A system of reporters for comparative investigation of EJC-independent and EJC-enhanced nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a network of pathways that degrades transcripts that undergo premature translation termination. In mammals, NMD can be divided into the exon junction complex (EJC)-enhanced and EJC-independent branches. Fluorescence- and luminescence-based reporters have long been effective tools to investigate NMD, yet existing reporters largely focus on the EJC-enhanced pathway. Here, we present a system of reporters for comparative studies of EJC-independent and EJC-enhanced NMD. This system also enables the study of NMD-associated outcomes such as premature termination codon (PTC) readthrough and truncated protein degradation. These reporters are compatible with fluorescence or luminescence-based readouts via transient transfection or stable integration. Using this reporter system, we show that EJC-enhanced NMD RNA levels are reduced by 2- or 9-fold and protein levels are reduced by 7- or 12-fold compared to EJC-independent NMD, depending on the reporter gene used. Additionally, the extent of readthrough induced by G418 and an NMD inhibitor (SMG1i), alone and in combination, varies across NMD substrates. When combined, G418 and SMG1i increase readthrough product levels in an additive manner for EJC-independent reporters, while EJC-enhanced reporters show a synergistic effect. We present these reporters as a valuable toolkit to deepen our understanding of NMD and its associated mechanisms.

P. aeruginosa tRNA-fMet halves secreted in outer membrane vesicles suppress lung inflammation in cystic fibrosis.

Although tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of Pseudomonas aeruginosa (P. aeruginosa) in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is not completely understood. Here, we demonstrate that tobramycin increases 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by laboratory and CF clinical isolates of P. aeruginosa. The 5' tRNA-fMet halves are transferred from OMVs into primary CF human bronchial epithelial cells (CF-HBEC), decreasing OMV-induced IL-8 and IP-10 secretion. In mouse lungs, increased expression of the 5' tRNA-fMet halves in OMVs attenuated KC (murine homolog of IL-8) secretion and neutrophil recruitment. Furthermore, there was less IL-8 and neutrophils in bronchoalveolar lavage fluid isolated from pwCF during the period of exposure to tobramycin versus the period off tobramycin. In conclusion, we have shown in mice and in vitro studies on CF-HBEC that tobramycin reduces inflammation by increasing 5' tRNA-fMet halves in OMVs that are delivered to CF-HBEC and reduce IL-8 and neutrophilic airway inflammation. This effect is predicted to improve lung function in pwCF receiving tobramycin for P. aeruginosa infection.NEW & NOTEWORTHY The experiments in this report identify a novel mechanism, whereby tobramycin reduces inflammation in two models of CF. Tobramycin increased the secretion of tRNA-fMet halves in OMVs secreted by P. aeruginosa, which reduced the OMV-LPS-induced inflammatory response in primary cultures of CF-HBEC and in mouse lung, an effect predicted to reduce lung damage in pwCF.

Let-7b-5p in vesicles secreted by human airway cells reduces biofilm formation and increases antibiotic sensitivity of P. aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that forms antibiotic-resistant biofilms, which facilitate chronic infections in immunocompromised hosts. We have previously shown that P. aeruginosa secretes outer-membrane vesicles that deliver a small RNA to human airway epithelial cells (AECs), in which it suppresses the innate immune response. Here, we demonstrate that interdomain communication through small RNA-containing membrane vesicles is bidirectional and that microRNAs (miRNAs) in extracellular vesicles (EVs) secreted by human AECs regulate protein expression, antibiotic sensitivity, and biofilm formation by P. aeruginosa Specifically, human EVs deliver miRNA let-7b-5p to P. aeruginosa, which systematically decreases the abundance of proteins essential for biofilm formation, including PpkA and ClpV1-3, and increases the ability of beta-lactam antibiotics to reduce biofilm formation by targeting the beta-lactamase AmpC. Let-7b-5p is bioinformatically predicted to target not only PpkA, ClpV1, and AmpC in P. aeruginosa but also the corresponding orthologs in Burkholderia cenocepacia, another notorious opportunistic lung pathogen, suggesting that the ability of let-7b-5p to reduce biofilm formation and increase beta-lactam sensitivity is not limited to P. aeruginosa Here, we provide direct evidence for transfer of miRNAs in EVs secreted by eukaryotic cells to a prokaryote, resulting in subsequent phenotypic alterations in the prokaryote as a result of this interdomain communication. Since let-7-family miRNAs are in clinical trials to reduce inflammation and because chronic P. aeruginosa lung infections are associated with a hyperinflammatory state, treatment with let-7b-5p and a beta-lactam antibiotic in nanoparticles or EVs may benefit patients with antibiotic-resistant P. aeruginosa infections.

