FELASA-AALAS Recommendations for Biosecurity in an Aquatic Facility, Including Prevention of Zoonosis, Introduction of New Fish Colonies, and Quarantine.

FELASA and AALAS established a joint working group to advise on good practices for the exchange of fish for research. In a first manuscript, the working group made recommendations for health monitoring and reporting of monitoring results. The focus of this second related manuscript is biosecurity in fish facilities. First, we define the risk of contamination of personnel by zoonotic pathogens from fish or from system water, including human mycobacteriosis. Preventive measures are recommended, such as wearing task-specific personal protective equipment. Then we discuss biosecurity, highlighting the establishment of biosecurity barriers to preserve the health status of a facility. A functional biosecurity program relies on integration of the entire animal facility organization, including the flow of staff and animals, water treatments, and equipment sanitation. Finally, we propose 4 steps for introducing new fish colonies: consideration of international trade and national restrictions; assessing risk according to fish source and developmental stage; establishing quarantine barriers; and the triage, screening, and treatment of newly imported fish. We then provide 3 realistic sample scenarios to illustrate practical biosecurity risk assessments and mitigation measures based on considerations of health status and quarantine conditions.

FELASA-AALAS Recommendations for Monitoring and Reporting of Laboratory Fish Diseases and Health Status, with an Emphasis on Zebrafish (Danio Rerio).

The exchange of fish for research may expose an aquatic laboratory to pathogen contamination as incoming fish can introduce bacteria, fungi, parasites, and viruses capable of affecting both experimental results and fish and personnel health and welfare. To develop risk mitigation strategies, FELASA and AALAS established a joint working group to recommend good practices for health monitoring of laboratory fish. The recommendations address all fish species used for research, with a particular focus on zebrafish (Danio rerio). First, the background of the working group and key definitions are provided. Next, fish diseases of high impact are described. Third, recommendations are made for health monitoring of laboratory fishes. The recommendations emphasize the importance of daily observation of the fish and strategies to determine fish colony health status. Finally, report templates are proposed for historical screening data and aquatic facility description to facilitate biohazard risk assessment when exchanging fish.

Predicting sepsis severity at first clinical presentation: The role of endotypes and mechanistic signatures.

BACKGROUND: Inter-individual variability during sepsis limits appropriate triage of patients. Identifying, at first clinical presentation, gene expression signatures that predict subsequent severity will allow clinicians to identify the most at-risk groups of patients and enable appropriate antibiotic use. METHODS: Blood RNA-Seq and clinical data were collected from 348 patients in four emergency rooms (ER) and one intensive-care-unit (ICU), and 44 healthy controls. Gene expression profiles were analyzed using machine learning and data mining to identify clinically relevant gene signatures reflecting disease severity, organ dysfunction, mortality, and specific endotypes/mechanisms. FINDINGS: Gene expression signatures were obtained that predicted severity/organ dysfunction and mortality in both ER and ICU patients with accuracy/AUC of 77-80%. Network analysis revealed these signatures formed a coherent biological program, with specific but overlapping mechanisms/pathways. Given the heterogeneity of sepsis, we asked if patients could be assorted into discrete groups with distinct mechanisms (endotypes) and varying severity. Patients with early sepsis could be stratified into five distinct and novel mechanistic endotypes, named Neutrophilic-Suppressive/NPS, Inflammatory/INF, Innate-Host-Defense/IHD, Interferon/IFN, and Adaptive/ADA, each based on ∼200 unique gene expression differences, and distinct pathways/mechanisms (e.g., IL6/STAT3 in NPS). Endotypes had varying overall severity with two severe (NPS/INF) and one relatively benign (ADA) groupings, consistent with reanalysis of previous endotype studies. A 40 gene-classification tool (accuracy=96%) and several gene-pairs (accuracy=89-97%) accurately predicted endotype status in both ER and ICU validation cohorts. INTERPRETATION: The severity and endotype signatures indicate that distinct immune signatures precede the onset of severe sepsis and lethality, providing a method to triage early sepsis patients.

Aquatic Models: Water Quality and Stability and Other Environmental Factors.

