The excavation of novel toxin-resistance proteins against trichothecenes toxins in Paramyrothecium roridum.

Trichothecene toxins cause serious hazard towards human health and economical crops. However, there are no sufficient molecular strategies to reduce the hazard of trichothecene toxins. Thus it is urgent to exploit novel approaches to control the hazard of trichothecenes. In this study, four trichothecene toxin-resistance genes including mfs1, GNAT1, TRP1 and tri12 in Paramyrothecium roridum were excavated based on genome sequencing results, and then expressed in toxin-sensitive Saccharomyces cerevisiae BJ5464, the toxin resistance genes pdr5, pdr10 and pdr15 of which were firstly knocked out simultaneously by the introduction of TAA stop codon employing CRISPR/Cas9 system. Therefore, three novel hazardous toxin-resistance genes mfs1, GNAT1, TRP1 in P. roridum were firstly excavated by the co-incubation of DON toxin and toxin resistant genes-containing BJ5464 strains. The in vitro function and properties of novel toxin-resistance genes coding proteins including GNAT1, MFS1 and TRP1 were identified by heterologous expression and cellular location analysis as well as in vitro biochemical reaction. The excavation of novel trichothecene toxin-resistance genes provide novel molecular clues for controlling the harm of trichothecenes, meanwhile, this study will also pave a new way for the yield improvement of trichothecenes by heterologous expression to facilitate the development of trichothecenes as anti-tumor lead compounds.

Depletion of Retinal Dopaminergic Activity in a Mouse Model of Rod Dysfunction Exacerbates Experimental Autoimmune Uveoretinitis: A Role for the Gateway Reflex.

The gateway reflex is a mechanism by which neural inputs regulate chemokine expression at endothelial cell barriers, thereby establishing gateways for the invasion of autoreactive T cells into barrier-protected tissues. In this study, we hypothesized that rod photoreceptor dysfunction causes remodeling of retinal neural activity, which influences the blood-retinal barrier and the development of retinal inflammation. We evaluated this hypothesis using Gnat1rd17 mice, a model of night blindness with late-onset rod-cone dystrophy, and experimental autoimmune uveoretinitis (EAU). Retinal remodeling and its effect on EAU development were investigated by transcriptome profiling, target identification, and functional validation. We showed that Gnat1rd17 mice primarily underwent alterations in their retinal dopaminergic system, triggering the development of an exacerbated EAU, which was counteracted by dopamine replacement with L-DOPA administered either systemically or locally. Remarkably, dopamine acted on retinal endothelial cells to inhibit NF-κB and STAT3 activity and the expression of downstream target genes such as chemokines involved in T cell recruitment. These results suggest that rod-mediated dopamine release functions in a gateway reflex manner in the homeostatic control of immune cell entry into the retina, and the loss of retinal dopaminergic activity in conditions associated with rod dysfunction increases the susceptibility to autoimmune uveitis.

Long Non-coding RNA lnc-GNAT1-1 Suppresses Liver Cancer Progression via Modulation of Epithelial-Mesenchymal Transition.

Recent studies have investigated the modulatory roles of long non-coding RNAs in the onset and progression of liver cancer. The present study aimed to elucidate the role of lnc-GNAT1-1 in liver cancer development and to explore the underlying mechanisms. Quantitative real-time polymerase chain reaction was performed to measure the expression levels of lnc-GNAT1-1 in cancerous tissues from patients with liver cancer and in liver cancer cell lines. The proliferative ability and apoptotic rates of liver cancer cells were measured using the counting kit-8 (CCK-8), colony formation, and flow cytometry assays. The abilities to invade and migrate were measured using Transwell assays. Epithelial-mesenchymal transition (EMT)-related proteins, E-cadherin, N-cadherin, and vimentin, were measured using western blotting. A nude mouse model was injected with xenografts to evaluate tumor growth in vivo. Downregulation of lnc-GNAT1-1 was observed in cancerous tissues from patients with liver cancer and in liver cancer cell lines, and low expression levels of lnc-GNAT1-1 were related to advanced TNM stage. Lnc-GNAT1-1 knockdown promoted invasion, migration, and proliferation of liver cancer cells and inhibited apoptosis, while lnc-GNAT1-1 upregulation exerted the opposite effects. The expression levels of lnc-GNAT1-1 negatively correlated with in vivo tumor growth in a xenograft nude mouse model. Mechanistic experiments revealed that lnc-GNAT1-1 exerted anti-tumor effects in liver cancer cells by inhibiting EMT. In conclusion, this study suggests that lnc-GNAT1-1 suppresses liver cancer progression by modulating EMT.

