Myxospore density of Kudoa inornata varies significantly within symmetrical white muscle tissue replicates of its fish host, the spotted seatrout, Cynoscion nebulosus.

The spotted seatrout, Cynoscion nebulosus, is a popular game fish in the southeastern USA. It is estimated that nearly 90% of the adult population in South Carolina estuaries are infected in their skeletal muscle by the myxosporean, Kudoa inornata. However, little is known about this parasite's biology, including the distribution and densities of myxospores within tissues of infected fish, which we expect affect the physiology of their hosts. In order to correlate densities with physiological parameters in future studies, we quantified the myxospores density in muscle and characterized the variation among individual fish. Naïve juvenile seatrout was experimentally infected via presumed K. inornata actinospores exposure to raw seawater. A plug of muscle was extracted from two bilaterally symmetrical regions in the epaxial fillet from fresh and frozen carcasses. Variation in density data was calculated both within and among individuals. Within individuals, density counts were compared between left- and right-side biopsies. There was no significant difference between fresh and frozen plugs, and variation among individuals accounted for the greatest proportion of variation at 68.8%, while variation within individuals was substantial at 25.6%. Simulation and correlation tests confirmed that bilaterally symmetrical replicates varied significantly within individuals. When sampled from areas surrounding the initial biopsies, myxospore density estimates were more similar than between sides. Our findings have important implications for sampling design, particularly for studies investigating physiological parameters at the cellular or molecular level in association with parasite infection.

Untargeted Metabolomics Analyses and Contaminant Chemistry of Dreissenid Mussels at the Maumee River Area of Concern in the Great Lakes.

Bivalves serve as an ideal ecological indicator; hence, their use by the NOAA Mussel Watch Program to monitor environmental health. This study aimed to expand the baseline knowledge of using metabolic end points in environmental monitoring by investigating the dreissenid mussel metabolome in the field. Dreissenids were caged at four locations along the Maumee River for 30 days. The mussel metabolome was measured using nuclear magnetic resonance spectroscopy, and mussel tissue chemical contaminants were analyzed using gas or liquid chromatography coupled with mass spectrometry. All Maumee River sites had a distinct mussel metabolome compared to the reference site and revealed changes in the energy metabolism and amino acids. Data also highlighted the importance of considering seasonality or handling effects on the metabolome at the time of sampling. The furthest upstream site presented a specific mussel tissue chemical signature of pesticides (atrazine and metolachlor), while a downstream site, located at Toledo's wastewater treatment plant, was characterized by polycyclic aromatic hydrocarbons and other organic contaminants. Further research into the dreissenid mussel's natural metabolic cycle and metabolic response to specific anthropogenic stressors is necessary before successful implementation of metabolomics in a biomonitoring program.

Metabolomics Analysis of Effects of Commercial Soy-based Protein Products in Red Drum (Sciaenops ocellatus).

We investigated the metabolic effects of four different commercial soy-based protein products on red drum fish (Sciaenops ocellatus) using nuclear magnetic resonance (NMR) spectroscopy-based metabolomics along with unsupervised principal component analysis (PCA) to evaluate metabolic profiles in liver, muscle, and plasma tissues. Specifically, during a 12 week feeding trial, juvenile red drum maintained in an indoor recirculating aquaculture system were fed four different commercially available soy formulations, containing the same amount of crude protein, and two reference diets as performance controls: a 60% soybean meal diet that had been used in a previous trial in our lab and a natural diet. Red drum liver, muscle, and plasma tissues were sampled at multiple time points to provide a more accurate snapshot of specific metabolic states during the grow-out. PCA score plots derived from NMR spectroscopy data sets showed significant differences between fish fed the natural diet and the soy-based diets, in both liver and muscle tissues. While red drum tolerated the inclusion of soy with good feed conversion ratios, a comparison to fish fed the natural diet revealed that the soy-fed fish in this study displayed a distinct metabolic signature characterized by increased protein and lipid catabolism, suggesting an energetic imbalance. Furthermore, among the soy-based formulations, one diet showed a more pronounced catabolic signature.

Evaluation of a hepatic biomarker of nutritional imbalance in juvenile red drum (Sciaenops ocellatus) fed 60% soybean meal-based diets using NMR-based metabolomics.

