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Image-based tracking for motion compensation is an important topic in image-guided interventions, as it enables physicians to operate in a less complex space. In this paper, we propose an automatic motion compensation scheme to boost image guidence power in transcatheter aortic valve implantation (TAVI). The proposed tracking algorithm automatically discovers reliable regions that correlate strongly with the target. These discovered regions can assist to estimate target motion under severe occlusion, even if target tracker fails. We evaluate the proposed method for pigtail tracking during TAVI. We obtain significant improvement (12 %) over the baseline in a clinical dataset. Calcification regions are automatically discovered during tracking, which would aid TAVI processes. In this work, we open a new paradigm to provide dynamic real-time guidance for TAVI without user interventions, specially in case of severe occlusion where conventional tracking methods are challenged.

The pigtail catheter is a type of catheter inserted into the human body during interventional surgeries such as the transcatheter aortic valve implantation (TAVI). The catheter is characterized by a tightly curled end in order to remain attached to a valve pocket during the intervention, and it is used to inject contrast agent for the visualization of the vessel in fluoroscopy. Image-based detection of this catheter is used during TAVI, in order to overlay a model of the aorta and enhance visibility during the surgery. Due to the different possible projection angles in fluoroscopy, the pigtail tip can appear in a variety of different shapes spanning from pure circular to ellipsoid or even line. Furthermore, the appearance of the catheter tip is radically altered when the contrast agent is injected during the intervention or when it is occluded by other devices. All these factors make the robust real-time detection and tracking of the pigtail catheter a challenging task. To address these challenges, this paper proposes a new tree-structured, hierarchical detection scheme, based on a shape categorization of the pigtail catheter tip, and a combination of novel Haar features. The proposed framework demonstrates improved detection performance, through a validation on a data set consisting of 272 sequences with more than 20,000 images. The detection framework presented in this paper is not limited to pigtail catheter detection, but it can also be applied successfully to any other shape-varying object with similar characteristics.

Presentation of detailed anatomical structures via 3-D models helps navigation and deployment of the prosthetic valve in TAVI procedures. Fast and automatic contrast detection in the aortic root on X-ray images facilitates a seamless workflow to utilize the 3-D models by triggering 2-D/3-D registration automatically when motion compensation is needed. In this paper, we propose a novel method for automatic detection of contrast injection in the aortic root on fluoroscopic and angiographic sequences. The proposed method is based on histogram analysis and likelihood ratio test, and is robust to variations in the background, the density and volume of the injected contrast, and the size of the aorta. The performance of the proposed algorithm was evaluated on 26 sequences from 5 patients and 3 clinical sites, with 16 out of 17 contrast injections correctly detected and zero false detections. The proposed method is of general form and can be extended for detection of contrast injection in other organs and/or applications.

With little or no negative impacton the activity of small interfering RNAs (siRNAs), regardless of the number of modifications or the positions within the strand, the 2′‐deoxy‐2′‐fluoro (2′‐F) modification is unique. Furthermore, the 2′‐F‐modified siRNA (see crystal structure) was thermodynamically more stable and more nuclease‐resistant than the parent siRNA, and produced no immunostimulatory response.

Eine vorteilhafte Variante: Die Modifikation 2′‐Desoxy‐2′‐fluor (2′‐F) ist insofern einzigartig, als sie die Aktivität kleiner interferierender RNAs (siRNAs) nicht oder nur sehr gering beeinträchtigt, und zwar unabhängig von der Zahl an Modifikationen oder der Position innerhalb des Strangs. Die 2′‐F‐modifizierte siRNA (siehe Kristallstruktur) war außerdem thermodynamisch stabiler und nucleaseresistenter als die Stamm‐siRNA, und sie erzeugte keine immunstimulierende Antwort.

The translocation t(8;21) yields the leukemic fusion gene AML1/MTG8 and is associated with 10%-15% of all de novo cases of acute myeloid leukemia. We demonstrate the efficient and specific suppression of AML1/MTG8 by small interfering RNAs (siRNAs) in the human leukemic cell lines Kasumi-1 and SKNO-1. siRNAs targeted against the fusion site of the AML1/MTG8 mRNA reduce the levels of AML1/MTG8 without affecting the amount of wild-type AML1. These data argue against a transitive RNA interference mechanism potentially induced by siRNAs in such leukemic cells. Depletion of AML1/MTG8 correlates with an increased susceptibility of both Kasumi-1 and SKNO-1 cells to tumor growth factor β1(TGFβ1)/vitamin D3–induced differentiation, leading to increased expression of CD11b, macrophage colony-stimulating factor (M-CSF) receptor, and C/EBPα (CAAT/enhancer binding protein). Moreover, siRNA-mediated AML1/MTG8 suppression results in changes in cell shape and, in combination with TGFβ1/vitamin D3, severely reduces clonogenicity of Kasumi-1 cells. These results suggest an important role for AML1/MTG8 in preventing differentiation, thereby propagating leukemic blast cells. Therefore, siRNAs are promising tools for a functional analysis of AML1/MTG8 and may be used in a molecularly defined therapeutic approach for t(8;21)-positive leukemia.

