Increased negative superhelical density in vivo enhances the genetic instability of triplet repeat sequences

The influence of negative superhelical density on the genetic instabilities of long GAA.TTC, CGG.CCG, and CTG.CAG repeat sequences was studied in vivo in topologically constrained plasmids in Escherichia coli. These repeat tracts are involved in the etiologies of Friedreich ataxia, fragile X syndrome, and myotonic dystrophy type 1, respectively. The capacity of these DNA tracts to undergo deletions-expansions was explored with three genetic-biochemical approaches including first, the utilization of topoisomerase I and/or DNA gyrase mutants, second, the specific inhibition of DNA gyrase by novobiocin, and third, the genetic removal of the HU protein, thus lowering the negative supercoil density (-sigma). All three strategies revealed that higher -sigma in vivo enhanced the formation of deleted repeat sequences. The effects were most pronounced for the Friedreich ataxia and the fragile X triplet repeat sequences. Higher levels of -sigma stabilize non-B DNA conformations (i.e. triplexes, sticky DNA, flexible and writhed DNA, slipped structures) at appropriate repeat tracts; also, numerous prior genetic instability investigations invoke a role for these structures in promoting the slippage of the DNA complementary strands. Thus, we propose that the in vivo modulation of the DNA structure, localized to the repeat tracts, is responsible for these behaviors. Presuming that these interrelationships are also found in humans, dynamic alterations in the chromosomal nuclear matrix may modulate the -sigma of certain DNA regions and, thus, stabilize/destabilize certain non-B conformations which regulate the genetic expansions-deletions responsible for the diseases.     

The myotonic dystrophy type 1 triplet repeat sequence induces gross deletions and inversions

The capacity of (CTG.CAG)n and (GAA.TTC)n repeat tracts in plasmids to induce mutations in DNA flanking regions was evaluated in Escherichia coli. Long repeats of these sequences are involved in the etiology of myotonic dystrophy type 1 and Friedreich's ataxia, respectively. Long (CTG.CAG)n (where n = 98 and 175) caused the deletion of most, or all, of the repeats and the flanking GFP gene. Deletions of 0.6-1.8 kbp were found as well as inversions. Shorter repeat tracts (where n = 0 or 17) were essentially inert, as observed for the (GAA.TTC)176-containing plasmid. The orientation of the triplet repeat sequence (TRS) relative to the unidirectional origin of replication had a pronounced effect, signaling the participation of replication and/or repair systems. Also, when the TRS was transcribed, the level of deletions was greatly elevated. Under certain conditions, 30-50% of the products contained gross deletions. DNA sequence analyses of the breakpoint junctions in 47 deletions revealed the presence of 1-8-bp direct or inverted homologies in all cases. Also, the presence of non-B folded conformations (i.e. slipped structures, cruciforms, or triplexes) at or near the breakpoints was predicted in all cases. This genetic behavior, which was previously unrecognized for a TRS, may provide the basis for a new type of instability of the myotonic dystrophy protein kinase (DMPK) gene in patients with a full mutation.     

Experimental lagochilascariosis: histopathological study of inflammatory response to larval migration in the Murine model

The goal of this study was to investigate the pattern of inflammatory response induced by Lagochilascaris minor in murine experimental model. For this purpose 115 mice were given 1000-3000 L. minor infective eggs "per os" and 51 uninfected mice were considered as controls. Four hours post-inoculation (PI), 3rd stage larvae were seen passing through the mucosa of terminal ends of small intestine. Six hours PI larvae were observed as an embolus inside the portal vein and also migrating through the liver parenchyma. During the first 24 h larvae-containing eggs of L. minor were observed in the lumen of intestinal tract. Two days PI larvae were seen migrating through lung parenchyma associated with an initial neutrophilic perivasculitis. From the 13th day of this experimental study, L. minor larvae were found mainly in skeletal muscles, in the center of granulomas. Concentric fibrosis with mixed inflammatory infiltrate involved the larvae after the 47th day PI, persistently. This experimental murine study with L. minor indicated that the 3rd stage larvae penetrated via ileum-cecal mucosa reaching the liver and probably other tissues through the hematogenic via. Throughout its pathway the larvae induced a granulomatous reaction, with abundant polimorphonuclear cells.     

Endothelial nitric oxide synthase,vascular integrity and human exceptional longevity

Aging is the sum of the deleterious changes that occur as time goes by. It is the main risk factor for the development of cardiovascular disease, and aging of the vasculature is the event that most often impacts on the health of elderly people. The “free-radical theory of aging” was proposed to explain aging as a consequence of the accumulation of reactive oxygen species (ROS). However, recent findings contradict this theory, and it now seems that mechanisms mediating longevity act through induction of oxidative stress. In fact, calorie restriction ? a powerful way of delaying aging ? increases ROS accumulation due to stimulation of the basal metabolic rate; moreover, reports show that antioxidant therapy is detrimental to healthy aging. We also now know that genetic manipulation of the insulin-like-growth-factor-1/insulin signal (IIS) has a profound impact on the rate of aging and that the IIS is modulated by calorie restriction and physical exercise. The IIS regulates activation of nitric oxide synthase (eNOS), the activity of which is essential to improving lifespan through calorie restriction, as demonstrated by experiments on eNOS knockout mice. Indeed, eNOS has a key role in maintaining vascular integrity during aging by activating vasorelaxation and allowing migration and angiogenesis. In this review, we will overview current literature on these topics and we will try to convince the reader of the importance of vascular integrity and nitric oxide production in determining healthy aging.     