A 12-week feeding trial with juvenile red drum (Sciaenops ocellatus) fed high-soybean meal (SBM) diets was conducted to investigate a putative biomarker of nutritional imbalance, N-formimino-L-glutamate (FIGLU). Three fishmeal-free, 60% SBM pelleted diets (named B12, Fol, and Met, respectively) were tested to evaluate the effects on growth performance and tissue metabolite profiles of supplementation of vitamin B12 (0.012 mg/kg), folate (10 mg/kg), methionine (1 g/kg) respectively, above basal supplementation levels. A fourth SBM-based diet (named B12/Fol/Met) was formulated with a combination of B12, folate, and methionine to attain the above-mentioned target concentrations. A fifth 60% SBM diet (named FWS) with methionine supplementation (1 g/kg above basal supplementation levels), enriched with taurine, lysine and threonine as well as minerals, was also tested. This diet contained formulation targets and additives which have allowed for replacing fishmeal with plant proteins in rainbow trout feeds. Control diets included a fishmeal-based diet (named FM), an unsupplemented basal 60% SBM diet (named SBM60), and a "natural" diet (named N) made up of equal parts of fish (cigar minnows), squid and shrimp as a positive reference for growth performance. Formulated feeds contained approximately 37% total crude protein, approximately 14% total crude lipid and were energetically balanced. Standard growth performance metrics were measured, and tissues (liver, muscle) were collected at week 12 to evaluate diet-induced metabolic changes using nuclear magnetic resonance (NMR)-based metabolomics. Our results show that the FWS diet outperformed all other SBM diets and the FM diet under all performance metrics (P < 0.05). FIGLU was not detected in fish fed the N diet but was detected in those fed the SBM diets and the FM diet. Fish fed the FWS diet and the Met diet showed lower hepatic levels of FIGLU compared with the other SBM-based diets (P < 0.05), suggesting that among the different supplementation regimes, methionine supplementation was associated with lower FIGLU levels. The FWS diet produced tissue metabolite profiles that were more similar to those of fish fed the N diet. Based on our results, the FWS diet constitutes a promising SBM-based alternative diet to fishmeal for red drum.

The arginine-rich RNA-binding motif of HIV-1 Rev is intrinsically disordered and folds upon RRE binding.

Arginine-rich motifs (ARMs) capable of binding diverse RNA structures play critical roles in transcription, translation, RNA trafficking, and RNA packaging. The regulatory HIV-1 protein Rev is essential for viral replication and belongs to the ARM family of RNA-binding proteins. During the early stages of the HIV-1 life cycle, incompletely spliced and full-length viral mRNAs are very inefficiently recognized by the splicing machinery of the host cell and are subject to degradation in the cell nucleus. These transcripts harbor the Rev Response Element (RRE), which orchestrates the interaction with the Rev ARM and the successive Rev-dependent mRNA export pathway. Based on established criteria for predicting intrinsic disorder, such as hydropathy, combined with significant net charge, the very basic primary sequences of ARMs are expected to adopt coil-like structures. Thus, we initiated this study to investigate the conformational changes of the Rev ARM associated with RNA binding. We used multidimensional NMR and circular dichroism spectroscopy to monitor the observed structural transitions, and described the conformational landscapes using statistical ensemble and molecular-dynamics simulations. The combined spectroscopic and simulated results imply that the Rev ARM is intrinsically disordered not only as an isolated peptide but also when it is embedded into an oligomerization-deficient Rev mutant. RRE recognition triggers a crucial coil-to-helix transition employing an induced-fit mechanism.

Chronic exposure to high-density polyethylene microplastic through feeding alters the nutrient metabolism of juvenile yellow perch (Perca flavescens).

