Changes in the topology of DNA replication intermediates: Important discrepancies between in vitro and in vivo.

The topology of DNA duplexes changes during replication and also after deproteinization in vitro. Here we describe these changes and then discuss for the first time how the distribution of superhelical stress affects the DNA topology of replication intermediates, taking into account the progression of replication forks. The high processivity of Topo IV to relax the left-handed (+) supercoiling that transiently accumulates ahead of the forks is not essential, since DNA gyrase and swiveling of the forks cooperate with Topo IV to accomplish this task in vivo. We conclude that despite Topo IV has a lower processivity to unlink the right-handed (+) crossings of pre-catenanes and fully replicated catenanes, this is indeed its main role in vivo. This would explain why in the absence of Topo IV replication goes-on, but fully replicated sister duplexes remain heavily catenated.

Cardioprotective and Antifibrotic Effects of Low-Dose Renin-Angiotensin-Aldosterone System Inhibitors in Type 1 Diabetic Rat Model.

Diabetic cardiovascular complications are associated with up to 50% mortality, and current therapies are not effective enough. Renin-angiotensin-aldosterone system inhibitors (RAASis) are the standard of care for diabetic patients with hypertension and albuminuria. Based on our previous studies reporting the renoprotective effects of low-dose RAASis, here, we hypothesized that low-dose RAASi treatment has cardioprotective and antifibrotic benefits in type 1 diabetes mellitus (T1DM). After five weeks of T1DM, adult male Wistar rats received low doses of ramipril, losartan, or eplerenone for two weeks. Heart rate, blood pressure, and pulse wave velocity (PWV) were recorded. Aortic intima-media thickness (IMT), collagen accumulation, and myocardial fibrosis were assessed. All RAASis reduced PWV elevation, prevented the progression of myocardial fibrosis, and normalized B-type natriuretic peptide, troponin I, and fibroblast growth factor 23 levels without affecting blood pressure. Interestingly, only eplerenone reversed the decline in Klotho levels and reduced IMT and fibrosis in the media of the aorta. Our comparative analysis suggests that mineralocorticoid receptor antagonists, particularly eplerenone, may offer superior efficacy in halting both the arterial and the myocardial injuries in T1DM compared to angiotensin-converting enzyme inhibitors or angiotensin II type 1 receptor blockers.

Replication Fork Barriers and Topological Barriers: Progression of DNA Replication Relies on DNA Topology Ahead of Forks.

During replication, the topology of DNA changes continuously in response to well-known activities of DNA helicases, polymerases, and topoisomerases. However, replisomes do not always progress at a constant speed and can slow-down and even stall at precise sites. The way these changes in the rate of replisome progression affect DNA topology is not yet well understood. The interplay of DNA topology and replication in several cases where progression of replication forks reacts differently to changes in DNA topology ahead is discussed here. It is proposed, there are at least two types of replication fork barriers: those that behave also as topological barriers and those that do not. Two-Dimensional (2D) agarose gel electrophoresis is the method of choice to distinguish between these two different types of replication fork barriers.

Closing the DNA replication cycle: from simple circular molecules to supercoiled and knotted DNA catenanes.

Due to helical structure of DNA, massive amounts of positive supercoils are constantly introduced ahead of each replication fork. Positive supercoiling inhibits progression of replication forks but various mechanisms evolved that permit very efficient relaxation of that positive supercoiling. Some of these mechanisms lead to interesting topological situations where DNA supercoiling, catenation and knotting coexist and influence each other in DNA molecules being replicated. Here, we first review fundamental aspects of DNA supercoiling, catenation and knotting when these qualitatively different topological states do not coexist in the same circular DNA but also when they are present at the same time in replicating DNA molecules. We also review differences between eukaryotic and prokaryotic cellular strategies that permit relaxation of positive supercoiling arising ahead of the replication forks. We end our review by discussing very recent studies giving a long-sought answer to the question of how slow DNA topoisomerases capable of relaxing just a few positive supercoils per second can counteract the introduction of hundreds of positive supercoils per second ahead of advancing replication forks.

Reduced O-GlcNAcylation and tubular hypoxia contribute to the antifibrotic effect of SGLT2 inhibitor dapagliflozin in the diabetic kidney.

