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Mathematical models of virus dynamics have not previously acknowledged spatial resolution at the intracellular level despite substantial arguments that favor the consideration of intracellular spatial dependence. The replication of the hepatitis C virus (HCV) viral RNA (vRNA) occurs within special replication complexes formed from membranes derived from endoplasmatic reticulum (ER). These regions, termed membranous webs, are generated primarily through specific interactions between nonstructural virus-encoded proteins (NSPs) and host cellular factors. The NSPs are responsible for the replication of the vRNA and their movement is restricted to the ER surface. Therefore, in this study we developed fully spatio-temporal resolved models of the vRNA replication cycle of HCV. Our simulations are performed upon realistic reconstructed cell structures—namely the ER surface and the membranous webs—based on data derived from immunostained cells replicating HCV vRNA. We visualized 3D simulations that reproduced dynamics resulting from interplay of the different components of our models (vRNA, NSPs, and a host factor), and we present an evaluation of the concentrations for the components within different regions of the cell. Thus far, our model is restricted to an internal portion of a hepatocyte and is qualitative more than quantitative. For a quantitative adaption to complete cells, various additional parameters will have to be determined through further in vitro cell biology experiments, which can be stimulated by the results deccribed in the present study.
Variants resistant to compounds specifically targeting HCV are observed in clinical trials. A multi-variant viral dynamic model was developed to quantify the evolution and in vivo fitness of variants in subjects dosed with monotherapy of an HCV protease inhibitor, telaprevir. Variant fitness was estimated using a model in which variants were selected by competition for shared limited replication space. Fitness was represented in the absence of telaprevir by different variant production rate constants and in the presence of telaprevir by additional antiviral blockage by telaprevir. Model parameters, including rate constants for viral production, clearance, and effective telaprevir concentration, were estimated from 1) plasma HCV RNA levels of subjects before, during, and after dosing, 2) post-dosing prevalence of plasma variants from subjects, and 3) sensitivity of variants to telaprevir in the HCV replicon. The model provided a good fit to plasma HCV RNA levels observed both during and after telaprevir dosing, as well as to variant prevalence observed after telaprevir dosing. After an initial sharp decline in HCV RNA levels during dosing with telaprevir, HCV RNA levels increased in some subjects. The model predicted this increase to be caused by pre-existing variants with sufficient fitness to expand once available replication space increased due to rapid clearance of wild-type (WT) virus. The average replicative fitness estimates in the absence of telaprevir ranged from 1% to 68% of WT fitness. Compared to the relative fitness method, the in vivo estimates from the viral dynamic model corresponded more closely to in vitro replicon data, as well as to qualitative behaviors observed in both on-dosing and long-term post-dosing clinical data. The modeling fitness estimates were robust in sensitivity analyses in which the restoration dynamics of replication space and assumptions of HCV mutation rates were varied.
Background: The development of robotic systems has provided an alternative to frame-based stereotactic procedures. The aim of this experimental phantom study was to compare the mechanical accuracy of the Robotic Surgery Assistant (ROSA) and the Leksell stereotactic frame by reducing clinical and procedural factors to a minimum.
Methods: To precisely compare mechanical accuracy, a stereotactic system was chosen as reference for both methods. A thin layer CT scan with an acrylic phantom fixed to the frame and a localizer enabling the software to recognize the coordinate system was performed. For each of the five phantom targets, two different trajectories were planned, resulting in 10 trajectories. A series of five repetitions was performed, each time based on a new CT scan. Hence, 50 trajectories were analyzed for each method. X-rays of the final cannula position were fused with the planning data. The coordinates of the target point and the endpoint of the robot- or frame-guided probe were visually determined using the robotic software. The target point error (TPE) was calculated applying the Euclidian distance. The depth deviation along the trajectory and the lateral deviation were separately calculated.
Results: Robotics was significantly more accurate, with an arithmetic TPE mean of 0.53 mm (95% CI 0.41–0.55 mm) compared to 0.72 mm (95% CI 0.63–0.8 mm) in stereotaxy (p < 0.05). In robotics, the mean depth deviation along the trajectory was −0.22 mm (95% CI −0.25 to −0.14 mm). The mean lateral deviation was 0.43 mm (95% CI 0.32–0.49 mm). In frame-based stereotaxy, the mean depth deviation amounted to −0.20 mm (95% CI −0.26 to −0.14 mm), the mean lateral deviation to 0.65 mm (95% CI 0.55–0.74 mm).
Conclusion: Both the robotic and frame-based approach proved accurate. The robotic procedure showed significantly higher accuracy. For both methods, procedural factors occurring during surgery might have a more relevant impact on overall accuracy.
Background: Acoustic Radiation Force Impulse (ARFI)-imaging is an ultrasound-based elastography method enabling quantitative measurement of tissue stiffness. The aim of the present study was to evaluate sensitivity and specificity of ARFI-imaging for differentiation of thyroid nodules and to compare it to the well evaluated qualitative real-time elastography (RTE).
