<p>Simplified view of the phylogenetic tree calculated in <a href="http://www.ploso... more <p>Simplified view of the phylogenetic tree calculated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193228#pone.0193228.g001" target="_blank">Fig 1</a>. The P5A group includes only ATP13A1-like sequences (grey), while the P5B group includes subclades belonging to the ATP13A2 (blue), P5B<sub>inv</sub> (purple, invertebrate P5B), ATP13A3 (yellow), ATP13A4 (red) and ATP13A5 (green) clades. Sequences from cnidaria, placozoa and ctenophore are marked in white dots. Invertebrate sequences are marked in yellow dots. Sequences from hemichordates and echinoderms are marked with light blue dots and sequences from higher vertebrates are marked in progressively darker shaded blue dots. Yeast Spf1p and Ypk9p are marked with red dots. The yellow numbers indicate the three major P5B gene duplications in vertebrate evolution as discussed in the main text.</p
<p>HeLa cells were transiently co-transfected with ATP13A1 wildtype (no tag, N- or C-termin... more <p>HeLa cells were transiently co-transfected with ATP13A1 wildtype (no tag, N- or C-terminal GFP-tag) or dead mutant (N-terminal GFP-tag) and SERCA2b-mCherry, an ER resident. <b>A-C.</b> ATP13A1 with N- (<b>A</b>) or C-terminal (<b>B</b>) GFP-tag or unlabeled ATP13A1 (<b>C</b>) co-localizes with SERCA2b. Unlabeled ATP13A1 was detected via immunocytochemistry with homemade ATP13A1 antibody (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193228#pone.0193228.s004" target="_blank">S4 Fig</a>) and Alexa Fluor 488. Scale bar represents 20 μm.</p
<p>HeLa cells were transiently co-transfected with hATP13A2, hATP13A3, mATP13A4 or mATP13A5... more <p>HeLa cells were transiently co-transfected with hATP13A2, hATP13A3, mATP13A4 or mATP13A5 (N- or C-terminal mCherry-tag, N-mCh and C-mCh, respectively). Scale bar represents 20 μm. <b>A-C.</b> Co-localization of hATP13A2 (N-terminal mCherry-tag) with various GFP-labeled markers, such as the early endosomal marker RAB5 (A), the late endo-/lysosomal marker RAB7 (B) and RAB11, a marker for the recycling endosomes (C). <b>D-F.</b> Immunocytochemistry of cells overexpressing N- or C-terminal mCherry labeled hATP13A2 with the endogenous markers of late endosomes (CD63, E-F) and lysosomes (lamp2, D). <b>G-I.</b> Co-localization of hATP13A3 with GFP-labeled RAB5 (G), RAB7 (H) and RAB11 (I). <b>J-L.</b> Immunocytochemistry of cells overexpressing N- or C-terminal mCherry labeled hATP13A3 with the endogenous markers of late endo-/lysosomes lamp1 (K, L) and of lysosomes lamp2 (J). <b>M-O.</b> Co-localization of hATP13A4 with GFP-labeled RAB5 (M), RAB7 (N) and RAB11 (O). <b>P-R.</b> Immunocytochemistry of cells overexpressing N- or C-terminal mCherry labeled hATP13A4 with the endogenous markers of late endosomes (CD63, Q-R) and lysosomes (lamp2, P). <b>S.</b> mATP13A5 overexpression is too weak for fluorescence microscopy. When a fluorescent signal is observed, an endosomal-like pattern was seen in 80% of the observations (two left panels), whereas in other images a reticular pattern was observed (right panel). <b>T-W.</b> Bar graphs depict Pearson’s coefficients (PCC) of ATP13A2-5 isoforms with various endosomal markers.</p