Diabetic kidney disease is a worldwide epidemic, and therapies are incomplete. Clinical data suggest that improved renal outcomes by Na+-glucose cotransporter 2 inhibitor (SGLT2i) are partly beyond their antihyperglycemic effects; however, the mechanisms are still elusive. Here, we investigated the effect of the SGLT2i dapagliflozin (DAPA) in the prevention of elevated O-GlcNAcylation and tubular hypoxia as contributors of renal fibrosis. Type 1 diabetes was induced by streptozotocin in adult male Wistar rats. After the onset of diabetes, rats were treated for 6 wk with DAPA or DAPA combined with losartan (LOS). The effect of hyperglycemia was tested in HK-2 cells kept under normal or high glucose conditions. To test the effect of hypoxia, cells were kept in 1% O2 for 2 h. Cells were treated with DAPA or DAPA combined with LOS. DAPA slowed the loss of renal function, mitigated renal tubular injury markers (kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin), and reduced tubulointerstitial fibrosis. DAPA diminished high glucose-induced protein O-GlcNAcylation and moderated the tubular response to hypoxia through the hypoxia-inducible factor pathway. DAPA alone was as effective as combined treatment with LOS in all outcome parameters. These data highlight the role of ameliorated O-GlcNAcylation and diminished tubular hypoxia as important benefits of SGLT2i treatment. Our results support the link between glucose toxicity, tubular hypoxia, and fibrosis, a vicious trio that could be targeted by SGLT2i in kidney diseases of other origins as well.

Novel therapeutic potential of angiotensin receptor 1 blockade in a rat model of diabetes-associated depression parallels altered BDNF signalling.

AIMS/HYPOTHESIS: Diabetes is a worldwide epidemic linked with diverse diseases of the nervous system, including depression. A few studies suggested a connection between renin-angiotensin-aldosterone system blockers and reduced depressive symptoms, although underlying mechanisms are unclear. Here we investigated the antidepressant effect and the mechanisms of action of the angiotensin receptor 1 blocker (ARB) losartan in an experiential model of diabetes-associated depression. METHODS: Experimental diabetes was induced by streptozotocin in adult male Wistar rats. After 5 weeks of diabetes, rats were treated for 2 weeks with a non-pressor oral dose of losartan (20 mg/kg). In protocol 1, cerebrovascular perfusion and glial activation were evaluated by single-photon emission computed tomography-MRI and immunohistochemistry. In protocol 2, behaviour studies were performed (forced swim test and open field test). Hippocampal proinflammatory response and brain-derived neurotrophic factor (BDNF) signalling were also assessed. RESULTS: Here, we show that diabetic rats exhibit depression-like behaviour, which can be therapeutically reversed by losartan. This action of losartan occurs via changes in diabetes-induced neuroinflammatory responses rather than altered cerebral perfusion. We also show that as a part of its protective effect losartan restores BDNF production in astrocytes and facilitates BDNF-tropomyosin receptor kinase B-cAMP response element-binding protein signalling in the diabetic brain. CONCLUSIONS/INTERPRETATION: We identified a novel effect of losartan in the nervous system that may be implemented to alleviate symptoms of diabetes-associated depression. These findings explore a new therapeutic horizon for ARBs as possible antidepressants and suggest that BDNF could be a target of future drug development in diabetes-induced complications.

The abundance of Fob1 modulates the efficiency of rRFBs to stall replication forks.

In eukaryotes, ribosomal genes (rDNA) are organized in tandem repeats localized in one or a few clusters. Each repeat encompasses a transcription unit and a non-transcribed spacer. Replication forks moving in the direction opposite to transcription are blocked at specific sites called replication fork barriers (rRFBs) in the non-transcribed spacer close to the 3' end of the transcription unit. Here, we investigated and quantified the efficiency of rRFBs in Saccharomyces cerevisiae and to this end transfected budding yeast cells that express dissimilar quantities of Fob1 with circular minichromosomes containing different copies of the minimal 20-bp DNA segment that bind Fob1. To identify fork stalling we used high-resolution 2D agarose gel electrophoresis. The results obtained indicated that neighbor DNA sequences and the relative abundance of Fob1 modulate the efficiency of rRFBs to stall replication forks.

A novel penicillin derivative induces antitumor effect in melanoma cells.