Methods: ARFI-imaging involves the mechanical excitation of tissue using acoustic pulses to generate localized displacements resulting in shear-wave propagation which is tracked using correlation-based methods and recorded in m/s. Inclusion criteria were: nodules $5 mm, and cytological/histological assessment. All patients received conventional ultrasound, real-time elastography (RTE) and ARFI-imaging.
Results: One-hundred-fifty-eight nodules in 138 patients were available for analysis. One-hundred-thirty-seven nodules were benign on cytology/histology, and twenty-one nodules were malignant. The median velocity of ARFI-imaging in the healthy thyroid tissue, as well as in benign and malignant thyroid nodules was 1.76 m/s, 1.90 m/s, and 2.69 m/s, respectively. While no significant difference in median velocity was found between healthy thyroid tissue and benign thyroid nodules, a significant difference was found between malignant thyroid nodules on the one hand and healthy thyroid tissue (p = 0.0019) or benign thyroid nodules (p = 0.0039) on the other hand. No significant difference of diagnostic accuracy for the diagnosis of malignant thyroid nodules was found between RTE and ARFI-imaging (0.74 vs. 0.69, p = 0.54). The combination of RTE with ARFI did not improve diagnostic accuracy.
Conclusions: ARFI can be used as an additional tool in the diagnostic work up of thyroid nodules with high negative predictive value and comparable results to RTE.
Acute cholecystitis – a cohort study in a real-world clinical setting (REWO study, NCT02796443)
(2018)
Background: For decades, the optimal timing of surgery for acute cholecystitis has been controversial. Recent meta-analyses and population-based studies favor early surgery. One recent large randomized trial has demonstrated that a delayed approach increases morbidity and cost compared to early surgery within 24 hours of hospital admission. Since cases of severe cholecystitis were excluded from this trial, we argue that these results do not reflect real-world clinical situations. From our point of view, these results were in contrast to the clinical experience with our patients; so, we decided to analyze critically all our patients with the null hypothesis that the patients treated with a delayed cholecystectomy after an acute cholecystitis have a similar or even better outcome than those treated with an early operative approach.
Patients and methods: We retrospectively analyzed clinical data from all patients with cholecystectomies in the period between January 2006 and September 2015. A total of 1,723 patients were categorized into four groups: early (n=138): urgent surgery of patients with acute cholecystitis within the first 72 hours of the onset of symptoms; intermediate (n=297): surgery of patients with acute cholecystitis within an average of 10 days after the onset of symptoms; delayed (n=427): initial non-surgical treatment of acute cholecystitis with surgery performed within 6–12 weeks of the onset of symptoms; and elective (n=868): cholecystectomy within a symptom-free interval of choice in patients with symptomatic cholecystolithiasis without signs of acute cholecystitis.
Results: In a real-world scenario, early/intermediate cholecystectomy in acute cholecystitis was associated with a significant increase in morbidity and mortality (Clavien–Dindo score) compared to a delayed approach with surgery performed 6–12 weeks after the onset of symptoms. The adjusted linear rank statistics showed a decrease in the complication score with values of 2.29 in the early group, 0.48 in the intermediate group, –0.26 in the delayed group and –2.12 in the elective group. The results translate into a continuous decrease of the complication score from early over intermediate and delayed to the elective group.
Conclusion: These results demonstrate that delayed cholecystectomy can be performed safely. In cases with severe cholecystitis, early and/or intermediate approaches still have a relatively high risk of morbidity and mortality.
Hepatitis C virus (HCV) naturally infects only humans and chimpanzees. The determinants responsible for this narrow species tropism are not well defined. Virus cell entry involves human scavenger receptor class B type I (SR-BI), CD81, claudin-1 and occludin. Among these, at least CD81 and occludin are utilized in a highly species-specific fashion, thus contributing to the narrow host range of HCV. We adapted HCV to mouse CD81 and identified three envelope glycoprotein mutations which together enhance infection of cells with mouse or other rodent receptors approximately 100-fold. These mutations enhanced interaction with human CD81 and increased exposure of the binding site for CD81 on the surface of virus particles. These changes were accompanied by augmented susceptibility of adapted HCV to neutralization by E2-specific antibodies indicative of major conformational changes of virus-resident E1/E2-complexes. Neutralization with CD81, SR-BI- and claudin-1-specific antibodies and knock down of occludin expression by siRNAs indicate that the adapted virus remains dependent on these host factors but apparently utilizes CD81, SR-BI and occludin with increased efficiency. Importantly, adapted E1/E2 complexes mediate HCV cell entry into mouse cells in the absence of human entry factors. These results further our knowledge of HCV receptor interactions and indicate that three glycoprotein mutations are sufficient to overcome the species-specific restriction of HCV cell entry into mouse cells. Moreover, these findings should contribute to the development of an immunocompetent small animal model fully permissive to HCV.