Microplastics are emergent contaminants threatening aquatic organisms including aquacultured fish. This study investigated the effects of high-density polyethylene (HDPE, 100 to 125 µm) on yellow perch (Perca flavescens) based on integrative evaluation including growth performance, nutritional status, nutrient metabolism, fish health, and gut microbial community. Five test diets (0, 1, 2, 4, or 8 g HDPE/100 g diet) containing 41% protein and 10.5% lipid were fed to juvenile perch (average body weight, 25.9 ± 0.2 g; n = 15) at a feeding rate of 1.5% to 2.0% body weight daily. The feeding trial was conducted in a flow-through water system for 9 wk with 3 tanks per treatment and 15 yellow perch per tank. No mortality or HDPE accumulation in the fish was found in any treatments. Weight gain and condition factor of fish were not significantly impacted by HDPE (P > 0.05). Compared to the control group, fish fed the 8% HDPE diet had significantly decreased levels of protein and ash (P < 0.05). In response to the increasing levels of HDPE exposure, the hepatosomatic index value, hepatocyte size, and liver glycogen level were increased, but lipid content was reduced in the liver tissues. Compared to the control treatment, fish fed the 8% HDPE diet had significant accumulations of total bile acids and different metabolism pathways such as bile acid biosynthesis, pyruvate metabolism, and carnitine synthesis. Significant enterocyte necrosis was documented in the foregut of fish fed the 2% or 8% HDPE diet; and significant cell sloughing was observed in the midgut and hindgut of fish fed the 8% HDPE diet. Fish fed the 2% HDPE diet harbored different microbiota communities compared to the control fish. This study demonstrates that HDPE ranging from 100 to 125 µm in feed can be evacuated by yellow perch with no impact on growth. However, dietary exposure to HDPE decreased whole fish nutrition quality, altered nutrient metabolism and the intestinal histopathology as well as microbiota community of yellow perch. The results indicate that extended exposure may pose a risk to fish health and jeopardize the nutrition quality of aquacultured end product. This hypothesis remains to be investigated further.

Investigation of graded levels of soybean meal diets for red drum, Sciaenops ocellatus, using quantitative PCR derived biomarkers.

A twelve-week feeding trial was conducted to examine potential metabolic and gene expression changes that occur in juvenile red drum, Sciaenops ocellatus, fed diets with increasing soybean meal inclusion. Significant reduction in fish performance characteristics (feed consumption, weight gain, final weight) was observed within the soybean meal based diets as soybean meal level increased (R, linear regression); however, all soybean meal based diets performed statistically equivalent in regards to performance characteristics (weight gain, feed conversion ratio, condition factor, etc.) to a commercial (45% crude protein and 16% crude lipid) reference diet (R, ANOVA). To better understand the underlying physiological responses and metabolic changes driving performance differences, traditional aquaculture metrics were paired with high throughput -omics techniques. Nuclear magnetic resonance (NMR) spectroscopy-based metabolomics data and RNA transcript abundance differences observed in liver tissue were utilized to select multiple sets of genes to target with quantitative polymerase chain reaction (qPCR), both for pathway activity validation and as rapid and accessible biomarkers of performance as a result of soybean meal. Genes selected based on the metabolic pathways most affected by soybean meal level corroborate the metabolite profile and performance data indicating an increase in gluconeogenic precursor production as soybean meal increased. The metabolomic and gene expression tools utilized in our study present a novel framework for diet and fish health evaluation that may provide more rapid and improved techniques for evaluating dietary manipulations and improving production of juvenile fish on alternative feeds.

Investigation of graded-level soybean meal diets in red drum (Sciaenops ocellatus) using NMR-based metabolomics analysis.

We investigated changes in the metabolome in juvenile red drum (Sciaenops ocellatus) induced by increasing amounts of soybean meal (0% to 60%) in extruded, fishmeal-free diets using a nuclear magnetic resonance spectroscopy (NMR)-based metabolomics approach in a 12-week feeding trial. All of the diets were composed of ≈40% total crude protein, ≈11% total crude lipid and were energetically balanced. A fishmeal-containing, commercial extruded diet was used as a control diet throughout the trial. Each week, liver, muscle, intestine and plasma samples were collected and analyzed by NMR to provide a "snapshot" of the metabolome at different time points. Results indicate significant time-dependence of the metabolic profiles in various tissues with stable metabolomic profiles forming after about 9-weeks on the experimental diets. We identify a previously unexploited biomarker of potential dietary stress (N­formimino­l­glutamate (FIGLU)) in the fish that may prove to be useful for optimization of alternative diet formulations.

Hyperbranched molecular structures with potential antiviral activity: derivatives of 5,6-dihydroxyindole-2-carboxylic Acid.

In the search of new HIV-1 integrase (IN) inhibitors, we synthesized a series of multimeric 5,6-dihydroxyindole-2-carboxylic acid (DHICA) derivatives. Preliminary results indicate that hyperbranched architectures could represent a peculiar molecular requisite for the development of new antiviral lead compounds.

Synthesis of 2'-substituted inosine analogs via unusual masking of the 6-hydroxyl group.

2'-Modified inosine analogs have been synthesized from 6-chloropurine riboside via 6-dimethylaminopurine or 6-benzyloxypurine intermediates. The dimethylaminopurine intermediate was obtained via an unusually facile dimethylamine transfer from dimethylformamide. Graphical Abstract: