TRIM29 modulates proteins involved in PTEN/AKT/mTOR and JAK2/STAT3 signaling pathway and suppresses the progression of hepatocellular carcinoma.

Tripartite motif-containing 29 (TRIM29), also known as the ataxia telangiectasia group D-complementing (ATDC) gene, has been reported to play an oncogenic or tumor suppressive role in developing different tumors. So far, its expression and biological functions in hepatocellular carcinoma (HCC) remain unclear. We investigated TRIM29 expression pattern in human HCC samples using quantitative RT-PCR and immunohistochemistry. Relationships between TRIM29 expression level, clinical prognostic indicators, overall survival (OS), and disease-free survival (DFS) were evaluated by Kaplan-Meier analysis and Cox proportional hazards model. A series of in vitro experiments and a xenograft tumor model were conducted to detect the functions of TRIM29 in HCC cells. RNA sequencing, western blotting, and immunochemical staining were performed to assess the molecular regulation of TRIM29 in HCC. We found that the mRNA and protein levels of TRIM29 were significantly reduced in HCC samples, compared with adjacent noncancerous tissues, and were negatively correlated with poor differentiation of HCC tissues. Survival analysis confirmed that lower TRIM29 expression significantly correlated with shorter OS and DFS of HCC patients. TRIM29 overexpression remarkably inhibited cell proliferation, migration, and EMT in HCC cells, whereas knockdown of TRIM29 reversed these effects. Moreover, deactivation of the PTEN/AKT/mTOR and JAK2/STAT3 pathways might be involved in the tumor suppressive role of TRIM29 in HCC. Our findings indicate that TRIM29 in HCC exerts its tumor suppressive effects through inhibition of the PTEN/AKT/mTOR and JAK2/STAT3 signaling pathways and may be used as a potential biomarker for survival in patients with HCC.

Two somatic mutations in the androgen receptor N-terminal domain are oncogenic drivers in hepatocellular carcinoma.

The androgen receptor (AR) plays an important role in male-dominant hepatocellular carcinoma, and specific acquired somatic mutations of AR have been observed in HCC patients. Our previous research have established the role of AR wild type as one of the key oncogenes in hepatocarcinogenesis. However, the role of hepatic acquired somatic mutations of AR remains unknown. In this study, we identify two crucial acquired somatic mutations, Q62L and E81Q, situated close to the N-terminal activation function domain-1 of AR. These mutations lead to constitutive activation of AR, both independently and synergistically with androgens, making them potent driver oncogene mutations. Mechanistically, these N-terminal AR somatic mutations enhance de novo lipogenesis by activating sterol regulatory element-binding protein-1 and promote glycogen accumulation through glycogen phosphorylase, brain form, thereby disrupting the AMPK pathway and contributing to tumorigenesis. Moreover, the AR mutations show sensitivity to the AMPK activator A769662. Overall, this study establishes the role of these N- terminal hepatic mutations of AR as highly malignant oncogenic drivers in hepatocarcinogenesis and highlights their potential as therapeutic targets for patients harboring these somatic mutations.

Electrochemical Valorization of Glycerol via Electrocatalytic Reduction into Biofuels: A Review.

Electrochemical conversion of underutilized biomass-based glycerol into high-value-added products provides a green approach for biomass and waste valorization. Plus, this approach offers an alternative to biofuel manufacturing procedure, under mild operating conditions, compared to the traditional thermochemical routes. Nevertheless, glycerol has been widely valorized via electrooxidation, with lower-value products generated at the cathode, ignoring the electroreduction. Here, a review of the efficient glycerol reduction into various products via the electrocatalytic reduction (ECR) process was presented. This review has been built upon the background of glycerol underutilization and theoretical knowledge about the state-of-the-art ECR. The experimental understanding of the processing parameter influences towards electrochemical efficiency, catalytic activity, and product selectivity are comprehensively reviewed, based on the recent glycerol ECR studies. We conclude by outlining present issues and highlighting potential future research avenues for enhanced ECR application.