<p>Phylogenetic tree of 146 protein sequences of P5-type ATPases as inferred from the combi... more <p>Phylogenetic tree of 146 protein sequences of P5-type ATPases as inferred from the combined output of two independent statistical methods of measurement (Bayesian inference and maximum likelihood analysis, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193228#sec002" target="_blank">materials and methods</a> for a detailed description). Yellow and blue shades represent sequences from protostomia and deuterostomia, respectively. Black dots represent Bayesian inference values of 1.00 and numbers noted at key nodes are represented with Bayesian inference statistical values on the <i>left</i> and maximum likelihood statistical values on the <i>right</i> of the dash (<i>left</i>/<i>right</i>). Accession numbers of named sequences can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193228#pone.0193228.s009" target="_blank">S1 Table</a>. Positions of human and model animal organisms (<i>C</i>. <i>elegans</i>, <i>D</i>. <i>melanogaster</i>, <i>D</i>. <i>rerio</i>, <i>M</i>. <i>musculus</i>) are marked by # and *, respectively. Yeast sequences are marked by ‡. The meaning of the colored arrows is provided in the description of the main text. The yellow numbers indicate the three major P5B gene duplications in vertebrate evolution as discussed in the main text.</p
<p><b>A-C.</b> Microsomal fractions of COS-8 cells, transiently overexpressing ... more <p><b>A-C.</b> Microsomal fractions of COS-8 cells, transiently overexpressing WT of dead mutants of the P5-isoforms (N-mCherry-tag) were analyzed on immunoblot or radiogram (EP<sup>32</sup>) to examine respectively expression levels and autophosphorylation. As a control, non-transfected cells (NT) were included. <b>A.</b> Immunoblot with mCherry antibody shows overexpression for ATP13A1 and ATP13A2 WT and dead mutants. GAPDH levels were determined as loading control. The radiogram (EP<sup>32</sup>) demonstrates phosphorylation for WT ATP13A1 and ATP13A2, but not for dead mutants. <b>B.</b> Radiogram indicates that ATP13A1 and ATP13A2 are both sensitive towards hydroxylamine (HA), a hallmark of P-type ATPases. HA quenches the phosphorylated Asp residues, but leaves phosphorylated Thr, Ser or Tyr residues intact. The lower panel shows a coomassie stained gel that depicts the equal loading for all lanes in the upper panel. <b>C.</b> Immunoblot and radiogram of the autophosphorylated intermediates (EP<sup>32</sup>) of the five P5 isoforms demonstrate overexpression of all isoforms, whereas phosphorylation was only seen for ATP13A1-4. <b>D.</b> Graph showing the autophosphorylated levels (EP<sup>32</sup> panel C) normalized to the relative expression levels of each isoform (mCherry immunoblot panel C). Results indicate strongest autophosphorylation for ATP13A1, ATP13A2 and ATP13A3. ATP13A4 displays low autophosphorylation levels, while ATP13A5 levels are negligible. Data are represented as average ± SD (n = 4). *, P ≤ 0.05 <i>versus</i> ATP13A1 and ATP13A2; #, P ≤ 0.05 <i>versus</i> ATP13A1, ATP13A2 and ATP13A3 (one-way ANOVA, Bonferroni post-hoc). <b>E.</b> Immunoblot demonstrating the expression of the N-terminal 10xHis-FLAG tagged versions of the yeast Ypk9p and Ypk9p (D781N) in yeast total membrane fractions. Antibody against the FLAG part of the tag was used to visualize protein expression and GAPDH was used as a loading control. The wildtype but not catalytic dead protein was able to complement loss of the native <i>YPK9</i> gene (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193228#pone.0193228.g006" target="_blank">Fig 6B</a>). Both Ypk9p variants were successfully purified using the 10xHis part of the tag (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193228#pone.0193228.s003" target="_blank">S3 Fig</a>). Proteins are expressed from the galactose inducible promotor when grown in rich media supplemented with galactose (YP-Gal) while glucose repress expression (YP-Glu). <b>F.</b> Quantification of Ypk9p autophosphorylation using scintillation counting, normalized to μg of purified Ypk9p. Purified Ypk9p was able to undergo autophosphorylation, which was abolished by mutating the Asp residue in the catalytic motif (D781N).</p
