Carbonic Anhydrase 5 Regulates Acid-Base Homeostasis in Zebrafish

The regulation of the acid-base balance in cells is essential for proper cellular homeostasis. Disturbed acid-base balance directly affects cellular physiology, which often results in various pathological conditions. In every living organism, the protein family of carbonic anhydrases regulate a broad variety of homeostatic processes. Here we describe the identification, mapping and cloning of a zebrafish carbonic anhydrase 5 (ca5) mutation, collapse of fins (cof), which causes initially a collapse of the medial fins followed by necrosis and rapid degeneration of the embryo. These phenotypical characteristics can be mimicked in wild-type embryos by acetazolamide treatment, suggesting that CA5 activity in zebrafish is essential for a proper development. In addition we show that CA5 regulates acid-base balance during embryonic development, since lowering the pH can compensate for the loss of CA5 activity. Identification of selective modulators of CA5 activity could have a major impact on the development of new therapeutics involved in the treatment of a variety of disorders

Characterization, mapping and cloning of the <i>cof</i> mutant.

<p>(A–D) Phenotypical comparison of wild-type and <i>cof</i> mutant embryos at 2 dpf (A, B) and 3 dpf (C, D). Asterisks mark the collapse of the medial fins in the <i>cof</i> mutant. (E) Summary of the linkage analysis and mapping of the <i>cof</i> locus at chromosome 25. The arrows mark the direction of the mutation. Red lines indicate the various transcripts in the genomic region. (F) Sequence chromatograms of wild-type and <i>cof</i> mutant cDNA. The corresponding amino acid residues are indicated below. (G) CA5 protein sequence alignment of zebrafish and human and other members of the human CA protein family. Arrow marks residue M280 that is substituted to a lysine in <i>cof</i> mutant embryos. (H) Detection of <i>ca5</i> mRNA in wild-type embryos at 24, 48 and 72 hpf by <i>in situ</i> hybridization. mRNA expression of <i>insulin</i> at 24 hpf marks the position of pancreatic β-cells. Upper panel shows dorsal view and lower panels lateral view. White arrowheads mark the expression in the lens and the pancreatic β-cells.</p

Rescue of the <i>ca5^cof</i> mutant and acetazolamide treatment phenocopies the <i>cof</i> mutation in wild-type embryos.

<p>(A, B) <i>ca5^cof</i> mutant embryos, (C–F) <i>ca5^cof</i> mutant embryos injected with 100 pg full-length wild-type <i>ca5</i> (C, D) or 100 pg <i>ca5^T839A</i> mutant RNA (E, F). (I–L) Wild-type embryos at 2 dpf (I–K) and 3 dpf (L) treated with 5 mM AZA. Treatment of wild-type and <i>cof</i> heterozygous embryos at 2 dpf with 2.5 mM AZA.</p

Ubiad1 Is an Antioxidant Enzyme that Regulates eNOS Activity by CoQ10 Synthesis

Protection against oxidative damage caused by excessive reactive oxygen species (ROS) by an antioxidant network is essential for the health of tissues, especially in the cardiovascular system. Here, we identified a gene with important antioxidant features by analyzing a null allele of zebrafish ubiad1, called barolo (bar). bar mutants show specific cardiovascular failure due to oxidative stress and ROS-mediated cellular damage. Human UBIAD1 is a nonmitochondrial prenyltransferase that synthesizes CoQ10 in the Golgi membrane compartment. Loss of UBIAD1 reduces the cytosolic pool of the antioxidant CoQ10 and leads to ROS-mediated lipid peroxidation in vascular cells. Surprisingly, inhibition of eNOS prevents Ubiad1-dependent cardiovascular oxidative damage, suggesting a crucial role for this enzyme and nonmitochondrial CoQ10 in NO signaling. These findings identify UBIAD1 as a nonmitochondrial CoQ10-forming enzyme with specific cardiovascular protective function via the modulation of eNOS activity

Inhaled nebulised unfractionated heparin for the treatment of hospitalised patients with COVID-19:A multicentre case series of 98 patients

AIMS To determine the safety and efficacy-potential of inhaled nebulised unfractionated heparin (UFH) in the treatment of hospitalised patients with COVID-19. METHODS Retrospective, uncontrolled multicentre single-arm case series of hospitalised patients with laboratory-confirmed COVID-19, treated with inhaled nebulised UFH (5000 IU q8h, 10 000 IU q4h, or 25 000 IU q6h) for 6 ± 3 (mean ± standard deviation) days. Outcomes were activated partial thromboplastin time (APTT) before treatment (baseline) and highest-level during treatment (peak), and adverse events including bleeding. Exploratory efficacy outcomes were oxygenation, assessed by ratio of oxygen saturation to fraction of inspired oxygen (FiO2 ) and FiO2 , and the World Health Organisation modified ordinal clinical scale. RESULTS There were 98 patients included. In patients on stable prophylactic or therapeutic systemic anticoagulant therapy but not receiving therapeutic UFH infusion, APTT levels increased from baseline of 34 ± 10 seconds to a peak of 38 ± 11 seconds (P < .0001). In 3 patients on therapeutic UFH infusion, APTT levels did not significantly increase from baseline of 72 ± 20 to a peak of 84 ± 28 seconds (P = .17). Two patients had serious adverse events: bleeding gastric ulcer requiring transfusion and thigh haematoma; both were on therapeutic anticoagulation. Minor bleeding occurred in 16 patients, 13 of whom were on therapeutic anticoagulation. The oxygen saturation/FiO2 ratio and the FiO2 worsened before and improved after commencement of inhaled UFH (change in slope, P < .001). CONCLUSION Inhaled nebulised UFH in hospitalised patients with COVID-19 was safe. Although statistically significant, inhaled nebulised UFH did not produce a clinically relevant increase in APTT (peak values in the normal range). Urgent randomised evaluation of nebulised UFH in patients with COVID-19 is warranted and several studies are currently underway

Apelin: A putative novel predictive biomarker for bevacizumab response in colorectal cancer

Bevacizumab (bvz) is currently employed as an anti-angiogenic therapy across several cancer indications. Bvz response heterogeneity has been well documented, with only 10-15% of colorectal cancer (CRC) patients benefitting in general. For other patients, clinical efficacy is limited and side effects are significant. This reinforces the need for a robust predictive biomarker of response. To identify such a biomarker, we performed a DNA microarray-based transcriptional profiling screen with primary endothelial cells (ECs) isolated from normal and tumour colon tissues. Thirteen separate populations of tumour-associated ECs and 10 of normal ECs were isolated using fluorescence-activated cell sorting. We hypothesised that VEGF-induced genes were overexpressed in tumour ECs; these genes could relate to bvz response and serve as potential predictive biomarkers. Transcriptional profiling revealed a total of 2,610 differentially expressed genes when tumour and normal ECs were compared. To explore their relation to bvz response, the mRNA expression levels of top-ranked genes were examined using quantitative PCR in 30 independent tumour tissues from CRC patients that received bvz in the adjuvant setting. These analyses revealed that the expression of MMP12 and APLN mRNA was significantly higher in bvz non-responders compared to responders. At the protein level, high APLN expression was correlated with poor progression-free survival in bvz-treated patients. Thus, high APLN expression may represent a novel predictive biomarker for bvz unresponsiveness.</p