HEARTBEAT4D: An Open-source Toolbox for Turning 4D Cardiac CT into VR/AR.

Four-dimensional data sets are increasingly common in MRI and CT. While clinical visualization often focuses on individual temporal phases capturing the tissue(s) of interest, it may be possible to gain additional insight through exploring animated 3D reconstructions of physiological motion made possible by augmented or virtual reality representations of 4D patient imaging. Cardiac CT acquisitions can provide sufficient spatial resolution and temporal data to support advanced visualization, however, there are no open-source tools readily available to facilitate the transformation from raw medical images to dynamic and interactive augmented or virtual reality representations. To address this gap, we developed a workflow using free and open-source tools to process 4D cardiac CT imaging starting from raw DICOM data and ending with dynamic AR representations viewable on a phone, tablet, or computer. In addition to assembling the workflow using existing platforms (3D Slicer and Unity), we also contribute two new features: 1. custom software which can propagate a segmentation created for one cardiac phase to all others and export to surface files in a fully automated fashion, and 2. a user interface and linked code for the animation and interactive review of the surfaces in augmented reality. Validation of the surface-based areas demonstrated excellent correlation with radiologists' image-based areas (R > 0.99). While our tools were developed specifically for 4D cardiac CT, the open framework will allow it to serve as a blueprint for similar applications applied to 4D imaging of other tissues and using other modalities. We anticipate this and related workflows will be useful both clinically and for educational purposes.

In situ hybridization for the detection of hepatitis C virus RNA in human liver tissue.

In situ hybridization (ISH) enables visualization of specific nucleic acid in morphologically preserved cells and tissue sections. Detection of the HCV genomes in clinical specimens is useful for differential diagnosis, particularly between recurrent HCV infection and acute cellular rejection in transplant specimens. We optimized an ISH protocol that demonstrated sensitivity and specificity for detecting genomic and replicative form of HCV RNA in tissue biopsies. Digoxigenin (Dig)-labelled sense and anti-sense riboprobes were synthesized using a plasmid containing a fragment of the highly conserved HCV noncoding region as a template. The efficiency of the Dig-labelled riboprobes in detecting genomic and replicative-intermediate HCV RNA was analysed in 30 liver biopsies from patients infected or uninfected with HCV in a blinded study. A Huh7 cell line that stably replicates genome-length HCV RNA was developed to be used as a positive control. Negative control riboprobes were used in parallel to evaluate and control for background staining. The anti-sense probe detected HCV RNA in 20/21 specimens from HCV-infected liver tissues obtained from patients and in 0/9 samples from patients with non-HCV-related liver diseases, resulting in a sensitivity and specificity of 95% and 100%, respectively. HCV genomic RNA was variably distributed in tissue sections and was located primarily in the perinuclear regions in hepatocytes. Detection of HCV RNA by our optimized ISH protocol appears to be a sensitive and specific method when processing clinical specimens. It may also be revealing when exploring the pathophysiology of HCV infection by verifying the presence of viral genetic material within heptocytes and other cellular elements of diseased liver tissue. This methodology might also evaluate the response to antiviral therapies by demonstrating the absence or alteration of genetic material in clinical specimens from successfully treated patients.

Role of a cytotoxic enterotoxin in Aeromonas-mediated infections: development of transposon and isogenic mutants.