In this study, we explored the in-vitro and in-vivo mechanism of antitumor action of a novel synthetic nonantibiotic triazolylpeptidyl penicillin derivative, named TAP7f, on B16-F0 murine melanoma cells. In-vitro assays showed that TAP7f caused an inhibition of S phase progression and a concomitant decrease of the percentage of cells in G0/G1 phase. We also found that TAP7f treatment induced an apoptotic response characterized by an increase of the sub-G1 fraction of B16-F0 hypodiploid cells, the occurrence of cells with picnotic nuclei, and the detection of phosphatidylserine exposure on the outer side of the plasma membrane. Apoptotic cell death was further characterized by the activation of caspase-8, caspase-9, and caspase-3; the increase in the proapoptotic/antiapoptotic ratio of Bcl-2 family proteins; the higher expression levels of Fas receptor and TRAIL ligand; and the cleavage of poly(ADP-ribose) polymerase, a caspase-3 substrate. The in-vivo effect of TAP7f was studied in a syngeneic C57BL/6J mouse melanoma model. Results showed that TAP7f inhibited melanoma cell proliferation in vivo, as determined by a decreased expression of proliferating cell nuclear antigen, inducing a significant reduction of tumor growth. Apoptosis in vivo was assessed by detecting active caspase-3 in tumor slices from treated mice and the expression levels of Fas, TRAIL, and Bcl-2 proteins in tumor lysates. The administration of 80 mg/kg of TAP7f to non-tumor-bearing mice showed no histopathological effects on different organ tissues. Our results suggest that TAP7f might be considered as a potential therapeutic agent for cancer treatment.

σ1-Receptor Agonism Protects against Renal Ischemia-Reperfusion Injury.

Mechanisms of renal ischemia-reperfusion injury remain unresolved, and effective therapies are lacking. We previously showed that dehydroepiandrosterone protects against renal ischemia-reperfusion injury in male rats. Here, we investigated the potential role of σ1-receptor activation in mediating this protection. In rats, pretreatment with either dehydroepiandrosterone or fluvoxamine, a high-affinity σ1-receptor agonist, improved survival, renal function and structure, and the inflammatory response after sublethal renal ischemia-reperfusion injury. In human proximal tubular epithelial cells, stimulation by fluvoxamine or oxidative stress caused the σ1-receptor to translocate from the endoplasmic reticulum to the cytosol and nucleus. Fluvoxamine stimulation in these cells also activated nitric oxide production that was blocked by σ1-receptor knockdown or Akt inhibition. Similarly, in the postischemic rat kidney, σ1-receptor activation by fluvoxamine triggered the Akt-nitric oxide synthase signaling pathway, resulting in time- and isoform-specific endothelial and neuronal nitric oxide synthase activation and nitric oxide production. Concurrently, intravital two-photon imaging revealed prompt peritubular vasodilation after fluvoxamine treatment, which was blocked by the σ1-receptor antagonist or various nitric oxide synthase blockers. In conclusion, in this rat model of ischemia-reperfusion injury, σ1-receptor agonists improved postischemic survival and renal function via activation of Akt-mediated nitric oxide signaling in the kidney. Thus, σ1-receptor activation might provide a therapeutic option for renoprotective therapy.

An Ibero-American inter-laboratory trial to evaluate serological tests for the detection of anti-Neospora caninum antibodies in cattle.

We carried out an inter-laboratory trial to compare the serological tests commonly used for the detection of specific Neospora caninum antibodies in cattle in Ibero-American countries. A total of eight laboratories participated from the following countries: Argentina (n = 4), Brazil (n = 1), Peru (n = 1), Mexico (n = 1), and Spain (n = 1). A blind panel of well-characterized cattle sera (n = 143) and sera representative of the target population (n = 351) was tested by seven in-house indirect fluorescent antibody tests (IFATs 1-7) and three enzyme-linked immunosorbent assays (ELISAs 1-3; two in-house and one commercial). Diagnostic performance of the serological tests was calculated and compared according to the following criteria: (1) the "Pre-test information," which uses previous epidemiological and serological data; (2) the "Majority of tests," which classifies a serum as positive or negative according to the results obtained by most tests evaluated. Unexpectedly, six tests showed either sensitivity (Se) or specificity (Sp) values lower than 90%. In contrast, the best tests in terms of Se, Sp, and area under the ROC curve (AUC) values were IFAT 1 and optimized ELISA 1 and ELISA 2. We evaluated a high number of IFATs, which are the most widely used tests in Ibero-America. The significant discordances observed among the tests regardless of the criteria employed hinder control programs and urge the use of a common test or with similar performances to either the optimized IFAT 1 and ELISAs 1 and 2.

