Radical-induced purine lesion formation is dependent on DNA helical topology

Abstract

Herein we report the quantification of purine lesions arising from gamma-radiation sourced hydroxyl radicals (HO^?) on tertiary dsDNA helical forms of supercoiled (SC), open circular (OC), and linear (L) conformation, along with single-stranded folded and non-folded sequences of guanine-rich DNA in selected G-quadruplex structures. We identify that DNA helical topology and folding plays major, and unexpected, roles in the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxo-dA), along with tandem-type purine lesions 5′,8-cyclo-2′-deoxyguanosine (5′,8-cdG) and 5′,8-cyclo-2′-deoxyadenosine (5′,8-cdA). SC, OC, and L dsDNA conformers together with folded and non-folded G-quadruplexes d[TGGGGT]₄ (TG4T), d[AGGG(TTAGGG)₃] (Tel22), and the mutated tel24 d[TTGGG(TTAGGG)₃A] (mutTel24) were exposed to HO^? radicals and purine lesions were then quantified via stable isotope dilution LC-MS/MS analysis. Purine oxidation in dsDNA follows L?>?OC???SC indicating greater damage towards the extended B-DNA topology. Conversely, G-quadruplex sequences were significantly more resistant toward purine oxidation in their unfolded states as compared with G-tetrad folded topologies; this effect is confirmed upon comparative analysis of Tel22 (～50% solution folded) and mutTel24 (～90% solution folded). In an effort to identify the accessibly of hydroxyl radicals to quadruplex purine nucleobases, G-quadruplex solvent cavities were then modeled at 1.33?Å with evidence suggesting that folded G-tetrads may act as potential oxidant traps to protect against chromosomal DNA damage.     

Blocking autophagy overcomes resistance to dual histone deacetylase and proteasome inhibition in gynecologic cancer

Histone deacetylase (HDAC) inhibitors and proteasome inhibitors have been approved by the FDA for the treatment of multiple myeloma and lymphoma, respectively, but have not achieved similar activity as single agents in solid tumors. Preclinical studies have demonstrated the activity of the combination of an HDAC inhibitor and a proteasome inhibitor in a variety of tumor models. However, the mechanisms underlying sensitivity and resistance to this combination are not well-understood. This study explores the role of autophagy in adaptive resistance to dual HDAC and proteasome inhibition. Studies focus on ovarian and endometrial gynecologic cancers, two diseases with high mortality and a need for novel treatment approaches. We found that nanomolar concentrations of the proteasome inhibitor ixazomib and HDAC inhibitor romidepsin synergistically induce cell death in the majority of gynecologic cancer cells and patient-derived organoid (PDO) models created using endometrial and ovarian patient tumor tissue. However, some models were not sensitive to this combination, and mechanistic studies implicated autophagy as the main mediator of cell survival in the context of dual HDAC and proteasome inhibition. Whereas the combination of ixazomib and romidepsin reduces autophagy in sensitive gynecologic cancer models, autophagy is induced following drug treatment of resistant cells. Pharmacologic or genetic inhibition of autophagy in resistant cells reverses drug resistance as evidenced by an enhanced anti-tumor response both in vitro and in vivo. Taken together, our findings demonstrate a role for autophagic-mediated cell survival in proteasome inhibitor and HDAC inhibitor-resistant gynecologic cancer cells. These data reveal a new approach to overcome drug resistance by inhibiting the autophagy pathway.Subject terms: Gynaecological cancer, Preclinical research     

Optic Neuritis in Multiple Sclerosis—A Review of Molecular Mechanisms Involved in the Degenerative Process

Multiple sclerosis is a central nervous system inflammatory demyelinating disease with a wide range of clinical symptoms, ocular involvement being frequently marked by the presence of optic neuritis (ON). The emergence and progression of ON in multiple sclerosis is based on various pathophysiological mechanisms, disease progression being secondary to inflammation, demyelination, or axonal degeneration. Early identification of changes associated with axonal degeneration or further investigation of the molecular processes underlying remyelination are current concerns of researchers in the field in view of the associated therapeutic potential. This article aims to review and summarize the scientific literature related to the main molecular mechanisms involved in defining ON as well as to analyze existing data in the literature on remyelination strategies in ON and their impact on long-term prognosis.     