Transposon and marker exchange mutagenesis were used to evaluate the role of Aeromonas cytotoxic enterotoxin (Act) in the pathogenesis of diarrheal diseases and deep wound infections. The transposon mutants were generated by random insertion of Tn5-751 in the chromosomal DNA of a diarrheal isolate SSU of Aeromonas hydrophila. Some of the transposon mutants had dramatically reduced hemolytic and cytotoxic activities, and such mutants exhibited reduced virulence in mice compared to wild-type Aeromonas when injected intraperitoneally (i.p.). Southern blot data indicated that transposition in these mutants did not occur within the cytotoxic enterotoxin gene (act). The transcription of the act gene was affected drastically in the transposon mutants, as revealed by Northern blot analysis. The altered virulence of these transposon mutants was confirmed by developing isogenic mutants of the wild-type Aeromonas by using a suicide vector. In these mutants, the truncated act gene was integrated in place of a functionally active act gene. The culture filtrates from isogenic mutants were devoid of hemolytic, cytotoxic, and enterotoxic activities associated with Act. These filtrates caused no damage to mouse small intestinal epithelium, as determined by electron microscopy, whereas culture filtrates from wild-type Aeromonas caused complete destruction of the microvilli. The 50% lethal dose of these mutants in mice was 1.0 x 10(8) when injected i. p., compared to 3.0 x 10(5) for the wild-type Aeromonas. Reintegration of the native act gene in place of the truncated toxin gene in isogenic mutants resulted in complete restoration of Act's biological activity and virulence in mice. The animals injected with a sublethal dose of wild-type Aeromonas or the revertant, but not the isogenic mutant, had circulating toxin-specific neutralizing antibodies. Taken together, these studies clearly established a role for Act in the pathogenesis of Aeromonas-mediated infections.

Hyperproduction, purification, and mechanism of action of the cytotoxic enterotoxin produced by Aeromonas hydrophila.

A gene encoding the cytotoxic enterotoxin (Act) from Aeromonas hydrophila was hyperexpressed with the pET, pTRX, and pGEX vector systems. Maximum toxin yield was obtained with the pTRX vector. Approximately 40 to 60% of Act was in a soluble form with the pTRX and pET vector systems. The toxin protein was purified to homogeneity by a combination of ammonium sulfate precipitation and fast protein liquid chromatography-based column chromatographies, including hydrophobic, anion-exchange, sizing, and hydroxylapatite chromatographies. Purified mature toxin migrated as a 52-kDa polypeptide on a sodium dodecyl sulfate (SDS)polyacrylamide gel that reacted with Act-specific antibodies in immunoblots. The minimal amount of toxin needed to cause fluid secretion in rat ileal loops was 200 ng, and the 50% lethal dose for mice was 27.5 ng when injected intravenously. Binding of the toxin to erythrocytes was temperature dependent, with no binding occurring at 4 degrees C. However, at 37 degrees C the toxin bound to erythrocytes within 1 to 2 min. It was determined that the mechanism of action of the toxin involved the formation of pores in erythrocyte membranes, and the diameter of the pores was estimated to be 1.14 to 2.8 nm, as determined by the use of saccharides of different sizes and by electron microscopy. Calcium chloride prevented lysis of erythrocytes by the toxin; however, it did not affect the binding and pore-forming capabilities of the toxin. A dose-dependent reduction in hemoglobin release from erythrocytes was observed when Act was preincubated with cholesterol, but not with myristylated cholesterol. With 14C-labeled cholesterol and gel filtration, the binding of cholesterol to Act was demonstrated. None of the other phospholipids and glycolipids tested reduced the hemolytic activity of Act. The toxin also appeared to undergo aggregation when preincubated with cholesterol, as determined by SDS-polyacrylamide gel electorphoresis. As a result of this aggregation, Act's capacity to form pores in the erythrocyte membrane was inhibited.

Amino-acid residues involved in biological functions of the cytolytic enterotoxin from Aeromonas hydrophila.

Some amino acid (aa) residues within the cytolytic enterotoxin (Act) of Aeromonas hydrophila essential for biological activity were identified. Act is a 52-kDa polypeptide, possessing hemolytic, cytotoxic and enterotoxic activities. By deletion analysis, generation of anti-peptide Ab, and site-directed mutagenesis we showed that two regions in Act (aa 245-274 and 361-405) were very important for biological functions. As shown by competitive inhibition assays, peptide 2 (aa 245-274) blocked cytotoxic activity of Act, and aa Tyr256, Trp270 and Gly274 were essential for cytotoxicity. Within peptide 3 (aa 361-405), Trp394 and Trp396 were important for biological activities. Mutations in other regions of the toxin (e.g., Gly169, Asp170, Gly171, Trp172, Asn177,178, Asp179 and His144,209,355) also decreased biological activity. The reactivity of these mutant toxins with Ab in immunoblots was not altered. Data reported in this study suggested the role of some aa residues in biological function(s) of Act.