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Steroid bile acid fused tetrazoles were screened for ability to inhibit aldo–keto reductase 1C3 (AKR1C3), a target for cancer treatment. The X-ray structure of AKR1C3 in complex with a bile acid tetrazole inhibitor is presented.

Here we show how different selection methods can be used for the discovery of a selective and potent carbonic anhydrase inhibitors. X-Ray and NMR structural studies were used to reveal the key binding interactions of the inhibitor with the enzyme.

Among non-covalent interactions, B-H⋯π and C-H⋯π hydrogen bonding is rather weak and less studied. Nevertheless, since both can affect the energetics of protein-ligand binding, their understanding is an important prerequisite for reliable predictions of affinities. Through a combination of high-resolution X-ray crystallography and quantum-chemical calculations on carbonic anhydrase II/carborane-based inhibitor systems, this paper provides the first example of B-H⋯π hydrogen bonding in a protein-ligand complex. It shows that the B-H⋯π interaction is stabilized by dispersion, followed by electrostatics. Furthermore, it demonstrates that the similar C-H⋯π interaction is twice as strong, with a slightly smaller contribution of dispersion and a slightly higher contribution of electrostatics. Such a detailed insight will facilitate the rational design of future protein ligands, controlling these types of non-covalent interactions.

Intraoperative indocyanine green (ICG) fluorescence angiography has gained popularity and acceptance in many surgical fields for the real-time assessment of tissue perfusion. Although vasopressors have the potential to preclude an accurate assessment of tissue perfusion, there is a lack of literature with regards to its effect on ICG fluorescence angiography. An experimental porcine model was used to expose the small bowel for quantitative tissue perfusion assessment. Three increasing doses of norepinephrine infusion (0.1, 0.5, and 1.0 µg/kg/min) were administered intravenously over a 25-min interval. Time-to-peak fluorescence intensity (TTP) was the primary outcome. Secondary outcomes included absolute fluorescence intensity and local capillary lactate (LCL) levels. Five large pigs (mean weight: 40.3 ± 4.24 kg) were included. There was no significant difference in mean TTP (in seconds) at baseline (4.23) as compared to the second (3.90), third (4.41), fourth (4.60), and fifth ICG assessment (5.99). As a result of ICG accumulation, the mean and the maximum absolute fluorescence intensity were significantly different as compared to the baseline assessment. There was no significant difference in LCL levels (in mmol/L) at baseline (0.74) as compared to the second (0.82), third (0.64), fourth (0.60), and fifth assessment (0.62). Increasing doses of norepinephrine infusion have no significant influence on bowel perfusion using ICG fluorescence angiography.

Since 2016, annually occurring species- specific die-offs of brown trout (Salmo trutta fario) occurred in the Thur river, situated in the Eastern part of Switzerland. These events lead to drastically reduced population densities in the impacted river regions. Clinical signs in brown trout and mortality were restricted to few weeks in August / September. To characterize the syndrome and to find possible causes, from end of March to November 2018, one year old brown trout (Salmo trutta fario) and rainbow trout (Oncorhynchus mykiss) were exposed to water from Thur river, fish were sampled regularly and screened for infectious agents, including viral metagenomics, and pathology was described. Starting approximately four months post exposure, brown trout showed severe lymphohistiocytic pancarditis and necrotising and haemorrhagic hepatitis. These lesions were recorded until the end of the experiment in November. Rainbow trout were not affected at any point in time. No infectious agents could be identified so far as cause of disease, especially no viral aetiology. Even if pathogenesis and pathology points in the direction of an infectious agent, a causative relationship could not be confirmed and aetiology remains unclear.

