Viral Bcl-2-Mediated Evasion of Autophagy Aids Chronic Infection of γHerpesvirus 68

γ-herpesviruses (γHVs) have developed an interaction with their hosts wherein they establish a life-long persistent infection and are associated with the onset of various malignancies. One critical virulence factor involved in the persistency of murine γ-herpesvirus 68 (γHV68) is the viral homolog of the Bcl-2 protein (vBcl-2), which has been implicated to counteract both host apoptotic responses and autophagy pathway. However, the relative significance of the two activities of vBcl-2 in viral persistent infection has yet to be elucidated. Here, by characterizing a series of loss-of-function mutants of vBcl-2, we have distinguished the vBcl-2-mediated antagonism of autophagy from the vBcl-2-mediated inhibition of apoptosis in vitro and in vivo. A mutant γHV68 virus lacking the anti-autophagic activity of vBcl-2 demonstrates an impaired ability to maintain chronic infections in mice, whereas a mutant virus lacking the anti-apoptotic activity of vBcl-2 establishes chronic infections as efficiently as the wild-type virus but displays a compromised ability for ex vivo reactivation. Thus, the vBcl-2-mediated antagonism of host autophagy constitutes a novel mechanism by which γHVs confer persistent infections, further underscoring the importance of autophagy as a critical host determinant in the in vivo latency of γ-herpesviruses.     

Proliferating cell nuclear antigen (PCNA) as a proliferative marker during embryonic and adult zebrafish hematopoiesis

We investigated the expression of proliferative cell nuclear antigen (PCNA) in zebrafish to delineate the proliferative hematopoietic component during adult and embryonic hematopoiesis. Immunostaining for PCNA and enhanced green fluorescence protein (eGFP) was performed in wild-type and fli1-eGFP (endothelial marker) and gata1-eGFP (erythroid cell marker) transgenic fish. Expression of PCNA mRNA was examined in wild-type and chordin morphant embryos. In adult zebrafish kidney, the renal tubules are surrounded by endothelial cells and it is separated into hematopoietic and excretory compartments. PCNA was expressed in hematopoietic progenitor cells but not in mature neutrophils, eosinophils or erythroid cells. Some PCNA+ cells are scattered in the hematopoietic compartment of the kidney while others are closely associated with renal tubular cells. PCNA was also expressed in spermatogonial stem cells and intestine crypts, consistent with its role in cell proliferation and DNA synthesis. In embryos, PCNA is expressed in the brain, spinal cord and intermediate cell mass (ICM) at 24 h-post fertilization. In chordin morphants, PCNA is significantly upregulated in the expanded ICM. Therefore, PCNA can be used to mark cell proliferation in zebrafish hematopoietic tissues and to identify a population of progenitor cells whose significance would have to be further investigated.     

Influence of the type of imaging on the delineation process during the treatment planning

AimThe aim of this study was to compare the intra- and interobserver contouring variability for structures with density of organ at risk in two types of tomography: kilovoltage computed tomography (KVCT) versus megavoltage computed tomography (MVCT). The intra- and interobserver differences were examined on both types of tomography for structures which simulate human tissue or organs.Materials and methodsSix structures with density of the liver, bone, trachea, lung, soft tissue and muscle were created and used. For the measurements, the special water phantom with all structures was designed. To evaluate interobserver variability, five observers delineated the structures in both types of computed tomography (CT).ResultsIntraobserver variability was in the range of 1–14% and was the largest for the liver. The observers segmented larger volumes on MVCT compared with KVCT for the trachea (79.56 ccm vs.74.91 ccm), lung (87.61 vs. 82.50), soft tissue (154.24 vs. 145.47) and muscle (164.01 vs. 157.89). For the liver (98.13 vs. 99.38) and bone (51.86 vs. 67.97), the volume on MVCT was smaller than KVCT. The statistically significant differences between observers were observed for structures with density of the liver, bone and soft tissue on KVCT and for the liver, lung and soft tissue on MVCT. For the structures with density of the trachea and muscles, there were no significant differences for both types of tomography.ConclusionsDuring the contouring process the interobserver and intraobserver contouring uncertainty was larger on MVCT, especially for structures with HU near 80, compared with KVCT.     

