Transcriptomic and Metabolomic Studies Reveal That Toll-like Receptor 2 Has a Role in Glucose-Related Metabolism in Unchallenged Zebrafish Larvae (Danio rerio).

Toll-like receptors (TLRs) have been implicated in the regulation of various metabolism pathways, in addition to their function in innate immunity. Here, we investigate the metabolic function of TLR2 in a larval zebrafish system. We studied larvae from a tlr2 mutant and the wild type sibling controls in an unchallenged normal developmental condition using transcriptomic and metabolomic analyses methods. RNAseq was used to evaluate transcriptomic differences between the tlr2 mutant and wild-type control zebrafish larvae and found a signature set of 149 genes to be significantly altered in gene expression. The expression level of several genes was confirmed by qPCR analyses. Gene set enrichment analysis (GSEA) revealed differential enrichment of genes between the two genotypes related to valine, leucine, and isoleucine degradation and glycolysis and gluconeogenesis. Using 1H nuclear magnetic resonance (NMR) metabolomics, we found that glucose and various metabolites related with glucose metabolism were present at higher levels in the tlr2 mutant. Furthermore, we confirmed that the glucose level is higher in tlr2 mutants by using a fluorometric assay. Therefore, we have shown that TLR2, in addition to its function in immunity, has a function in controlling metabolism during vertebrate development. The functions are associated with transcriptional regulation of various enzymes involved in glucose metabolism that could explain the different levels of glucose, lactate, succinate, and malate in larvae of a tlr2 mutant.

Aberrant glial activation and synaptic defects in CaMKIIα-iCre and nestin-Cre transgenic mouse models.

Current scientific research is driven by the ability to manipulate gene expression by utilizing the Cre/loxP system in transgenic mouse models. However, artifacts in Cre-driver mouse lines that introduce undesired effects and confound results are increasingly being reported. Here, we show aberrant neuroinflammation and synaptic changes in two widely used Cre-driver mouse models. Neuroinflammation in CaMKIIα-iCre mice was characterized by the activation and proliferation of microglia and astrocytes in synaptic layers of the hippocampus. Increased GFAP and Iba1 levels were observed in hippocampal brain regions of 4-, 8- and 22-month-old CaMKIIα-iCre mice compared to WT littermates. Synaptic changes in NMDAR, AMPAR, PSD95 and phosphorylated CaMKIIα became apparent in 8-month-old CaMKIIα-iCre mice but were not observed in 4-month-old CaMKIIα-iCre mice. Synaptophysin and synaptoporin were unchanged in CaMKIIα-iCre compared to WT mice, suggesting that synaptic alterations may occur in excitatory postsynaptic regions in which iCre is predominantly expressed. Finally, hippocampal volume was reduced in 22-month-old CaMKIIα-iCre mice compared to WT mice. We tested the brains of mice of additional common Cre-driver mouse models for neuroinflammation; the nestin-Cre mouse model showed synaptic changes and astrocytosis marked by increased GFAP+ astrocytes in cortical and hippocampal regions, while the original CaMKIIα-Cre T29-1 strain was comparable to WT mice. The mechanisms underlying abnormal neuroinflammation in nestin-Cre and CaMKIIα-iCre are unknown but may be associated with high levels of Cre expression. Our findings are critical to the scientific community and demonstrate that the correct Cre-driver controls must be included in all studies using these mice.

Farnesyltransferase inhibitor LNK-754 attenuates axonal dystrophy and reduces amyloid pathology in mice.

BACKGROUND: Amyloid plaque deposition and axonal degeneration are early events in AD pathogenesis. Aß disrupts microtubules in presynaptic dystrophic neurites, resulting in the accumulation of impaired endolysosomal and autophagic organelles transporting ß-site amyloid precursor protein cleaving enzyme (BACE1). Consequently, dystrophic neurites generate Aß42 and significantly contribute to plaque deposition. Farnesyltransferase inhibitors (FTIs) have recently been investigated for repositioning toward the treatment of neurodegenerative disorders and block the action of farnesyltransferase (FTase) to catalyze farnesylation, a post-translational modification that regulates proteins involved in lysosome function and microtubule stability. In postmortem AD brains, FTase and its downstream signaling are upregulated. However, the impact of FTIs on amyloid pathology and dystrophic neurites is unknown. METHODS: We tested the effects of the FTIs LNK-754 and lonafarnib in the 5XFAD mouse model of amyloid pathology. RESULTS: In 2-month-old 5XFAD mice treated chronically for 3 months, LNK-754 reduced amyloid plaque burden, tau hyperphosphorylation, and attenuated the accumulation of BACE1 and LAMP1 in dystrophic neurites. In 5-month-old 5XFAD mice treated acutely for 3 weeks, LNK-754 reduced dystrophic neurite size and LysoTracker-Green accumulation in the absence of effects on Aß deposits. Acute treatment with LNK-754 improved memory and learning deficits in hAPP/PS1 amyloid mice. In contrast to LNK-754, lonafarnib treatment was less effective at reducing plaques, tau hyperphosphorylation and dystrophic neurites, which could have resulted from reduced potency against FTase compared to LNK-754. We investigated the effects of FTIs on axonal trafficking of endolysosomal organelles and found that lonafarnib and LNK-754 enhanced retrograde axonal transport in primary neurons, indicating FTIs could support the maturation of axonal late endosomes into lysosomes. Furthermore, FTI treatment increased levels of LAMP1 in mouse primary neurons and in the brains of 5XFAD mice, demonstrating that FTIs stimulated the biogenesis of endolysosomal organelles. CONCLUSIONS: We show new data to suggest that LNK-754 promoted the axonal trafficking and function of endolysosomal compartments, which we hypothesize decreased axonal dystrophy, reduced BACE1 accumulation and inhibited amyloid deposition in 5XFAD mice. Our results agree with previous work identifying FTase as a therapeutic target for treating proteinopathies and could have important therapeutic implications in treating AD.