Background/purpose Few clinically useful biomarkers are known to predict prognosis in patients with hepatocellular carcinoma (HCC). The aim of this study was to investigate the correlation between PPAR activity and ALDH7A1 expression and their prognostic significance using RNA sequencing in patients undergoing liver resection for HCC. Methods We included patients undergoing liver resection for HCC at a tertiary referral center for hepato-pancreato-biliary surgery between May 2014 and January 2018. PPAR activity and ALDH7A1 expression were evaluated by RNA sequencing and correlated with overall survival, recurrence and histological features. Results We included 52 patients with a median follow-up of 20.9 months, predominantly males (88.5%) with a single tumor (84.6%) in a non-cirrhotic liver (73.1%). Three-year overall survival was 48.6% in patients with a specific PPAR target gene expression profile (cancer cluster 3) compared with 81.7% in controls (P = .04, Log-rank test). This remained significant (odds ratio 14.02, 95% confidence interval 1.92-102.22, P = .009) when adjusted for age, cirrhosis, microvascular invasion, number of tumors and free resection margins. ALDH7A1 expression was not correlated with PPAR or any outcomes. Conclusion PPAR activity in a subset of tumor samples was associated with reduced overall survival indicating that PPAR may be a valuable prognostic biomarker.

Background: Several members of the aldehyde dehydrogenase (ALDH) isoenzyme family have been suggested as prognostic biomarkers in patients with hepatocellular carcinoma (HCC). The aim of the study was to evaluate overall ALDH family member expression by RNA sequencing and hierarchical clustering in tumor and adjacent liver tissue to predict survival and evaluate correlation with liver cirrhosis in patients undergoing liver resection for HCC. Methods: We included patients having undergone liver resection for HCC between May 2014 and January 2018 at a tertiary referral university hospital (Copenhagen University Hospital, Rigshospitalet, Denmark). ALDH family member expression was evaluated by RNA sequencing of tumor and non-tumor liver tissue. Hierarchical clustering of ALDH genes was used to identify patient groups and correlations were established with overall survival, recurrence and histological features. Results: Fifty-two patients were included with 88.5% males, 84.6% with only one HCC and 73.1% with a non-cirrhotic background liver. Median follow-up was 45.7 months. Patients in one cluster defined by its ALDH expression in the tumor tissue showed significantly worse overall survival (log-rank p = 0.015), also when adjusted for age, cirrhosis, microvascular invasion, resection margins and tumor number (hazard ratio 4.2, 95% confidence interval (CI) 1.5–11.9, p = 0.007). When evaluated individually, the isoenzyme ALDH1L1 may be of particular importance. Several clusters in non-tumor tissue were correlated with cirrhosis. Especially one cluster had a high discriminative ability (area under receiver operating characteristic curve of 0.839) and remained significantly associated with cirrhosis when corrected for age, microvascular invasion, resection margins and tumor number (odds ratio 44.2, 95% CI 5.5–352.0, p < 0.001). The combination of ALDH and a previously identified candidate biomarker (expression signature of the transcriptional targets of the peroxisome proliferator-activated receptors (PPARs)) may add additional prognostic value. Conclusion: The expression of ALDH family members in HCC was correlated with overall survival. Moreover, ALDH expression in non-tumor liver tissue was correlated with cirrhosis. Members of the ALDH family of enzymes may serve as a prognostic biomarker as well as potential targets for systemic treatment.

These are the supplementary files for G3/2020/401038: RhoBTB proteins regulate the Hippo pathway by antagonizing ubiquitination of LKB1Also included are the source file of the revised manuscript, including the updated references, and a formatted early access PDF

The Hippo pathway regulates growth and apoptosis. We identify RhoBTB proteins as novel regulators of Hippo signaling. RhoBTB depletion in the Drosophila wing disc epithelium cooperated with Yki to drive hyperplasia into neoplasia. Depletion of RhoBTB2 caused elevated YAP activity in human cells. RhoBTB2 deficiency resulted in increased colony formation in assays for anchorage-independent growth. We provide evidence that RhoBTBs acts on Hippo signaling through regulation of the kinase LKB1. LKB1 protein levels were reduced upon RhoBTB2 depletion, which correlated with increased LKB1 ubiquitination. Restoring LKB1 levels rescued loss of RhoBTB in Drosophila. Our results suggest that RhoBTB-dependent LKB1 regulation may contribute to its tumor-suppressive function.