<p>The genome of <i>S</i>. <i>cerevisiae</i> contains a single P5A ... more <p>The genome of <i>S</i>. <i>cerevisiae</i> contains a single P5A ATPase gene (<i>SPF1</i>) and a single P5B ATPase gene (<i>YPK9</i>). <b>A.</b> Gene knockout of <i>SPF1</i> results in increased sensitivity to caffeine which can be rescued by expression of untagged Spf1p and mATP13A1 respectively, but not by the untagged catalytically dead SPF1 D487N or by untagged mammalian ATP13A2-5. (e.v.–empty vector) <b>B.</b> Deletion of <i>ypk9</i><sup><i>-</i></sup> results in increased sensitivity to MnCl<sub>2</sub>, which can be rescued by expression of untagged Ypk9p, but not by the untagged and catalytically dead Ypk9p D781N. None of the untagged mammalian P5 ATPase genes showed rescue of <i>ypk9</i><sup><i>-</i></sup> (not shown). <b>C.</b> ATP13A1 complements the caffeine phenotype of the <i>spf1</i><sup><i>-</i></sup> knockout strain, whereas the catalytically dead mutant D530N fails to complement. <b>D.</b> Expression of Ypk9p and catalytically dead Ypk9p D781N proteins was confirmed by both N-terminal GFP fusion constructs. Ypk9p tagged with GFP show vacuolar localization. <b>E.</b> Expression of mammalian ATP13A1-5 proteins was similarly confirmed by N- and C-terminal GFP fusion constructs. N- or C-terminal positioning of GFP showed no apparent difference in localization patterns. ATP13A1 shows ER localization and is absent in vacuoles, ATP13A2 and ATP13A4 show vacuolar localization, ATP13A3 and ATP13A5 show localization to cytosolic spots which could represent a pre-vacuolar compartment or early endosomes. Scale bars represent 2,5 μm.</p
The core retrieved from Lake Van consists of seismites that were possibly deposited during the ea... more The core retrieved from Lake Van consists of seismites that were possibly deposited during the earthquakes around the Van region. Deformed parts of the core sediments display folded laminations that can be attributed to seismites. The problem arises that if the fold axis is deposited perpendicular to the liner and, if the hinge line is far enough, describing the true laminations might be impossible related to real age of basin evolution because extra laminae seem deposited to the area. Scientist must pay attention such problem that dating method like varve counting and basin evolution estimates can totally change due to extra laminae that explained before. For eliminate to wrong interpretations considering reversal reflected anomalies even with angularity effects to one package of pair can show significant difference than other symmetric one due to angle of the hinge line or soft sediment deformation. Considering the situation explained, p-wave is not enough to support the idea however; chemical analyses (x-ray florescence), ICP-MS (asdasd) analysis can provide appropriate results to identify laminae that appear on the limbs of the reversed micro folds. New easy designed extra U-Channel drive tray framework prepared by us. U-Channels are prepared well conditioned, saturated enough to well contact between sediment surface and plastic shield of u-channel samples from cores. Physical parameters are measured by Multi sensor core logger (MSCL) with high resolution step ratio fixed to 1mm. At the p-wave and gamma ray results, we observed together stair upwards form and reverse reflected downward data graphics, thus our interpretation of identifying the fold limbs are now visible. We understand that laminae packages are exactly the same. XRF and MSCL are totally supporting to origin of pairs generated after their sedimentation age with mechanical forces. For this reason, in this study, we attended to solve such problem to analyze deformed folded laminations that must be documented for paleo-climate studies in Lake Van.