Comparison between chronic spontaneous urticaria and chronic induced urticaria on the efficacy of omalizumab treatment: A systematic review and meta-analysis.

This meta-analysis aimed to assess the efficacy of omalizumab in the treatment of refractory-to-antihistamines chronic induced urticaria (CIndU) in comparison with that of refractory-to-antihistamines chronic spontaneous urticaria (CSU). We retrieved interventional studies and observational studies on omalizumab efficacy to CIndU patients and efficacy comparison between CSU and CIndU both refractory to H1-antihistamines in electronic databases (accessed till May 2022). The odd ratio (OR) and 95% confidence interval (CI) was calculated with a random-effect model in this meta-analysis. The majority of patients with different CIndU subtypes gained complete or partial response and good safety after omalizumab treatment. A total of five studies with 355 CSU patients and 103 CIndU patients were included for the meta-analysis. There was no significant difference in the efficacy of omalizumab in the treatment of CSU and CIndU (OR -0.83, 95% CI [0.84, 2.21], P > 0.05). Based on the validity of omalizumab in the treatment of various CIndU subtypes and non-differential efficacy between CSU and CIndU, it is reasonable to list omalizumab as a third-line treatment of refractory CIndU.

Potential molecular mechanism of the Xiexin capsule in the intervention of dyslipidemia based on bioinformatics and molecular docking / Posible mecanismo molecular de la cápsula Xiexin en la intervención de la dislipidemia basada en la bioinformática y el acoplamiento molecular

Objective: bioinformatic methods and molecular docking technology were used to predict the active components, targets, and related biological pathways of the Xiexin capsule in the intervention for dyslipidemia, exploring its mechanism. Methods: the active components and targets of the Xiexin capsule were screened by the TCMSP (Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform )database. Genecards (The Human Gene Database), OMIM (Online Mendelian Inheritance in Man), PharmGkb (Pharmacogenomics Knowledge Base database), TTD (Therapeutic Target Database), and Drugbank platforms were used to search the disease targets of dyslipidemia. The Cytoscape 3.8.0 software was used to construct the 'component-target' network diagram, and the STRING (functional protein association networks) platform was used to analyze protein-protein interaction (PPI). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomics (KEGG) enrichment analyses were performed by R language data packets to predict the mechanism of action. The AutoDockVina and PyMol software were used to dock the key active components in the Xiexin capsule and the core proteins in PPI. Results: a total of 66 effective components were screened, involving 114 targets; 87 key active compounds were screened from the 'drug-component-target' diagram. The PPI network mainly involved core proteins such as PTGS2 (prostaglandin-endoperoxide synthase 2), PTGS1 (prostaglandin-endoperoxide synthase 1), and HSP90AA1 (heat shock protein 90 alpha family class A member 1). GO and KEGG enrichment analysis results of common targets mainly involved hormone-mediated signaling pathway, steroid hormone response, lipid transport and metabolism, regulation of cholesterol storage, cyclooxygenase pathway, and other biological pathways, as well asMM PPAR (peroxisome proliferators-activated receptor) signaling pathway, IL-17 (interleukin 17) signaling pathway (AU)