We introduce a new algorithm to merge several overlapping independent volumic meshes. Considering the different treated meshes, the biggest one is defined as the “main-mesh”, while the other ones are defined as the “sub-meshes”, and will be treated iteratively in order to provide a new unique mesh entity with a specific node distribution integrating the initial meshes geometry and precision. The “sub-meshes” are geometrically localised within the “main-mesh” into which the associated elements are refined. The proposed algorithm stops when the refined size of the “main-mesh” elements is close to the original “sub-mesh” elements size. The present algorithm preserves the mesh quality, initial geometry and precision of the different meshes, and optimises the number of elements produced, in order to keep a further Finite Element Model (FEM) calculation time as low as possible. The algorithm efficiency is validated on simple geometries and real life cases for medical applications and compared with refinement using the Brute Force Approach (BFA). Results indicate that only 3 iterative refinement steps are necessary to produce a new mesh presenting good integrated geometrical precision compared with BFA while optimising the calculation time by reducing the number of elements by 90%.

Medical applications require the numerical models to be both precise and quickly computed. In the context of liver surgery, this study aims to develop a homogenized mechanical model of the liver accounting for both hepatic tissue properties and macroscopic level blood flow impact. For this, a fluid analysis is carried out to simulate the blood flow inside the liver vessels and extract the pressure on the liver vascularization walls. This pressure is then integrated through a homogenization study, based first on alternative Eshelby type approach, then through a Mori-Tanaka scheme to compute the equivalent material rigidity. Once the equivalent mechanical properties identified, they are integrated into the macroscopic liver model, allowing a light quickly computed model integrating the underlying physics relying on the blood flow mechanical impact. Keywords: Multilevel homogenization, Eshelby type estimatore, Liver and blood vessels geometry

International audience; Background and objective: This paper focuses on computer simulation aspects of Digital Twin models in the medical framework. In particular, it addresses the need of fast and accurate simulators for the mechanical response at tissue and organ scale and the capability of integrating patient-specific anatomy from medical images to pinpoint the individual variations from standard anatomical models.Methods: We propose an automated procedure to create mechanical models of the human liver with patient-specific geometry and real time capabilities. The method hinges on the use of Statistical Shape Analysis to extract the relevant anatomical features from a database of medical images and Model Order Reduction to compute an explicit parametric solution for the mechanical response as a function of such features. The Sparse Subspace Learning, coupled with a Finite Element solver, was chosen to create low-rank solutions using a non-intrusive sparse sampling of the feature space.Results: In the application presented in the paper, the statistical shape model was trained on a database of 385 three dimensional liver shapes, extracted from medical images, in order to create a parametrized representation of the liver anatomy. This parametrization and an additional parameter describing the breathing motion in linear elasticity were then used as input in the reduced order model. Results show a consistent agreement with the high fidelity Finite Element models built from liver images that were excluded from the training dataset. However, we evidence in the discussion the difficulty of having compact shape parametrizations arising from the extreme variability of the shapes found in the dataset and we propose potential strategies to tackle this issue.Conclusions: A method to represent patient-specific real-time liver deformations during breathing is proposed in linear elasticity. Since the proposed method does not require any adaptation to the direct Finite Element solver used in the training phase, the procedure can be easily extended to more complex non-linear constitutive behaviors - such as hyperelasticity - and more general load cases. Therefore it can be integrated with little intrusiveness to generic simulation software including more sophisticated and realistic models.

International audience; The main objective of this study is to combine the statistical shape analysis (SSA) with a morphing procedure in order to generate shape-parametric finite element models of tissues and organs and to explore the reliability and the limitations of this approach when applied to databases of real medical images. As classical statistical shape models are not always adapted to the morphing procedure, a new registration method was developed in order to maximize the morphing efficiency. The method was compared to the traditional iterative thin plate spline (iTPS). Two data sets of 33 proximal femora shapes and 385 liver shapes were used for the comparison. The principal component analysis (PCA) was used to get the principal morphing modes. In terms of anatomical shape reconstruction (evaluated through the criteria of generalization, compact-ness and specificity), our approach compared fairly well to the iTPS method, while performing remarkably better in terms of mesh quality, since it was less prone to generate invalid meshes in the interior. This was particularly true in the liver case. Such methodology offers a potential application for the generation of automated finite element (FE) models from medical images. Parametrized anatomical models can also be used to assess the influence of inter-patient variability on the biomechanical response of the tissues. Indeed, thanks to the shape parametrization the user would easily have access to a valid FE model for any shape belonging to the parameters subspace.