The translocation t(8;21) yields the leukemic fusion gene AML1/MTG8 and is associated with 10%-15% of all de novo cases of acute myeloid leukemia. We demonstrate the efficient and specific suppression of AML1/MTG8 by small interfering RNAs (siRNAs) in the human leukemic cell lines Kasumi-1 and SKNO-1. siRNAs targeted against the fusion site of the AML1/MTG8 mRNA reduce the levels of AML1/MTG8 without affecting the amount of wild-type AML1. These data argue against a transitive RNA interference mechanism potentially induced by siRNAs in such leukemic cells. Depletion of AML1/MTG8 correlates with an increased susceptibility of both Kasumi-1 and SKNO-1 cells to tumor growth factor β1 (TGFβ1)/vitamin D3–induced differentiation, leading to increased expression of CD11b, macrophage colony-stimulating factor (M-CSF) receptor, and C/EBPα (CAAT/enhancer binding protein). Moreover, siRNA-mediated AML1/MTG8 suppression results in changes in cell shape and, in combination with TGFβ1/vitamin D3, severely reduces clonogenicity of Kasumi-1 cells. These results suggest an important role for AML1/MTG8 in preventing differentiation, thereby propagating leukemic blast cells. Therefore, siRNAs are promising tools for a functional analysis of AML1/MTG8 and may be used in a molecularly defined therapeutic approach for t(8;21)-positive leukemia.

Background. Tracheal reconstruction is the treatment of choice in nontumorous tracheal stenoses, but recurrences and concomitant medical conditions limit this approach. We investigated the outcome after balloon dilatation and silicone stent implantation.

Abstract: Enterochromaffin-like (ECL) cells regulate gastric acid secretion through vesicular release of histamine. Until now, the molecular machinery of human ECL cells involved in the formation and release of vesicles is largely unknown. We analyzed tissue samples obtained from normal human gastric mucosa (n=4) and ECLomas (n=5) immunohistochemically using the APAAP method or double immunofluorescence confocal laser microscopy. Human pheochromocytomas (n=5) were investigated in parallel and compared to ECL cells. Secretory pathways were characterized using antibodies specific for marker proteins of large dense-core vesicles (LDCVs; islet cell antigen 512, chromogranin A, pancreastatin, and vesicular monoamine transporter 2) and small synaptic vesicle (SSV) analogues (synaptophysin). Tissues were also analyzed for expression of the peptide hormone processing enzymes, carboxypeptidase E and prohormone convertase 1, as well as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, 25-kDa synaptosome-associated protein (SNAP25), syntaxin, and synaptobrevin. Immunoreactivity for markers of LDCVs and SSV analogues were detected in normal ECL cells and ECLomas. Both tissues also showed expression of carboxypeptidase E and prohormone convertase 1. Analysis of vesicular SNARE (v-SNARE) and target membrane SNARE (t-SNARE) proteins revealed the presence of SNAP25, syntaxin, and synaptobrevin in normal and neoplastic ECL cells. Our data suggest that ECL cells possess the two vesicle types of regulated neuroendocrine secretory pathways, LDCVs and SSV analogues. Since ECL cells also contain typical SNARE proteins, the molecular machinery underlying secretory processes in this cell type appears to be identical to the secretory apparatus of neuroendocrine cells and neurons. In addition, our findings suggest that the secretory apparatus of ECL cells is maintained during neoplastic transformation.

Mishra: CRISPR Therapeutics: Employment. Chou:CRISPR Therapeutics: Employment. Lin:CRISPR Therapeutics: Employment. Paik:CRISPR Therapeutics: Employment. Zhang:CRISPR Therapeutics: Employment, Equity Ownership. Liang:CRISPR Therapeutics: Employment. Tomkinson:CRISPR Therapeutics: Employment. Pettiglio:CRISPR Therapeutics: Employment. Sanginario:CRISPR Therapeutics: Employment. Allen:CRISPR Therapeutics: Employment. Cradick:CRISPR Therapeutics: Employment. John:CRISPR Therapeutics: Employment. Chakraborty:CRISPR Therapeutics: Employment. Cowan:CRISPR Therapeutics: Employment, Equity Ownership. Novak:CRISPR Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Lundberg:CRISPR Therapeutics: Employment, Equity Ownership.

