and 5 points (decreases of 33% and 16.7%, respectively). The difference in the early (immediately after) post-treatment VAS score between the TFESI and TFOOT groups was statistically significant (p = 0.0152). At the third and sixth months after treatment, the median VAS score was five points in all groups, without a statistically significant difference (p > 0.05). Additionally, there were no significant differences in the Oswestry disability index (ODI) values among the groups at any of the follow-up visits. Finally, there were no significant effects of age or body mass index (BMI) on both treatment outcomes (maximum absolute value of Spearman's rank correlation coefficient = 0.193). Conclusions: Although the three methods are equally efficient in reducing pain over the entire follow-up, we observed that TFESI (a corticosteroid with a local anaesthetic) proved to be the most effective method for early post-treatment pain relief.

Ligase 3-mediated end-joining maintains genome stability of human embryonic stem cells.

Maintenance of human embryonic stem cells (hESCs) with stable genome is important for their future use in cell replacement therapy and disease modeling. Our understanding of the mechanisms maintaining genomic stability of hESC and our ability to modulate them is essential in preventing unwanted mutation accumulation during their in vitro cultivation. In this study, we show the DNA damage response mechanism in hESCs is composed of known, yet unlikely components. Clustered oxidative base damage is converted into DNA double-strand breaks (DSBs) by base excision repair (BER) and then quickly repaired by ligase (Lig)3-mediated end-joining (EJ). If there is further induction of clustered oxidative base damage by irradiation, then BER-mediated DSBs become essential in triggering the checkpoint response in hESCs. hESCs limit the mutagenic potential of Lig3-mediated EJ by DNA break end protection involving p53 binding protein 1 (53BP1), which results in fast and error-free microhomology-mediated repair and a low mutant frequency in hESCs. DSBs in hESCs are also repaired via homologous recombination (HR); however, DSB overload, together with massive end protection by 53BP1, triggers competition between error-free HR and mutagenic nonhomologous EJ.-Kohutova, A., Raska, J., Kruta, M., Seneklova, M., Barta, T., Fojtik, P., Jurakova, T., Walter, C. A., Hampl, A., Dvorak, P., Rotrekl, V. Ligase 3-mediated end-joining maintains genome stability of human embryonic stem cells.

Reptodigitus Chapmanii (Nostocales, Hapalosiphonaceae) Gen. Nov.: A Unique Nostocalean (Cyanobacteria) Genus Based on a Polyphasic Approach1.

The Nostocales is a monophyletic, heterocytous lineage of cyanobacteria capable of akinete production and division in multiple planes, depending upon family-level clade. While present in a variety of ecosystems, the diversity of the Nostocales has been poorly elucidated. Due to environmentally -induced phenotypic plasticity, morphology alone is often insufficient to determine the true phylogenetic placement of these taxa. In order to bridge this gap, taxonomists now employ the polyphasic approach, combining methods such as morphological analysis, phylogenetic analysis based on DNA sequence and genetic identity based on ribosomal genes, and secondary structure of the 16S-23S ITS and 16S rRNA gene sequences, as well as ecological characterization. Using this combined approach, a new genus and species (Reptodigitus chapmanii gen. et sp. nov.) isolated from the St. Johns River (Jacksonville, Florida, USA) within the Nostocales is herein described. Phylogenetic analyses place this taxon within the Hapalosiphonaceae, sister to the clade containing Fischerella, Hapalosiphon, and Westiellopsis. The 16S-23S ITS secondary folding structure analysis also supports the erection of this new genus.

High-Resolution Quantitative Phase Imaging of Plasmonic Metasurfaces with Sensitivity down to a Single Nanoantenna.

Optical metasurfaces have emerged as a new generation of building blocks for multifunctional optics. Design and realization of metasurface elements place ever-increasing demands on accurate assessment of phase alterations introduced by complex nanoantenna arrays, a process referred to as quantitative phase imaging. Despite considerable effort, the widefield (nonscanning) phase imaging that would approach resolution limits of optical microscopy and indicate the response of a single nanoantenna still remains a challenge. Here, we report on a new strategy in incoherent holographic imaging of metasurfaces, in which unprecedented spatial resolution and light sensitivity are achieved by taking full advantage of the polarization selective control of light through the geometric (Pancharatnam-Berry) phase. The measurement is carried out in an inherently stable common-path setup composed of a standard optical microscope and an add-on imaging module. Phase information is acquired from the mutual coherence function attainable in records created in broadband spatially incoherent light by the self-interference of scattered and leakage light coming from the metasurface. In calibration measurements, the phase was mapped with the precision and spatial background noise better than 0.01 and 0.05 rad, respectively. The imaging excels at the high spatial resolution that was demonstrated experimentally by the precise amplitude and phase restoration of vortex metalenses and a metasurface grating with 833 lines/mm. Thanks to superior light sensitivity of the method, we demonstrated for the first time to our knowledge the widefield measurement of the phase altered by a single nanoantenna while maintaining the precision well below 0.15 rad.

Functional Divergence of Microtubule-Associated TPX2 Family Members in Arabidopsis thaliana.