The use of aquatic animals in ecotoxicology, genetic, and biomedical research has grown immensely in recent years, especially due to the increased use of zebrafish in the laboratory setting. Because water is the primary environment of most aquatic species, the composition and management of this water is paramount to ensuring their health and welfare. In this publication, we will describe the important variables in water quality that can influence animal health and research results, using the zebrafish model for detailed specifics of optimal conditions. Wherever possible, recommendations are provided to reduce the potential impact of poor or highly variable water quality, and standards are given which can be used as institutional goals to maximize animal health and welfare and reduce research variability. It is increasingly important that authors of publications describing work done using aquatic models characterize water quality and other environmental conditions of the animal environment so that the work can be repeated and understood in context of these important factors. It is clear that there are a great many extrinsic factors which may influence research outcomes in the aquatics model laboratory setting, and consequently, an increased level of funding will be essential to support continued research exploring these and other important husbandry conditions. References from a large body of literature on this subject are provided.

Influence of Commercial and Laboratory Diets on Growth, Body Composition, and Reproduction in the Zebrafish Danio rerio.

The value of the zebrafish (Danio rerio) as a model organism continues to expand. In developing the model, current feeding practice in zebrafish laboratories includes the use of commercially available diets. In this study, we compared outcomes in growth, body composition, and reproduction among zebrafish fed five highly utilized commercial diets and one formulated chemically defined reference diet. Wild-type zebrafish larvae were raised on live feed until 21 days postfertilization and then fed diets for 16 weeks. All fish received a daily ration of >5% of body weight (adjusted biweekly). Growth varied among diets throughout the feeding trial, and at study termination (week 16), significant differences among diets were observed for terminal weight gain, body condition index, body fat deposition, and reproductive outcomes. In addition, the proportion of viable embryos produced from females fed the formulated reference diet was high relative to the commercial diets. These data suggest that metabolic profiles, most likely reflecting nutrient/energy availability, utilization, and allocation, vary relative to diet in zebrafish. Undefined differences in metabolic profiles could result in erroneous predictions of health outcomes and make comparisons among laboratories more challenging. We recommend that dietary standards should be defined for zebrafish to support their common utility in biomedical research.

Temporary fetal tracheal occlusion using a gel plug in a rabbit model of congenital diaphragmatic hernia.

PURPOSE: Temporary tracheal occlusion induces lung growth in congenital diaphragmatic hernia (CDH) but has significant drawbacks because the device must be removed in utero. We devised a gel plug (GP) that can be placed in the fetal trachea in a rabbit model of CDH to provide temporary tracheal occlusion and evaluated its effect on lung growth and postnatal ventilation mechanics. METHODS: In each of 16 pregnant rabbits, experimental CDH was created in 4 fetuses. These were randomized to intratracheal instillation of a fibrin GP, tracheal suture ligation, intratracheal instillation of normal saline, or sham amniotomy. Unmanipulated fetuses of the litter without CDH served as controls. Fetuses were harvested at gestational day 29 and mechanically ventilated to determine lung compliance and airway resistance. Fetal lung-to-body weight was compared among the groups. RESULTS: Mean fetal lung-to-body weight was higher in GP-treated fetuses than in the normal saline group, although not as high as that in fetuses subjected to tracheal ligation. Gel plug-treated fetuses had the highest airway resistance, whereas non-CDH control fetuses had the most compliant lungs. CONCLUSIONS: Prenatal instillation of an intratracheal GP leads to increased postnatal lung mass in rabbit fetuses with CDH but also increases airway resistance.

Screening and characterization of surface-tethered cationic peptides for antimicrobial activity.

There is an urgent need to coat the surfaces of medical devices, including implants, with antimicrobial agents to reduce the risk of infection. A peptide array technology was modified to permit the screening of short peptides for antimicrobial activity while tethered to a surface. Cellulose-amino-hydroxypropyl ether (CAPE) linker chemistry was used to synthesize, on a cellulose support, peptides that remained covalently bound during biological assays. Among 122 tested sequences, the best surface-tethered 9-, 12-, and 13-mer peptides were found to be highly antimicrobial against bacteria and fungi, as confirmed using alternative surface materials and coupling strategies as well as coupling through the C and N termini of the peptides. Structure-activity modeling of the structural features determining the activity of tethered peptides indicated that the extent and positioning of positive charges and hydrophobic residues were influential in determining activity.