Fetal hemoglobin (HbF) is abundant in the late stage fetus and newborns, but is progressively lost over the first 6 months of life as the genes encoding the g-globin subunit of HbF are repressed. HbF expression can provide clinical benefit to patients with deficient or defective b-globin, for example in β-thalassemia and Sickle Cell Disease (SCD), respectively. CRISPR-Cas9 technology offers a unique treatment modality that can be used to ex vivoedit regulatory DNA sequences in patient CD34+hematopoietic stem and progenitor cells (HSPC) containing hematopoietic stem and progenitor cells in order to upregulate HbF. Reinfusion of edited CD34+HSPC would be expected to lead to the production of erythrocytes expressing high levels of HbF, and amelioration of disease.

This unit provides information how to use short interfering RNA (siRNA) for sequence specific gene silencing in mammalian cells. Several ways for siRNA generation and optimisation, as well as recommendations for cell transfection are presented.

We constructed plasmids encoding the sequences for the bZip modules of c-Jun and c-Fos which could then be expressed as soluble proteins in Escherichia coli. The purified bZip modules were tested for their binding capacities of synthetic oligonucleotides containing either TRE or CRE recognition sites in electrophoretic mobility shift assays and circular dichroism (CD). Electrophoretic mobility shift assays showed that bZip Jun homodimers and bZip Jun/Fos heterodimers bind a collagenase-like TRE (CTGACTCAT) with dissociation constants of respectively 1.4 × 10−7 M and 5 × 10−8 M. As reported earlier [Patel et al. (1990) Nature 347, 572–575], DNA binding induces a marked change of the protein structure. However, we found that the DNA also undergoes a conformational change. This is most clearly seen with small oligonucleotides of 13 or 14 bp harboring respectively a TRE (TGACTCA) or a CRE (TGACGTCA) sequence. In this case, the positive DNA CD signal at 280 nm increases almost two-fold with a concomitant blue-shift of 3–4 nm. Within experimental error the same spectral changes are observed for TRE and CRE containing DNA fragments. The spectral changes observed with a non-specific DNA fragment are weaker and the signal of free DNA is recovered upon addition of much smaller salt concentrations than required for a specific DNA fragment. Surprisingly the spectral changes induced by Jun/Jun homodimers are not identical to those induced by Jun/Fos hetero-dimers. However, in both cases the increase of the positive CD band and the concomitant blue shift would be compatible with a B to A-transition of part of the binding site or a DNA conformation intermediate between the canonical A and B structures.

The mammalian Fos and Fos-related proteins are unable to form homodimers and to bind DNA in the absence of a second protein, like c-Jun for example. In order to study the implications of hydrophobic point mutations in the c-Fos leucine zipper on DNA binding of the entire c-Fos protein, we have constructed and purified a set of Fos mutant proteins harboring one or several isoleucine or leucine residues in the five Fos zipper a positions. We show that a single point mutation in the hydrophobic interface of the c-Fos leucine zipper enables the c-Fos mutant protein to bind specifically to an oligonucleotide duplex harboring the TRE consensus sequence TGA(C/G)TCA. This point mutation (Thr169→Ile) is situated in the a position of the second heptade (a2) of the Fos zipper. The introduction of additional isoleucine residues in the other a positions progressively increases the DNA binding affinity of these homodimerizing Fos zipper variants. Heterodimerization of these c-Fos variants with c-Jun reveals a complex behaviour, in that the DNA binding affinity of these heterodimers does not simply increase with the number of isoleucine side chains in position a. For example, a c-Fos variant harboring a wild-type Thr in position a1 and Ile in the four other a positions (c-Fos4I) interacts more tightly with c-Jun than a variant harboring Ile in all five a positions (c-Fos5I). The same holds true for the corresponding leucine variants, suggesting that the wild-type a1 residue of the Fos zipper (Thr162) is thermodynamically relevant for Fos-Jun heterodimer formation and DNA binding. The c-Fos4I variant forms heterodimers with c-Jun slightly better than the wild-type zipper protein, suggesting that the driving force for Fos-Jun heterodimerization is not the simple fact that the Fos protein is unable to form homodimers. These c-Fos variants were further tested for their transactivation properties in F9 and NIH3T3 cells. At low expression levels the most efficiently homodimerizing variant (c-Fos5I) activates transcription in F9 cells about 6-fold. However part of this activation may be due to the formation of heterodimers with a member of the Jun family (like JunD for example), since a wild-type c-Fos expression vector confers a 3-fold activation under these conditions. In the case of the homodimerizing c-Fos variants however, this activation is abrogated at higher expression levels due to a strong inhibition of basal transcription activity.