Preliminary Report of Three-Dimensional Reconstructive Intraoperative C-Arm in Percutaneous Vertebroplasty

Objective: Percutaneous vertebroplasty (PVP) is usually carried out under three-dimensional (2D) fluoroscopic guidance. However, operative complications or bone cement distribution might be difficult to assess on the basis of only 2D radiographic projection images. We evaluated the feasibility of performing an intraoperative and postoperative examination in patients undergoing PVP by using three-dimensional (3D) reconstructive C-arm. Methods: Standard PVP procedures were performed on 14 consecutive patients by using a Siremobil Iso-C 3D and a multidetector computed tomography machine. Post-processing of acquired volumetric datasets included multiplanar reconstruction (MPR) and surface shaded display (SSD). We analyzed intraoperative and immediate postoperative evaluation of the needle trajectory and bone cement distribution. Results: The male: female ratio was 2: 12; mean age of patients, 70 (range, 77-54) years; and mean T score,-3.4. The mean operation time was 52.14 min, but the time required to perform and post-process the rotational acquisitions was 7.76 min. The detection of bone cement distribution and leakage after PVP by using MPR and SSD was possible in all patients. However, detection of the safe trajectory for needle insertion was not possible. Conclusion: 3D rotational image acquisition can enable intra- or post-procedural assessment of vertebroplasty procedures for the detection of bone cement distribution and leakage. However, it is difficult to assess the safe trajectory for needle insertion. Key Words: Vertebroplasty · Intraoperative 3D imaging · Complication · Feasibility

Funktionelle Kooperation der Transmembransegmente S3 und S4 beim Schaltverhalten von TRPM8

TRP channels are polymodal receptors that are involved in many substantial physiological processes. TRPM8 is a sensor for cold temperatures, but can additionally be activated by substances that mediate a cold feeling such as menthol. Furthermore, the TRPM8 channel is voltage-dependent, whereby the voltage sensitivity can be positively modulated both by, cold as well as by various TRPM8 channel agonist. In contrast to the well studied "classical" voltage-dependent cation channels, there is no generally accepted model for TRPM8, which describes the gating mechanism in detail so far. Based on a recently published computer simulated model for TRPM8 gating, we used site directed mutagenesis to systematically analyze potential interactions between transmembrane domain S3 and S4, which has been demonstrated to be essential for voltage sensitivity. Furthermore, the structural and functional importance of S3 and S4 for gating and folding of TRPM8 was analyzed. In the present work, the evidence of a functional cooperation between the transmembrane segment S3 and S4 in TRPM8 was proofed. The charge distribution in the central region of these protein domains plays a crucial role for a proper folding and for posttranslational maturation (glycosylation) of the channel and therefor is essential for its functionality. Furthermore, the highly conserved sequence motif N-x-x-D located in S3, is not only for TRPM8 but also for the closely related TRPM2 channel of functional relevance. In addition, new insights could be gained about the voltage dependence of TRPM8 by systematic changes of charged amino acids within the S4 segment, which question the so far favored model of an autonomous mobile S4 voltage sensor in TRPM8. Taken together, the results of this study indicate a protein domain corporately formed by S3 and S4 in TRPM8. This domain conduces i.a. the interaction of the channel with different TRPM8 agonists and additionally is of crucial importance for the voltage sensitivity of the channel, possibly through an interaction with the negatively charged phospholipid PIP2

The Cell Adhesion Molecules Roughest, Hibris, Kin of Irre and Sticks and Stones Are Required for Long Range Spacing of the Drosophila Wing Disc Sensory Sensilla