TPX2 (Targeting Protein for Xklp2) is an evolutionary conserved microtubule-associated protein important for microtubule nucleation and mitotic spindle assembly. The protein was described as an activator of the mitotic kinase Aurora A in humans and the Arabidopsis AURORA1 (AUR1) kinase. In contrast to animal genomes that encode only one TPX2 gene, higher plant genomes encode a family with several TPX2-LIKE gene members (TPXL). TPXL genes of Arabidopsis can be divided into two groups. Group A proteins (TPXL2, 3, 4, and 8) contain Aurora binding and TPX2_importin domains, while group B proteins (TPXL1, 5, 6, and 7) harbor an Xklp2 domain. Canonical TPX2 contains all the above-mentioned domains. We confirmed using in vitro kinase assays that the group A proteins contain a functional Aurora kinase binding domain. Transient expression of Arabidopsis TPX2-like proteins in Nicotiana benthamiana revealed preferential localization to microtubules and nuclei. Co-expression of AUR1 together with TPX2-like proteins changed the localization of AUR1, indicating that these proteins serve as targeting factors for Aurora kinases. Taken together, we visualize the various localizations of the TPX2-LIKE family in Arabidopsis as a proxy to their functional divergence and provide evidence of their role in the targeted regulation of AUR1 kinase activity.

Speciation in protists: Spatial and ecological divergence processes cause rapid species diversification in a freshwater chrysophyte.

Although eukaryotic microorganisms are extremely numerous, diverse and essential to global ecosystem functioning, they are largely understudied by evolutionary biologists compared to multicellular macroscopic organisms. In particular, very little is known about the speciation mechanisms which may give rise to the diversity of microscopic eukaryotes. It was postulated that the enormous population sizes and ubiquitous distribution of these organisms could lead to a lack of population differentiation and therefore very low speciation rates. However, such assumptions have traditionally been based on morphospecies, which may not accurately reflect the true diversity, missing cryptic taxa. In this study, we aim to articulate the major diversification mechanisms leading to the contemporary molecular diversity by using a colonial freshwater flagellate, Synura sphagnicola, as an example. Phylogenetic analysis of five sequenced loci showed that S. sphagnicola differentiated into two morphologically distinct lineages approximately 15.4 million years ago, which further diverged into several evolutionarily recent haplotypes during the late Pleistocene. The most recent haplotypes are ecologically and biogeographically much more differentiated than the old lineages, presumably because of their persistent differentiation after the allopatric speciation events. Our study shows that in microbial eukaryotes, species diversification via the colonization of new geographical regions or ecological resources occurs much more readily than was previously thought. Consequently, divergence times of microorganisms in some lineages may be equivalent to the estimated times of speciation in plants and animals.

A polyphasic approach to the delimitation of diatom species: a case study for the genus Pinnularia (Bacillariophyta).

Diatoms are one of the most abundant and arguably the most species-rich group of protists. Diatom species delimitation has often been based exclusively on the recognition of morphological discontinuities without investigation of other lines of evidence. Even though DNA sequences and reproductive experiments have revealed several examples of (pseudo)cryptic diversity, our understanding of diatom species boundaries and diversity remains limited. The cosmopolitan pennate raphid diatom genus Pinnularia represents one of the most taxon-rich diatom genera. In this study, we focused on the delimitation of species in one of the major clades of the genus, the Pinnularia subgibba group, based on 105 strains from a worldwide origin. We compared genetic distances between the sequences of seven molecular markers and selected the most variable pair, the mitochondrial cox1 and nuclear encoded LSU rDNA, to formulate a primary hypothesis on the species limits using three single-locus automated species delimitation methods. We compared the DNA-based primary hypotheses with morphology and with other available lines of evidence. The results indicate that our data set comprised 15 species of the P. subgibba group. The vast majority of these taxa have an uncertain taxonomic identity, suggesting that several may be unknown to science and/or members of (pseudo)cryptic species complexes within the P. subgibba group.

Tyrosine Kinase Expressed in Hepatocellular Carcinoma, TEC, Controls Pluripotency and Early Cell Fate Decisions of Human Pluripotent Stem Cells via Regulation of Fibroblast Growth Factor-2 Secretion.

Human pluripotent stem cells (hPSC) require signaling provided by fibroblast growth factor (FGF) receptors. This can be initiated by the recombinant FGF2 ligand supplied exogenously, but hPSC further support their niche by secretion of endogenous FGF2. In this study, we describe a role of tyrosine kinase expressed in hepatocellular carcinoma (TEC) kinase in this process. We show that TEC-mediated FGF2 secretion is essential for hPSC self-renewal, and its lack mediates specific differentiation. Following both short hairpin RNA- and small interfering RNA-mediated TEC knockdown, hPSC secretes less FGF2. This impairs hPSC proliferation that can be rescued by increasing amounts of recombinant FGF2. TEC downregulation further leads to a lower expression of the pluripotency markers, an improved priming towards neuroectodermal lineage, and a failure to develop cardiac mesoderm. Our data thus demonstrate that TEC is yet another regulator of FGF2-mediated hPSC pluripotency and differentiation. Stem Cells 2017;35:2050-2059.

Computer-assisted engineering of hyperstable fibroblast growth factor 2.

Fibroblast growth factors (FGFs) serve numerous regulatory functions in complex organisms, and their corresponding therapeutic potential is of growing interest to academics and industrial researchers alike. However, applications of these proteins are limited due to their low stability. Here we tackle this problem using a generalizable computer-assisted protein engineering strategy to create a unique modified FGF2 with nine mutations displaying unprecedented stability and uncompromised biological function. The data from the characterization of stabilized FGF2 showed a remarkable prediction potential of in silico methods and provided insight into the unfolding mechanism of the protein. The molecule holds a considerable promise for stem cell research and medical or pharmaceutical applications.