Geochemical and sedimentological analyses and radionuclide (Pb and Cs) dating of three cores from... more Geochemical and sedimentological analyses and radionuclide (Pb and Cs) dating of three cores from the Bosporus outlet area of the Black Sea, north of Istanbul, were conducted to assess the sources and history of heavy metal pollution. The sedimentary succession in the shelf core KD12-01 consists mainly of clay (49-80%) and silt (15-41%). Radionuclide dating of the core indicates that it consists of old sediments that are uncontaminated with heavy metals. In contrast, cores KD12-04 and KD12-07 recovered from -350 m and -304 mm in the upper slope area represent sediments consisting of silt and clay that were deposited since at least the last 120 years and 60 years, respectively. The latter core contains two mass-flow units represented by relatively old sedimentary material according to the low Pb activity and relatively low heavy metal contents. The upper 40 and 48 cm of cores KD 12-04 and KD 12-07 represent sediments deposited since 1970s and 1980s that are significantly polluted wit...
Friction between archwires and labial brackets has received considerable attention; however, info... more Friction between archwires and labial brackets has received considerable attention; however, information on the frictional behaviour of commercially available lingual brackets is limited. The aim of this study was to investigate the frictional resistance resulting from a combination of lingual orthodontic brackets (7th Generation, STb, Magic, and In-Ovation L) and stainless steel archwires at 0, 5, and 10 degrees of second-order angulation. Each bracket type (n = 30) was tested with three different sizes of archwires. Static and kinetic frictional forces were evaluated with a universal testing machine. Statistical analysis of the data was performed with non-parametric Kruskal-Wallis and Dunn's multiple comparison tests. All tested brackets showed higher frictional forces as the wire size and second-order angulation increased. The lowest friction was found with In-Ovation L brackets and 0.016 inch archwires at 0 degrees angulation, and the greatest friction with a combination of STb brackets and 0.017 × 0.025 inch archwires at 10 degrees angulation. For all combinations, Magic and In-Ovation L brackets showed lower frictional resistance when compared with 7th Generation and STb brackets. The slot width (occluso-gingival dimension) of the brackets, measured using the optics of a microhardness machine, showed that all brackets were oversized and that Magic brackets had the largest slot width. Surface roughness of the brackets investigated using atomic force microscopy and scanning electron microscopy, demonstrated that the 7th Generation brackets had the greatest surface roughness.
Several human P5-type transport ATPases are implicated in neurological disorders, but little is k... more Several human P5-type transport ATPases are implicated in neurological disorders, but little is known about their physiological function and properties. Here, we investigated the relationship between the five mammalian P5 isoforms ATP13A1-5 in a comparative study. We demonstrated that ATP13A1-4 isoforms undergo autophosphorylation, which is a hallmark P-type ATPase property that is required for substrate transport. A phylogenetic analysis of P5 sequences revealed that ATP13A1 represents clade P5A, which is highly conserved between fungi and animals with one member in each investigated species. The ATP13A2-5 isoforms belong to clade P5B and diversified from one isoform in fungi and primitive animals to a maximum of four in mammals by successive gene duplication events in vertebrate evolution. We revealed that ATP13A1 localizes in the endoplasmic reticulum (ER) and experimentally demonstrate that ATP13A1 likely contains 12 transmembrane helices. Conversely, ATP13A2-5 isoforms reside in overlapping compartments of the endosomal system and likely contain 10 transmembrane helices, similar to what was demonstrated earlier for ATP13A2. ATP13A1 complemented a deletion of the yeast P5A ATPase SPF1, while none of ATP13A2-5 could complement either the loss of SPF1 or that of the single P5B ATPase YPK9 in yeast. Thus, ATP13A1 carries out a basic ER function similar to its yeast counterpart Spf1p that plays a role in ER related processes like protein folding and processing. ATP13A2-5 isoforms diversified in mammals and are expressed in the endosomal system where they may have evolved novel complementary or partially redundant functions. While most P5-type ATPases are widely expressed, some P5B-type ATPases (ATP13A4 and ATP13A5) display a more limited tissue distribution in the brain and epithelial glandular cells, where they may exert specialized functions. At least some P5B isoforms are of vital importance for the nervous system, since ATP13A2 and ATP13A4 are linked to respectively Parkinson disease and autism spectrum disorders.