Objetivo: se utilizaron métodos bioinformáticos y técnicas de acoplamiento molecular para predecir los componentes efectivos, los objetivos y las vías biológicas relacionadas de la cápsula Xiexin en la intervención de la dislipidemia y explorar su mecanismo. Métodos: los componentes activos y los objetivos de la cápsula Xiexin fueron seleccionados por la base de datos TCMSP. Se utilizaron las plataformas Genecards, OMIM, PharmGkb, TTD (Therapeutic Target Database) y Drugbank para buscar las dianas de la enfermedad en la dislipidemia. El diagrama reticular “componente-diana” fue construido por el software Cytoscape 3.7.0, y la interacción proteína-proteína (PPI) fue analizada por la plataforma STRING. Los análisis de enriquecimiento de Gene Ontology (GO) y Kyoto Encyclopedia of Genes and Genomics (KEGG) se realizaron mediante paquetes de datos en lenguaje R para predecir el mecanismo de acción. El software AutoDockVina y PyMol se utilizó para unir los componentes activos clave de la cápsula Xiexin y las proteínas clave de la PPI. Resultados: se seleccionaron 65 componentes activos y 114 dianas. Veintitrés compuestos activos clave fueron seleccionados a partir de la tabla “componentes farmacéuticos-dianas”. Las redes PPI incluyen principalmente proteínas básicas como PTGS2, PTGS1 y HSP90AA1. Los resultados del análisis de enriquecimiento de GO y KEGG en los objetivos comunes se refieren principalmente a la vía de señalización mediada por esteroides, la respuesta hormonal esteroidea, el transporte y metabolismo lipídicos, la regulación del almacenamiento de colesterol, la vía de la ciclooxigenasa y otras vías biológicas, así como la vía de señalización de PPAR, la vía de señalización de IL-17, la vía de señalización de PI3K-Akt (AU)

Machine learning based analysis of reaction phenomena in catalytic lignin depolymerization.

Heterogeneously catalyzed lignin solvolysis opens the possibility of transforming low value biomass into high value, useful aromatic chemicals, however, its reaction behavior is poorly understood due to the many possible interactions between reaction parameters. In this study, a novel predictive model for bio-oil yield, char yield and reaction time is developed using Random Forest (RF) regression method using data available from the literature to study the impact of surface properties of the catalyst and the weight averaged molecular weight of the lignin (Mw) used in the reaction. The models achieved a coefficient of determination (R2) score of 0.9062, 0.9428 and 0.8327, respectively, and feature importance for each case was explained and tied to studies that provide a mechanistic explanation for the performance of the model. Surface properties and lignin Mw showed no importance to the prediction of bio-oil yield and average pore diameter contributed 3% of feature importance to reaction time.

Zinc attenuates ferroptosis and promotes functional recovery in contusion spinal cord injury by activating Nrf2/GPX4 defense pathway.

AIM: Spinal cord injury (SCI) involves multiple pathological processes. Ferroptosis has been shown to play a critical role in the injury process. We wanted to explore whether zinc can inhibit ferroptosis, reduce inflammation, and then exert a neuroprotective effect. METHODS: The Alice method was used to establish a spinal cord injury model. The Basso Mouse Scale (BMS), Nissl staining, hematoxylin-eosin staining, and immunofluorescence analysis were used to investigate the protective effect of zinc on neurons on spinal cord neurons and the recovery of motor function. The regulation of the nuclear factor E2/heme oxygenase-1 (NRF2/HO-1) pathway was assessed, the levels of essential ferroptosis proteins were measured, and the changes in mitochondria were confirmed by transmission electron microscopy and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide (JC-1) staining. In vitro experiments using VSC4.1 (spinal cord anterior horn motor neuroma cell line), 4-hydroxynonenal (4HNE), reactive oxygen species (ROS), superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), lipid peroxides, and finally the levels of inflammatory factors were detected to assess the effect of zinc. RESULTS: Zinc reversed behavioral and structural changes after SCI. Zinc increased the expression of NRF2/HO-1, thereby increasing the content of glutathione peroxidase 4 (GPX4), SOD, and GHS and reducing the levels of lipid peroxides, MDA, and ROS. Zinc also rescued injured mitochondria and effectively reduced spinal cord injury and the levels of inflammatory factors, and the NRF2 inhibitor Brusatol reversed the effects of zinc. CONCLUSION: Zinc promoted the degradation of oxidative stress products and lipid peroxides through the NRF2/HO-1 and GPX4 signaling pathways to inhibit ferroptosis in neurons.