Characteristics of headache during and after digital substraction angiography: A critical re-appraisal of the ICHD-3 criteria.

Introduction Headache is a frequent but neglected side effect of angiography, and the criteria for angiography related headache have been based on only a few studies. Methods One-hundred and thirty nine patients who underwent cerebral angiography and 30 controls who underwent peripheral angiography participated in this prospective, non-randomized, case-control study. Participants were instructed to tell the angiography staff in case a headache developed and were questioned about their headache just after, 24 hours after, and one week after angiography. Results In the cerebral angiography group 42 patients (30.2%) had procedural headache compared to three of the controls (10%). Two different types of headache related to angiography were observed: one during angiography, which was related to contrast injection, and the other occuring within 24 hours. Both headaches were more frequent in patients with a history of primary headache, but were distinctly different from the premorbid headache. Headache characteristics are described in detail. Discussion Both types of headache related to angiography did not match the ICHD-3beta criteria for angiography headache, but were similar to headaches reported in studies that were not included in the current criteria. The timing and characteristics of these headaches are discussed in the light of our and previous reports. We suggest that the present ICHD-3 beta criteria are inadequate in properly defining angiography headache and should be revised in the light of the present literature.

Electrophoretic mobility of supercoiled, catenated and knotted DNA molecules.

We systematically varied conditions of two-dimensional (2D) agarose gel electrophoresis to optimize separation of DNA topoisomers that differ either by the extent of knotting, the extent of catenation or the extent of supercoiling. To this aim we compared electrophoretic behavior of three different families of DNA topoisomers: (i) supercoiled DNA molecules, where supercoiling covered the range extending from covalently closed relaxed up to naturally supercoiled DNA molecules; (ii) postreplicative catenanes with catenation number increasing from 1 to ∼15, where both catenated rings were nicked; (iii) knotted but nicked DNA molecules with a naturally arising spectrum of knots. For better comparison, we studied topoisomer families where each member had the same total molecular mass. For knotted and supercoiled molecules, we analyzed dimeric plasmids whereas catenanes were composed of monomeric forms of the same plasmid. We observed that catenated, knotted and supercoiled families of topoisomers showed different reactions to changes of agarose concentration and voltage during electrophoresis. These differences permitted us to optimize conditions for their separation and shed light on physical characteristics of these different types of DNA topoisomers during electrophoresis.

National-Level Wetland Policy Specificity and Goals Vary According to Political and Economic Indicators.

Growing recognition of the importance of wetlands to human and ecosystem well-being has led countries worldwide to implement wetland protection policies. Different countries have taken different approaches to wetland protection by implementing various policies, including territorial exclusion, market-based offsetting, and incentive programs for land users. Our objective was to describe the relationship between components of national-level wetland protection policies and national characteristics, including natural resource, economic, social, and political factors. We compiled data on the wetland policies of all 193 countries recognized by the U.N. and described the relationships among wetland policy goals and wetland protection mechanisms using non-metric multidimensional scaling. The first non-metric multidimensional scaling axis strongly correlated with whether a country had a wetland-specific environmental policy in place. Adoption of a comprehensive, wetland-specific policy was positively associated with degree of democracy and a commitment to establishing protected areas. The second non-metric multidimensional scaling axis defined a continuum of policy goals and mechanisms by which wetlands are protected, with goals to protect wetland ecosystem services on one end of the spectrum and goals to protect biodiversity on the other. Goals for protecting ecosystem services were frequently cited in policy documents of countries with agriculture-based economies, whereas goals associated with wetland biodiversity tended to be associated with tourism-based economies. We argue that the components of a country's wetland policies reflect national-level resource and economic characteristics. Understanding the relationship between the type of wetland policy countries adopt and national-level characteristics is critical for international efforts to protect wetlands.

Direct Evidence for the Formation of Precatenanes during DNA Replication.