The hepatitis C virus (HCV) RNA replication cycle is a dynamic intracellular process occurring in three-dimensional space (3D), which is difficult both to capture experimentally and to visualize conceptually. HCV-generated replication factories are housed within virus-induced intracellular structures termed membranous webs (MW), which are derived from the Endoplasmatic Reticulum (ER). Recently, we published 3D spatiotemporal resolved diffusion–reaction models of the HCV RNA replication cycle by means of surface partial differential equation (sPDE) descriptions. We distinguished between the basic components of the HCV RNA replication cycle, namely HCV RNA, non-structural viral proteins (NSPs), and a host factor. In particular, we evaluated the sPDE models upon realistic reconstructed intracellular compartments (ER/MW). In this paper, we propose a significant extension of the model based upon two additional parameters: different aggregate states of HCV RNA and NSPs, and population dynamics inspired diffusion and reaction coefficients instead of multilinear ones. The combination of both aspects enables realistic modeling of viral replication at all scales. Specifically, we describe a replication complex state consisting of HCV RNA together with a defined amount of NSPs. As a result of the combination of spatial resolution and different aggregate states, the new model mimics a cis requirement for HCV RNA replication. We used heuristic parameters for our simulations, which were run only on a subsection of the ER. Nevertheless, this was sufficient to allow the fitting of core aspects of virus reproduction, at least qualitatively. Our findings should help stimulate new model approaches and experimental directions for virology.
Background/aims: Hepatocellular carcinoma (HCC) is a leading indication for liver transplantation (LT) worldwide. Early identification of patients at risk for HCC recurrence is of paramount importance since early treatment of recurrent HCC after LT may be associated with increased survival. We evaluated incidence of and predictors for HCC recurrence, with a focus on the course of AFP levels.
Methods: We performed a retrospective, single-center study of 99 HCC patients who underwent LT between January 28th, 1997 and May 11th, 2016. A multi-stage proportional hazards model with three stages was used to evaluate potential predictive markers, both by univariate and multivariable analysis, for influences on 1) recurrence after transplantation, 2) mortality without HCC recurrence, and 3) mortality after recurrence.
Results: 19/99 HCC patients showed recurrence after LT. Waiting time was not associated with overall HCC recurrence (HR = 1, p = 0.979). Similarly, waiting time did not affect mortality in LT recipients both with (HR = 0.97, p = 0.282) or without (HR = 0.99, p = 0.685) HCC recurrence. Log10-transformed AFP values at the time of LT (HR 1.75, p = 0.023) as well as after LT (HR 2.07, p = 0.037) were significantly associated with recurrence. Median survival in patients with a ratio (AFP at recurrence divided by AFP 3 months before recurrence) of 0.5 was greater than 70 months, as compared to a median of only 8 months in patients with a ratio of 5.
Conclusion: A rise in AFP levels rather than an absolute threshold could help to identify patients at short-term risk for HCC recurrence post LT, which may allow intensification of the surveillance strategy on an individualized basis.
Estimating the age of the developmental stages of the blow fly Calliphora vicina (Diptera: Calliphoridae) is of forensic relevance for the determination of the minimum post-mortem interval (PMImin). Fly eggs and larvae can be aged using anatomical and morphological characters and their modification during development. However, such methods can only hardly be applied for aging fly pupae. Previous study described age estimation of C. vicina pupae using gene expression, but just when reared at constant temperatures, but fluctuating temperatures represent a more realistic scenario at a crime scene. Therefore, age-dependent gene expression of C. vicina pupae were compared at 3 fluctuating and 3 constant temperatures, the latter representing the mean values of the fluctuating profiles. The chosen marker genes showed uniform expression patterns during metamorphosis of C. vicina pupae bred at different temperature conditions (constant or fluctuating) but the same mean temperature (e.g. constant 10 °C vs. fluctuating 5–15 °C). We present an R-based statistical tool, which enables estimation of the age of the examined pupa based on the analysed gene expression data.
Altered mucosal immune response after acute lung injury in a murine model of Ataxia Telangiectasia
(2014)
Background: Ataxia telangiectasia (A-T) is a rare but devastating and progressive disorder characterized by cerebellar dysfunction, lymphoreticular malignancies and recurrent sinopulmonary infections. In A-T, disease of the respiratory system causes significant morbidity and is a frequent cause of death.
Methods: We used a self-limited murine model of hydrochloric acid-induced acute lung injury (ALI) to determine the inflammatory answer due to mucosal injury in Atm (A-T mutated)- deficient mice (Atm−/−).
Results: ATM deficiency increased peak lung inflammation as demonstrated by bronchoalveolar lavage fluid (BALF) neutrophils and lymphocytes and increased levels of BALF pro-inflammatory cytokines (e.g. IL-6, TNF). Furthermore, bronchial epithelial damage after ALI was increased in Atm−/− mice. ATM deficiency increased airway resistance and tissue compliance before ALI was performed.
Conclusions: Together, these findings indicate that ATM plays a key role in inflammatory response after airway mucosal injury.