Functional validation of mouse tyrosinase non-coding regulatory DNA elements by CRISPR–Cas9-mediated mutagenesis

Newly developed genome-editing tools, such as the clustered regularly interspaced short palindromic repeat (CRISPR)–Cas9 system, allow simple and rapid genetic modification in most model organisms and human cell lines. Here, we report the production and analysis of mice carrying the inactivation via deletion of a genomic insulator, a key non-coding regulatory DNA element found 5′ upstream of the mouse tyrosinase (Tyr) gene. Targeting sequences flanking this boundary in mouse fertilized eggs resulted in the efficient deletion or inversion of large intervening DNA fragments delineated by the RNA guides. The resulting genome-edited mice showed a dramatic decrease in Tyr gene expression as inferred from the evident decrease of coat pigmentation, thus supporting the functionality of this boundary sequence in vivo, at the endogenous locus. Several potential off-targets bearing sequence similarity with each of the two RNA guides used were analyzed and found to be largely intact. This study reports how non-coding DNA elements, even if located in repeat-rich genomic sequences, can be efficiently and functionally evaluated in vivo and, furthermore, it illustrates how the regulatory elements described by the ENCODE and EPIGENOME projects, in the mouse and human genomes, can be systematically validated.     

Variations in heart rate and rhythm of harbor seal pups during rehabilitation

Heart rate and rhythm is regulated by the autonomic nervous system, which matures during the first months of life. Little is known about heart rate and rhythm development and potential arrhythmias in seal pups during rehabilitation in seal centers. Using an iPhone ECG device, 1 min ECGs were obtained from harbor seal pups admitted to a seal rehabilitation facility. ECGs were taken from 55 seals after admission, 53 seals after 14 d, and 52 seals prior to release. From 24 seal pups additional ECGs were taken daily for the first week of rehabilitation. At admission sinus rhythm with a median heart rate of 148 complexes per minute was detected, prior to release sinus bradycardia or sinus arrhythmia with a median heart rate of 104 complexes minute was present. P wave morphology was highly variable and single supra‐ and ventricular premature complexes were recorded in individual animals. The first 14 d were characterized by highly variable heart rates and rhythms, including episodes of sinus tachycardia and 2nd degree atrioventricular blocks. The reduction in heart rates and development of a regular heart rhythm during rehabilitation suggest adaptation to the unfamiliar environment, resolution of disease, and/or maturation of the autonomic nervous system.     

Population structure of Squatina guggenheim (Squatiniformes,Squatinidae) from the south‐western Atlantic Ocean

Population genetic analyses based on both mitochondrial cytochrome b and the internal transcribed spacer 2 of recombinant (r)DNA genes were implemented to examine hypotheses of population differentiation in the angular angel shark Squatina guggenheim, one of the four most‐widespread endemic species inhabiting coastal ecosystems in the south‐western Atlantic Ocean. A total of 82 individuals of S. guggenheim from 10 sampling sites throughout the Río de la Plata mouth, its maritime front, the outer shelf at the subtropical confluence and the coastal areas of the south‐west Atlantic Ocean, were included. The analysis of molecular variance (AMOVA) based on the second internal transcribed spacer (its‐2) region supports that the samples from the outer shelf represent an isolated group from other sites. Historical gene flow in a coalescent‐based approach revealed significant immigration and emigration asymmetry between sampling sites. Based on the low level of genetic diversity, the existence of a long‐term population decline or a past recent population expansion following a population bottleneck could be proposed in S. guggenheim. This demographic differentiation suggests a degree of vulnerability to overexploitation in this endemic and endangered south‐west Atlantic Ocean shark, given its longevity and low reproductive potential.     

Maintaining Specimen Integrity for G6PD Screening by Cytofluorometric Assays

Cytochemical staining remains an efficient way of identifying females who are heterozygous for the X chromosome-linked glucose-6-phosphate dehydrogenase (G6PD) gene. G6PD is highly polymorphic with certain alleles resulting in low intracellular G6PD activity in red blood cells. Low intracellular G6PD activity is associated with a risk of severe hemolysis when exposed to an oxidative stress such as fava beans, certain drugs and infections. Heterozygous females express the enzyme from both X-chromosome alleles resulting in two red blood cell populations each with G6PD enzyme characteristics representative of each allele; for example, normal and deficient. Cytochemical staining is the only way to determine the relative representation of each allele in red blood cells, a feature that is critical when assessing the risk for severe hemolysis when exposed to an oxidant such as the anti-malarial drug primaquine. This letter discusses red blood cell integrity with respect to the cytofluorometric assays for G6PD activity. An approach to making this test more robust is suggested. The approach makes this test more reliable and extends its use to a broader range of blood specimens.     