Contamination and health risks brought by arsenic, lead and cadmium in a water-soil-plant system nearby a non-ferrous metal mining area.

Heavy metal(loid)s contamination prevails in the water-soil-plant system around non-ferrous metal mining areas. The present study aimed to evaluate the heavy metal(loid)s contamination in Nandan Pb-Zn mining area (Guangxi, China). A total of 36 river water samples, 75 paired paddy soil and rice samples, and 128 paired upland soil and plant samples were collected from this area. The concentrations of arsenic (As), lead (Pb), and cadmium (Cd) in these samples were measured. Results showed that the average water quality indexes (WQIs) at the 12 sampling sites along the main river ranged from 41 to 5008, indicating the water qualities decreasing from "Excellent" to "Undrinkable". The WQIs nearby tailings or industrial park were significantly higher than those at the other sites. 34.0% and 64.5% of soil samples exceeded the risk screening values for As and Cd. The Pb and Cd concentrations in all rice samples exceeded the Chinese food safety limits by 18.7% and 82.7%, respectively. Leafy vegetables had a higher concentration of As, Pb, and Cd than other vegetables, exceeding the maximum permissible limits by 14.1%, 61.2%, and 40.0%, respectively. The biological accumulation coefficient (BAC) of Cd was the highest in rice and lettuce leaves. The hazard quotients (HQs) of As and Cd, indicating non-carcinogenic risks, were 4.15 and 1.76 in adult males, and 3.40 and 1.45 in adult females, all higher than the permitted level (1.0). The carcinogenic probabilities of As and Cd from rice and leafy vegetables consumption were all higher than 1 × 10-4. We conclude that metal(loid)s contamination of the water-soil-plant system has posed great non-carcinogenic and carcinogenic risks to the local population.

Shear Adhesion of Ice on Large-Sized Grooved Aluminum Surfaces.

Ice adhesion is important when designing aircraft anti-icing/de-icing systems. Major and minor grooves are common in the skin of aircraft. However, the effects of millimeter-scale grooves on ice adhesive strength have not been given due attention. Specimens with varying depths, widths, and numbers of grooves were fabricated by machining to investigate the ice adhesive characteristics of large-sized grooved aluminum surfaces. After the ice cube was frozen on the surface using a silicon mold, the adhesive force was measured using a self-assembled shear adhesive force setup. A correlation between groove size and apparent adhesive strength in the perpendicular loading direction was established based on the experimental results. Every 1% increase in the groove width ratio was associated with an 18.7 kPa increase in apparent adhesive strength. The increasing speed of the adhesion rapidly decayed as the groove depth increased. The increase in adhesion reached 99% of the maximum increase when the groove depth reached 0.8 times the width. The number of grooves had little effect on the adhesion when the total width of the grooves was kept constant. Stress distribution analysis was conducted using the finite element method, and the results were in accordance with the cracking phenomena in the experiments. The adhesive strength in the parallel loading direction was 30% lower than that in the perpendicular loading direction for all six chosen surfaces. This study is the first to propose a quantitative relationship between the surface textures of millimeter-sized grooves and ice adhesive strength. The loading orientation also had a substantial influence on adhesion. The results will serve as a valuable reference for future studies on ice adhesion on textured surfaces and for improving the performance of anti-icing/de-icing systems.

[Animal experimental study on the effects of different levels of amputation on cardiovascular system].

Vascular injury resulting from lower limb amputation leads to the redistribution of blood flow and changes in vascular terminal resistance, which can affect the cardiovascular system. However, there was no clear understanding of how different amputation levels affect the cardiovascular system in animal experiments. Therefore, this study established two animal models of above-knee amputation (AKA) and below-knee amputation (BKA) to explore the effects of different amputation levels on the cardiovascular system through blood and histopathological examinations. The results showed that amputation caused pathological changes in the cardiovascular system of animals, including endothelial injury, inflammation, and angiosclerosis. The degree of cardiovascular injury was higher in the AKA group than in the BKA group. This study sheds light on the internal mechanisms of amputation's impact on the cardiovascular system. Based on the amputation level of patients, the findings recommend more comprehensive and targeted monitoring after surgery and necessary interventions to prevent cardiovascular diseases.