Regulatory Effect of Modified Liu Junzitang on Immune Function, Nutritional Status and Intestinal Microecology in Advanced Gastric Cancer Patients with Syndrome of Deficiency of Qi and Blood / 中国实验方剂学杂志

Objective:To explore the regulatory effect of modified Liu Junzitang on the immune function， nutritional status and intestinal microecology in advanced gastric cancer patients with syndrome of deficiency of Qi and blood. Method:The 86 advanced gastric cancer patients with syndrome of deficiency of Qi and blood were randomly divided into control group and observation group according to their admission numbers， with 43 cases in each group. The control group was given Yiqi Yangxue oral liquid on the basis of basic treatment while the observation group was given modified Liu Junzitang. After 4 weeks， compare the clinical efficacy of two groups of patients， traditional Chinese medicine （TCM） syndrome score， gastrointestinal function recovery， adverse reaction and quality of life， immune function， T cell subsets CD3⁺， CD4⁺， CD8⁺， C₃ and C₄ levels， immunoglobulin A （IgA）， immunoglobulin G （IgG）， immunoglobulin M （IgM）， nutritional status： albumin （propagated）， prealbumin （PA）， serum total protein （TP） and hemoglobin （Hb） content changes， the intestinal micro ecology： <italic>Bifidobacterium</italic>， <italic>Lactobacillus</italic>， <italic>Enterococcus aureus</italic>， <italic>Escherichia coli</italic> content changes. Result:The total effective rate of the observation group was 95.35% （41/43）， which was significantly higher than 79.07% （34/43） of the control group （<italic>χ²</italic>=5.108，<italic>P</italic><0.05）， after treatment， the TCM syndromes such as dizziness， pale complexion， palpitation， shortness of breath and fatigue in the observation group were significantly lower than those in the control group （<italic>P</italic><0.05）， the bowel sound recovery， exhaust and defecation time of the observation group were significantly lower than those of the control group （<italic>P</italic><0.05）， the quality of life scores in the observation group， such as the nature-to-human correspondence， form and spirit integration， specific modules， functional areas， and overall health score， were significantly higher than those in control group （<italic>P</italic><0.05）， the CD3⁺， CD4⁺， CD4⁺/CD8⁺， C₃， C₄， IgA， immune function indexes such as IgG and IgM were significantly higher than those of the control group， and the CD8⁺ level was lower than which of control group （<italic>P</italic><0.05）， the nutritional status levels such as Alb， PA， TP and Hb in the observation group were significantly higher than those of the control group （<italic>P</italic><0.05）， <italic>Bifidobacterium</italic>， <italic>Lactobacillus</italic>， and <italic>E. faecalis </italic>in the observation group were higher than those in the control group， and <italic>E. coli</italic> was lower than the control group （<italic>P</italic><0.05）， the adverse reaction rate of the observation group was 11.63% （5/43） and the control group was 16.28% （7/43） ， and there was no statistical difference between two groups. Conclusion:Modified Liu Junzitang has a good clinical effect on advanced gastric cancer patients with syndrome of deficiency of Qi and blood. It can improve TCM syndromes and gastrointestinal function， improve quality of life， and its mechanism is related to improving immune function， enhancing nutritional status， and improving intestinal microecology， and it has good safety.

Rac1 Modulates Excitatory Synaptic Transmission in Mouse Retinal Ganglion Cells / 神经科学通报·英文版

Ras-related C3 botulinum toxin substrate 1 (Rac1), a member of the Rho GTPase family which plays important roles in dendritic spine morphology and plasticity, is a key regulator of cytoskeletal reorganization in dendrites and spines. Here, we investigated whether and how Rac1 modulates synaptic transmission in mouse retinal ganglion cells (RGCs) using selective conditional knockout of Rac1 (Rac1-cKO). Rac1-cKO significantly reduced the frequency of AMPA receptor-mediated miniature excitatory postsynaptic currents, while glycine/GABA receptor-mediated miniature inhibitory postsynaptic currents were not affected. Although the total GluA1 protein level was increased in Rac1-cKO mice, its expression in the membrane component was unchanged. Rac1-cKO did not affect spine-like branch density in single dendrites, but significantly reduced the dendritic complexity, which resulted in a decrease in the total number of dendritic spine-like branches. These results suggest that Rac1 selectively affects excitatory synaptic transmission in RGCs by modulating dendritic complexity.