The dynamics of DNA topology during replication are still poorly understood. Bacterial plasmids are negatively supercoiled. This underwinding facilitates strand separation of the DNA duplex during replication. Leading the replisome, a DNA helicase separates the parental strands that are to be used as templates. This strand separation causes overwinding of the duplex ahead. If this overwinding persists, it would eventually impede fork progression. In bacteria, DNA gyrase and topoisomerase IV act ahead of the fork to keep DNA underwound. However, the processivity of the DNA helicase might overcome DNA gyrase and topoisomerase IV. It was proposed that the overwinding that builds up ahead of the fork could force it to swivel and diffuse this positive supercoiling behind the fork where topoisomerase IV would also act to maintain replicating the DNA underwound. Putative intertwining of sister duplexes in the replicated region are called precatenanes. Fork swiveling and the formation of precatenanes, however, are still questioned. Here, we used classical genetics and high resolution two-dimensional agarose gel electrophoresis to examine the torsional tension of replication intermediates of three bacterial plasmids with the fork stalled at different sites before termination. The results obtained indicated that precatenanes do form as replication progresses before termination.

Abnormal high-energy phosphate molecule metabolism during regional brain activation in patients with bipolar disorder.

Converging evidence suggests bioenergetic abnormalities in bipolar disorder (BD). In the brain, phosphocreatine (PCr) acts a reservoir of high-energy phosphate (HEP) bonds, and creatine kinases (CK) catalyze the transfer of HEP from adenosine triphosphate (ATP) to PCr and from PCr back to ATP, at times of increased need. This study examined the activity of this mechanism in BD by measuring the levels of HEP molecules during a stimulus paradigm that increased local energy demand. Twenty-three patients diagnosed with BD-I and 22 healthy controls (HC) were included. Levels of phosphorus metabolites were measured at baseline and during visual stimulation in the occipital lobe using (31)P magnetic resonance spectroscopy at 4T. Changes in metabolite levels showed different patterns between the groups. During stimulation, HC had significant reductions in PCr but not in ATP, as expected. In contrast, BD patients had significant reductions in ATP but not in PCr. In addition, PCr/ATP ratio was lower at baseline in patients, and there was a higher change in this measure during stimulation. This pattern suggests a disease-related failure to replenish ATP from PCr through CK enzyme catalysis during tissue activation. Further studies measuring the CK flux in BD are required to confirm and extend this finding.

Topo IV is the topoisomerase that knots and unknots sister duplexes during DNA replication.

DNA topology plays a crucial role in all living cells. In prokaryotes, negative supercoiling is required to initiate replication and either negative or positive supercoiling assists decatenation. The role of DNA knots, however, remains a mystery. Knots are very harmful for cells if not removed efficiently, but DNA molecules become knotted in vivo. If knots are deleterious, why then does DNA become knotted? Here, we used classical genetics, high-resolution 2D agarose gel electrophoresis and atomic force microscopy to show that topoisomerase IV (Topo IV), one of the two type-II DNA topoisomerases in bacteria, is responsible for the knotting and unknotting of sister duplexes during DNA replication. We propose that when progression of the replication forks is impaired, sister duplexes become loosely intertwined. Under these conditions, Topo IV inadvertently makes the strand passages that lead to the formation of knots and removes them later on to allow their correct segregation.

[Endemic and epidemic bovine neosporosis: description of two events in beef cattle]. / Neosporosis epidémica y endémica: descripción de dos eventos en bovinos para cría.

The aim of this study was to describe two events in which Neospora caninum was involved in bovine abortions in beef cattle. In the first event, 11 abortions in 57 heifers were recorded in 45 days. One aborted heifer was 5 times more likely to be seropositive than a non-aborted heifer (OR=4.9; IC 1.2-19.9) (p<0.05). In the second event, no association between serological results and abortions were observed (OR=0,69; 0,06-7,31) (p>0.05). Neither antibodies nor isolation of other pathogens were achieved in any case. On the contrary, antibodies and pathognomonic histopathological lesions were observed in the four fetuses from both cases. Interestingly, the findings in the first event suggest the epidemic behavior of the disease. In contrast, in the second event it appears that few abortions were due to N. caninum, suggesting the presence of endemic neosporosis.