Most animal tissues and organ systems are comprised of highly ordered arrays of varying cell types. The development of external sensory organs requires complex cell-cell communication in order to give each cell a specific identity and to ensure a regular distributed pattern of the sensory bristles. This involves both long and short range signaling mediated by either diffusible or cell anchored factors. In a variety of processes the heterophilic Irre Cell Recognition Module, consisting of the Neph-like proteins: Roughest, Kin of irre and of the Nephrin-like proteins: Sticks and Stones, Hibris, plays key roles in the recognition events of different cell types throughout development. In the present study these proteins are apically expressed in the adhesive belt of epithelial cells participating in sense organ development in a partially exclusive and asymmetric manner. Using mutant analysis the GAL4/UAS system, RNAi and gain of function we found an involvement of all four Irre Cell Recognition Module-proteins in the development of a highly structured array of sensory organs in the wing disc. The proteins secure the regular spacing of sensory organs showing partial redundancy and may function in early lateral inhibition events as well as in cell sorting processes. Comparisons with other systems suggest that the Irre Cell Recognition module is a key organizer of highly repetitive structures.status: publishe

The Cell Adhesion Molecules <i>Roughest</i>, <i>Hibris</i>, <i>Kin of Irre</i> and <i>Sticks and Stones</i> Are Required for Long Range Spacing of the <i>Drosophila</i> Wing Disc Sensory Sensilla

<div><p>Most animal tissues and organ systems are comprised of highly ordered arrays of varying cell types. The development of external sensory organs requires complex cell-cell communication in order to give each cell a specific identity and to ensure a regular distributed pattern of the sensory bristles. This involves both long and short range signaling mediated by either diffusible or cell anchored factors. In a variety of processes the heterophilic <u>I</u>rre Cell <u>R</u>ecognition <u>M</u>odule, consisting of the Neph-like proteins: Roughest, Kin of irre and of the Nephrin-like proteins: Sticks and Stones, Hibris, plays key roles in the recognition events of different cell types throughout development. In the present study these proteins are apically expressed in the adhesive belt of epithelial cells participating in sense organ development in a partially exclusive and asymmetric manner. Using mutant analysis the <i>GAL4/UAS</i> system, RNAi and gain of function we found an involvement of all four Irre Cell Recognition Module-proteins in the development of a highly structured array of sensory organs in the wing disc. The proteins secure the regular spacing of sensory organs showing partial redundancy and may function in early lateral inhibition events as well as in cell sorting processes. Comparisons with other systems suggest that the Irre Cell Recognition module is a key organizer of highly repetitive structures.</p></div

Hbs acts cooperatively with SNS to secure the bristle pattern in the anterior wing margin.

<p>(A-L) Projection views of IRM-protein immunoreactivity in late third instar larvae. Rst is shown in red (A, E and I), Hbs in green (B, F and N), Kirre in blue (C, G and K) and SNS in yellow (D, H and L). (A-D) Global <i>hbs-RNAi</i> using <i>MZ1369-GAL4</i> reduces the staining for Rst (A) and Kirre (C) in all membranes that are not in contact to the SOPs (B) Hbs immunoreactivity is reduced and no clear membrane localization is detectable. (D) SNS immunoreactivity is only mildly affected, but SOPs stand significantly nearer to each other. (E-H) <i>MZ1369-GAL4</i>><i>UAS-sns-RNAi</i> shows mildly reduced Rst staining (E). Hbs (F) and Kirre (K) immunoreactivity is unchanged. SNS (H) is not detectable. (I-L) In the double RNAi <i>MZ1369>hbs-RNAi</i>, <i>SNS-RNAi</i> only the two adhesive belts with Rst (I) and Kirre (K) are visible, but no obvious SOPs are marked. Hbs (J) and SNS (L) are not detectable. (M) In the adult <i>MZ1369-GAL4</i> driven <i>hbs-RNAi</i> shows only a mild spacing phenotype with 0 to 7 intervening cells. (N) The global <i>sns-RNAi</i> in the entire wing disc shows a very mild spacing phenotype with spacing ranging from 1 to 5. (O) <i>MZ1369>hbs-RNAi</i>, <i>sns-RNAi</i> shows a significant disturbance of the spacing of recurved bristles with spacing ranging from 0 to 8. (P) <i>MZ1369-GAL4</i> driven misexpression of <i>hbs</i> has a strong impact on the spacing of recurved bristles with spacing ranging from 0 to 12. Additionally, clustered recurved bristles are frequently observed. (Q) Quantitative analysis of the recurved bristle spacing, as measured by the number of slender bristles between the recurved bristles. The distribution of <i>MZ1369>GFP</i> differs significantly from <i>MZ1369>hbs-RNAi</i> in the following spacing value: 3: p-value = 0.009. <i>MZ1369>SNS-RNAi</i> differs significantly in the following spacing value: < = 1: p-value = 0.035. The double RNAi for <i>hbs</i> and <i>SNS</i> is significantly different for: < = 1: p-value = 0.0002, 3: p-value = 0.014. <i>hbs</i> overexpression differs for: < = 1: p-value = 0.0011, 3: p-value = 0.0013, > = 6: p-value = <0.001. Scale bars correspond to 10μm in all images.</p

