A comparison of commercially-available automated and manual extraction kits for the isolation of total RNA from small tissue samples.

BACKGROUND: This study compared the performance of five commercially available kits in extracting total RNA from small eukaryotic tissue samples (<15 mg). Total RNA was isolated from fathead minnow (Pimephales promelas) tissues (spleen, blood, kidney, embryo, and larvae) using the Qiagen RNeasy® Plus Mini, Qiagen RNeasy® Plus Universal, Promega Maxwell® 16 LEV simplyRNA, Ambion MagMAX™-96 and Promega SimplyRNA HT kits. Kit performance was evaluated via measures of RNA quantity (e.g., total RNA amount) and quality (e.g., ratio of absorbance at 260 and 280 nm, RNA integrity number (RIN), presence of gDNA). RESULTS: With the exception of embryos, each kit generally extracted ≥5 µg of total RNA from each sample. With regard to RNA quality, the RINs of RNA samples isolated via the Plus Mini and Maxwell® 16 kits were consistently higher than those of samples extracted via the remaining three kits and for all tissues, these kits produced intact RNA with average RIN values ≥7. The Plus Universal and SimplyRNA HT kits produced moderately degraded (RIN values <7, but ≥5), while the RNA recovered via the MagMAX™ kit tended to exhibit a high degree of degradation (RIN values <5). CONCLUSIONS: Each kit was generally capable of extracting the amount of RNA required for most downstream gene expression applications suggesting that RNA yield is unlikely to be a limiting factor for any of the kits evaluated. However, differences in the quality of RNA extracted via each of the kits indicate that these kits may differ in their ability to yield RNA acceptable for some applications. Overall, the findings of this study demonstrate that there are practical differences between commercially available RNA extraction kits that should be taken into account when selecting extraction methods to be used for isolating RNA designated for gene expression analysis.

Effect of oral tryptamines on the gut microbiome of rats-a preliminary study.

Background: Psilocybin and related tryptamines have come into the spotlight in recent years as potential therapeutics for depression. Research on the mechanisms of these effects has historically focused on the direct effects of these drugs on neural processes. However, in addition to such neural effects, alterations in peripheral physiology may also contribute to their therapeutic effects. In particular, substantial support exists for a gut microbiome-mediated pathway for the antidepressant efficacy of other drug classes, but no prior studies have determined the effects of tryptamines on microbiota. Methods: To address this gap, in this preliminary study, male Long Evans rats were treated with varying dosages of oral psilocybin (0.2 or 2 mg/kg), norbaeocystin (0.25 or 2.52 mg/kg), or vehicle and their fecal samples were collected 1 week and 3 weeks after exposure for microbiome analysis using integrated 16S ribosomal DNA sequencing to determine gut microbiome composition. Results: We found that although treatment with neither psilocybin nor norbaeocystin significantly affected overall microbiome diversity, it did cause significant dose- and time-dependent changes in bacterial abundance at the phylum level, including increases in Verrucomicrobia and Actinobacteria, and decreases in Proteobacteria. Conclusion and Implications: These preliminary findings support the idea that psilocybin and other tryptamines may act on the gut microbiome in a dose- and time-dependent manner, potentially identifying a novel peripheral mechanism for their antidepressant activity. The results from this preliminary study also suggest that norbaeocystin may warrant further investigation as a potential antidepressant, given the similarity of its effects to psilocybin.

Subregion-specific transcriptomic profiling of rat brain reveals sex-distinct gene expression impacted by adolescent stress.

Stress during adolescence clearly impacts brain development and function. Sex differences in adolescent stress-induced or exacerbated emotional and metabolic vulnerabilities could be due to sex-distinct gene expression in hypothalamic, limbic, and prefrontal brain regions. However, adolescent stress-induced whole-genome expression changes in key subregions of these brain regions were unclear. In this study, female and male adolescent Sprague Dawley rats received one-hour restraint stress daily from postnatal day (PD) 32 to PD44. Corticosterone levels, body weights, food intake, body composition, and circulating adiposity and sex hormones were measured. On PD44, brain and blood samples were collected. Using RNA-sequencing, sex-specific differences in stress-induced differentially expressed (DE) genes were identified in subregions of the hypothalamus, limbic system, and prefrontal cortex. Canonical pathways reflected well-known sex-distinct maladies and diseases, substantiating the therapeutic potential of the DE genes found in the current study. Thus, we proposed specific sex distinct, adolescent stress-induced transcriptional changes found in the current study as examples of the molecular bases for sex differences witnessed in stress induced or exacerbated emotional and metabolic disorders. Future behavioral studies and single-cell studies are warranted to test the implications of the DE genes identified in this study in sex-distinct stress-induced susceptibilities.