Tomato bifunctional nuclease 1 (TBN1) is a polyfunctional protein with anticancerogenic activity originally isolated as an overexpressed protein from viroid-infected tomato. Its molecular farming in plant cells could be a non-expensive source for its biotechnology preparation. So we analysed TBN1 expression in Agrobacterium-infiltrated leaf sectors of Nicotiana benthamiana and in transformed suspension culture of tobacco BY-2 cells. During its transient expression, TBN1 mRNA was strongly degraded within a hot spot localized in the 3′ region. This early degradation process was inhibited by PTGS suppressors p19 and p38 resulting in increased TBN1 mRNA and protein yield. In parallel to degradation of TBN1 mRNA, high mRNA levels of two RNA-dependent RNA polymerases were detected in infiltrated leaf sectors, as well as in the transformed tobacco suspension culture BY-2, where low expression of the nuclease was stably maintained. Higher TBN1 mRNA and nuclease activity levels were found during its molecular farming in RDR6-deficient N. benthamiana plants. By fluorescent microscopy of infiltrated and transformed plant cells, the nuclease-GFP fusion protein was shown to be organized in filament-like structures.

Introduction: In the Thur, a river situated in the Eastern part of Switzerland, massive mortalities have occurred regularly in brown trout since 2016. Dead fish were recorded in a period of a few weeks in July/August and with a sharp demarcation between affected and unaffected river stretches. The majority of affected animals were 1-year old. Materials and Methods: From June to November 2018, brown and rainbow trout were sampled from the Thur. For each species, five fish were sampled every 2 weeks. Parasitology, bacteriology, virology and histology were performed. The water temperature was measured regularly. Water samples were taken every 10 minutes and pooled for daily samples for a period of 6 months. Non-target screening, target screening and suspect screening were performed. Results: Macroscopically, clinically ill fish showed dark colouration and apathy. No consistent pathogenic agent was isolated. By histology, extensive liver necrosis, haemorrhage and severe lymphohistiocytic myocarditis were diagnosed exclusively in brown trout. The first lesions were found in mid-August, while the highest degree of alterations occurred from the end of August to October. Full recovery did not occur until final sampling at the end of November. As alterations were in agreement with those recently described for brown trout proliferative darkening syndrome (PDS) in Germany, severely affected specimens were analysed for piscine reovirus 1 and 3, agents proposed to be involved in PDS. None of the examined samples were found to be positive. Discussion: Up to now, the cause of the described syndrome remains unclear. Results from water analyses are still outstanding.

The Hippo pathway serves as a key barrier for oncogenic transformation. It acts by limiting the activity of the proto-oncogenes YAP and TAZ. Reduced Hippo signaling and elevated YAP/TAZ activities are frequently observed in various types of tumors. Emerging evidence suggests that the ubiquitin system plays an important role in regulating Hippo pathway activity. Deregulation of ubiquitin ligases and of deubiquitinating enzymes has been implicated in increased YAP/TAZ activity in cancer. In this article, we review recent insights into the ubiquitin-mediated regulation of the mammalian Hippo pathway, its deregulation in cancer, and possibilities for targeting the Hippo pathway through the ubiquitin system.