Rational design of alpha-helical antimicrobial peptides with enhanced activities and specificity/therapeutic index.

In the present study, the 26-residue peptide sequence Ac-KWKSFLKTFKSAVKTVLHTALKAISS-amide (V681) was utilized as the framework to study the effects of peptide hydrophobicity/hydrophilicity, amphipathicity, and helicity (induced by single amino acid substitutions in the center of the polar and nonpolar faces of the amphipathic helix) on biological activities. The peptide analogs were also studied by temperature profiling in reversed-phase high performance liquid chromatography, from 5 to 80 degrees C, to evaluate the self-associating ability of the molecules in solution, another important parameter in understanding peptide antimicrobial and hemolytic activities. A higher ability to self-associate in solution was correlated with weaker antimicrobial activity and stronger hemolytic activity of the peptides. Biological studies showed that strong hemolytic activity of the peptides generally correlated with high hydrophobicity, high amphipathicity, and high helicity. In most cases, the D-amino acid substituted peptides possessed an enhanced average antimicrobial activity compared with L-diastereomers. The therapeutic index of V681 was improved 90- and 23-fold against Gram-negative and Gram-positive bacteria, respectively. By simply replacing the central hydrophobic or hydrophilic amino acid residue on the nonpolar or the polar face of these amphipathic derivatives of V681 with a series of selected D-/L-amino acids, we demonstrated that this method has excellent potential for the rational design of antimicrobial peptides with enhanced activities.

The tortoise and the hare II: relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis.

Chloroplast DNA sequences are a primary source of data for plant molecular systematic studies. A few key papers have provided the molecular systematics community with universal primer pairs for noncoding regions that have dominated the field, namely trnL-trnF and trnK/matK. These two regions have provided adequate information to resolve species relationships in some taxa, but often provide little resolution at low taxonomic levels. To obtain better phylogenetic resolution, sequence data from these regions are often coupled with other sequence data. Choosing an appropriate cpDNA region for phylogenetic investigation is difficult because of the scarcity of information about the tempo of evolutionary rates among different noncoding cpDNA regions. The focus of this investigation was to determine whether there is any predictable rate heterogeneity among 21 noncoding cpDNA regions identified as phylogenetically useful at low levels. To test for rate heterogeneity among the different cpDNA regions, we used three species from each of 10 groups representing eight major phylogenetic lineages of phanerogams. The results of this study clearly show that a survey using as few as three representative taxa can be predictive of the amount of phylogenetic information offered by a cpDNA region and that rate heterogeneity exists among noncoding cpDNA regions.

Optimization of microbial specificity in cyclic peptides by modulation of hydrophobicity within a defined structural framework.

In the present study we have utilized the structural framework of the analog GS14K4 (cyclo(VKLd-KVd-YPL KVKLd-YP, where d denotes a d-amino acid)), to examine the role of hydrophobicity in microbial activity and specificity. The hydrophobicity of GS14K4 was systematically altered by residue replacements in the hydrophobic sites of the molecule to produce a series of analogs that were either less or more hydrophobic than the parent compound. Circular dichroism spectroscopy and reversed-phase high performance liquid chromatography analysis showed that the molecules were structurally similar and only differed in overall hydrophobicity. The hydrophobicity of GS14K4 was found to be the midpoint for hemolytic activity, with more hydrophobic analogs exhibiting increased hemolytic activity and less hydrophobic analogs showing decreased hemolytic activity. For antimicrobial activity there were differences between the hydrophobicity requirements against Gram-positive and Gram-negative microorganisms. The hydrophobicity of GS14K4 was sufficient for maximum activity against Gram-negative microorganisms and yeast, with no further increases in activity occurring with increasing hydrophobicity. With Gram-positive microorganisms significant increases in activity with increasing hydrophobicity were seen in three of the six microorganisms tested. A therapeutic index (calculated as a measure of specificity of the peptides for the microorganisms over human erythrocytes) served to define the boundaries of a therapeutic window within which lay the optimum peptide hydrophobicity for each microorganism. The therapeutic window was found to be at a lower hydrophobicity level for Gram-negative microorganisms than for Gram-positive microorganisms, although the limits were more variable for the latter. Our results show that the balance between activity and specificity in the present cyclic peptides can be optimized for each microorganism by systematic modulation of hydrophobicity.