Differential consumption of alcohol, caffeine, and caffeinated alcohol by adolescent rats, and effects on post-adolescent gene expression signatures in the nucleus accumbens and orbitofrontal cortex.

Caffeinated alcoholic beverages (CABs) are widely consumed despite little known about their behavioral and biological effects. Furthermore, CABs are also popular among adolescents, a particularly vulnerable and maturing demographic. In this preliminary study, we compared levels of daily adolescent voluntary consumption of caffeine (0.03%), alcohol (10%), caffeinated alcohol (0.03% + 10%), or vehicle and evaluated the effects of this on mRNA expression in brain regions associated with addiction and known to be affected by each drug. Beginning on postnatal day 30, rats were allowed unrestricted access to gelatin combined with one, both, or neither drug for twenty days. Compared to vehicle-consuming animals, consumption of gelatin was significantly attenuated when alcohol was included. The addition of caffeine to alcohol increased alcohol consumption in the early days of access compared to alcohol alone; however, after two weeks, alcohol consumption between these groups reached comparable levels. Compared to animals consuming caffeine alone, combining caffeine with alcohol significantly reduced caffeine intake. Targeted mRNA analysis of tissue collected from the nucleus accumbens and orbitofrontal cortex after the consumption period identified unique patterns of differentially expressed genes between treatment groups, across a broad array of neurotransmitter systems. Of particular note were genes related to a number of solute transporters and serotonergic functions. This preliminary work suggests unique pharmacological and behavioral effects from consuming caffeinated alcohol during adolescence. Since CABs are widely consumed by adolescents, these results suggest that more research into the pharmacological and behavioral effects elicited by CABs is warranted.

Small-angle X-ray scattering studies of the intact eye lens: effect of crystallin composition and concentration on microstructure.

BACKGROUND: The cortex and nucleus of eye lenses are differentiated by both crystallin protein concentration and relative distribution of three major crystallins (alpha, beta, and gamma). Here, we explore the effects of composition and concentration of crystallins on the microstructure of the intact bovine lens (37 degrees C) along with several lenses from Antarctic fish (-2 degrees C) and subtropical bigeye tuna (18 degrees C). METHODS: Our studies are based on small-angle X-ray scattering (SAXS) investigations of the intact lens slices where we study the effect of crystallin composition and concentration on microstructure. RESULTS: We are able to distinguish the nuclear and cortical regions by the development of a characteristic peak in the intensity of scattered X-rays. For both the bovine and fish lenses, the peak corresponds to that expected for dense suspensions of alpha-crystallins. CONCLUSIONS: The absence of the scattering peak in the nucleus indicates that there is no characteristic wavelength for density fluctuations in the nucleus although there is liquid-like order in the packing of the different crystallins. The loss in peak is due to increased polydispersity in the sizes of the crystallins and due to the packing of the smaller gamma-crystallins in the void space of alpha-crystallins. GENERAL SIGNIFICANCE: Our results provide an understanding for the low turbidity of the eye lens that is a mixture of different proteins. This will inform design of optically transparent suspensions that can be used in a number of applications (e.g., artificial liquid lenses) or to better understand human diseases pathologies such as cataract.

The protective role of aquaporins in the freeze-tolerant insect Eurosta solidaginis: functional characterization and tissue abundance of EsAQP1.