[Effect of lower limb amputation level on aortic hemodynamics: a numerical study].

It has been found that the incidence of cardiovascular disease in patients with lower limb amputation is significantly higher than that in normal individuals, but the relationship between lower limb amputation and the episodes of cardiovascular disease has not been studied from the perspective of hemodynamics. In this paper, numerical simulation was used to study the effects of amputation on aortic hemodynamics by changing peripheral impedance and capacitance. The final results showed that after amputation, the aortic blood pressure increased, the time averaged wall shear stress of the infrarenal abdominal aorta decreased and the oscillatory shear index of the left and right sides was asymmetrically distributed, while the time averaged wall shear stress of the iliac artery decreased and the oscillatory shear index increased. The changes above were more significant with the increase of amputation level, which will result in a higher incidence of atherosclerosis and abdominal aortic aneurysm. These findings preliminarily revealed the influence of lower limb amputation on the occurrence of cardiovascular diseases, and provided theoretical guidance for the design of rehabilitation training and the optimization of cardiovascular diseases treatment.

[Establishment of an animal model to study the effects of amputation on the cardiovascular system].

Lower limb amputation is a significant change in body structure. Loss of muscle, blood vessels, and blood leads to a redistribution of blood flow and changes in resistance at the end of blood vessels. In view of the significant increase in the prevalence of cardiovascular disease after lower limb amputation, the mechanism of which is still unclear, this study aims to establish an animal research model that can verify and explore the effects of amputation on cardiovascular system, and provide the experimental basis for subsequent animal experiments when exploring the effect of different amputation levels on the cardiovascular system. SPF New Zealand rabbits were divided into normal group ( n = 6) and amputation group ( n = 6). The amputation group was treated with above-knee amputation. The changes of low-density liptein cholesterol (LDL-C) and total cholesterol (TC) in serum of all the rabbits were monitored regularly after the surgery. The arterial pathological examination was conducted after the experimental rabbits were executed. The results showed that compared with the normal group, serum LDL-C content and TC content in the amputation group were significantly increased ( P<0.05); The blood vessels of the amputated rabbits had pathological changes such as degeneration and necrosis of smooth muscle cells in the middle membrane layer and rupture of elastic fibers. At the abdominal aorta and aortic arch, the elastic fiber area expression percentage (EFEP) of the experimental group was significantly lower than that of the normal group. The results suggest that the cardiovascular system of rabbits has the tendency of decreased arterial elasticity and lipid deposition in blood after amputation, indicating that the animal research model on the effect of amputation on the cardiovascular system has been successfully established, and can provide an experimental platform for further study on the mechanism of the effect of amputation on the cardiovascular system.

CF monocyte-derived macrophages have an attenuated response to extracellular vesicles secreted by airway epithelial cells.

Mutations in CFTR alter macrophage responses, for example, by reducing their ability to phagocytose and kill bacteria. Altered macrophage responses may facilitate bacterial infection and inflammation in the lungs, contributing to morbidity and mortality in cystic fibrosis (CF). Extracellular vesicles (EVs) are secreted by multiple cell types in the lungs and participate in the host immune response to bacterial infection, but the effect of EVs secreted by CF airway epithelial cells (AEC) on CF macrophages is unknown. This report examines the effect of EVs secreted by primary AEC on monocyte-derived macrophages (MDM) and contrasts responses of CF and wild type (WT) MDM. We found that EVs generally increase pro-inflammatory cytokine secretion and expression of innate immune genes in MDM, especially when EVs are derived from AEC exposed to Pseudomonas aeruginosa and that this effect is attenuated in CF MDM. Specifically, EVs secreted by P. aeruginosa exposed AEC (EV-PA) induced immune response genes and increased secretion of proinflammatory cytokines, chemoattractants, and chemokines involved in tissue repair by WT MDM, but these effects were less robust in CF MDM. We attribute attenuated responses by CF MDM to differences between CF and WT macrophages because EVs secreted by CF AEC or WT AEC elicited similar responses in CF MDM. Our findings demonstrate the importance of AEC EVs in macrophage responses and show that the Phe508del mutation in CFTR attenuates the innate immune response of MDM to EVs.

