mRNA-encoded Cas13 treatment of Influenza via site-specific degradation of genomic RNA.

The CRISPR-Cas13 system has been proposed as an alternative treatment of viral infections. However, for this approach to be adopted as an antiviral, it must be optimized until levels of efficacy rival or exceed the performance of conventional approaches. To take steps toward this goal, we evaluated the influenza viral RNA degradation patterns resulting from the binding and enzymatic activity of mRNA-encoded LbuCas13a and two crRNAs from a prior study, targeting PB2 genomic and messenger RNA. We found that the genome targeting guide has the potential for significantly higher potency than originally detected, because degradation of the genomic RNA is not uniform across the PB2 segment, but it is augmented in proximity to the Cas13 binding site. The PB2 genome targeting guide exhibited high levels (>1 log) of RNA degradation when delivered 24 hours post-infection in vitro and maintained that level of degradation over time, with increasing multiplicity of infection (MOI), and across modern influenza H1N1 and H3N2 strains. Chemical modifications to guides with potent LbuCas13a function, resulted in nebulizer delivered efficacy (>1-2 log reduction in viral titer) in a hamster model of influenza (Influenza A/H1N1/California/04/09) infection given prophylactically or as a treatment (post-infection). Maximum efficacy was achieved with two doses, when administered both pre- and post-infection. This work provides evidence that mRNA-encoded Cas13a can effectively mitigate Influenza A infections opening the door to the development of a programmable approach to treating multiple respiratory infections.

Heteropolyacid-Containing Ionic Liquid-Catalyzed Multicomponent Synthesis of Bridgehead Nitrogen Heterocycles: Mechanisms and Mitochondrial Staining.

The current manuscript describes the use of a heteropolyacid-containing task-specific ionic liquid, supported in imidazolium-based ionic liquids, as the catalyst for an efficient multicomponent synthesis of hexahydroimidazo[1,2-α]pyridine derivatives. The reactions conditions were fully optimized, and the bridgehead nitrogen heterocycle derivatives could be obtained in just 1 h exclusively as a single isomer ( trans). Single crystal X-ray analysis confirmed the trans derivative as the only isomer. The mechanism of the reaction was investigated by ESI(+)-MS(/MS), and the use of the elegant charge-tag strategy allowed a catalytic cycle to be proposed for the current transformation and revealed the possibility of at least two reaction pathways. One derivative bearing a coumarin scaffold was synthesized, and its fluorescent properties allowed it to be tested as a probe for live-cell imaging experiments with a preference for mitochondria.

Effects of gestational and breastfeeding caffeine exposure in adenosine A1 agonist-induced antinociception of infant rats.

OBJECTIVES: Caffeine is extensively consumed as a psychostimulant drug, acting on A1 and A2A adenosine receptors blockade. Chronic exposure to caffeine during gestation and breast-feeding may be involved in infant rat's behavioral and biochemical alterations. Our goal was to evaluate the effect of chronic caffeine exposure during gestation and breast-feeding in the functionality of adenosine A1 receptors in infant rats at P14. NTPDase and 5'-nucleotidase activities were also evaluated. METHODS: Mating of adult female Wistar rats was confirmed by presence of sperm in vaginal smears. Rats were divided into three groups on the first day of pregnancy: (1) control: tap water, (2) caffeine: 0.3 g/L until P14, and (3) washout caffeine: caffeine was changed to tap water at P7. Evaluation of nociceptive response was performed at P14 using hot plate (HP) and tail-flick latency (TFL) tests. A1 receptor involvement was assessed using caffeine agonist (CPA) and antagonist (DPCPX). Enzymatic activities assays were conducted in the spinal cord. RESULTS: Gestational and breastfeeding exposure to caffeine (caffeine and washout groups) did not induce significant alterations in thermal nociceptive thresholds (HP and TF tests). Both caffeine groups did not show analgesic response induced by CPA when compared to the control group at P14, indicating chronic exposure to caffeine in the aforementioned periods inhibits the antinociceptive effects of the systemic A1 receptor agonist administration. No effect was observed upon ectonucleotidase activities. CONCLUSIONS: Our results demonstrate that chronic caffeine exposure in gestational and breastfeeding alters A1-mediated analgesic response in rats.

Static Magnetic Stimulation Induces Cell-type Specific Alterations in the Viability of SH-SY5Y Neuroblastoma Cell Line.

BACKGROUND/AIM: Magnetic stimulation is used in the treatment of a diversity of diseases, but a complete understanding of the underlying mechanisms of action requires further investigation. We examined the effect of static magnetic stimulation (SMS) in different cell lines. MATERIALS AND METHODS: A culture plate holder with attached NeFeB magnets was developed. Different magnetic field intensities and periods were tested in tumoral and non-tumoral cell lines. To verify the cellular responses to SMS, cell viability, cell death, cell cycle and BDNF expression were evaluated. RESULTS: Exposure of SH-SY5Y cells to SMS for 24 hours led to a decrease in cell viability. Analysis 24 h after stimulation revealed a decrease in apoptotic and double-positive cells, associated with an increase in the number of necrotic cells. CONCLUSION: The effects of SMS on cell viability are cell type-specific, inducing a decrease in cell viability in SH-SY5Y cells. This suggests that SMS may be a potential tool in the treatment of neuronal tumors.

From Live Cells to Caenorhabditis elegans: Selective Staining and Quantification of Lipid Structures Using a Fluorescent Hybrid Benzothiadiazole Derivative.

The current article describes the synthesis, characterization, and application of a designed hybrid fluorescent BTD-coumarin (2,1,3-benzothiadiazole-coumarin) derivative (named BTD-Lip). The use of BTD-Lip for live-cells staining showed excellent results, and lipid droplets (LDs) could be selectively stained. When compared with the commercially available dye (BODIPY) for LD staining, it was noted that the designed hybrid fluorescence was capable of staining a considerable larger number of LDs in both live and fixed cells (ca. 40% more). The new dye was also tested on live Caenorhabditis elegans (complex model) and showed an impressive selectivity inside the worm, whereas the commercial dye showed no selectivity in the complex model.