Metformin protects against doxorubicin-induced cardiotoxicity: involvement of the adiponectin cardiac system

Doxorubicin has cardiotoxic effects that limit its clinical benefit in cancer patients. Metformin exerts cardioprotective actions via AMP-activated protein kinase (AMPK) and increases the expression of adiponectin and its receptors (adipoR1 and adipoR2) in skeletal muscle and adipose tissue, but its effect on cardiac tissue is still unknown. This work aimed to study whether metformin exerts any protective action against the cardiotoxicity of doxorubicin and whether the cardiac system of adiponectin is involved in any such action. The addition of doxorubicin (5μM) to adult mouse cardiomyocytes (HL-1 cell line) induced apoptosis, which was characterized by a loss of cell viability, activation of caspases, and fragmentation of the genetic material. Doxorubicin treatment also caused a decrease in the activity of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase. Pretreatment with metformin (4mM, 24h) provided protection against doxorubicin-induced damage. This pretreatment significantly increased cell viability, attenuated the activation of caspases and the fragmentation of genetic material, and restored the antioxidant activity. In addition, metformin up-regulated the expression of adiponectin and its receptors, adipoR1 and adipoR2, in cardiomyocytes. In contrast, silencing either adipoR1 or adipoR2 with siRNA inhibited the AMPK activation and the protective effects of metformin. Taken together, these results demonstrate that metformin protects cardiomyocytes from doxorubicin-induced damage and that the cardiac adiponectin system plays an important role in this protective action.     

Biofiltration of reduced sulphur compounds and community analysis of sulphur-oxidizing bacteria

The present work aims to use a two-stage biotrickling filters for simultaneous treatment of hydrogen sulphide (H₂S), methyl mercaptan (MM), dimethyl sulphide (DMS) and dimethyl disulphide (DMDS). The first biofilter was inoculated with Acidithiobacillus thiooxidans (BAT) and the second one with Thiobacillus thioparus (BTT). For separate feeds of reduced sulphur compounds (RSC), the elimination capacity (EC) order was DMDS > DMS > MM. The EC values were 9.8 g_MM-S/m³/h (BTT; 78% removal efficiency (RE); empty bed residence time (EBRT) 58 s), 36 g_DMDS-S/m³/h (BTT; 94.4% RE; EBRT 76 s) and 57.5 g_H2S-S/m³/h (BAT; 92% RE; EBRT 59 s). For the simultaneous removal of RSC in BTT, an increase in the H₂S concentration from 23 to 293 ppmv (EBRT of 59 s) inhibited the RE of DMS (97-84% RE), DMDS (86-76% RE) and MM (83-67% RE). In the two-stage biofiltration, the RE did not decrease on increasing the H₂S concentration from 75 to 432 ppmv.     

Viral genome segmentation can result from a trade-off between genetic content and particle stability

The evolutionary benefit of viral genome segmentation is a classical, yet unsolved question in evolutionary biology and RNA genetics. Theoretical studies anticipated that replication of shorter RNA segments could provide a replicative advantage over standard size genomes. However, this question has remained elusive to experimentalists because of the lack of a proper viral model system. Here we present a study with a stable segmented bipartite RNA virus and its ancestor non-segmented counterpart, in an identical genomic nucleotide sequence context. Results of RNA replication, protein expression, competition experiments, and inactivation of infectious particles point to a non-replicative trait, the particle stability, as the main driver of fitness gain of segmented genomes. Accordingly, measurements of the volume occupation of the genome inside viral capsids indicate that packaging shorter genomes involves a relaxation of the packaging density that is energetically favourable. The empirical observations are used to design a computational model that predicts the existence of a critical multiplicity of infection for domination of segmented over standard types. Our experiments suggest that viral segmented genomes may have arisen as a molecular solution for the trade-off between genome length and particle stability. Genome segmentation allows maximizing the genetic content without the detrimental effect in stability derived from incresing genome length.