Acute Hemodynamic Improvement by Thermal Vasodilation inside the Abdominal and Iliac Arterial Segments of Young Sedentary Individuals.

OBJECTIVE: To study the hemodynamic response to lower leg heating intervention (LLHI) inside the abdominal and iliac arterial segments (AIAS) of young sedentary individuals. METHODS: A Doppler measurement of blood flow was conducted for 5 young sedentary adults with LLHI. Heating durations of 0, 20, and 40 min were considered. A lumped parameter model (LPM) was used to ascertain the hemodynamic mechanism. The hemodynamics were determined via numerical approaches. RESULTS: Ultrasonography revealed that the blood flow waveform shifted upwards under LLHI; in particular, the mean flow increased significantly (p < 0.05) with increasing heating duration. The LPM showed that its mechanism depends on the reduction in afterload resistance, not on the inertia of blood flow and arterial compliance. The time-averaged wall shear stress, time-averaged production rate of nitric oxide, and helicity in the external iliac arteries increased more significantly than in other segments as the heating duration increased, while the oscillation shear index (OSI) and relative residence time (RRT) in the AIAS declined with increasing heating duration. There was a more obvious helicity response in the bilateral external iliac arteries than the OSI and RRT responses. CONCLUSION: LLHI can effectively induce a positive hemodynamic environment in the AIAS of young sedentary individuals.

Hemodynamics and Oxygen Transport through Pararenal Aortic Aneurysm Treated with Multilayer Stent: A Numerical Study.

BACKGROUND: As opposed to an endoluminal stent graft, a multilayer stent (MS) consists of a porous mesh, which allows for the possibility of treating pararenal aortic aneurysms (PRAAs) that involve a significant branch vessel. However, the choice of the density of the MS plays a vital role in isolating the aneurysm and allowing unobstructed blood flow in the branch vessel. METHOD: In the present study, we examined 3 cases (without a stent and with single-layer and double-layer stents) via numerical simulations to explore the feasibility of the MSs used in the treatment of such aneurysms and estimate whether there is a more appropriate or optimal stent density. RESULTS: With stent intervention, the velocity of blood flow in the sac decreased, but the pressure on the surface of the aneurysm did not decrease although it became more uniform. In addition, the "region of double low" (with low wall shear stress and a low Sherwood number) enlarged after stent implantation. Even with the double-layer stent, however, the flux of the branch vessel was still above normal, and we could predict that the optimal stent porosity was approximately 49.9%. CONCLUSIONS: Unlike in previous studies, an MS could not be feasibly applied to high-risk PRAAs. However, an MS can induce sac thrombosis in the later stages while maintaining visceral vessel patency, and our results suggest that the optimal stent may be a 4-layer stent.

Cdkn2a suppresses metastasis in squamous cell carcinomas induced by the gain-of-function mutant p53(R172H).

p53 (TP53) is the most frequently mutated gene in squamous cell carcinomas (SCCs) of the skin and head and neck. Certain p53 mutations are oncogenic and promote invasion and metastasis in SCCs. However, it is unclear how the oncogenic function of mutant p53 is modulated by other molecular alterations that co-exist in SCCs. Here, we show that deletion of the p53 gene and activation of an endogenous p53(R172H) gain-of-function mutation in the skin induce carcinomas with similar kinetics and penetrance. Deletion of p53 induced primarily well-differentiated SCCs. However, most of the tumours induced by p53(R172H) were poorly differentiated SCCs, the only metastatic tumours in this model. These tumours expressed higher levels of cyclin D1 than the well-differentiated SCCs and spindle carcinomas that developed in these mice. Unexpectedly, metastasis was not observed in mice that developed spindle carcinomas, which expressed high levels of the tumour suppressors p16(Ink4a) and p19(Arf) , encoded by Cdkn2a, a gene frequently deleted in human SCCs. Remarkably, deletion of the Cdkn2a gene in p53(R172H) -induced SCCs promoted a dramatic increase in metastasis rates and a shorter survival in mice that developed these tumours, compared with those observed in mice with tumours in which Cdkn2a was deleted in the presence of a p53 loss-of-function mutation or wild-type p53. Accordingly, the survival of patients with head and neck SCCs bearing co-occurring high-risk p53 mutations and CDKN2A homozygous deletions was much shorter than that of patients with tumours in which high-risk p53 mutations did not contain CDKN2A homozygous deletions, or that of patients with tumours in which homozygous CDKN2A deletions co-existed with either low-risk p53 mutations or potential loss-of-function mutations in p53. These findings genetically identify a population of SCC patients with worst outcomes and will help to predict outcomes according to the p53 status and alterations in CDKN2A. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Clinical outcome and expression of mutant P53, P16, and Smad4 in lung adenocarcinoma: a prospective study.