<p>Simplified view of the phylogenetic tree calculated in <a href="http://www.ploso... more <p>Simplified view of the phylogenetic tree calculated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193228#pone.0193228.g001" target="_blank">Fig 1</a>. The P5A group includes only ATP13A1-like sequences (grey), while the P5B group includes subclades belonging to the ATP13A2 (blue), P5B<sub>inv</sub> (purple, invertebrate P5B), ATP13A3 (yellow), ATP13A4 (red) and ATP13A5 (green) clades. Sequences from cnidaria, placozoa and ctenophore are marked in white dots. Invertebrate sequences are marked in yellow dots. Sequences from hemichordates and echinoderms are marked with light blue dots and sequences from higher vertebrates are marked in progressively darker shaded blue dots. Yeast Spf1p and Ypk9p are marked with red dots. The yellow numbers indicate the three major P5B gene duplications in vertebrate evolution as discussed in the main text.</p
<p>HeLa cells were transiently co-transfected with ATP13A1 wildtype (no tag, N- or C-termin... more <p>HeLa cells were transiently co-transfected with ATP13A1 wildtype (no tag, N- or C-terminal GFP-tag) or dead mutant (N-terminal GFP-tag) and SERCA2b-mCherry, an ER resident. <b>A-C.</b> ATP13A1 with N- (<b>A</b>) or C-terminal (<b>B</b>) GFP-tag or unlabeled ATP13A1 (<b>C</b>) co-localizes with SERCA2b. Unlabeled ATP13A1 was detected via immunocytochemistry with homemade ATP13A1 antibody (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193228#pone.0193228.s004" target="_blank">S4 Fig</a>) and Alexa Fluor 488. Scale bar represents 20 μm.</p
<p>HeLa cells were transiently co-transfected with hATP13A2, hATP13A3, mATP13A4 or mATP13A5... more <p>HeLa cells were transiently co-transfected with hATP13A2, hATP13A3, mATP13A4 or mATP13A5 (N- or C-terminal mCherry-tag, N-mCh and C-mCh, respectively). Scale bar represents 20 μm. <b>A-C.</b> Co-localization of hATP13A2 (N-terminal mCherry-tag) with various GFP-labeled markers, such as the early endosomal marker RAB5 (A), the late endo-/lysosomal marker RAB7 (B) and RAB11, a marker for the recycling endosomes (C). <b>D-F.</b> Immunocytochemistry of cells overexpressing N- or C-terminal mCherry labeled hATP13A2 with the endogenous markers of late endosomes (CD63, E-F) and lysosomes (lamp2, D). <b>G-I.</b> Co-localization of hATP13A3 with GFP-labeled RAB5 (G), RAB7 (H) and RAB11 (I). <b>J-L.</b> Immunocytochemistry of cells overexpressing N- or C-terminal mCherry labeled hATP13A3 with the endogenous markers of late endo-/lysosomes lamp1 (K, L) and of lysosomes lamp2 (J). <b>M-O.</b> Co-localization of hATP13A4 with GFP-labeled RAB5 (M), RAB7 (N) and RAB11 (O). <b>P-R.</b> Immunocytochemistry of cells overexpressing N- or C-terminal mCherry labeled hATP13A4 with the endogenous markers of late endosomes (CD63, Q-R) and lysosomes (lamp2, P). <b>S.</b> mATP13A5 overexpression is too weak for fluorescence microscopy. When a fluorescent signal is observed, an endosomal-like pattern was seen in 80% of the observations (two left panels), whereas in other images a reticular pattern was observed (right panel). <b>T-W.</b> Bar graphs depict Pearson’s coefficients (PCC) of ATP13A2-5 isoforms with various endosomal markers.</p
