Cardiovascular effects of dietary salt intake in aged healthy cats: a 2-year prospective randomized, blinded, and controlled study

High salt dry expanded diets are commercially available for cats to increase water intake and urine volume, as part of the prevention or treatment of naturally occurring urinary stone formation (calcium oxalates and struvites). However, chronic high salt intake may have potential cardiovascular adverse effects in both humans, especially in aging individuals, and several animal models. The objective of this prospective, randomized, blinded, and controlled study was to assess the long-term cardiovascular effects of high salt intake in healthy aged cats. Twenty healthy neutered cats (10.1±2.4 years) were randomly allocated into 2 matched groups. One group was fed a high salt diet (3.1 g/Mcal sodium, 5.5 g/Mcal chloride) and the other group a control diet of same composition except for salt content (1.0 g/Mcal sodium, 2.2 g/Mcal chloride). Clinical examination, systolic and diastolic arterial blood pressure measurements, standard transthoracic echocardiography and conventional Doppler examinations were repeatedly performed on non-sedated cats by trained observers before and over 24 months after diet implementation. Radial and longitudinal velocities of the left ventricular free wall and the interventricular septum were also assessed in systole and diastole using 2-dimensional color tissue Doppler imaging. Statistics were performed using a general linear model. No significant effect of dietary salt intake was observed on systolic and diastolic arterial blood pressure values. Out of the 33 tested imaging variables, the only one affected by dietary salt intake was the radial early on late diastolic velocity ratio assessed in the endocardium of the left ventricular free wall, statistically lower in the high salt diet group at 12 months only (P = 0.044). In conclusion, in this study involving healthy aged cats, chronic high dietary salt intake was not associated with an increased risk of systemic arterial hypertension and myocardial dysfunction, as observed in some elderly people, salt-sensitive patients and animal models

Base-editing-mediated dissection of a γ-globin cis-regulatory element for the therapeutic reactivation of fetal hemoglobin expression

: Sickle cell disease and β-thalassemia affect the production of the adult β-hemoglobin chain. The clinical severity is lessened by mutations that cause fetal γ-globin expression in adult life (i.e., the hereditary persistence of fetal hemoglobin). Mutations clustering ~200 nucleotides upstream of the HBG transcriptional start sites either reduce binding of the LRF repressor or recruit the KLF1 activator. Here, we use base editing to generate a variety of mutations in the -200 region of the HBG promoters, including potent combinations of four to eight γ-globin-inducing mutations. Editing of patient hematopoietic stem/progenitor cells is safe, leads to fetal hemoglobin reactivation and rescues the pathological phenotype. Creation of a KLF1 activator binding site is the most potent strategy - even in long-term repopulating hematopoietic stem/progenitor cells. Compared with a Cas9-nuclease approach, base editing avoids the generation of insertions, deletions and large genomic rearrangements and results in higher γ-globin levels. Our results demonstrate that base editing of HBG promoters is a safe, universal strategy for treating β-hemoglobinopathies

Attenuation of Notch and Hedgehog Signaling Is Required for Fate Specification in the Spinal Cord

During the development of the spinal cord, proliferative neural progenitors differentiate into postmitotic neurons with distinct fates. How cells switch from progenitor states to differentiated fates is poorly understood. To address this question, we studied the differentiation of progenitors in the zebrafish spinal cord, focusing on the differentiation of Kolmer-Agduhr″ (KA″) interneurons from lateral floor plate (LFP) progenitors. In vivo cell tracking demonstrates that KA″ cells are generated from LFP progenitors by both symmetric and asymmetric cell divisions. A photoconvertible reporter of signaling history (PHRESH) reveals distinct temporal profiles of Hh response: LFP progenitors continuously respond to Hh, while KA″ cells lose Hh response upon differentiation. Hh signaling is required in LFP progenitors for KA″ fate specification, but prolonged Hh signaling interferes with KA″ differentiation. Notch signaling acts permissively to maintain LFP progenitor cells: activation of Notch signaling prevents differentiation, whereas inhibition of Notch signaling results in differentiation of ectopic KA″ cells. These results indicate that neural progenitors depend on Notch signaling to maintain Hh responsiveness and rely on Hh signaling to induce fate identity, whereas proper differentiation depends on the attenuation of both Notch and Hh signaling

Cooperation of Mtmr8 with PI3K Regulates Actin Filament Modeling and Muscle Development in Zebrafish