The involvement of non-B DNA structures in gross chromosomal rearrangements

Non-B DNA conformations adopted by certain types of DNA sequences promote genetic instabilities, especially gross rearrangements including translocations. We conclude the following: (a) slipped (hairpin) structures, cruciforms, triplexes, tetraplexes and i-motifs, and left-handed Z-DNA are formed in chromosomes and elicit profound genetic consequences via recombination-repair, (b) repeating sequences, probably in their non-B conformations, cause gross genomic rearrangements (translocations, deletions, insertions, inversions, and duplications), and (c) these rearrangements are the genetic basis for numerous human diseases including polycystic kidney disease, adrenoleukodystrophy, follicular lymphomas, and spermatogenic failure.     

Role of neural NO synthase (nNOS) uncoupling in the dysfunctional nitrergic vasorelaxation of penile arteries from insulin-resistant obese Zucker rats

Objective

Erectile dysfunction (ED) is considered as an early sign of vascular disease due to its high prevalence in patients with cardiovascular risk factors. Endothelial and neural dysfunction involving nitric oxide (NO) are usually implicated in the pathophysiology of the diabetic ED, but the underlying mechanisms are unclear. The present study assessed the role of oxidative stress in the dysfunctional neural vasodilator responses of penile arteries in the obese Zucker rat (OZR), an experimental model of metabolic syndrome/prediabetes.

Methods and Results

Electrical field stimulation (EFS) under non-adrenergic non-cholinergic (NANC) conditions evoked relaxations that were significantly reduced in penile arteries of OZR compared with those of lean Zucker rats (LZR). Blockade of NO synthase (NOS) inhibited neural relaxations in both LZR and OZR, while saturating concentrations of the NOS substrate L-arginine reversed the inhibition and restored relaxations in OZR to levels in arteries from LZR. nNOS expression was unchanged in arteries from OZR compared to LZR and nNOS selective inhibition decreased the EFS relaxations in LZR but not in OZR, while endothelium removal did not alter these responses in either strain. Superoxide anion production and nitro-tyrosine immunostaining were elevated in the erectile tissue from OZR. Treatment with the NADPH oxidase inhibitor apocynin or acute incubation with the NOS cofactor tetrahydrobiopterin (BH4) restored neural relaxations in OZR to levels in control arteries, while inhibition of the enzyme of BH4 synthesis GTP-cyclohydrolase (GCH) reduced neural relaxations in arteries from LZR but not OZR. The NO donor SNAP induced decreases in intracellular calcium that were impaired in arteries from OZR compared to controls.

Conclusions

The present study demonstrates nitrergic dysfunction and impaired neural NO signalling due to oxidative stress and nNOS uncoupling in penile arteries under conditions of insulin resistance. This dysfunction likely contributes to the metabolic syndrome-associated ED, along with the endothelial dysfunction also involving altered NO signalling.     

Relevance of the College of American Pathologists guideline for validating whole slide imaging for diagnostic purposes to cytopathology

Whole slide imaging (WSI) allows pathologists to view virtual versions of slides on computer monitors. With increasing adoption of digital pathology, laboratories have begun to validate their WSI systems for diagnostic purposes according to reference guidelines. Among these the College of American Pathologists (CAP) guideline includes three strong recommendations (SRs) and nine good practice statements (GPSs). To date, the application of WSI to cytopathology has been beyond the scope of the CAP guideline due to limited evidence. Herein we systematically reviewed the published literature on WSI validation studies in cytology. A systematic search was carried out in PubMed-MEDLINE and Embase databases up to November 2021 to identify all publications regarding validation of WSI in cytology. Each article was reviewed to determine if SRs and/or GPSs recommended by the CAP guideline were adequately satisfied. Of 3963 retrieved articles, 25 were included. Only 4/25 studies (16%) satisfied all three SRs, with only one publication (1/25, 4%) fulfilling all three SRs and nine GPSs. Lack of a suitable validation dataset was the main missing SR (16/25, 64%) and less than a third of the studies reported intra-observer variability data (7/25, 28%). Whilst the CAP guideline for WSI validation in clinical practice helped the widespread adoption of digital pathology, more evidence is required to routinely employ WSI for diagnostic purposes in cytopathology practice. More dedicated validation studies satisfying all SRs and/or GPSs recommended by the CAP are needed to help expedite the use of WSI for primary diagnosis in cytopathology.