The IRM-proteins Rst, Hbs, Kirre and SNS are expressed in distinct patterns during the development of sensory bristles in the anterior wing margin.

<p>(A) The <i>Drosophila</i> IRM-proteins consist of the Neph-like proteins Rst and Kirre and of the Nephrin-like proteins Hbs and SNS. (B) Representative image showing the three bristle rows of the anterior wing margin [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128490#pone.0128490.ref005" target="_blank">5</a>]. The dorsal row consists of recurved bristles marked with an arrow. The medial row consists of mechanosensory stout bristles. The ventral row is composed of mechanosensory slender bristles and recurved bristles marked with an arrow. (C) Representative image of the presumptive wing margin of a late third instar larvae. Kirre in blue can be found in the presumptive posterior wing margin (pwm), anterior wing margin (awm) and the wing veins L3, L4 and L5. SNS in yellow can be only found in the SOPs of the awm. Six example SOPs are marked with an arrow. The ventral (v) side faces up and dorsal (d) faces down. (D-E) The apical localization of the IRM-protein Rst (red) is shown in third instar larvae of the genotype <i>neur-GAL4</i>><i>UAS-mCD8-GFP</i> (green). The SOPs are specifically marked by GFP. The images show a 3D reconstruction in (D) and a lateral view (E). (F-I) Localization of the IRM-proteins in the awm of third instar larvae. Rst (F) in red is localized in two adhesive belts in the awm and is enriched at the border to the SOPs of the recurved bristles. <i>neur-GAL4</i>><i>UAS-rst-RNAi</i> shows no effect on the enrichment of staining around the SOPs (F’). A similar staining pattern in green can be seen for Hbs in wild type (G), but the SOP specific RNAi shows a reduction of staining around the SOP borders (G’), indicating Hbs expression inside the SOPs. Kirre in blue (H) shows a similar pattern as Rst and SOP specific RNAi has no effect (H’). SNS (I) in yellow can only be found at the border of the SOPs. <i>neur-GAL4</i>><i>UAS-sns-RNAi</i> reduces SNS in the SOPs (I’). Scale bars correspond to 10μm in all images.</p

Bristle numbers on dorsal and ventral side of the anterior wing margin.

<p>The table shows the mean and standard error (SE) of the bristle counts from several genotypes used in this study. The first column shows the genotypes. The data columns are named as follows: dorsal triple row (dTR), middle triple row (mTr), dTR/mTr, ventral triple row recurved bristles (vTr r), ventral triple row slender bristles (vTr s) and vTr r/vTr s. Mean numbers shown are always half male half female as no sex differences were observed. Values that differ significantly from the controls are marked with an asterisk (*) (T-test < 0.05) and with two asterisks for (T-test < 0.01) and three for (T-test <0.001).</p><p>Bristle numbers on dorsal and ventral side of the anterior wing margin.</p

The Nephrin-like proteins Hbs and SNS affect other IRM-proteins asymmetrically and induce filopodial extensions.