The movement of water and small solutes is integral to the survival of freezing and desiccation in insects, yet the underlying mechanisms of these processes are not fully known. Recent evidence suggests that aquaporin (AQP) water channels play critical roles in protecting cells from osmotic damage during freezing and desiccation. Our study sequenced, functionally characterized and measured the tissue abundance of an AQP from freeze-tolerant larvae of the gall fly, Eurosta solidaginis (Diptera: Tephritidae). The newly characterized EsAQP1 contains two NPA motifs and six transmembrane regions, and is phylogenetically related to an AQP from the anhydrobiotic chironomid Polypedilum vanderplanki. Using a Xenopus laevis oocyte swelling assay, we demonstrated that EsAQP1 increases water permeability to nine times that of simple diffusion through the membrane. In contrast to its high water permeability, EsAQP1 was impermeable to both glycerol and urea. The abundance of EsAQP1 increased from October to December in all tissues tested and was most abundant in the brain of winter larvae. Because the nervous system is thought to be the primary site of freezing injury, EsAQP1 may cryoprotect the brain from damage associated with water imbalance. The sequence, phylogenetic relationship, osmotic permeability, tissue distribution and seasonal abundance of EsAQP1 further support the role of AQPs in promoting freezing tolerance.

Seasonal variation in the hepatoproteome of the dehydration and freeze-tolerant wood frog, Rana sylvatica.

Winter's advent invokes physiological adjustments that permit temperate ectotherms to cope with stresses such as food shortage, water deprivation, hypoxia, and hypothermia. We used liquid chromatography (LC) in combination with tandem mass spectrometry (MS/MS) quantitative isobaric (iTRAQ™) peptide mapping to assess variation in the abundance of hepatic proteins in summer- and winter-acclimatized wood frogs (Rana sylvatica), a northerly-distributed species that tolerates extreme dehydration and tissue freezing during hibernation. Thirty-three unique proteins exhibited strong seasonal lability. Livers of winter frogs had relatively high levels of proteins involved in cytoprotection, including heat-shock proteins and an antioxidant, and a reduced abundance of proteins involved in cell proliferation, protein synthesis, and mitochondrial function. They also exhibited altered levels of certain metabolic enzymes that participate in the biochemical reorganization associated with aphagia and reliance on energy reserves, as well as the freezing mobilization and post-thaw recovery of glucose, an important cryoprotective solute in freezing adaptation.

Validation and deployment of a direct saliva real-time RT-PCR test on pooled samples for COVID-19 surveillance testing.

A direct, real-time reverse transcriptase PCR test on pooled saliva was validated in 2,786 participants against oropharyngeal swabs. Among asymptomatic/pre-symptomatic participants, the test was found to be in 99.21% agreement and 45% more sensitive than contemporaneous oropharyngeal swabs. The test was then used for surveillance testing on 44,242 saliva samples from asymptomatic participants. Those whose saliva showed evidence of SARS-CoV-2 within 50 cycles of amplification were referred for confirmatory testing, with 87% of those tested by nasal swab within 72 hours receiving a positive diagnostic result on Abbott ID NOW or real-time PCR platforms. Median Ct values on the saliva PCR for those with a positive and negative confirmatory tests was 30.67 and 35.92 respectively, however, binary logistic regression analysis of the saliva Ct values indicates that Ct thresholds as high as 47 may be useful in a surveillance setting. Overall, data indicate that direct RT-PCR testing of pooled saliva samples is an effective method of SARS-CoV-2 surveillance.

Why does the zebrafish cloche mutant develop lens cataract?

The zebrafish has become a valuable model for examining ocular lens development, physiology and disease. The zebrafish cloche mutant, first described for its loss of hematopoiesis, also shows reduced eye and lens size, interruption in lens cell differentiation and a cataract likely caused by abnormal protein aggregation. To facilitate the use of the cloche mutant for studies on cataract development and prevention we characterized variation in the lens phenotype, quantified changes in gene expression by qRT-PCR and RNA-Seq and compared the ability of two promoters to drive expression of introduced proteins into the cloche lens. We found that the severity of cloche embryo lens cataract varied, while the decrease in lens diameter and retention of nuclei in differentiating lens fiber cells was constant. We found very low expression of both αB-crystallin genes (cryaba and cryabb) at 4 days post fertilization (dpf) by both qRT-PCR and RNA-Seq in cloche, cloche sibling and wildtype embryos and no significant difference in αA-crystallin (cryaa) expression. RNA-Seq analysis of 4 dpf embryos identified transcripts from 25,281 genes, with 1,329 showing statistically significantly different expression between cloche and wildtype samples. Downregulation of eight lens ß- and γM-crystallin genes and 22 retinal related genes may reflect a general reduction in eye development and growth. Six stress response genes were upregulated. We did not find misregulation of any known components of lens development gene regulatory networks. These results suggest that the cloche lens cataract is not caused by loss of αA-crystallin or changes to lens gene regulatory networks. Instead, we propose that the cataract results from general physiological stress related to loss of hematopoiesis. Our finding that the zebrafish αA-crystallin promoter drove strong GFP expression in the cloche lens demonstrates its use as a tool for examining the effects of introduced proteins on lens crystallin aggregation and cataract prevention.