Figure S1. ALDH7A1 depletion promotes tumor formation in vivo. Describes effect of depleting Drosophila ALDH on tumor formation in vivo. Figure S2. ALDH7A1 mRNA level in human cancers. (A) Compares ALDH7A1 expression levels in TCGA datasets for 19 human cancers and compares survival outcome in low middle and high expressing patient groups. (B) Heatmap of the correlation between ALDH7A1 mRNA expression and EGFR RNA and EGFR phosphorylation in all cancer types. (C) Cox proportional hazard regression analysis of the association between ALDH7A1 mRNA and EGFR levels for liver and kidney cancer. Figure S3. Pathway analysis. (A) Gene set and pathway analysis comparing low vs high ALDH7A1 tumors. (B) Effects of low ALDH7A1 on pathways in KIRC. Figure S4. Metabolite profiles on cancer cell lines. Figure S5. Assessment of correlation between PPAR activity and ALDH7A1 on other cancers. Shows shows survival outcome for patients groups by PPAR target signature groups, and comparison with ALDH7A1 expression. Figure S6. Effects of PPAR agonists. Shows the effects of PPAR agonist treatment on ALDH7A1 protein levels, would healing assays, invasive migration (transwell) assays and PPAR target gene expression levels. Figure S7. Assays on cancer cell lines. Summarizes assays carried out on cancer cell lines. Figure S8. Clinical characteristics of the patients included in the study Summarizes TCGA clinical data. (PDF 40809 kb)

Representing one of the world’s leading causes of death, cancer accounted for 22% of the recorded fatalities in the United States in 2015 (1). Cancer arises from a complex interplay of genetic and epigenetic alterations that can result in dysregulation of signalling networks (2).

Pattern formation during epithelial development requires the coordination of multiple signaling pathways. Here, we investigate the functions of an ovary-enriched miRNA, miR-318, in epithelial development during Drosophila oogenesis. mir-318 maternal loss-of-function mutants were female-sterile and laid eggs with abnormal morphology. Removal of mir-318 disrupted the dorsal–anterior follicle cell patterning, resulting in abnormal dorsal appendages. mir-318 mutant females also produced thin and fragile eggshells due to impaired chorion gene amplification. We provide evidence that the ecdysone signaling pathway activates expression of miR-318 and that miR-318 cooperates with Tramtrack69 to control the switch from endocycling to chorion gene amplification during differentiation of the follicular epithelium. The multiple functions of miR-318 in oogenesis illustrate the importance of miRNAs in maintaining cell fate and in promoting the developmental transition in the female follicular epithelium.

SummaryMicroRNAs are abundant in animal genomes, yet little is known about their functions in vivo. Here, we report the production of 80 new Drosophila miRNA mutants by targeted homologous recombination. These mutants remove 104 miRNAs. Together with 15 previously reported mutants, this collection includes 95 mutants deleting 130 miRNAs. Collectively, these genes produce over 99% of all Drosophila miRNAs, measured by miRNA sequence reads. We present a survey of developmental and adult miRNA phenotypes. Over 80% of the mutants showed at least one phenotype using a p < 0.01 significance threshold. We observed a significant correlation between miRNA abundance and phenotypes related to survival and lifespan, but not to most other phenotypes. miRNA cluster mutants were no more likely than single miRNA mutants to produce significant phenotypes. This mutant collection will provide a resource for future analysis of the biological roles of Drosophila miRNAs.

Mini-invasive surgery restricts the surgeon information to two-dimensional digital representation without the corresponding physical information obtained in previous open surgery. To overcome these drawbacks, real time augmented reality interfaces including the true mechanical behaviour of organs depending on their internal microstructure need to be developed. For the case of tumour resection, we present here a finite element numerical study of the liver mechanical behaviour including the effects of its own vascularisation through numerical indentation tests in order extract the corresponding macroscopic behaviour. The obtained numerical results show excellent correlation of the corresponding force-displacement curves when compared with macroscopic experimental data available in the literature.

The Hippo pathway, which acts to repress the activity of YAP and TAZ trancriptional co-activators, serve as a barrier for oncogenic transformation. Unlike other oncoproteins, YAP and TAZ are rarely activated by mutations or amplified in cancer. However, elevated YAP/TAZ activity is frequently observed in cancer and often correlates with worse survival. The activity and stability of Hippo pathway components, including YAP/TAZ, AMOT and LATS1/2, are regulated by ubiquitin-mediated protein degradation. Aberrant expression of ubiquitin ligase complexes that regulate the turnover of Hippo components and deubiquitylating enzymes that counteract these ubiquitin ligases have been implicated in human cancer. Here we identify the USP21 deubiquitylating enzyme as a novel regulator of Hippo pathway activity. We provide evidence that USP21 regulates YAP/TAZ activity by controlling the stability of MARK kinases, which promote Hippo signaling. Low expression of USP21 in early stage renal clear cell carcinoma suggests that USP21 may be a useful biomarker.