<p>(A-L) Projection view of IRM immunoreactivity in late third instar larvae. Rst is shown in red (A, E, I and M), Hbs in green (B, F, J and N), Kirre in blue (C, G, K and O) and SNS in yellow (D, H, L and P). (A-D) Misexpression of <i>hbs</i> using <i>MZ1369-GAL4</i> leads to a significant enlargement of Rst (A) and Kirre (B) immunoreactivity positive areas. Specific enrichment of immunoreactivity around the SOPs is lost. Hbs (C) is ubiquitously located in all membranes. SNS (D) staining shows that the SOPs have lost their regular pattern already in this developmental stage. (E-H) Ubiquitous misexpression of <i>sns</i> via <i>MZ1369-GAL4</i> leads to reduced staining of Rst (E), Hbs (F) and Kirre (G). SNS (H) staining shows patched expression. (I-L) Overexpression of <i>hbs</i> via <i>neur-GAL4</i> leads to strongly Rst stained membranes in contact with the SOPs (I). Membranes not in contact to the SOPs show reduced staining. This is also evident in an enlargement of the Rst staining (I’). (J) Hbs staining can be found in the entire SOPs without profound apical basal polarity. (K) Kirre is similar to Rst enriched at the membranes in contact to the SOPs. Hardly any staining can be found in other membranes of the adhesive belt. (L) Cellular localization of SNS is only mildly affected by the <i>hbs</i> misexpression as the protein is mainly, but not exclusively localized at the SOP membrane. Additionally, filopodial outgrowths are frequently observed in this genotype as marked with arrowheads in (I), (K) and (L) and in the enlargements (K’) and (L’). (M-P) Overexpression of <i>sns</i> using <i>neur-GAL4</i> leads to strongly enriched staining of Rst (M), Hbs (N) and Kirre (O) around the SOPs. (P) SNS can be found along all membranes of the SOPs due to the overexpression. Similar to hbs overexpression are filopodial outgrowth frequently observed. Scale bars correspond to 10μm in all images.</p

The Neph-like proteins Rst and Kirre affect the localization of other IRM members in <i>cis</i> and <i>trans</i>.

<p>(A-M, O, Q-X) Projection views of IRM immunoreactivity in third instar larvae. Rst is shown in red (A, E, I, M-Q and U-V), Hbs in green (B, F, J, M-P, R and U-V), Kirre in blue (C, G, K, S and W-Z) and SNS in yellow (D, H, L, T and W-Z). (A-D) Misexpression of <i>rst</i> using <i>MZ1369-GAL4</i> leads to ubiquitous Rst staining (A) in the entire wing disc. Hbs (B) and Kirre (C) are significantly reduced. SNS (D) staining is unaffected in strength, but the localization is not limited to the apical contact zone of the SOPs. Instead it is found in the entire cell. Additionally, the order of the SOPs is severely disturbed. (E-H) Misexpression of <i>kirre</i> via <i>MZ1369-GAL4</i> leads to wider stripes of Rst (E) and Hbs (F) staining. Kirre (G) can be ubiquitously detected in the entire wing disc. SNS (H) staining is strong on all membranes in contact with Kirre positive membranes. Spacing of SOPs is already disrupted at this developmental stage. (I-L) Misexpression of <i>rst</i> using <i>neur-GAL4</i> leads to strongly stained Rst (I) positive SOPs. Hbs (J) is found only around the SOPs and staining of membranes not in contact to the SOPs is reduced. (K) Kirre staining is further enriched around the SOPs. (L) SNS is relocated and it is not specifically located at the adherens junction any more. (M) Magnification of three SOPs of a wild type control stained for Rst and Hbs. The dashed line shows the approximate area of cut for the Z-projection in (N). Hbs can be detected inside the SOPs (arrowhead) and also in the basal appendix (arrow). (O) Magnification of three SOPs in <i>neur-GAL4</i>><i>UAS-rst</i>. The approximate area of cut for the Z-projection in (P) is given with a dashed line. Arrowheads mark the immunopositive interior of the SOPs showing strong Rst staining. Arrows marks the basal appendix of the SOPs. Asterisk mark the co-localization of Rst and Hbs immunoreactivity at the Border of SOPs. (Q-T) Misexpression of <i>kirre</i> using <i>neur-GAL4</i> leads to strong Rst staining around the SOPs. Hbs (R) is found much stronger around or in the SOPs and is strongly reduced on the membranes not in contact with any SOPs. (S) Kirre staining is strongly found in all membranes of the SOPs, showing no apical-basal polarity. (T) SNS localization inside the SOPs is disrupted and a possible degradation product can be found in vesicles basal of the adherens junction. (U) Magnification of three SOPs of a wild type control stained for Rst and Hbs. (V) Magnification of three SOPs in <i>neur-GAL4</i>><i>UAS-kirre</i>. Strong accumulation of Hbs immunoreactivity is evident around the SOPs. (W) Magnification of three SOPs of a wild type control stained for Kirre and SNS. (X) The magnification of three SOPs of <i>neur-GAL4</i>><i>UAS-kirre</i> shows the strong Kirre staining in the entire SOP and the mislocalization of SNS. A dotted line in (W and X) shows the approximate area of a Z-cut that is shown in (Y and Z). In wild type (Y) both proteins interact with each other only in a defined apical contact zone. The distribution of the proteins is clearly visible. SNS is inside the SOPs and Kirre in the surrounding cells. In (Z) the distribution of Kirre and SNS is shown in the mutant situation. Mislocalization and vesicular degradation products of SNS can be seen basal of the adherens junction in this z-axis view. Scale bars correspond to 10μm in all images.</p