Acute Pericarditis Occurring Three Days after Intravesical Instillation of Mitomycin C after Transurethral Bladder Tumor Resection in a 64-Year-Old Woman.

We present a 64-year-old woman who developed symptoms of acute pericarditis three days after undergoing intravesical instillation of mitomycin C following transurethral bladder tumor resection. Mitomycin C is a chemotherapeutic agent which acts by alkylation of DNA and is known to be cardiotoxic when systemically administered. Despite classic pericarditis symptoms, the patient underwent an urgent coronary angiogram due to elevated cardiac troponin I level, EKG changes, and wall motion abnormalities on her echocardiogram. During her angiogram, it was found that she had multiple stenotic coronary artery lesions, with no acute total coronary occlusions, and percutaneous coronary intervention (PCI) was done with placement of a single drug-eluting stent for a 95% stenotic lesion in the left anterior descending artery. The patient was discharged after an uneventful hospitalization on dual antiplatelet therapy with aspirin and prasugrel, and colchicine for pericarditis. It is likely that the patient's presentation was the result of a perimyocardial inflammatory process secondary to intravesically administered mitomycin C, rather than an acute coronary syndrome. While the pathophysiological basis of cardiotoxicity of systemically administered mitomycin C is well documented, more studies are needed to determine whether intravesical MMC may cause cardiotoxicity.

Novel blood pressure estimation method using single photoplethysmography feature.

Continuous blood pressure (BP) monitoring has a significant meaning to the prevention and early diagnosis of cardiovascular disease. However, existing continuous BP monitoring approaches, especially cuff-less BP monitoring approaches, are all contraptions which complex and huge computation required. For example, for the most sophisticated cuff-less BP monitoring method using pulse transit time (PTT), the simultaneous record of photoplethysmography (PPG) signal and electrocardiography (ECG) are required, and various measurement of characteristic points are needed. These issues hindered widely application of cuff less BP measurement in the wearable devices. In this study, a novel BP estimation method using single PPG signal feature was proposed and its performance in BP estimation was also tested. The results showed that the new approach proposed in this study has a mean error -0.91 ± 3.84 mmHg for SBP estimation and -0.36 ± 3.36 mmHg for DBP estimation respectively. This approach performed better than the traditional PTT based BP estimation, which mean error for SBP estimation was -0.31 ± 4.78 mmHg, and for DBP estimation was -0.18 ± 4.32 mmHg. Further investigation revealed that this new BP estimation approach only required measurement of one characteristic point, reducing much computation when implementing. These results demonstrated that this new approach might be more suitable implemented in the wearable BP monitoring devices.

The effect of flowering time and distance between pollen source and recipient on maize.

Field experiments were conducted in Central Taiwan for 2 crop seasons to examine the effect of non-coincidence flowering on the cross-pollination (CP) rate of maize at various distances. Four local maize hybrid varieties with different flowering dates and one local maize variety were sown as the pollen sources and recipient, respectively. All varieties were sown on the same day to simulate the real situation of coexistence in which adjacent fields are sown with different genetically modified (GM) and non-GM varieties of maize. The CP rate was <0.2% at a distance of 3 m for the first crop season when the flowering time for the recipient was 5 d later than that of the pollen source variety. The CP rate was <0.02% at all distances for the second season when the flowering time for the recipient was 7 d later than that of the pollen source variety. The CP rate was <1% at a distance of 0.75 m when the flowering time was 3 d later. However, varieties with closer synchrony may result in a CP rate of >1% at a distance of 1.5 m and <1% at 2.25 m. Temporal separation and isolation distances can work together in Taiwan with fragmented landscapes to minimize the adventitious presence of one crop with another.