Light deprivation reduces the severity of experimental diabetic retinopathy.

Illumination of the retina is a major determinant of energy expenditure by its neurons. However, it remains unclear whether light exposure significantly contributes to the pathophysiology of common retinal disease. Driven by the premise that light exposure reduces the metabolic demand of the retina, recent clinical trials failed to demonstrate a benefit for constant illumination in the treatment of diabetic retinopathy. Here, we instead ask whether light deprivation or blockade of visual transduction could modulate the severity of this common cause of blindness. We randomized adult mice with two different models of diabetic retinopathy to 1-3 months of complete dark housing. Unexpectedly, we find that diabetic mice exposed to short or prolonged light deprivation have reduced diabetes-induced retinal pathology, using measures of visual function, compared to control animals in standard lighting conditions. To corroborate these results, we performed assays of retinal vascular health in diabetic Gnat1-/- and Rpe65-/- mice, which lack phototransduction. Both mutants displayed less diabetes-associated retinal vascular disease compared to respective wild-type controls. Collectively, these results suggest that light-induced visual transduction promotes the development of diabetic retinopathy and implicate photoreceptors as an early source of visual pathology in diabetes.

Coexistence of GNAT1 and ABCA4 variants associated with Nougaret-type congenital stationary night blindness and childhood-onset cone-rod dystrophy.

PURPOSE: A single variant (p.G38D) in the GNAT1 gene, encoding the rod-specific transducin α-subunit in phototransduction, has been reported only in one French family with Nougaret-type autosomal dominant congenital stationary night blindness (CSNB). We identified a Japanese family with Nougaret-type CSNB and cone-rod dystrophy (CORD). METHODS: Five patients with CSNB and two patients with childhood-onset CORD were recruited. We performed a comprehensive ophthalmic examination including electroretinography (ERG). Disease-causing variants were identified by whole exome sequencing, with candidates confirmed by Sanger sequencing in nine family members. RESULTS: The GNAT1 variant (p.G38D) was identified in all four CSNB patients, whereas the two CORD patients carried biallelic truncated known ABCA4 variants as well as the GNAT1 variant. Clinically, no remarkable findings were observed in fuduscopy, fundus autofluorescence, or optical coherence tomography images from the CSNB patients. No response was detectable by rod ERG. The a-waves of standard and bright flash ERG were delayed and broadened rather than biphasic, and b/a-wave amplitude ratio was negative. Cone and 30-Hz flicker responses were normal, and overall, the ERG findings were compatible with previous descriptions of Nougaret-type CSNB. ERG of the CORD patients with macular atrophy showed non-recordable rod response and severely decreased standard flash, cone and 30-Hz flicker responses. CONCLUSIONS: This is the second report of a Nougaret-type CSNB family with the GNAT1 variant. Our novel findings suggest that coexistence of the GNAT1 and biallelic ABCA4 variants is associated with an overlapping phenotype with both Nougaret-type CSNB and CORD.

Novel homozygous in-frame deletion of GNAT1 gene causes golden appearance of fundus and reduced scotopic ERGs similar to that in Oguchi disease in Japanese family.

Background: The GNAT1 gene encodes the alpha-subunit of transducin in rod photoreceptors and is an important part of the phototransduction cascade. Defects in GNAT1 are very rare but have been identified in autosomal dominant and recessive congenital stationary night blindness (CSNB) and autosomal recessive rod-cone dystrophy. The purpose of this study was to determine the phenotype-genotype relationship in a non-consanguineous Japanese family with a GNAT1 mutation.Methods: Detailed ophthalmic examinations were performed on the patients and their family members. Whole exome sequencing (WES) was applied to the DNA obtained from the family members. Sanger sequencing and co-segregation analyses were performed to identify the most likely pathogenic variant.Results: Two female (13- and 11-years) and one male (15-years) patients from a family had night blindness from their childhood. The fundus had a mild golden appearance regardless of the state of light- or dark-adaptation. Electroretinographic (ERG) analyses showed that the scotopic a-wave was extinguished, and the mixed rod-cone responses were severely reduced with an electronegative form in patients. The shapes of the dark-adapted ERGs were similar to those recorded from patients with Oguchi disease. We identified a homozygous in-frame deletion c.818_820delAGA, p.Lys273del in the GNAT1 gene. Variants were verified by Sanger sequencing and co-segregated with the disease in five members of the family.Conclusions: Our findings indicate that a recessive GNAT1 mutation found in this family could be the cause of the golden appearance of the fundus and negative ERGs with reduced a-waves, and nearly absent b-waves in the mixed rod-cone ERGs.