Anxiety-like behavior and whole-body cortisol responses to components of energy drinks in zebrafish (Danio rerio).

The current study investigated the independent and combined effects of caffeine and taurine on anxiety-like behavior and neuroendocrine responses in adult zebrafish (Danio rerio). Caffeine (1,3,7-trimethylpurine-2,6-dione), the world's most commonly used psychoactive drug, acts as an adenosine receptor blocker and a mild central nervous system stimulant. However, excessive use of caffeine is associated with heightened anxiety levels. Taurine (2-aminoethanesulfonic acid), a semi-essential amino acid synthesized within the human brain, has been hypothesized to play a role in regulating anxiolytic behavior. Caffeine and taurine are two common additives in energy drinks and are often found in high concentrations in these beverages. However, few studies have investigated the interaction of these two chemicals with regards to anxiety measures. A suitable vertebrate to examine anxiety-like behavior and physiological stress responses is the zebrafish, which has shown promise due to substantial physiological and genetic homology with humans. Anxiety-like behavior in zebrafish can be determined by analyzing habituation to novelty when fish are placed into a novel tank and scototaxis (light avoidance) behavior in the light-dark test. Stress-related neuroendocrine responses can be measured in zebrafish by analyzing whole-body cortisol levels. The goal of this study was to determine if exposure to caffeine, taurine, or a combination of the two compounds altered anxiety-like behavior and whole-body cortisol levels in zebrafish relative to control. Zebrafish were individually exposed to either caffeine (100 mg/L), taurine (400 mg/L), or both for 15 min. Zebrafish in the control group were handled in the same manner but were only exposed to system tank water. After treatment, fish were transferred to the novel tank test or the light-dark test. Behavior was tracked for the first 6 min in the novel tank and 15 min in the light-tark test. Fifteen min after introduction to the behavioral task, fish were euthanized for the analysis of whole-body cortisol levels. The results demonstrate that caffeine treatment decreased the amount of exploration in the top of the novel tank and increased scototaxis behavior in the light-dark test, which supports the established anxiogenic effect of acute exposure to caffeine. Taurine alone did not alter basal levels of anxiety-like behavioral responses nor ameliorated the anxiogenic effects of caffeine on behavior when the two compounds were administered concurrently. None of the drug treatments altered basal levels of whole-body cortisol. The current results of this study suggest that, at least at this dose and time of exposure, taurine does not mitigate the anxiety-producing effects of caffeine when administered in combination, such as with energy drink consumption.

The contribution of telemedicine to hepatitis C elimination in a correctional facility.

BACKGROUND: micro-elimination has been recently proposed as an efficient strategy to achieve global hepatitis C virus (HCV) elimination. The Spanish Health Ministry Strategic Plan for hepatitis C infection highlighted intervention in prisons as a priority action. However, there are important barriers associated with the specialized care provision to the penitentiary population. AIMS: to assess the contribution of telemedicine for HCV elimination in a correctional facility in Spain. METHODS: an open label program of HCV elimination via telemedicine was started on February 3rd, 2015 in a large penitentiary of 1,200 inmates, as an alternative to referring patients to specialists. An anonymous satisfaction survey was performed among a random sample of inmates and all participating doctors. RESULTS: the prevalence of HCV viremia prior to program initiation was 12.4%. One hundred and thirty-one patients received DAA HCV treatment during the period 2015-2018; 42.74% had a HCV-HIV co-infection. Overall, 97% achieved a sustained virological response (SVR). A second regime of DAA successfully rescued non-responder patients and the HCV prevalence was zero at the end of the program. Satisfaction was high or very high according to 67% of inmates and all participating doctors. CONCLUSION: telemedicine is an effective tool for HCV elimination in penitentiary correctional facilities where referral to specialists is difficult. The extensive use of this technology should be recommended in this setting in order to facilitate equitable access to specialized care.