Multienergetic verification of dynamic wedge angles in medical accelerators using multichannel linear array

Background

The aim of the modern radiotherapy is to get a homogenous dose distribution in PTV, which is obtained by using for example physical or dynamic wedges. The using of a physical wedge has provided such isodose distributions but their use resulted in detrimental dosimetric consequences, for example beam hardening effects and practical consequences of filter handling or possible misalignment. Linear accelerators are now equipped with collimator jaws systems and controlled by modern computers and it is possible to generate wedge shaped isodose distributions dynamically. Because of a more comfortable use of a dynamic wedge, there are alternatives to the standard physical wedge. During the treatment, different segments of the treatment field can be exposed to the primary beam at different intervals of time. This process of shrinking the field while modulating the collimator jaw velocity and dose rate creates the desired wedge-shaped isodose gradient across the treatment field. Dynamic wedges can replace physical wedges but they need more precise dosimetry and quality control procedures.

Aim

The aim of this study was to perform a multienergetic verification of dynamic wedge angles using the multichannel detector PTW LA48 linear array.

Material and methods

The measurements of angle value of dynamic wedges were performed for Clinac 2300 C/D accelerators (Varian). The accelerator was equipped with the EDW option for 6 MV and 15 MV photon beams. In this case, 7 wedge angle values were used: 10°, 15°, 20°, 25°, 30°, 45° and 60°. The dynamic wedges are realized by continuous movement of one collimator jaw. The field size is gradually reduced until the collimator is almost completely closed or the field increases, while the beam is on. The measurements were divided in two steps: in the first step, the dynamic wedges were verified with the recommended values and in the second step there the planned and measured angles of dynamic wedges were compared. Measurements were made by means of LA48 linear array of ionization chambers (PTW). The results of the measurements were compared with the reference profile produced by the treatment planning system ECLIPSE 8.5 (Varian).

Results

The results showed differences between measured and calculated angle of dynamic wedges. The differences were observed for both energies in the case of a small angle value. For energies 6 MV and 15 MV, almost all percentage difference between the measured and calculated profile was lower than 5%. The biggest difference was observed in the first step of measurements when the angle of Dynamic Wedge was verified. The comparison between the planned and measured angle value of Dynamic Wedge showed the difference between 0.1% and 4.5%.The difference for 6 MV for the angle value of 10° in orientation IN was 1.1% and for energy 15 MV in the same case the difference was 3.8%. Thinner wedges exhibit less difference.

Conclusion

It is necessary to provide comprehensive quality control procedure for enhanced dynamic wedges. Verification measurements should be an obligatory procedure in the recommendation for the testing of medical accelerators. These results are the preliminary results to provide measurements in other Polish Cancer Centres.     

A search for factors specifying tonotopy implicates DNER in hair-cell development in the chick's cochlea

The accurate perception of sound frequency by vertebrates relies upon the tuning of hair cells, which are arranged along auditory organs according to frequency. This arrangement, which is termed a tonotopic gradient, results from the coordination of many cellular and extracellular features. Seeking the mechanisms that orchestrate those features and govern the tonotopic gradient, we used expression microarrays to identify genes differentially expressed between the high- and low-frequency cochlear regions of the chick (Gallus gallus). Of the three signaling systems that were represented extensively in the results, we focused on the notch pathway and particularly on DNER, a putative notch ligand, and PTPζ, a receptor phosphatase that controls DNER trafficking. Immunohistochemistry confirmed that both proteins are expressed more strongly in hair cells at the cochlear apex than in those at the base. At the apical surface of each hair cell, the proteins display polarized, mutually exclusive localization patterns. Using morpholinos to decrease the expression of DNER or PTPζ as well as a retroviral vector to overexpress DNER, we observed disturbances of hair-bundle morphology and orientation. Our results suggest a role for DNER and PTPζ in hair-cell development and possibly in the specification of tonotopy.