Catalytic activities of Werner protein are affected by adduction with 4-hydroxy-2-nonenal

4-Hydroxy-2-nonenal (HNE) is a reactive α,β-unsaturated aldehyde generated during oxidative stress and subsequent peroxidation of polyunsaturated fatty acids. Here, Werner protein (WRN) was identified as a novel target for modification by HNE. Werner syndrome arises through mutations in the WRN gene that encodes the RecQ DNA helicase which is critical for maintaining genomic stability. This hereditary disease is associated with chromosomal instability, premature aging and cancer predisposition. WRN appears to participate in the cellular response to oxidative stress and cells devoid of WRN display elevated levels of oxidative DNA damage. We demonstrated that helicase/ATPase and exonuclease activities of HNE-modified WRN protein were inhibited both in vitro and in immunocomplexes purified from the cell extracts. Sites of HNE adduction in human WRN were identified at Lys577, Cys727, His1290, Cys1367, Lys1371 and Lys1389. We applied in silico modeling of the helicase and RQC domains of WRN protein with HNE adducted to Lys577 and Cys727 and provided a potential mechanism of the observed deregulation of the protein catalytic activities. In light of the obtained results, we postulate that HNE adduction to WRN is a post-translational modification, which may affect WRN conformational stability and function, contributing to features and diseases associated with premature senescence.     

Establishment of a New Murine Elastase-Induced Aneurysm Model Combined with Transplantation

Introduction

The aim of our study was to develop a reproducible murine model of elastase-induced aneurysm formation combined with aortic transplantation.

Methods

Adult male mice (n = 6–9 per group) underwent infrarenal, orthotopic transplantation of the aorta treated with elastase or left untreated. Subsequently, both groups of mice were monitored by ultrasound until 7 weeks after grafting.

Results

Mice receiving an elastase-pretreated aorta developed aneurysms and exhibited a significantly increased diastolic vessel diameter compared to control grafted mice at 7 week after surgery (1.11±0.10 mm vs. 0.75±0.03 mm; p≤0,001). Histopathological examination revealed disruption of medial elastin, an increase in collagen content and smooth muscle cells, and neointima formation in aneurysm grafts.

Conclusions

We developed a reproducible murine model of elastase-induced aneurysm combined with aortic transplantation. This model may be suitable to investigate aneurysm-specific inflammatory processes and for use in gene-targeted animals.     

Hfq assists small RNAs in binding to the coding sequence of ompD mRNA and in rearranging its structure

The bacterial protein Hfq participates in the regulation of translation by small noncoding RNAs (sRNAs). Several mechanisms have been proposed to explain the role of Hfq in the regulation by sRNAs binding to the 5′-untranslated mRNA regions. However, it remains unknown how Hfq affects those sRNAs that target the coding sequence. Here, the contribution of Hfq to the annealing of three sRNAs, RybB, SdsR, and MicC, to the coding sequence of Salmonella ompD mRNA was investigated. Hfq bound to ompD mRNA with tight, subnanomolar affinity. Moreover, Hfq strongly accelerated the rates of annealing of RybB and MicC sRNAs to this mRNA, and it also had a small effect on the annealing of SdsR. The experiments using truncated RNAs revealed that the contributions of Hfq to the annealing of each sRNA were individually adjusted depending on the structures of interacting RNAs. In agreement with that, the mRNA structure probing revealed different structural contexts of each sRNA binding site. Additionally, the annealing of RybB and MicC sRNAs induced specific conformational changes in ompD mRNA consistent with local unfolding of mRNA secondary structure. Finally, the mutation analysis showed that the long AU-rich sequence in the 5′-untranslated mRNA region served as an Hfq binding site essential for the annealing of sRNAs to the coding sequence. Overall, the data showed that the functional specificity of Hfq in the annealing of each sRNA to the ompD mRNA coding sequence was determined by the sequence and structure of the interacting RNAs.     

Analysis of adrenocortical tumors morphology as regards their structure and potential malignancy

INTRODUCTION: A consequence of diagnosis of adrenocortical carcinoma (ACC) is introduction of pharmacological therapy, precise monitoring of the patients and in some cases re-operation. The aim of the study is to analyse morphology of adrenocortical tumours as regards their malignancy by use of criteria proposed by Weiss. MATERIAL AND METHODS: 110 adrenocortical tumours in 107 patients were analysed (M 27.1%, F 72.9%; age 32 to 77 years, mean 55.2 +/- 9.7). Conn syndrome was diagnosed in 16 patients (14.9%), Cushing syndrome in 12 (11.2%), and virilisation in 3 (2.8%). In 76 patients (71.0%) biochemical tests did not reveal hormonal hyperactivity of the tumour. RESULTS: In routine histopatological examination ACC was diagnosed in 6 tumours (5.4%), adrenocortical adenoma (ACA) in 92 (83.6%) and adrenocortical hyperplasia in 12 (10.9%). Nuclear grade III or IV was observed in 8 tumours (7.3%), mitotic rate > 5/50 high power fields in 6 (5.4%), atypical mitoses in 5 (4.5%), clear cells constituting < 25% of the tumour in 10 (9.1%), diffuse architecture in 8 (7.3%), necrosis in 16 (14.5%), veins infiltration in 4 (3.6%), sinusoids infiltration in 7 (6.3%), and tumour capsule infiltration in 5 (4.5%). Among ACC tumours 4-9 features of malignancy were present, among ACA--0-3 features. Statistical analysis revealed correlation between number of criteria proposed by Weiss and maximal tumour size (p < 0.05). CONCLUSION: The structure and cell arrangement in adrenocortical adenoma are heterogeneous. Application of criteria proposed by Weiss in histopathological examination of adrenocortical tumours can be useful in differentiating adrenocortical adenoma from carcinoma.     