[The progression of liver fibrosis in prison inmates co-infected by HIV and HCV who started on boosted protease inhibitor therapy]. / Evolución de la fibrosis hepática en reclusos coinfectados por VIH y VHC que inician tratamiento con inhibidores de la proteasa potenciados.

OBJECTIVES: To analyse the progression of liver fibrosis as measured by elastography and biochemical testing in prisoninmates co-infected by HIV and HCVwho started on ritonavir-boosted protease inhibitor (PI) therapy. METHODS: A prospective, observational and multi-centre study. The progression of liver fibrosis as measured by transient elastography (FibroScan) and biochemical testing was monitored for 48 weeks in a Spanish prison population co-infected with HIV and HCV. RESULTS: Of the 94 patients included, 54 (57.4%) were followed-up for 48 weeks. At week 48, no significant changes were seen in the grade of fibrosis measured using FibroScan (8.1 kPa vs. 8.3 kPa; p=0.20) or the Forns index (5.6 vs. 5.1; p= 0.50), although significant changes were detected using the APRI (0.7 vs. 0.6; p=0.05) and the FIB-4 indexes (p= 0.02).When measurement was done compared to baseline fibrosis, it was seen that therapy reduced the percentage of patients with fibrosis ≥3 but <4 (50% vs. 15%; p=0.001), but no change was seen in those found to have grade 4 fibrosis at baseline (20.4% vs. 20.4%). CONCLUSION: The inmates co-infected with HIV and HCV who were started on antiretroviral therapy with the boosted protease inhibitor (PI) showed stasterilizationbilisation of the liver fibrosis as measured with FibroScan after one year of follow-up. Overall, the therapy improved fibrosis when measured using the APRI or FIB-4 indexes, but not when using the Forns index or elastography.

Large-scale analysis of acute ethanol exposure in zebrafish development: a critical time window and resilience.

BACKGROUND: In humans, ethanol exposure during pregnancy causes a spectrum of developmental defects (fetal alcohol syndrome or FAS). Individuals vary in phenotypic expression. Zebrafish embryos develop FAS-like features after ethanol exposure. In this study, we ask whether stage-specific effects of ethanol can be identified in the zebrafish, and if so, whether they allow the pinpointing of sensitive developmental mechanisms. We have therefore conducted the first large-scale (>1500 embryos) analysis of acute, stage-specific drug effects on zebrafish development, with a large panel of readouts. METHODOLOGY/PRINCIPAL FINDINGS: Zebrafish embryos were raised in 96-well plates. Range-finding indicated that 10% ethanol for 1 h was suitable for an acute exposure regime. High-resolution magic-angle spinning proton magnetic resonance spectroscopy showed that this produced a transient pulse of 0.86% concentration of ethanol in the embryo within the chorion. Survivors at 5 days postfertilisation were analysed. Phenotypes ranged from normal (resilient) to severely malformed. Ethanol exposure at early stages caused high mortality (≥88%). At later stages of exposure, mortality declined and malformations developed. Pharyngeal arch hypoplasia and behavioral impairment were most common after prim-6 and prim-16 exposure. By contrast, microphthalmia and growth retardation were stage-independent. CONCLUSIONS: Our findings show that some ethanol effects are strongly stage-dependent. The phenotypes mimic key aspects of FAS including craniofacial abnormality, microphthalmia, growth retardation and behavioral impairment. We also identify a critical time window (prim-6 and prim-16) for ethanol sensitivity. Finally, our identification of a wide phenotypic spectrum is reminiscent of human FAS, and may provide a useful model for studying disease resilience.

Photo-CIDNP MAS NMR in intact cells of Rhodobacter sphaeroides R26: molecular and atomic resolution at nanomolar concentration.

Photochemically induced dynamic nuclear polarization (photo-CIDNP) is observed in photosynthetic reaction centers of the carotenoid-less strain R26 of the purple bacterium Rhodobacter sphaeroides by (13)C solid-state NMR at three different magnetic fields (4.7, 9.4, and 17.6 T). The signals of the donor appear enhanced absorptive (positive) and of the acceptor emissive (negative). This spectral feature is in contrast to photo-CIDNP data of reactions centers of Rhodobacter sphaeroides wildtype reported previously (Prakash, S.; Alia; Gast, P.; de Groot, H. J. M.; Jeschke, G.; Matysik, J. J. Am. Chem. Soc. 2005, 127, 14290-14298) in which all signals appear emissive. The difference is due to an additional mechanism occurring in RCs of R26 in the long-living triplet state of the donor, allowing for spectral editing by different enhancement mechanisms. The overall shape of the spectra remains independent of the magnetic field. The strongest enhancement is observed at 4.7 T, enabling the observation of photo-CIDNP enhanced NMR signals from reaction center cofactors in entire bacterial cells allowing for detection of subtle changes in the electronic structure at nanomolar concentration of the donor cofactor. Therefore, we establish in this paper photo-CIDNP MAS NMR as a method to study the electronic structure of photosynthetic cofactors at the molecular and atomic resolution as well as at cellular concentrations.