BACKGROUND: Whole-exome sequencing has shown that lung adenocarcinoma (LAC) can be driven by mutant genes, including TP53, P16, and Smad4. The aim of this study was to clarify protein alterations of P53, P16, and Smad4 and to explore their correlations between the protein alterations and clinical outcome. METHODS: We investigated associations among P53 mutant (P53(Mut)) expression, and P16 and Smad4 loss-of-expression, with clinical outcome in 120 LAC patients who underwent curative resection, using immunohistochemical (IHC) methods. RESULTS: Of the 120 patients, 76 (63.3%) expressed P53(Mut) protein, whereas 54 (45.0%) loss of P16 expressed and 75 (62.5%) loss of Smad4 expressed. P53(Mut) expression was associated with tumor size (P = 0.041) and pathological stage (P = 0.025). Loss of P16 expression was associated with lymph node metastasis (P = 0.001) and pathological stage (P < 0.001). Loss of Smad4 expression was associated with tumor size (P = 0.033), lymph node metastasis (P = 0.014), pathological stage (P = 0.017), and tumor differentiation (P = 0.022). Kaplan-Meier survival analysis showed that tumor size (P = 0.031), lymph node metastasis (P < 0.001), pathological stage (P < 0.001), P53(Mut) protein expression (P = 0.038), and loss of p16 or Smad4 expression (P < 0.001) were significantly associated with shorter overall survival(OS), whereas multivariate analysis indicated that lymph node metastasis (P = 0.014) and loss of p16 or Smad4 expression (P < 0.001) were independent prognostic factors. Analysis of protein combinations showed patients with more alterations had poorer survival (P < 0.001). Spearman correlation analysis showed that loss of Smad4 expression inversely correlated with expression of P53(Mut) (r = (-)0.196, P = 0.032) and positively with lost P16 expression (r =0.182, P = 0.047). CONCLUSIONS: The findings indicate that IHC status of P53(Mut), P16, and Smad4 may predict patient outcomes in LAC.

Uncovering the characteristics of the gut microbiota in patients with ischemic stroke and hemorrhagic stroke.

This study aimed to explore the gut microbiota characteristics of ischemic and hemorrhagic stroke patients. A case-control study was conducted, and high-throughput sequencing of the V4-V5 region of 16S rRNA was used to analyze the differences in gut microbiota. The results showed that Proteobacteria was significantly increased in the ischemic stroke group compared with the healthy control group, while Fusobacteria was significantly increased in the hemorrhagic stroke group. In the ischemic stroke group, Butyricimonas, Alloprevotella, and Escherichia were significantly more abundant than in the healthy control group. In the hemorrhagic stroke group, Atopobium, Hungatella, Eisenbergiella, Butyricimonas, Odonbacter, Lachnociostridium, Alistipes, Parabacteroides, and Fusobacterium were significantly more abundant than in the healthy control group. Additionally, Alloprevotella, Ruminococcus, and Prevotella were significantly more abundant in the ischemic stroke group than in the hemorrhagic stroke group. The gut microbiota of ischemic and hemorrhagic stroke patients has significant diversity characteristics. These results provide new theoretical basis for exploring the prevention and treatment of different types of stroke through gut microbiota research.