Rst acts cooperatively with Kirre to secure the bristle pattern in the anterior wing margin.

<p>(A-L) Projection views of IRM-protein immunoreactivity in late third instar larvae. Rst is shown in red (A, E, and I), Hbs in green (B, F and J), Kirre in blue (C, G and K) and SNS in yellow (D, H and L). (A-D) The <i>rst</i> allele <i>rst</i>^<i>1R34</i> shows no detectable Rst staining (A). (B) Hbs staining is reduced in the membranes surrounding the SOPs and is mainly detected in SOP membranes. Kirre (C) and SNS (D) show no significant pattern change. (E-H) <i>MZ1369-GAL4</i>><i>UAS-kirre-RNAi</i> shows no significant changes of the Rst (E) and Hbs pattern (F). Kirre immunoreactivity is hardly detectable (G) while SNS (H) is mildly reduced. (I-L) In the <i>rst</i>, <i>kirre</i> double RNAi hardly any Rst (I) and Kirre (K) can be detected. Enrichment of Hbs (J) around SOPs is reduced and the SOP arrangement as seen with SNS (L) is severely disrupted. (M) In the adult <i>MZ1369>rst-RNAi</i> shows a mild spacing phenotype with spacing ranging from 2 to 5 intervening bristles. (N) <i>MZ1369>kirre-RNAi</i> shows a mild spacing phenotype with spacing ranging from 1 to 7 (similar data was obtained for the mutant <i>rst</i>^<i>1R34</i>, data not shown). (O) <i>MZ1369>rst-RNAi</i>, <i>kirre-RNAi</i> shows a significant disturbance of the spacing of recurved bristles with 0 to 8 intervening cells. (P) <i>MZ1369-GAL4</i> misexpression of <i>rst</i> has a strong impact on the spacing of recurved bristles with spacing ranging from 0 to 13 intervening cells. Clustered recurved bristles are frequently observed and similarly long areas without any chemosensory bristles are seen. (Q) shows the quantitative analysis of the recurved bristle spacing as measured by the number of slender bristles between the recurved bristles. The distribution of <i>MZ1369>GFP</i> differs significantly from <i>MZ1369>rst-RNAi</i>, <i>kirre-RNAi</i> in the following spacing values: < = 1: p-value = 0.034, 5: p-value = 0.041. <i>MZ1369>rst</i> differs significantly in the following spacing values: < = 1: p-value = 0.0002, 3: p-value = 0.0002, > = 6: p-value = 0.0001. Scale bars correspond to 10μm in all images.</p