Controlling Liquid-Liquid Phase Separation of Cold-Adapted Crystallin Proteins from the Antarctic Toothfish.

Liquid-liquid phase separation (LLPS) of proteins is important to a variety of biological processes both functional and deleterious, including the formation of membraneless organelles, molecular condensations that sequester or release molecules in response to stimuli, and the early stages of disease-related protein aggregation. In the protein-rich, crowded environment of the eye lens, LLPS manifests as cold cataract. We characterize the LLPS behavior of six structural γ-crystallins from the eye lens of the Antarctic toothfish Dissostichus mawsoni, whose intact lenses resist cold cataract in subzero waters. Phase separation of these proteins is not strongly correlated with thermal stability, aggregation propensity, or cross-species chaperone protection from heat denaturation. Instead, LLPS is driven by protein-protein interactions involving charged residues. The critical temperature of the phase transition can be tuned over a wide temperature range by selective substitution of surface residues, suggesting general principles for controlling this phenomenon, even in compactly folded proteins.

Functional validation of hydrophobic adaptation to physiological temperature in the small heat shock protein αA-crystallin.

Small heat shock proteins (sHsps) maintain cellular homeostasis by preventing stress and disease-induced protein aggregation. While it is known that hydrophobicity impacts the ability of sHsps to bind aggregation-prone denaturing proteins, the complex quaternary structure of globular sHsps has made understanding the significance of specific changes in hydrophobicity difficult. Here we used recombinant protein of the lenticular sHsp α A-crystallin from six teleost fishes environmentally adapted to temperatures ranging from -2°C to 40°C to identify correlations between physiological temperature, protein stability and chaperone-like activity. Using sequence and structural modeling analysis we identified specific amino acid differences between the warm adapted zebrafish and cold adapted Antarctic toothfish that could contribute to these correlations and validated the functional consequences of three specific hydrophobicity-altering amino acid substitutions in αA-crystallin. Site directed mutagenesis of three residues in the zebrafish (V62T, C143S, T147V) confirmed that each impacts either protein stability or chaperone-like activity or both, with the V62T substitution having the greatest impact. Our results indicate a role for changing hydrophobicity in the thermal adaptation of α A-crystallin and suggest ways to produce sHsp variants with altered chaperone-like activity. These data also demonstrate that a comparative approach can provide new information about sHsp function and evolution.

Comparative analysis of crystallins and lipids from the lens of Antarctic toothfish and cow.

Animal model systems of senile cataract and lens crystallin stability are essential to understand the complex nature of lens transparency. Our aim in this study was to assess the long-lived Antarctic toothfish Dissostichus mawsoni (Norman) as a model system to understand long-term lens clarity in terms of solubility changes that occur to crystallins. We compared the toothfish with the mammalian model cow lens, dissecting each species' lens into a cortex and nuclear region. In addition to crystallin distribution, we also assayed fatty acid (FA) composition by negative ion electrospray ionization mass spectrometry (ESI-MS). The majority of toothfish lens crystallins from cortex (90.4%) were soluble, whereas only a third (31.8%) from the nucleus was soluble. Crystallin solubility analysis by SDS-PAGE and immunoblots revealed that relative proportions of crystallins in both soluble and urea-soluble fractions were similar within each species examined and in agreement with previous reports for bovine lens. From our data, we found that both toothfish and cow crystallins follow patterns of insolubility that mirror each animals lens composition with more γ crystallin aggregation seen in the toothfish lens nucleus than in cow. Toothfish lens lipids had a large amount of polyunsaturated fatty acids that were absent in cow resulting in an unsaturation index (I(U)) four-fold higher than that of cow. We identified a novel FA with a molecular mass of 267 mass units in the lens epithelial layer of the toothfish that accounted for well over 50% of the FA abundance. The unidentified lipid in the toothfish lens epithelia corresponds to either an odd-chain (17 carbons) FA or a furanoid. We conclude that long-lived fishes are likely good animal models of lens crystallin solubility and may model post-translational modifications and solubility changes better than short-lived animal models.