P. aeruginosa tRNA-fMet halves secreted in outer membrane vesicles suppress lung inflammation in Cystic Fibrosis.

Although tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of Pseudomonas aeruginosa (P. aeruginosa) in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is not completely understood. Here, we demonstrate that tobramycin increases 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by laboratory and CF clinical isolates of P. aeruginosa . The 5' tRNA-fMet halves are transferred from OMVs into primary CF human bronchial epithelial cells (CF-HBEC), decreasing OMV-induced IL-8 and IP-10 secretion. In mouse lung, increased expression of the 5' tRNA-fMet halves in OMVs attenuated KC secretion and neutrophil recruitment. Furthermore, there was less IL-8 and neutrophils in bronchoalveolar lavage fluid isolated from pwCF during the period of exposure to tobramycin versus the period off tobramycin. In conclusion, we have shown in mice and in vitro studies on CF-HBEC that tobramycin reduces inflammation by increasing 5' tRNA-fMet halves in OMVs that are delivered to CF-HBEC and reduce IL-8 and neutrophilic airway inflammation. This effect is predicted to improve lung function in pwCF receiving tobramycin for P. aeruginosa infection. New and noteworthy: The experiments in this report identify a novel mechanim whereby tobramycin reduces inflammation in two models of CF. Tobramycin increased the secretion of tRNA-fMet haves in OMVs secreted by P. aeruginiosa , which reduced the OMV-LPS induced inflammatory response in primary cultures of CF-HBEC and in mouse lung, an effect predicted to reduce lung damage in pwCF. Graphical abstract: The anti-inflammatory effect of tobramycin mediated by 5' tRNA-fMet halves secreted in P. aeruginosa OMVs. (A) P. aeruginosa colonizes the CF lungs and secrets OMVs. OMVs diffuse through the mucus layer overlying bronchial epithelial cells and induce IL-8 secretion, which recruits neutrophils that causes lung damage. ( B ) Tobramycin increases 5' tRNA-fMet halves in OMVs secreted by P. aeruginosa . 5' tRNA-fMet halves are delivered into host cells after OMVs fuse with lipid rafts in CF-HBEC and down-regulate protein expression of MAPK10, IKBKG, and EP300, which suppresses IL-8 secretion and neutrophils in the lungs. A reduction in neutrophils in CF BALF is predicted to improve lung function and decrease lung damage.

Remodeling of the cardiovascular hemodynamic environment by lower limb heat exposure: A computational fluid dynamic study.

BACKGROUND: Lower limb heat exposure (LLHE) is a promising strategy for the daily management of cardiovascular health because of its non-pharmaceutical advantages. To support the application of this strategy in cardiovascular protection, we examined its impact on the global hemodynamic environment. METHODS: Skin blood flow (SBF) of eight locations on the lower limbs was measured before and after LLHE (40 °C and 44 °C) in ten healthy subjects by using a laser Doppler flowmeter. A closed-loop model of circulation uses changes in SBF to quantify the influence of LLHE on the blood flow of the arterial trunk (from ascending aorta to the femoral artery) and visceral branches (coronary, celiac, renal, and mesenteric arteries). RESULTS: The SBF in all locations tested on the lower limbs increased significantly (p<0.001) with LLHE and a 3.39-fold and 7.40-fold increase in mean SBF were observed under 40 °C and 44 °C conditions, respectively. In the model, the peak (3.9-25.1%), end-diastolic (13.7-107.3%), and mean blood flow (8.5-86.5%) in the arterial trunk increased with the increase in temperature, but the retrograde flow in the thoracic aorta and abdominal aorta Ⅰ increased at least twice in the diastolic period. Furthermore, LLHE also increased the blood flow of the visceral branches (2.5-20.7%). CONCLUSION: These findings suggest that LLHE is expected to be a daily strategy for enhancing the functions of both the arterial trunk and visceral arteries, but the increased blood flow reversal in the thoracic and abdominal aortas warrants further investigation.

A system of reporters for comparative investigation of EJC-independent and EJC-enhanced nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a network of pathways that degrades transcripts that undergo premature translation termination. In mammals, NMD can be divided into the exon junction complex (EJC)-enhanced and EJC-independent branches. Fluorescence- and luminescence-based reporters have long been effective tools to investigate NMD, yet existing reporters largely focus on the EJC-enhanced pathway. Here, we present a system of reporters for comparative studies of EJC-independent and EJC-enhanced NMD. This system also enables the study of NMD-associated outcomes such as premature termination codon (PTC) readthrough and truncated protein degradation. These reporters are compatible with fluorescence or luminescence-based readouts via transient transfection or stable integration. Using this reporter system, we show that EJC-enhanced NMD RNA levels are reduced by 2- or 9-fold and protein levels are reduced by 7- or 12-fold compared to EJC-independent NMD, depending on the reporter gene used. Additionally, the extent of readthrough induced by G418 and SMG1i, alone and in combination, varies across NMD substrates. When combined, G418 and SMG1i increase readthrough product levels in an additive manner for EJC-independent reporters, while EJC-enhanced reporters show a synergistic effect. We present these reporters as a valuable toolkit to deepen our understanding of NMD and its associated mechanisms.

