Type I Diabetes in Zebrafish Reduces Sperm Quality and Increases Insulin and Glucose Transporter Transcripts.

Type I diabetes is a prominent human pathology with increasing incidence in the population; however, its cause is still unknown. This disease promotes detrimental effects on reproduction, such as lower sperm motility and DNA integrity. Hence, the investigation of the underlying mechanisms of this metabolic disturbance in reproduction and its transgenerational consequences is of the utmost importance. The zebrafish is a useful model for this research considering its high homology with human genes as well as its fast generation and regeneration abilities. Therefore, we aimed to investigate sperm quality and genes relevant to diabetes in the spermatozoa of Tg(ins:nfsb-mCherry) zebrafish, a model for type I diabetes. Diabetic Tg(ins:nfsb-mCherry) males showed significantly higher expression of transcripts for insulin a (insa) and glucose transporter (slc2a2) compared to controls. Sperm obtained from the same treatment group showed significantly lower sperm motility, plasma membrane viability, and DNA integrity compared to that from the control group. Upon sperm cryopreservation, sperm freezability was reduced, which could be a consequence of poor initial sperm quality. Altogether, the data showed similar detrimental effects related to type I diabetes in zebrafish spermatozoa at the cellular and molecular levels. Therefore, our study validates the zebrafish model for type I diabetes research in germ cells.

Approach and anesthetic management for kidney transplantation in a patient with bilateral lung transplantation: case report

Abstract Lung transplantation is the last resort for end-stage lung disease treatment. Due to increased survival, lung recipients present an increased likelihood to be submitted to anesthesia and surgery. This case report describes a 23-year-old female patient with history of lung transplantation for cystic fibrosis, with multiple complications, and chronic kidney disease, and who underwent kidney transplantation under general anesthesia. Understanding the pathophysiology and changes related to immunosuppressive therapy is essential to anesthetic technique planning and safety, and for perioperative management. The success of both anesthesia and surgery requires a qualified multidisciplinary team due to the rarity of the clinical scenario and high incidence of associated morbidity and mortality.

Multidimensional chromatin profiling of zebrafish pancreas to uncover and investigate disease-relevant enhancers.

The pancreas is a central organ for human diseases. Most alleles uncovered by genome-wide association studies of pancreatic dysfunction traits overlap with non-coding sequences of DNA. Many contain epigenetic marks of cis-regulatory elements active in pancreatic cells, suggesting that alterations in these sequences contribute to pancreatic diseases. Animal models greatly help to understand the role of non-coding alterations in disease. However, interspecies identification of equivalent cis-regulatory elements faces fundamental challenges, including lack of sequence conservation. Here we combine epigenetic assays with reporter assays in zebrafish and human pancreatic cells to identify interspecies functionally equivalent cis-regulatory elements, regardless of sequence conservation. Among other potential disease-relevant enhancers, we identify a zebrafish ptf1a distal-enhancer whose deletion causes pancreatic agenesis, a phenotype previously found to be induced by mutations in a distal-enhancer of PTF1A in humans, further supporting the causality of this condition in vivo. This approach helps to uncover interspecies functionally equivalent cis-regulatory elements and their potential role in human disease.

Novel protein carrier system based on cyanobacterial nano-sized extracellular vesicles for application in fish.

Aquaculture has been one of the fastest-growing food industry sectors, expanding at the pace of consumers' demands. To promote safe and effective fish growth performance strategies, and to stimulate environmentally friendly solutions to protect fish against disease outbreaks, new approaches are needed to safeguard fish welfare, as well as farmers and consumers interests. Here, we tested the use of cyanobacterial extracellular vesicles (EVs) as a novel nanocarrier system of heterologous proteins for applications in fish. We started by incubating zebrafish larvae with Synechocystis sp. PCC6803 EVs, isolated from selected mutant strains with different cell envelope characteristics. Results show that Synechocystis EVs are biocompatible with fish larvae, regardless of their structural composition, as EVs neither induced fish mortality nor triggered significant inflammatory responses. We establish also that cyanobacteria are amenable to engineering heterologous protein expression and loading into EVs, for which we used the reporter sfGFP. Moreover, upon immersion treatment, we successfully demonstrate that sfGFP-loaded Synechocystis EVs accumulate in the gastrointestinal tract of zebrafish larvae. This work opens the possibility of using cyanobacterial EVs as a novel biotechnological tool in fish, with prospective applications in carrying proteins/enzymes, for example for modulating their nutritional status or stimulating specific adaptive immune responses.

Approach and anesthetic management for kidney transplantation in a patient with bilateral lung transplantation: case report.