<p>Phylogenetic tree of 146 protein sequences of P5-type ATPases as inferred from the combi... more <p>Phylogenetic tree of 146 protein sequences of P5-type ATPases as inferred from the combined output of two independent statistical methods of measurement (Bayesian inference and maximum likelihood analysis, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193228#sec002" target="_blank">materials and methods</a> for a detailed description). Yellow and blue shades represent sequences from protostomia and deuterostomia, respectively. Black dots represent Bayesian inference values of 1.00 and numbers noted at key nodes are represented with Bayesian inference statistical values on the <i>left</i> and maximum likelihood statistical values on the <i>right</i> of the dash (<i>left</i>/<i>right</i>). Accession numbers of named sequences can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193228#pone.0193228.s009" target="_blank">S1 Table</a>. Positions of human and model animal organisms (<i>C</i>. <i>elegans</i>, <i>D</i>. <i>melanogaster</i>, <i>D</i>. <i>rerio</i>, <i>M</i>. <i>musculus</i>) are marked by # and *, respectively. Yeast sequences are marked by ‡. The meaning of the colored arrows is provided in the description of the main text. The yellow numbers indicate the three major P5B gene duplications in vertebrate evolution as discussed in the main text.</p
<p><b>A-C.</b> Microsomal fractions of COS-8 cells, transiently overexpressing ... more <p><b>A-C.</b> Microsomal fractions of COS-8 cells, transiently overexpressing WT of dead mutants of the P5-isoforms (N-mCherry-tag) were analyzed on immunoblot or radiogram (EP<sup>32</sup>) to examine respectively expression levels and autophosphorylation. As a control, non-transfected cells (NT) were included. <b>A.</b> Immunoblot with mCherry antibody shows overexpression for ATP13A1 and ATP13A2 WT and dead mutants. GAPDH levels were determined as loading control. The radiogram (EP<sup>32</sup>) demonstrates phosphorylation for WT ATP13A1 and ATP13A2, but not for dead mutants. <b>B.</b> Radiogram indicates that ATP13A1 and ATP13A2 are both sensitive towards hydroxylamine (HA), a hallmark of P-type ATPases. HA quenches the phosphorylated Asp residues, but leaves phosphorylated Thr, Ser or Tyr residues intact. The lower panel shows a coomassie stained gel that depicts the equal loading for all lanes in the upper panel. <b>C.</b> Immunoblot and radiogram of the autophosphorylated intermediates (EP<sup>32</sup>) of the five P5 isoforms demonstrate overexpression of all isoforms, whereas phosphorylation was only seen for ATP13A1-4. <b>D.</b> Graph showing the autophosphorylated levels (EP<sup>32</sup> panel C) normalized to the relative expression levels of each isoform (mCherry immunoblot panel C). Results indicate strongest autophosphorylation for ATP13A1, ATP13A2 and ATP13A3. ATP13A4 displays low autophosphorylation levels, while ATP13A5 levels are negligible. Data are represented as average ± SD (n = 4). *, P ≤ 0.05 <i>versus</i> ATP13A1 and ATP13A2; #, P ≤ 0.05 <i>versus</i> ATP13A1, ATP13A2 and ATP13A3 (one-way ANOVA, Bonferroni post-hoc). <b>E.</b> Immunoblot demonstrating the expression of the N-terminal 10xHis-FLAG tagged versions of the yeast Ypk9p and Ypk9p (D781N) in yeast total membrane fractions. Antibody against the FLAG part of the tag was used to visualize protein expression and GAPDH was used as a loading control. The wildtype but not catalytic dead protein was able to complement loss of the native <i>YPK9</i> gene (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193228#pone.0193228.g006" target="_blank">Fig 6B</a>). Both Ypk9p variants were successfully purified using the 10xHis part of the tag (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193228#pone.0193228.s003" target="_blank">S3 Fig</a>). Proteins are expressed from the galactose inducible promotor when grown in rich media supplemented with galactose (YP-Gal) while glucose repress expression (YP-Glu). <b>F.</b> Quantification of Ypk9p autophosphorylation using scintillation counting, normalized to μg of purified Ypk9p. Purified Ypk9p was able to undergo autophosphorylation, which was abolished by mutating the Asp residue in the catalytic motif (D781N).</p