Lgi1 null mutant mice exhibit myoclonic seizures and CA1 neuronal hyperexcitability.

LGI1 in humans is responsible for a predisposition to autosomal dominant partial epilepsy with auditory features (ADPEAF). However, mechanisms of how LGI1 mutations cause epilepsy remain unclear. We have used a mouse chromosome engineering strategy to create a null mutation for the gene ortholog encoding LGI1. The Lgi1 null mutant mice show no gross overall developmental abnormalities from routine histopathological analysis. After 12-18 days of age, the homozygous mutant mice all exhibit myoclonic seizures accompanied by rapid jumping and running and die shortly thereafter. The heterozygous mutant mice do not develop seizures. Electrophysiological analysis demonstrates an enhanced excitatory synaptic transmission by increasing the release of the excitatory neurotransmitter glutamate, suggesting a basis for the seizure phenotype. This mouse model, therefore, provides novel insights into the mechanism behind ADPEAF and offers a new opportunity to study the mechanism behind the role of LGI1 in susceptibility to myoclonic seizures.

A mouse model of Down syndrome trisomic for all human chromosome 21 syntenic regions.

Down syndrome (DS) is caused by the presence of an extra copy of human chromosome 21 (Hsa21) and is the most common genetic cause for developmental cognitive disability. The regions on Hsa21 are syntenically conserved with three regions located on mouse chromosome 10 (Mmu10), Mmu16 and Mmu17. In this report, we describe a new mouse model for DS that carries duplications spanning the entire Hsa21 syntenic regions on all three mouse chromosomes. This mouse mutant exhibits DS-related neurological defects, including impaired cognitive behaviors, reduced hippocampal long-term potentiation and hydrocephalus. These results suggest that when all the mouse orthologs of the Hsa21 genes are triplicated, an abnormal cognitively relevant phenotype is the final outcome of the elevated expressions of these orthologs as well as all the possible functional interactions among themselves and/or with other mouse genes. Because of its desirable genotype and phenotype, this mutant may have the potential to serve as one of the reference models for further understanding the developmental cognitive disability associated with DS and may also be used for developing novel therapeutic interventions for this clinical manifestation of the disorder.

Gain-of-function mutant p53 but not p53 deletion promotes head and neck cancer progression in response to oncogenic K-ras.

Mutations in p53 occur in over 50% of the human head and neck squamous cell carcinomas (SCCHN). The majority of these mutations result in the expression of mutant forms of p53, rather than deletions in the p53 gene. Some p53 mutants are associated with poor prognosis in SCCHN patients. However, the molecular mechanisms that determine the poor outcome of cancers carrying p53 mutations are unknown. Here, we generated a mouse model for SCCHN and found that activation of the endogenous p53 gain-of-function mutation p53$^{\rm{R172H}}$, but not deletion of p53, cooperates with oncogenic K-ras during SCCHN initiation, accelerates oral tumour growth, and promotes progression to carcinoma. Mechanistically, expression profiling of the tumours that developed in these mice and studies using cell lines derived from these tumours determined that mutant p53 induces the expression of genes involved in mitosis, including cyclin B1 and cyclin A, and accelerates entry in mitosis. Additionally, we discovered that this oncogenic function of mutant p53 was dependent on K-ras because the expression of cyclin B1 and cyclin A decreased, and entry in mitosis was delayed, after suppressing K-ras expression in oral tumour cells that express p53$^{\rm{R172H}}$. The presence of double-strand breaks in the tumours suggests that oncogene-dependent DNA damage resulting from K-ras activation promotes the oncogenic function of mutant p53. Accordingly, DNA damage induced by doxorubicin also induced increased expression of cyclin B1 and cyclin A in cells that express p53$^{\rm{R172H}}$. These findings represent strong in vivo evidence for an oncogenic function of endogenous p53 gain-of-function mutations in SCCHN and provide a mechanistic explanation for the genetic interaction between oncogenic K-ras and mutant p53.