The YAP and TAZ transcriptional coactivators promote oncogenic transformation. Elevated YAP/TAZ activity has been documented in human tumors. YAP and TAZ are negatively regulated by the Hippo tumor suppressor pathway. The activity and stability of several Hippo pathway components, including YAP/TAZ, is regulated by ubiquitin mediated protein turnover and several ubiquitin ligase complexes have been implicated in human cancer. However, little is known about the deubiquitylating enzymes that counteract these ubiquitin ligases in regulation of the Hippo pathway. Here we identify the DUB3 family deubiquitylating enzymes as regulators of Hippo pathway activity. We provide evidence that DUB3 proteins regulate YAP/TAZ activity by controlling the stability of the E3 ligase ITCH, the LATS kinases and the AMOT family proteins. As a novel Hippo pathway regulator, DUB3 has the potential to act a tumor suppressor by limiting YAP activity.

MicroRNAs (miRNAs) are posttranscriptional regulators of gene expression that may act as buffering agents to stabilize gene-regulatory networks. Here, we identify two miRNAs that are maternally required for normal embryonic primordial germ cell development in Drosophila melanogaster. Embryos derived from miR-969 and miR-9c mutant mothers had, on average, reduced germ cell numbers. Intriguingly, this reduction correlated with an increase in the variance of this quantitative phenotypic trait. Analysis of an independent set of maternal mutant genotypes suggests that reduction of germ cell number need not lead to increased variance. Our observations are consistent with the hypothesis that miR-969 and miR-9c contribute to stabilizing the processes that control germ number, supporting phenotypic robustness.

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Dosage-sensitive modifier screening is a powerful tool for linking genes to biological processes. Use of chromosomal deletions permits sampling the effects of removing groups of genes related by position on the chromosome. Here, we explore the use of inducible microRNA transgenes as a complement to deficiency-based modifier screens. miRNAs are predicted to have hundreds of targets. miRNA overexpression provides an efficient means to reduces expression of large gene sets. A collection of transgenes was prepared to allow overexpression of 89 miRNAs or miRNA clusters. These transgenes and a set of genomic deficiencies were screened for their ability to modify the bristle phenotype of the cell-cycle regulator minus. Sixteen miRNAs were identified as dominant suppressors, while the deficiency screen uncovered four genomic regions that contain a dominant suppressor. Comparing the genes uncovered by the deletions with predicted miRNA targets uncovered a small set of candidate suppressors. Two candidates were identified as suppressors of the minus phenotype, Cullin-4 and CG5199/Cut8. Additionally, we show that Cullin-4 acts through its substrate receptor Cdt2 to suppress the minus phenotype. We suggest that inducible microRNA transgenes are a useful complement to deficiency-based modifier screens.

Bicaudal-C (Bic-C) is a multiple KH-domain RNA-binding protein required for Drosophila oogenesis and, maternally, for embryonic patterning. In early oogenesis, Bic-C negatively regulates target mRNAs, including Bic-C, by recruiting the CCR4 deadenylase through a direct association with its NOT3 subunit. Here, we identify a novel function for Bic-C in secretion of the TGF-α homolog Gurken (Grk). In Bic-C mutant egg chambers, Grk is sequestered within actin-coated structures during mid-oogenesis. As a consequence, Egfr signalling is not efficiently activated in the dorsal-anterior follicle cells. This phenotype is strikingly similar to that of trailer hitch (tral) mutants. Consistent with the idea that Bic-C and Tral act together in Grk secretion, Bic-C co-localizes with Tral within cytoplasmic granules, and can be co-purified with multiple protein components of a Tral mRNP complex. Taken together, our results implicate translational regulation by Bic-C and Tral in the secretory pathway.