<p>The genome of <i>S</i>. <i>cerevisiae</i> contains a single P5A ... more <p>The genome of <i>S</i>. <i>cerevisiae</i> contains a single P5A ATPase gene (<i>SPF1</i>) and a single P5B ATPase gene (<i>YPK9</i>). <b>A.</b> Gene knockout of <i>SPF1</i> results in increased sensitivity to caffeine which can be rescued by expression of untagged Spf1p and mATP13A1 respectively, but not by the untagged catalytically dead SPF1 D487N or by untagged mammalian ATP13A2-5. (e.v.–empty vector) <b>B.</b> Deletion of <i>ypk9</i><sup><i>-</i></sup> results in increased sensitivity to MnCl<sub>2</sub>, which can be rescued by expression of untagged Ypk9p, but not by the untagged and catalytically dead Ypk9p D781N. None of the untagged mammalian P5 ATPase genes showed rescue of <i>ypk9</i><sup><i>-</i></sup> (not shown). <b>C.</b> ATP13A1 complements the caffeine phenotype of the <i>spf1</i><sup><i>-</i></sup> knockout strain, whereas the catalytically dead mutant D530N fails to complement. <b>D.</b> Expression of Ypk9p and catalytically dead Ypk9p D781N proteins was confirmed by both N-terminal GFP fusion constructs. Ypk9p tagged with GFP show vacuolar localization. <b>E.</b> Expression of mammalian ATP13A1-5 proteins was similarly confirmed by N- and C-terminal GFP fusion constructs. N- or C-terminal positioning of GFP showed no apparent difference in localization patterns. ATP13A1 shows ER localization and is absent in vacuoles, ATP13A2 and ATP13A4 show vacuolar localization, ATP13A3 and ATP13A5 show localization to cytosolic spots which could represent a pre-vacuolar compartment or early endosomes. Scale bars represent 2,5 μm.</p
The core retrieved from Lake Van consists of seismites that were possibly deposited during the ea... more The core retrieved from Lake Van consists of seismites that were possibly deposited during the earthquakes around the Van region. Deformed parts of the core sediments display folded laminations that can be attributed to seismites. The problem arises that if the fold axis is deposited perpendicular to the liner and, if the hinge line is far enough, describing the true laminations might be impossible related to real age of basin evolution because extra laminae seem deposited to the area. Scientist must pay attention such problem that dating method like varve counting and basin evolution estimates can totally change due to extra laminae that explained before. For eliminate to wrong interpretations considering reversal reflected anomalies even with angularity effects to one package of pair can show significant difference than other symmetric one due to angle of the hinge line or soft sediment deformation. Considering the situation explained, p-wave is not enough to support the idea however; chemical analyses (x-ray florescence), ICP-MS (asdasd) analysis can provide appropriate results to identify laminae that appear on the limbs of the reversed micro folds. New easy designed extra U-Channel drive tray framework prepared by us. U-Channels are prepared well conditioned, saturated enough to well contact between sediment surface and plastic shield of u-channel samples from cores. Physical parameters are measured by Multi sensor core logger (MSCL) with high resolution step ratio fixed to 1mm. At the p-wave and gamma ray results, we observed together stair upwards form and reverse reflected downward data graphics, thus our interpretation of identifying the fold limbs are now visible. We understand that laminae packages are exactly the same. XRF and MSCL are totally supporting to origin of pairs generated after their sedimentation age with mechanical forces. For this reason, in this study, we attended to solve such problem to analyze deformed folded laminations that must be documented for paleo-climate studies in Lake Van.