Clinician impression versus prescription drug monitoring program criteria in the assessment of drug-seeking behavior in the emergency department.

STUDY OBJECTIVE: We compare emergency provider impression of drug-seeking behavior with objective criteria from a state prescription drug monitoring program, assess change in opioid pain reliever prescribing after prescription drug monitoring program review, and examine clinical factors associated with suspected drug-seeking behavior. METHODS: This was a prospective observational study of emergency providers assessing a convenience sample of patients aged 18 to 64 years who presented to either of 2 academic medical centers with chief complaint of back pain, dental pain, or headache. Drug-seeking behavior was objectively defined as present when a patient had greater than or equal to 4 opioid prescriptions by greater than or equal to 4 providers in the 12 months before emergency department evaluation. Emergency providers completed data forms recording their impression of the likelihood of drug-seeking behavior, patient characteristics, and plan for prescribing pre- and post-prescription drug monitoring program review. Descriptive statistics were generated. We calculated agreement between emergency provider impression of drug-seeking behavior and prescription drug monitoring program definition, and sensitivity, specificity, and positive predictive value of emergency provider impression, using prescription drug monitoring program criteria as the criterion standard. A multivariate logistic regression analysis was conducted to determine clinical factors associated with drug-seeking behavior. RESULTS: Thirty-eight emergency providers with prescription drug monitoring program access participated. There were 544 patient visits entered into the study from June 2011 to January 2013. There was fair agreement between emergency provider impression of drug-seeking behavior and prescription drug monitoring program (κ=0.30). Emergency providers had sensitivity 63.2% (95% confidence interval [CI] 54.8% to 71.7%), specificity 72.7% (95% CI 68.4% to 77.0%), and positive predictive value 41.2% (95% CI 34.4% to 48.2%) for identifying drug-seeking behavior. After exposure to prescription drug monitoring program data, emergency providers changed plans to prescribe opioids at discharge in 9.5% of cases (95% CI 7.3% to 12.2%), with 6.5% of patients (n=35) receiving opioids not previously planned and 3.0% (n=16) no longer receiving opioids. Predictors for drug-seeking behavior by prescription drug monitoring program criteria were patient requests opioid medications by name (odds ratio [OR] 1.91; 95% CI 1.13 to 3.23), multiple visits for same complaint (OR 2.5; 95% CI 1.49 to 4.18), suspicious history (OR 1.88; 95% CI 1.1 to 3.19), symptoms out of proportion to examination (OR 1.83; 95% CI 1.1 to 3.03), and hospital site (OR 3.1; 95% CI 1.76 to 5.44). CONCLUSION: Emergency providers had fair agreement with objective criteria from the prescription drug monitoring program in suspecting drug-seeking behavior. Program review changed management plans in a small number of cases. Multiple clinical factors were predictive of drug-seeking behavior.

Genetic diversity of sweet potato begomoviruses in the United States and identification of a natural recombinant between sweet potato leaf curl virus and sweet potato leaf curl Georgia virus.