Autophagy in Xenopus laevis rod photoreceptors is independently regulated by phototransduction and misfolded RHOP23H.

We previously reported autophagic structures in rod photoreceptors expressing a misfolding RHO (rhodopsin) mutant (RHOP23H), suggesting that autophagy may play a role in degrading the mutant RHO and/or be involved in photoreceptor cell death. To further examine autophagy in normal and diseased rods, we generated transgenic Xenopus laevis tadpoles expressing the dually fluorescent autophagy marker mRFP-eGFP-LC3 in rods, which changes from green to yellow and finally red as autophagic structures develop and mature. Using transgenic lines with constitutive and inducible expression, we determined the time-course of autophagy in rod photoreceptors: autophagosomes last for 6 to 8 hours before fusing with lysosomes, and acidified autolysosomes last for about 28 hours before being degraded. Autophagy was diurnally regulated in normal rods, with more autophagic structures generated during periods of light, and this regulation was non-circadian. We also found that more autophagosomes were produced in rods expressing the misfolding RHOP23H mutant. The RHO chromophore absorbs photons to initiate phototransduction, and is consumed in this process; it also promotes RHO folding. To determine whether increased autophagy in light-exposed normal rods is caused by increased RHO misfolding or phototransduction, we used CRISPR/Cas9 to knock out the RPE65 and GNAT1 genes, which are essential for chromophore biosynthesis and phototransduction respectively. Both knockouts suppressed light-induced autophagy, indicating that although light and misfolded rhodopsin can both induce autophagy in rods, light-induced autophagy is not due to misfolding of RHO, but rather due to phototransduction. Abbreviations: CYCS: cytochrome c; bRHOP23H: bovine RHOP23H; Cas9: CRISPR associated protein 9; dpf: days post-fertilization; eGFP: enhanced green fluorescent protein; GNAT1: guanine nucleotide-binding protein G(t) subunit alpha-1 aka rod alpha-transducin; HSPA1A/hsp70: heat shock protein of 70 kilodaltons; LAMP1: lysosomal-associated membrane protein 1; LC3: microtubule-associated protein 1A/1B light chain 3; mRFP: monomeric red fluorescent protein; RHO: rhodopsin; RP: retinitis pigmentosa; RPE65: retinal pigment epithelium-specific 65 kDa protein: sfGFP: superfolding GFP; sgRNA: single guide RNA; WGA: wheat germ agglutinin; RHOp: the Xenopus laevis RHO.2.L promoter.

Riggs-type dominant congenital stationary night blindness: ERG findings, a new GNAT1 mutation and a systemic association.

PURPOSE: Complete congenital stationary night blindness (CSNB) is most often x-linked or recessive, and associated with a transmission defect from photoreceptors to bipolar cells. This produces a characteristic "negative" Schubert-Bornschein type of scotopic rod-cone electroretinogram (ERG) with a large a-wave and minimal b-wave. CSNB from abnormalities in phototransduction can be recessive or dominant and is much less common. This produces a Riggs type of ERG with loss of the rod a-wave as well as the b-wave. We report the clinical and ERG findings from a family with autosomal dominant CSNB that was shown previously to have a new GNAT1 mutation with a novel mechanism of action. They provide a classic demonstration of the Riggs-type ERG and have an unusual systemic association. METHODS: Clinical case report of a father and daughter. RESULTS: A Chinese father and daughter presented with good visual acuity, moderate myopia, and lifelong night blindness. Both show normal fundi except for mild myopia, and fundus autofluorescence and OCT images are normal. Their ERGs illustrate the typical Riggs-type ERG with no rod a-wave (they have only a small cone-dominated combined response). They also have postural orthostatic tachycardia syndrome (POST), which is an autonomic dysfunction disorder thought usually to be sporadic. The retinal gene analyses revealed no abnormalities that might account for POST. CONCLUSIONS: Our family's ERG showed essentially no rod response, consistent with a Danish GNAT1 pedigree but different from the Nougaret GNAT1 pedigree that shows partial preservation of rod signal. A genetic connection between CSNB and POST would be intriguing, but we found no evidence for this.