Linguistic evidence supports date for Homeric epics

The Homeric epics are among the greatest masterpieces of literature, but when they were produced is not known with certainty. Here we apply evolutionary‐linguistic phylogenetic statistical methods to differences in Homeric, Modern Greek and ancient Hittite vocabulary items to estimate a date of approximately 710–760 BCE for these great works. Our analysis compared a common set of vocabulary items among the three pairs of languages, recording for each item whether the words in the two languages were cognate – derived from a shared ancestral word – or not. We then used a likelihood‐based Markov chain Monte Carlo procedure to estimate the most probable times in years separating these languages given the percentage of words they shared, combined with knowledge of the rates at which different words change. Our date for the epics is in close agreement with historians' and classicists' beliefs derived from historical and archaeological sources.     

Molecular analysis of phylogeographic subspecies in three Ponto-Caspian sturgeon species

Sturgeons (Order Acipenseriformes) represent an extremely valuable natural resource that is now facing depletion. In the current study we evaluate if the traditional classification in subspecies of Acipenser gueldenstaedtii, Acipenser stellatus and Huso huso, endemic to Ponto-Caspian region is sustained by molecular analysis and if these represent Evolutionary Significant Units (ESUs) that should be managed separately in conservation programs. To examine the classification of taxonomic entities we sequenced a fragment of the mitochondrial control region in case of three sturgeon species that inhabit the North-western of Black Sea and migrate for reproduction in the Lower Danube. Beside these sequences, we used previously published sequences from sturgeon individuals sampled in the Black Sea, Azov Sea and Caspian Sea. We determined the genetic diversity and genetic differentiation, conducted a Population Aggregation Analysis (PAA) and inferred an intraspecific molecular phylogeny and haplotype network. The results indicated a low level of genetic differentiation between the geographically designated subspecies and did not support a significant divergence or reciprocal monophyly between them. Our results confirm previous genetic studies with smaller samples sizes, but additional analyses including nuclear markers should be conducted for proper recommendations aiming at the development of conservation programs.     

Angiotensin II induces skin fibrosis: a novel mouse model of dermal fibrosis

Introduction

Systemic sclerosis (SSc) is an autoimmune inflammatory disorder of unknown etiology characterized by fibrosis of the skin and internal organs. Ang II (angiotensin II), a vasoconstrictive peptide, is a well-known inducer of kidney, heart, and liver fibrosis. The goal of this study was to investigate the profibrotic potential of Ang II in the mouse skin.

Methods

Ang II was administered by subcutaneous osmotic mini pumps to C57BL/6 male mice. Collagen-content measurements were performed with Gomori Trichrome staining and hydroxyproline assay. The mRNA expression level of collagens, TGF-β1, TGF-β2, TGF-β3, CTGF, αSMA, CD3, Emr1, CD45/B220, MCP1, and FSP1 were quantified with real-time polymerase chain reaction (PCR). Immunostaining was performed for markers of inflammation and fibrosis, including, phospho-Smad2, αSMA, CD3, Mac3, CD45/B220, and CD163B. Fibrocytes were identified by double staining with CD45/FSP1 and CD45/PH4. Endothelial cells undergoing endothelial-to-mesenchymal transition (EndoMT) were identified by double staining with VE-cadherin/FSP1.

Results

Ang II-infused mice develop prominent dermal fibrosis in the area proximal to the pump, as shown by increased collagen and CTGF mRNA levels, increased hydroxyproline content, and more tightly packed collagen fibers. In addition, elevated mRNA levels of TGF-β2 and TGF-β3 along with increased expression of pSmad2 were observed in the skin of Ang II-treated mice. Dermal fibrosis was accompanied by an increased number of infiltrating fibrocytes, and an increased number of αSMA-positive cells, as well as CD163B⁺ macrophages in the upper dermis. This correlated with significantly increased mRNA levels of αSMA, Emr1, and MCP1. Infiltration of CD3-, CD45/B220-, and Mac3-positive cells was observed mainly in the hypodermis. Furthermore, an increased number of double-positive VE-cadherin/FSP1 cells were detected in the hypodermis only.