In the United States, two sweet potato begomoviruses, sweet potato leaf curl virus (SPLCV) and sweet potato leaf curl Georgia virus (SPLCGV), were previously identified in Louisiana. In recent years, at least seven additional sweet potato begomoviruses have been identified in other parts of the world. In an effort to determine the genetic diversity and distribution of sweet potato begomoviruses in the U.S., we focused our efforts on molecular characterization of field-collected begomovirus isolates in two states: Mississippi and South Carolina. Using rolling-circle amplification, a total of 52 clones of the full genome were obtained. Initial inspection of alignments of the end sequences in these clones revealed a strong genetic diversity. Overall, 10 genotypes could be assigned. A majority of the isolates (50/52) in eight genotypes were shown to be closely related to SPLCV. A representative clone of each genotype was fully sequenced and analyzed. Among them, four genotypes from South Carolina with 91-92% sequence identity to the type member of SPLCV were considered a new strain, whereas four other genotypes from Mississippi with >95% sequence identity to SPLCV were considered variants. In addition, a member of a proposed new begomovirus species was identified after comparative sequence analysis of the isolate [US:SC:646B-9] from South Carolina with less than 89% sequence identity to any known begomovirus. Hence, the provisional name Sweet potato leaf curl South Carolina virus (SPLCSCV) is proposed. Moreover, a natural recombinant consisting of two distinct parental genomic sequences from SPLCV and SPLCGV was identified in the sample [US:MS:1B-3] from Mississippi. Two recombinant breakpoints were identified, one in the origin of replication and the other between C2 and C4. This knowledge about the genetic diversity of begomoviruses infecting sweet potato will likely have a major impact on PCR-based virus detection and on disease management practice through breeding for virus resistance.

Alternative splicing and differential subcellular localization of the rat FGF antisense gene product.

BACKGROUND: GFG/NUDT is a nudix hydrolase originally identified as the product of the fibroblast growth factor-2 antisense (FGF-AS) gene. While the FGF-AS RNA has been implicated as an antisense regulator of FGF-2 expression, the expression and function of the encoded GFG protein is largely unknown. Alternative splicing of the primary FGF-AS mRNA transcript predicts multiple GFG isoforms in many species including rat. In the present study we focused on elucidating the expression and subcellular distribution of alternatively spliced rat GFG isoforms. RESULTS: RT-PCR and immunohistochemistry revealed tissue-specific GFG mRNA isoform expression and subcellular distribution of GFG immunoreactivity in cytoplasm and nuclei of a wide range of normal rat tissues. FGF-2 and GFG immunoreactivity were co-localized in some, but not all, tissues examined. Computational analysis identified a mitochondrial targeting sequence (MTS) in the N-terminus of three previously described rGFG isoforms. Confocal laser scanning microscopy and subcellular fractionation analysis revealed that all rGFG isoforms bearing the MTS were specifically targeted to mitochondria whereas isoforms and deletion mutants lacking the MTS were localized in the cytoplasm and nucleus. Mutation and deletion analysis confirmed that the predicted MTS was necessary and sufficient for mitochondrial compartmentalization. CONCLUSION: Previous findings strongly support a role for the FGF antisense RNA as a regulator of FGF2 expression. The present study demonstrates that the antisense RNA itself is translated, and that protein isoforms resulting form alternative RNA splicing are sorted to different subcellular compartments. FGF-2 and its antisense protein are co-expressed in many tissues and in some cases in the same cells. The strong conservation of sequence and genomic organization across animal species suggests important functional significance to the physical association of these transcript pairs.

Alternative splicing of the FGF antisense gene: differential subcellular localization in human tissues and esophageal adenocarcinoma.