A novel long non-coding RNA lnc-GNAT1-1 is low expressed in colorectal cancer and acts as a tumor suppressor through regulating RKIP-NF-κB-Snail circuit.

BACKGROUND: The role of long non-coding RNAs (lncRNAs) in colorectal cancer (CRC) progression has not fully been elucidated. This study was designed to report the identification of a novel lncRNA, lnc-GNAT1-1, and its functional role in CRC progression. METHODS: lncRNA expression profile microarray was performed in three paired primary and liver metastatic tissues of CRC, and a novel lncRNA, lnc-GNAT1-1, was identified to be a potential functional lncRNA. Quantitative real-time PCR was used to detect its expression in CRC tissues, cell lines, and patients' plasma, cell fractionation was used to evaluate its subcellular location. lnc-GNAT1-1 was knockdown by siRNA or overexpressed by a lentivirus vector, then in vitro an vivo experiments were performed to evaluate its biological role and the underlying mechanisms in CRC. RESULTS: Expression of lnc-GNAT1-1 was decreased in liver metastasis than the primary tumor, while the later one is lower than the paired normal mucosa. Decreased lnc-GNAT1-1 expression was associated unfavorable clinicopathological features and a poor prognosis of CRC patients. In multivariate analysis, lnc-GNAT1-1 was proved to be an independent prognostic factor. In plasma, lnc-GNAT1-1 was significant decreased in CRC patients than healthy donors, and with the TNM stages advanced, the plasma lnc-GNAT1-1 level decreased; Receiver operating characteristic curve (ROC curve) showed that plasma lnc-GNAT1-1 had a moderate to well diagnostic efficiency for CRC. In vitro experiments showed that knockdown of lnc-GNAT1-1 could inhibit the aggressive phenotypes of CRC cell lines. In vivo study showed that overexpression of lnc-GNAT1-1 could suppress the liver metastasis of CRC cells. Finally, we explored the underlying mechanism of the role lnc-GNAT1-1 plays in CRC, and found a positive correlation between lnc-GNAT1-1 and Raf kinase inhibitor protein (RKIP) expression both in cells and in patients' tissues. We further found that lnc-GNAT1-1 could regulate the RKIP-NF-κB-Snail circuit in CRC. CONCLUSIONS: We have demonstrated in this study that a novel lncRNA, lnc-GNAT1-1, is low expressed in colorectal cancer tissues and plasma, and acts as a tumor suppressor through regulating RKIP-NF-κB-Snail circuit.

Contributions of Rod and Cone Pathways to Retinal Direction Selectivity Through Development.

UNLABELLED: Direction selectivity is a robust computation across a broad stimulus space that is mediated by activity of both rod and cone photoreceptors through the ON and OFF pathways. However, rods, S-cones, and M-cones activate the ON and OFF circuits via distinct pathways and the relative contribution of each to direction selectivity is unknown. Using a variety of stimulation paradigms, pharmacological agents, and knockout mice that lack rod transduction, we found that inputs from the ON pathway were critical for strong direction-selective (DS) tuning in the OFF pathway. For UV light stimulation, the ON pathway inputs to the OFF pathway originated with rod signaling, whereas for visible stimulation, the ON pathway inputs to the OFF pathway originated with both rod and M-cone signaling. Whole-cell voltage-clamp recordings revealed that blocking the ON pathway reduced directional tuning in the OFF pathway via a reduction in null-side inhibition, which is provided by OFF starburst amacrine cells (SACs). Consistent with this, our recordings from OFF SACs confirmed that signals originating in the ON pathway contribute to their excitation. Finally, we observed that, for UV stimulation, ON contributions to OFF DS tuning matured earlier than direct signaling via the OFF pathway. These data indicate that the retina uses multiple strategies for computing DS responses across different colors and stages of development. SIGNIFICANCE STATEMENT: The retina uses parallel pathways to encode different features of the visual scene. In some cases, these distinct pathways converge on circuits that mediate a distinct computation. For example, rod and cone pathways enable direction-selective (DS) ganglion cells to encode motion over a wide range of light intensities. Here, we show that although direction selectivity is robust across light intensities, motion discrimination for OFF signals is dependent upon ON signaling. At eye opening, ON directional tuning is mature, whereas OFF DS tuning is significantly reduced due to a delayed maturation of S-cone to OFF cone bipolar signaling. These results provide evidence that the retina uses multiple strategies for computing DS responses across different stimulus conditions.