It has been shown that mutations in at least four myotubularin family genes (MTM1, MTMR1, 2 and 13) are causative for human neuromuscular disorders. However, the pathway and regulative mechanism remain unknown.Here, we reported a new role for Mtmr8 in neuromuscular development of zebrafish. Firstly, we cloned and characterized zebrafish Mtmr8, and revealed the expression pattern predominantly in the eye field and somites during early somitogenesis. Using morpholino knockdown, then, we observed that loss-of-function of Mtmr8 led to defects in somitogenesis. Subsequently, the possible underlying mechanism and signal pathway were examined. We first checked the Akt phosphorylation, and observed an increase of Akt phosphorylation in the morphant embryos. Furthermore, we studied the PH/G domain function within Mtmr8. Although the PH/G domain deletion by itself did not result in embryonic defect, addition of PI3K inhibitor LY294002 did give a defective phenotype in the PH/G deletion morphants, indicating that the PH/G domain was essential for Mtmr8's function. Moreover, we investigated the cooperation of Mtmr8 with PI3K in actin filament modeling and muscle development, and found that both Mtmr8-MO1 and Mtmr8-MO2+LY294002 led to the disorganization of the actin cytoskeleton. In addition, we revealed a possible participation of Mtmr8 in the Hedgehog pathway, and cell transplantation experiments showed that Mtmr8 worked in a non-cell autonomous manner in actin modeling.The above data indicate that a conserved functional cooperation of Mtmr8 with PI3K regulates actin filament modeling and muscle development in zebrafish, and reveal a possible participation of Mtmr8 in the Hedgehog pathway. Therefore, this work provides a new clue to study the physiological function of MTM family members

In the Absence of Sonic Hedgehog, p53 Induces Apoptosis and Inhibits Retinal Cell Proliferation, Cell-Cycle Exit and Differentiation in Zebrafish

Background: Sonic hedgehog (Shh) signaling regulates cell proliferation during vertebrate development via induction of cell-cycle regulator gene expression or activation of other signalling pathways, prevents cell death by an as yet unclear mechanism and is required for differentiation of retinal cell types. Thus, an unsolved question is how the same signalling molecule can regulate such distinct cell processes as proliferation, cell survival and differentiation. Methodology/Principal Findings: Analysis of the zebrafish shh 2/2 mutant revealed that in this context p53 mediates elevated apoptosis during nervous system and retina development and interferes with retinal proliferation and differentiation. While in shh 2/2 mutants there is activation of p53 target genes and p53-mediated apoptosis, an increase in Hedgehog (Hh) signalling by over-expression of dominant-negative Protein Kinase A strongly decreased p53 target gene expression and apoptosis levels in shh 2/2 mutants. Using a novel p53 reporter transgene, I confirm that p53 is active in tissues that require Shh for cell survival. Proliferation assays revealed that loss of p53 can rescue normal cell-cycle exit and the mitotic indices in the shh 2/2 mutant retina at 24, 36 and 48 hpf. Moreover, generation of amacrine cells and photoreceptors was strongly enhanced in the double p53 2/2 shh 2/2 mutant retina suggesting the effect of p53 on retinal differentiation. Conclusions: Loss of Shh signalling leads to the p53-dependent apoptosis in the developing nervous system and retina

A ubiquitous enhancer shared by two promoters in the human aldolase A gene.

The human aldolase A gene is transcribed from three different promoters, which are all clustered within a 1.6 kbp DNA domain. Two of these, PN and PH, are ubiquitous and seem to be co-regulated in most tissues while the third one, PM, is specific to adult skeletal muscle. We investigated the sequences involved in the ubiquitous activity of the PN and PH promoters of the human aldolase A gene. Deletion analysis, performed by transient expression assays of chloramphenicol acetyltransferase reporter genes in human HepG2 hepatoma cells, indicated that PH activity results from the interaction of an upstream activating region with two distinct core promoters. The upstream activating region was able to stimulate transcription from the HSV tk promoter as efficiently as the SV40 enhancer in all cell types tested. It appears, therefore, to be a strong ubiquitous enhancer. DNAsel footprinting revealed protections covering sequences scattered along the enhancer, including Sp1 and AP1 motifs. Importantly, we found that this enhancer was also necessary to activity of the other ubiquitous promoter of the aldolase A gene, PN. These studies demonstrate that expression of the human aldolase A gene is mediated by a complex interplay of enhancer and promoter elements

An opportunistic promoter sharing regulatory sequences with either a muscle-specific or a ubiquitous promoter in the human aldolase A gene.

The human aldolase A gene is transcribed from three different promoters, pN, pM, and pH, all of which are clustered within a small 1.6-kbp DNA domain. pM, which is highly specific to adult skeletal muscle, lies in between pN and pH, which are ubiquitous but particularly active in heart and skeletal muscle. A ubiquitous enhancer, located just upstream of pH start sites, is necessary for the activity of both pH and pN in transient transfection assays. Using transgenic mice, we studied the sequence controlling the muscle-specific promoter pM and the relations between the three promoters and the ubiquitous enhancer. A 4.3-kbp fragment containing the three promoters and the ubiquitous enhancer showed an expression pattern consistent with that known in humans. In addition, while pH was active in both fast and slow skeletal muscles, pM was active only in fast muscle. pM activity was unaltered by the deletion of a 1.8-kbp region containing the ubiquitous enhancer and the pH promoter, whereas pN remained active only in fast skeletal muscle. These findings suggest that in fast skeletal muscle, a tissue-specific enhancer was acting on both pN and pM, whereas in other tissues, the ubiquitous enhancer was necessary for pN activity. Finally, a 2.6-kbp region containing the ubiquitous enhancer and only the pH promoter was sufficient to bring about high-level expression of pH in cardiac and skeletal muscle. Thus, while pH and pM function independently of each other, pN, remarkably, shares regulatory elements with each of them, depending on the tissue. Importantly, expression of the transgenes was independent of the integration site, as originally described for transgenes containing the beta-globin locus control region