Overexpression of FGF-2 is associated with tumor recurrence and reduced survival after surgical resection of esophageal cancer, and these risks are reduced in tumors co-expressing the FGF antisense (FGF-AS) RNA. The aim of this study was to characterize the expression of alternatively spliced FGF-AS transcripts and encoded nudix-motif proteins in normal human tissues and in esophageal adenocarcinoma, and to correlate their expression with clinicopathologic findings and outcome. Three alternatively spliced FGF-AS transcripts encoding GFG/NUDT6 isoforms with distinct N termini were detected in various human tissues including esophageal adenocarcinoma. Expression of each isoform as a fusion protein with enhanced green fluorescent protein revealed differential subcellular trafficking: hGFGa is localized to mitochondria by an N-terminal targeting sequence (MTS), whereas hGFGb and hGFGc were localized in the cytoplasm and nucleus. Mutation/deletion analysis confirmed that the predicted MTS was necessary and sufficient for mitochondrial compartmentalization. The predominant FGF-AS mRNA expressed in esophageal tumors was splice variant b. GFG immunoreactivity was detected in the cytoplasm of all esophageal adenocarcinomas and in 88% of tumor cell nuclei. Although we found a trend towards reduced disease-free survival in patients with FGF-2 overexpressing esophageal adenocarcinomas, significantly worse disease-free survival was noted among patients whose tumors did not also overexpress the FGF-AS b isoform (p = 0.03). Tetracycline-inducible FGF-AS b expression in stably transfected human Seg-1 esophageal adenocarcinoma cells resulted in a significant suppression of steady state FGF-2 mRNA content and cell proliferation. Our data implicate the FGF-AS b isoform in modulation of FGF-2 expression and clinical outcome in esophageal adenocarcinoma.

Evidence that insertion of Tomato ringspot nepovirus NTB-VPg protein in endoplasmic reticulum membranes is directed by two domains: a C-terminal transmembrane helix and an N-terminal amphipathic helix.

The NTB-VPg protein of Tomato ringspot nepovirus is an integral membrane protein found in association with endoplasmic reticulum (ER)-derived membranes active in virus replication. A transmembrane helix present in a hydrophobic region at the C terminus of the NTB domain was previously shown to traverse the membranes, resulting in the translocation of the VPg domain in the lumen. We have now conducted an in planta analysis of membrane-targeting domains within NTB-VPg using in-frame fusions to the green fluorescent protein (GFP). As expected, the entire NTB-VPg protein directed the GFP fluorescence to ER membranes. GFP fusion proteins containing the C-terminal 86 amino acids of NTB-VPg also associated with ER membranes, resulting in ER-specific glycosylation at a naturally occurring glycosylation site in the VPg domain. Deletion of the hydrophobic region prevented the membrane association. The N-terminal 80 amino acids of NTB were also sufficient to direct the GFP fluorescence to intracellular membranes. A putative amphipathic helix in this region was necessary and sufficient to promote membrane association of the fusion proteins. Using in vitro membrane association assays and glycosylation site mapping, we show that the N terminus of NTB can be translocated in the lumen at least in vitro. This translocation was dependent on the presence of the putative amphipathic helix, suggesting that oligomeric forms of this helix traverse the membrane. Taken together, our results suggest that at least two distinct elements play a key role in the insertion of NTB-VPg in the membranes: a C-terminal transmembrane helix and an N-terminal amphipathic helix. An updated model of the topology of the protein in the membrane is presented.

Inhibitory effects of cystatins on proteolytic activities of the Plum pox potyvirus cysteine proteinases.

In an effort to develop new antiviral strategies effective against potyviruses, several cystatins were evaluated for their ability to inhibit the cysteine proteinases of Plum pox potyvirus (PPV) using in vitro proteolytic assays. The following cystatins were purified as GST fusion proteins and shown to be active against papain:oryzacystatins I and II (OCI and OCII), corn cystatin II (CCII), human stefin A (HSA), the domain 8 of tomato multicystatin (TMC-8) and a large 24kDa tomato cystatin (LTCyst). These cystatins did not inhibit the activity of purified recombinant PPV NIa proteinase, a serine-like cysteine proteinases related to the 3C proteinases of picornaviruses and to chymotrypsin. The cystatins were shown to inhibit slightly the activity of the PPV HC-Pro proteinase with CCII being the best inhibitor. However a large excess of the cystatins was required to observe any inhibition. Based on these results and on the documented pleiotropic effects of cystatins on the metabolism of plants, we conclude that they are not the best candidates for antiviral strategies targeted to viral cysteine proteinases. The availability of soluble active recombinant PPV NIa proteinase will be instrumental for the selection of other proteinase inhibitors with increased affinity and specificity for this proteinase.