Conclusions

This work demonstrates that Ang II induces both inflammation and fibrosis in the skin via MCP1 upregulation and accumulation of activated fibroblasts. Additionally, our data suggest that populations of these fibroblasts originate from circulating blood cells. Ang II infusion via osmotic minipumps could serve as a useful mouse model of skin fibrosis to gain new insights into pathogenic mechanisms and to test new antifibrotic therapies.     

Fluidization, resolidification, and reorientation of the endothelial cell in response to slow tidal stretches

Mechanical stretch plays an important role in regulating shape and orientation of the vascular endothelial cell. This morphological response to stretch is basic to angiogenesis, neovascularization, and vascular homeostasis, but mechanism remains unclear. To elucidate mechanisms, we used cell mapping rheometry to measure traction forces in primary human umbilical vein endothelial cells subjected to periodic uniaxial stretches. Onset of periodic stretch of 10% strain amplitude caused a fluidization response typified by attenuation of traction forces almost to zero. As periodic stretch continued, the prompt fluidization response was followed by a slow resolidification response typified by recovery of the traction forces, but now aligned along the axis perpendicular to the imposed stretch. Reorientation of the cell body lagged reorientation of the traction forces, however. Together, these observations demonstrate that cellular reorientation in response to periodic stretch is preceded by traction attenuation by means of cytoskeletal fluidization and subsequent traction recovery transverse to the stretch direction by means of cytoskeletal resolidification.     

Adiposity and hand osteoarthritis: the Netherlands Epidemiology of Obesity study

Introduction

Obesity, usually characterized by the body mass index (BMI), is a risk factor for hand osteoarthritis (OA). We investigated whether adipose tissue and abdominal fat distribution are associated with hand OA.

Methods

The Netherlands Epidemiology of Obesity (NEO) study is a population-based cohort aged 45 to 65 years, including 5315 participants (53% women, median BMI 29.9 kg/m²). Fat percentage and fat mass (FM) (kg) were estimated using bioelectrical impedance analysis. The waist-to-hip ratio (WHR) was calculated. In 1721 participants, visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) (cm²) were assessed using abdominal MR imaging. Hand OA was defined according to the ACR criteria.Odds ratios (OR) with 95% confidence intervals (CI) were calculated for the association of fat percentage, FM, WHR, VAT and SAT with hand OA using logistic regression analyses per standard deviation, stratified by sex and adjusted for age.

Results

Hand OA was present in 8% of men and 20% of women. Fat percentage was associated with hand OA in men (OR 1.34 (95% CI 1.11 to 1.61)) and women (OR 1.26 (1.05 to 1.51)), as was FM. WHR was associated with hand OA in men (OR 1.45 (1.13 to 1.85)), and to a lesser extent in women (OR 1.17 (1.00 to 1.36)). Subgroup analysis revealed that VAT was associated with hand OA in men (OR1.33 (1.01 to 1.75)). This association increased after additional adjustment for FM (OR 1.51 (1.13 to 2.03)).

Conclusions

Fat percentage, FM and WHR were associated with hand OA. VAT was associated with hand OA in men, suggesting involvement of visceral fat in hand OA.     

Controversial behavior of aminoguanidine in the presence of either reducing sugars or soluble glycated bovine serum albumin

The elucidation of the controversial inhibitory effect of aminoguanidine (AG) on the cross-linking and fluorescent advanced glycation end products (AGEs) formation during long-term in vitro glycation of type I collagen with 250 mM reducing sugars or 0.5 mg/ml soluble glycated bovine serum albumin (AGE-BSA) was researched.Chromatographic and SDS–PAGE analyses revealed the formation of aggregates during collagen glycation. AG at all concentrations (5–80 mM) prevented the cross-linking of collagen peptides with monosaccharides but an increase in fluorescence with a maximum value at 10 mM AG was noticed. In the presence of AGE-BSA, AG prevented the cross-linking process and decreased the fluorescence levels in a concentration-dependent manner.Our results suggest that AG is an efficient inhibitor of collagen cross-linking and the highest increase in fluorescence due to reducing sugars and AG can be explained by the competition between guanidine group of AG and arginine residues of some protein-bound dideoxyosones, which could form fluorescent compounds.