Lung transplantation is the last resort for end-stage lung disease treatment. Due to increased survival, lung recipients present an increased likelihood to be submitted to anesthesia and surgery. This case report describes a 23-year-old female patient with history of lung transplantation for cystic fibrosis, with multiple complications, and chronic kidney disease, and who underwent kidney transplantation under general anesthesia. Understanding the pathophysiology and changes related to immunosuppressive therapy is essential to anesthetic technique planning and safety, and for perioperative management. The success of both anesthesia and surgery requires a qualified multidisciplinary team due to the rarity of the clinical scenario and high incidence of associated morbidity and mortality.

In Vivo Reporter Assays Uncover Changes in Enhancer Activity Caused by Type 2 Diabetes-Associated Single Nucleotide Polymorphisms.

Many single nucleotide polymorphisms (SNPs) associated with type 2 diabetes overlap with putative endocrine pancreatic enhancers, suggesting that these SNPs modulate enhancer activity and, consequently, gene expression. We performed in vivo mosaic transgenesis assays in zebrafish to quantitatively test the enhancer activity of type 2 diabetes-associated loci. Six out of 10 tested sequences are endocrine pancreatic enhancers. The risk variant of two sequences decreased enhancer activity, while in another two incremented it. One of the latter (rs13266634) locates in an SLC30A8 exon, encoding a tryptophan-to-arginine substitution that decreases SLC30A8 function, which is the canonical explanation for type 2 diabetes risk association. However, other type 2 diabetes-associated SNPs that truncate SLC30A8 confer protection from this disease, contradicting this explanation. Here, we clarify this incongruence, showing that rs13266634 boosts the activity of an overlapping enhancer and suggesting an SLC30A8 gain of function as the cause for the increased risk for the disease. We further dissected the functionality of this enhancer, finding a single nucleotide mutation sufficient to impair its activity. Overall, this work assesses in vivo the importance of disease-associated SNPs in the activity of endocrine pancreatic enhancers, including a poorly explored case where a coding SNP modulates the activity of an enhancer.

Selection Criteria of Zebrafish Male Donors for Sperm Cryopreservation.

Selection criteria for sperm cryopreservation are highly relevant in zebrafish since sperm quality is particularly variable in this species. Successful cryopreservation depends on high-quality sperm, which can only be ensured by the selection of breeders. Consequently, male selection and management are a priority to improve cryopreservation, and therefore, this study aimed to characterize optimal age and sperm collection frequency in zebrafish. For this purpose, males from wild type (AB) and from a transgenic line [Tg(runx2:eGFP)] were sampled at 6, 8, 12, and 14 months. For each age, sperm were collected at time 0 followed by samplings at 2, 7, and 14 days of rest. Sperm quality was assessed according to motility and membrane viability parameters. Quality assessment showed that Tg(runx2:eGFP) displayed significantly higher motility than AB and younger males showed higher motility in both lines. Sperm collection frequency affected membrane viability. While AB fish recovered sperm viability after 14 days of rest, Tg(runx2:eGFP) could not recover. Consequently, it may be important to study the sperm quality of each zebrafish line before sperm cryopreservation. Taking into consideration the results achieved in both lines, sperm collection should be performed between 6 and 8 months of age with a minimum collection interval of 14 days.

Airway management in a patient with Crouzon syndrome proposed to orthognathic surgery.

Airway management is one of the most important anaesthetist's skills as major complications of airway management, although rare, can be among the most life threatening in medicine. Crouzon syndrome is a rare condition with physical characteristics that can result in difficult airway manipulation. A correct preanaesthetic evaluation and a planned preinduction strategy should be designed to facilitate intubation.

Bcl-2 overexpression protects against amyloid-beta and prion toxicity in GT1-7 neural cells.

In this study we analysed the effect of Bcl-2 on the cytotoxicity induced by the amyloid-beta (Abeta(25-35)) and prion (PrP(106-126)) peptides by using GT1-7puro and GT1-7bcl-2 (overexpressing the anti-apoptotic protein Bcl-2) neural cells. Exposure to Abeta(25-35) (1-5 microM) and PrP(106-126) (25 microM) caused a decrease in cell viability, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. These data were correlated with Abeta(25-35) and PrP(106-126)-induced activation of caspase-9, which is linked to the mitochondrial death pathway, and the activation of the effector caspase-3, suggesting cell death by apoptosis. Furthermore, Bcl-2 overexpression protected from loss of cell viability and caspase-9 and -3 activation induced by Abeta(25-35) and PrP(106-126), showing that Bcl-2 is neuroprotective against apoptotic cell death caused by amyloidogenic peptides.