Geochemical and sedimentological analyses and radionuclide (Pb and Cs) dating of three cores from... more Geochemical and sedimentological analyses and radionuclide (Pb and Cs) dating of three cores from the Bosporus outlet area of the Black Sea, north of Istanbul, were conducted to assess the sources and history of heavy metal pollution. The sedimentary succession in the shelf core KD12-01 consists mainly of clay (49-80%) and silt (15-41%). Radionuclide dating of the core indicates that it consists of old sediments that are uncontaminated with heavy metals. In contrast, cores KD12-04 and KD12-07 recovered from -350 m and -304 mm in the upper slope area represent sediments consisting of silt and clay that were deposited since at least the last 120 years and 60 years, respectively. The latter core contains two mass-flow units represented by relatively old sedimentary material according to the low Pb activity and relatively low heavy metal contents. The upper 40 and 48 cm of cores KD 12-04 and KD 12-07 represent sediments deposited since 1970s and 1980s that are significantly polluted wit...
Friction between archwires and labial brackets has received considerable attention; however, info... more Friction between archwires and labial brackets has received considerable attention; however, information on the frictional behaviour of commercially available lingual brackets is limited. The aim of this study was to investigate the frictional resistance resulting from a combination of lingual orthodontic brackets (7th Generation, STb, Magic, and In-Ovation L) and stainless steel archwires at 0, 5, and 10 degrees of second-order angulation. Each bracket type (n = 30) was tested with three different sizes of archwires. Static and kinetic frictional forces were evaluated with a universal testing machine. Statistical analysis of the data was performed with non-parametric Kruskal-Wallis and Dunn's multiple comparison tests. All tested brackets showed higher frictional forces as the wire size and second-order angulation increased. The lowest friction was found with In-Ovation L brackets and 0.016 inch archwires at 0 degrees angulation, and the greatest friction with a combination of STb brackets and 0.017 × 0.025 inch archwires at 10 degrees angulation. For all combinations, Magic and In-Ovation L brackets showed lower frictional resistance when compared with 7th Generation and STb brackets. The slot width (occluso-gingival dimension) of the brackets, measured using the optics of a microhardness machine, showed that all brackets were oversized and that Magic brackets had the largest slot width. Surface roughness of the brackets investigated using atomic force microscopy and scanning electron microscopy, demonstrated that the 7th Generation brackets had the greatest surface roughness.
Several human P5-type transport ATPases are implicated in neurological disorders, but little is k... more Several human P5-type transport ATPases are implicated in neurological disorders, but little is known about their physiological function and properties. Here, we investigated the relationship between the five mammalian P5 isoforms ATP13A1-5 in a comparative study. We demonstrated that ATP13A1-4 isoforms undergo autophosphorylation, which is a hallmark P-type ATPase property that is required for substrate transport. A phylogenetic analysis of P5 sequences revealed that ATP13A1 represents clade P5A, which is highly conserved between fungi and animals with one member in each investigated species. The ATP13A2-5 isoforms belong to clade P5B and diversified from one isoform in fungi and primitive animals to a maximum of four in mammals by successive gene duplication events in vertebrate evolution. We revealed that ATP13A1 localizes in the endoplasmic reticulum (ER) and experimentally demonstrate that ATP13A1 likely contains 12 transmembrane helices. Conversely, ATP13A2-5 isoforms reside in overlapping compartments of the endosomal system and likely contain 10 transmembrane helices, similar to what was demonstrated earlier for ATP13A2. ATP13A1 complemented a deletion of the yeast P5A ATPase SPF1, while none of ATP13A2-5 could complement either the loss of SPF1 or that of the single P5B ATPase YPK9 in yeast. Thus, ATP13A1 carries out a basic ER function similar to its yeast counterpart Spf1p that plays a role in ER related processes like protein folding and processing. ATP13A2-5 isoforms diversified in mammals and are expressed in the endosomal system where they may have evolved novel complementary or partially redundant functions. While most P5-type ATPases are widely expressed, some P5B-type ATPases (ATP13A4 and ATP13A5) display a more limited tissue distribution in the brain and epithelial glandular cells, where they may exert specialized functions. At least some P5B isoforms are of vital importance for the nervous system, since ATP13A2 and ATP13A4 are linked to respectively Parkinson disease and autism spectrum disorders.
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