Mathematical modelling of the influence of heat shock proteins on cancer invasion of tissue

Tumour cell invasion is crucial for cancer metastasis, which is the main cause of cancer mortality. An important group of proteins involved in cancer invasion are the Heat Shock Proteins (HSPs). According to experimental data, inhibition of one of these proteins, Hsp90, slows down cancer cells while they are invading tissue, but does not affect the synthesis of matrix metalloproteinases (MMP2 and MMP9), which are very important for cancer metastasis, acting as extracellular matrix (ECM) degrading enzymes. To test different biological hypotheses regarding how precisely Hsp90 influences tumour invasion, in this paper we use a model of solid tumour growth which accounts for the interactions between Hsp90 dynamics and the migration of cancer cells and, alternatively, between Hsp90 dynamics and the synthesis of matrix degrading enzymes (MDEs). The model consists of a system of reaction-diffusion-taxis partial differential equations describing interactions between cancer cells, MDE, and the host tissue (ECM). Using numerical simulations we investigate the effects of the administration of Hsp90 inhibitors on the dynamics of tumour invasion. Alternative mechanisms of reduction of cancer invasiveness result in different simulated patterns of the invading tumour cells. Therefore, predictions of the model suggest experiments which might be performed to develop a deeper understanding of the tumour invasion process.     

Tissue clearing‐based method for unobstructed three‐dimensional imaging of mouse penis with subcellular resolution

Although mice are widely used to elucidate factors contributing to penile disorders and develop treatment options, quantification of tissue changes upon intervention is either limited to minuscule tissue volume (histology) or acquired with limited spatial resolution (MRI/CT). Thus, imaging method suitable for expeditious acquisition of the entire mouse penis with subcellular resolution is described that relies on both aqueous‐ (clear, unobstructed brain imaging cocktails and computational analysis) and solvent‐based (fluorescence‐preserving capability imaging of solvent‐cleared organs) tissue optical clearing (TOC). The combined TOC approach allows to image mouse penis innervation and vasculature with unprecedented detail and, for the first time, reveals the three‐dimensional structure of murine penis fibrocartilage.     

The circadian gene Cryptochrome 2 influences stress-induced brain activity and depressive-like behavior in mice

Cryptochrome 2 (Cry2) is a core clock gene important for circadian regulation. It has also been associated with anxiety and depressive-like behaviors in mice, but the previous findings have been conflicting in terms of the direction of the effect. To begin to elucidate the molecular mechanisms of this association, we carried out behavioral testing, PET imaging, and gene expression analysis of Cry2^−/− and Cry2^+/+ mice. Compared to Cry2^+/+ mice, we found that Cry2^−/− mice spent less time immobile in the forced swim test, suggesting reduced despair-like behavior. Moreover, Cry2^−/− mice had lower saccharin preference, indicative of increased anhedonia. In contrast, we observed no group differences in anxiety-like behavior. The behavioral changes were accompanied by lower metabolic activity of the ventro-medial hypothalamus, suprachiasmatic nuclei, ventral tegmental area, anterior and medial striatum, substantia nigra, and habenula after cold stress as measured by PET imaging with a glucose analog. Although the expression of many depression-associated and metabolic genes was upregulated or downregulated by cold stress, we observed no differences between Cry2^−/− and Cry2^+/+ mice. These findings are consistent with other studies showing that Cry2 is required for normal emotional behavior. Our findings confirm previous roles of Cry2 in behavior and extend them by showing that the effects on behavior may be mediated by changes in brain metabolism.     

High performance anion exchange chromatography purification of probiotic bacterial extracellular vesicles enhances purity and anti-inflammatory efficacy

Bacterial extracellular vesicles (BEVs), including outer membrane vesicles, have emerged as a promising new class of vaccines and therapeutics to treat cancer and inflammatory diseases, among other applications. However, clinical translation of BEVs is hindered by a current lack of scalable and efficient purification methods. Here, we address downstream BEV biomanufacturing limitations by developing a method for orthogonal size- and charge-based BEV enrichment using tangential flow filtration (TFF) in tandem with high performance anion exchange chromatography (HPAEC). The data show that size-based separation coisolated protein contaminants, whereas size-based TFF with charged-based HPAEC dramatically improved purity of BEVs produced by probiotic Gram-negative Escherichia coli and Gram-positive lactic acid bacteria (LAB). Escherichia coli BEV purity was quantified using established biochemical markers while improved LAB BEV purity was assessed via observed potentiation of anti-inflammatory bioactivity. Overall, this work establishes orthogonal TFF + HPAEC as a scalable and efficient method for BEV purification that holds promise for future large-scale biomanufacturing of therapeutic BEV products.