Molecular diversity and genomic organisation of the alpha, beta and gamma eye lens crystallins from the Antarctic toothfish Dissostichus mawsoni.

The eye lens of the Antarctic toothfish living in the -2 degrees C Southern Ocean is cold-stable. To investigate the molecular basis of this cold stability, we isolated, cloned and sequenced 22 full length crystallin cDNAs. We found two alpha crystallins (alphaA, alphaB), six beta crystallins (betaA1, betaA2, betaA4, betaB1, betaB2, betaB3) and 14 gamma crystallins (gammaN, gammaS1, gammaS2, gammaM1, gammaM3, gammaM4, gammaM5, gammaM7, gammaM8a, gammaM8b, gammaM8c, gammaM8d, gammaM8e, and gammaM9). Alignments of alpha, beta and gamma with other known crystallin sequences indicate that toothfish alpha and beta crystallins are relatively conserved orthologues of their vertebrate counterparts, but the toothfish and other fish gammaM crystallins form a distinct group that are not orthologous to mammalian gamma crystallins. A preliminary Fingerprinted Contig analysis of clones containing crystallin genes screened from a toothfish BAC library indicated alpha crystallin genes occurred in a single genomic region of ~266 kbp, beta crystallin genes in ~273 kbp, while the gamma crystallin gene family occurred in two separate regions of ~180 and ~296 kbp. In phylogenetic analysis, the gammaM isoforms of the ectothermic toothfish displayed a diversity not seen with endothermic mammalian gamma crystallins. Similar to other fishes, several toothfish gamma crystallins are methionine-rich (gammaM isoforms) which may have predisposed the toothfish lens to biochemically attenuate gamma crystallin hydrophobicity allowing for cold adaptation. In addition to high methionine content, conservation of alphabeta crystallins both in sequence and abundance suggests greater functional constraints relative to gamma crystallins. Conversely, reduced constraints upon gamma crystallins could have allowed for greater evolutionary plasticity resulting in increased polydispersity of gamma crystallins contributing to the cold-stability of the Antarctic toothfish lens.

Cold-stable eye lens crystallins of the Antarctic nototheniid toothfish Dissostichus mawsoni Norman.

The eye lenses of the Antarctic nototheniid fishes that inhabit the perennially freezing Antarctic seawater are transparent at -2 degrees C, whereas the cold-sensitive mammalian and tropical fish lenses display cold-induced cataract at 20 degrees C and 7 degrees C, respectively. No cold-cataract occurs in the giant Antarctic toothfish Dissostichus mawsoni lens when cooled to temperatures as low as -12 degrees C, indicating highly cold-stable lens proteins. To investigate this cold stability, we characterised the lens crystallin proteins of the Antarctic toothfish, in parallel with those of the sub-tropical bigeye tuna Thunnus obesus and the endothermic cow Bos taurus, representing three disparate thermal climes (-2 degrees C, 18 degrees C and 37 degrees C, respectively). Sizing chromatography resolved their lens crystallins into three groups, alpha/betaH, beta and gamma, with gamma crystallins being the most abundant (>40%) lens proteins in fish, in contrast to the cow lens where they comprise only 19%. The upper thermal stability of these crystallin components correlated with the body temperature of the species. In vitro chaperone assays showed that fish alpha crystallin can protect same-species gamma crystallins from heat denaturation, as well as lysozyme from DTT-induced unfolding, and therefore are small Heat Shock Proteins (sHSP) like their mammalian counterparts. Dynamic light scattering measured an increase in size of alphagamma crystallin mixtures upon heating, which supports formation of the alphagamma complex as an integral part of the chaperone process. Surprisingly, in cross-species chaperone assays, tuna alpha crystallins only partly protected toothfish gamma crystallins, while cow alpha crystallins completely failed to protect, indicating partial and no alphagamma interaction, respectively. Toothfish gamma was likely to be the component that failed to interact, as the supernatant from a cow alpha plus toothfish gamma incubation could chaperone cow gamma crystallins in a subsequent heat incubation, indicating the presence of uncomplexed cow alpha. This suggests that the inability of toothfish gamma crystallins to fully complex with tuna alpha, and not at all with the cow alpha crystallins, may have its basis in adaptive changes in the protein that relate to the extreme cold-stability of the toothfish lens.