Electrophoretic Mobility Assay to Separate Supercoiled, Catenated, and Knotted DNA Molecules.

Two-dimensional (2D) agarose gel electrophoresis is the method of choice to analyze DNA topology. The possibility to use E. coli strains with different genetic backgrounds in combination with nicking enzymes and different concentrations of norfloxacin improves the resolution of 2D gels to study the electrophoretic behavior of three different families of DNA topoisomers: supercoiled DNA molecules, post-replicative catenanes, and knotted DNA molecules. Here, we describe the materials and procedures required to optimize their separation by 2D gels. Understanding the differences in their electrophoretic behavior can help explain some important physical characteristics of these different types of DNA topoisomers. Key features • Preparative method to enrich DNA samples of supercoiled, catenated, and knotted families of topoisomers, later analyzed by 2D gels (or other techniques, e.g., microscopy). • 2D gels facilitate the separation of the topoisomers of any given circular DNA molecule. • Separation of DNA molecules with the same molecular masses but different shapes can be optimized by modifying the conditions of 2D gels. • Evaluating the roles of electric field and agarose concentration on the electrophoretic mobility of DNA topoisomers sheds light on their physical characteristics.

FEMaLe: The use of machine learning for early diagnosis of endometriosis based on patient self-reported data-Study protocol of a multicenter trial.

INTRODUCTION: Endometriosis is a chronic disease that affects up to 190 million women and those assigned female at birth and remains unresolved mainly in terms of etiology and optimal therapy. It is defined by the presence of endometrium-like tissue outside the uterine cavity and is commonly associated with chronic pelvic pain, infertility, and decreased quality of life. Despite the availability of various screening methods (e.g., biomarkers, genomic analysis, imaging techniques) intended to replace the need for invasive surgery, the time to diagnosis remains in the range of 4 to 11 years. AIMS: This study aims to create a large prospective data bank using the Lucy mobile health application (Lucy app) and analyze patient profiles and structured clinical data. In addition, we will investigate the association of removed or restricted dietary components with quality of life, pain, and central pain sensitization. METHODS: A baseline and a longitudinal questionnaire in the Lucy app collects real-world, self-reported information on symptoms of endometriosis, socio-demographics, mental and physical health, economic factors, nutritional, and other lifestyle factors. 5,000 women with confirmed endometriosis and 5,000 women without diagnosed endometriosis in a control group will be enrolled and followed up for one year. With this information, any connections between recorded symptoms and endometriosis will be analyzed using machine learning. CONCLUSIONS: We aim to develop a phenotypic description of women with endometriosis by linking the collected data with existing registry-based information on endometriosis diagnosis, healthcare utilization, and big data approach. This may help to achieve earlier detection of endometriosis with pelvic pain and significantly reduce the current diagnostic delay. Additionally, we may identify dietary components that worsen the quality of life and pain in women with endometriosis, upon which we can create real-world data-based nutritional recommendations.

The fission yeast rDNA-binding protein Reb1 regulates G1 phase under nutritional stress.

Yeast Reb1 and its mammalian ortholog TTF1 are conserved Myb-type DNA-binding proteins that bind to specific sites near the 3'-end of rRNA genes (rDNA). Here, they participate in the termination of transcription driven by RNA polymerase I and block DNA replication forks approaching in the opposite direction. We found that Schizosaccharomyces pombe Reb1 also upregulates transcription of the ste9(+) gene that is required for nitrogen-starvation-induced growth arrest with a G1 DNA content and sexual differentiation. Ste9 activates the anaphase-promoting complex or cyclosome ('APC/C') in G1, targeting B-cyclin for proteasomal degradation in response to nutritional stress. Reb1 binds in vivo and in vitro to a specific DNA sequence at the promoter of ste9(+), similar to the sequence recognized in the rDNA, and this binding is required for ste9(+) transcriptional activation and G1 arrest. This suggests that Reb1 acts as a link between rDNA metabolism and cell cycle control in response to nutritional stress. In agreement with this new role for Reb1 in the regulation of the G1-S transition, reb1Δ and wee1(ts) mutations are synthetically lethal owing to the inability of these cells to lengthen G1 before entering S phase. Similarly, reb1Δ cdc10(ts) cells are unable to arrest in G1 and die at the semi-permissive temperature.