MIR145 Core Promoter Methylation in Pretreatment Cell-Free DNA: A Liquid Biopsy Tool for Muscle-Invasive Bladder Cancer Treatment Outcome.

PURPOSE: The lack of personalized management of bladder cancer (BlCa) results in patients' lifelong post-treatment monitoring with invasive interventions, underlying the urgent need for tailored and minimally invasive health care services. On the basis of our previous findings on miR-143/145 cluster methylation in bladder tumors, we evaluated its clinical significance in pretreatment cell-free DNA (cfDNA) of patients with BlCa. MATERIALS AND METHODS: Methylation analysis was performed in our screening cohort (120 patients with BlCa; 20 age-matched healthy donors) by bisulfite-based pyrosequencing. Tumor recurrence/progression for patients with non-muscle-invasive bladder cancer, and progression and mortality for patients with muscle-invasive bladder cancer (MIBC) were used as clinical end point events in survival analysis. Bootstrap analysis was applied for internal validation of Cox regression models and decision curve analysis for assessment of clinical benefit on disease prognosis. RESULTS: Decreased methylation of MIR145 core promoter in pretreatment cfDNA was associated with short-term disease progression (multivariate Cox: hazard ratio [HR], 2.027 [95% CI, 1.157 to 3.551]; P = .010) and poor overall survival (multivariate Cox: HR, 2.098 [95% CI, 1.154 to 3.817]; P = .009) of patients with MIBC after radical cystectomy (RC). Multivariate models incorporating MIR145 promoter methylation in cfDNA with tumor stage clearly ameliorated patients' risk stratification, highlighting superior clinical benefit in MIBC prognostication. CONCLUSION: Reduced pretreatment cfDNA methylation of MIR145 core promoter was markedly correlated with increased risk for short-term progression and worse survival of patients with MIBC after RC and adjuvant therapy, supporting modern personalized and minimally invasive prognosis. Methylation profiling of MIR145 core promoter in pretreatment cfDNA could serve as a minimally invasive and independent predictor of MIBC treatment outcome and emerge as a promising marker for blood-based test in BlCa.

L-Dopa decarboxylase modulates autophagy in hepatocytes and is implicated in dengue virus-caused inhibition of autophagy completion.

The enzyme L-Dopa Decarboxylase (DDC) synthesizes the catecholamine dopamine and the indolamine serotonin. Apart from its role in the brain as a neurotransmitter biosynthetic enzyme, DDC has been detected also in the liver and other peripheral organs, where it is implicated in cell proliferation, apoptosis, and host-virus interactions. Dengue virus (DENV) suppresses DDC expression at the later stages of infection, during which DENV also inhibits autophagosome-lysosome fusion. As dopamine affects autophagy in neuronal cells, we investigated the possible association of DDC with autophagy in human hepatocytes and examined whether DDC mediates the relationship between DENV infection and autophagy. We performed DDC silencing/overexpression and evaluated autophagic markers upon induction of autophagy, or suppression of autophagosome-lysosome fusion. Our results showed that DDC favored the autophagic process, at least in part, through its biosynthetic function, while knockdown of DDC or inhibition of DDC enzymatic activity prevented autophagy completion. In turn, autophagy induction upregulated DDC, while autophagy reduction by chemical or genetic (ATG14L knockout) ways caused the opposite effect. This study also implicated DDC with the cellular energetic status, as DDC silencing reduced the oxidative phosphorylation activity of the cell. We also report that upon DDC silencing, the repressive effect of DENV on the completion of autophagy was enhanced, and the inhibition of autolysosome formation did not exert an additive effect on viral proliferation. These data unravel a novel role of DDC in the autophagic process and suggest that DENV downregulates DDC expression to inhibit the completion of autophagy, reinforcing the importance of this protein in viral infections.

Expression of Human L-Dopa Decarboxylase (DDC) under Conditions of Oxidative Stress.

Oxidative stress is known to influence mRNA levels, translation, and proteolysis. The importance of oxidative stress has been demonstrated in several human diseases, including neurodegenerative disorders. L-Dopa decarboxylase (DDC) is the enzyme that converts L-Dopa to dopamine (DA). In spite of a large number of studies, little is known about the biological significance of the enzyme under physiological and pathological conditions. Here, we investigated the relationship between DDC expression and oxidative stress in human neural and non-neural cells. Oxidative stress was induced by treatment with H2O2. Our data indicated that mRNA and protein expression of DDC was enhanced or remained stable under conditions of ROS induction, despite degradation of total RNA and increased cytotoxicity and apoptosis. Moreover, DDC silencing caused an increase in the H2O2-induced cytotoxicity. The current study suggests that DDC is involved in the mechanisms of oxidative stress.

Unraveling the Concealed Transcriptomic Landscape of PTEN in Human Malignancies.

Background: Phosphatase and tensin homolog, widely known as PTEN, is a major negative regulator of the PI3K/AKT/mTOR signaling pathway, involved in the regulation of a variety of important cellular processes, including cell proliferation, growth, survival, and metabolism. Since most of the molecules involved in this biological pathway have been described as key regulators in cancer, the study of the corresponding genes at several levels is crucial. Objective: Although previous studies have elucidated the physiological role of PTEN under normal conditions and its involvement in carcinogenesis and cancer progression, the transcriptional profile of PTEN has been poorly investigated. Methods: In this study, instead of conducting the "gold-standard" direct RNA sequencing that fails to detect less abundant novel mRNAs due to the decreased sequencing depth, we designed and implemented a multiplexed PTEN-targeted sequencing approach that combined both short- and long-read sequencing. Results: Our study has highlighted a broad spectrum of previously unknown PTEN mRNA transcripts and assessed their expression patterns in a wide range of human cancer and non-cancer cell lines, shedding light on the involvement of PTEN in cell cycle dysregulation and thus tumor development. Conclusion: The identification of the described novel PTEN splice variants could have significant implications for understanding PTEN regulation and function, and provide new insights into PTEN biology, opening new avenues for monitoring PTEN-related diseases, including cancer.

Heat and Cold-Stressed Individuals of Pistacia lentiscus (Mastic Tree) Do Modify Their Secreting Profile.

Seedlings from the germinated seeds of Pistacia lentiscus were cultured in plant growth chambers for three months. Then, the plants were separated into three groups. Each group was cultured under different conditions. The first group was left to grow under normal Mediterranean conditions, as those recorded in spring. The other group was subjected to a ten-day heat stress while the last one also suffered a cold stress for ten days. The anatomical features of the leaves (leaf thickness, epidermal cell thickness, number of palisade layers, and development) between these three groups differed. The stressed plants accumulated large amounts of phenolics within their mesophyll cells. The biomass of the cold-stressed plants was minor, while it was high for the control plants. The oxidative stress was hardly detectable in the leaves of the control plants, while their heat-stressed counterparts suffered the highest concentration of reactive oxygen species. Differences concerning the absorption spectra of the three groups of leaves were not significant. An interesting incompatibility between the three groups concerned the expression of L-Dopa Decarboxylase, which climbed significantly in the heat-stressed plants. Finally, an interesting variation was observed concerning the concentrations of some biogenic amines/amino acids. This variation can be correlated to the other stress-induced reactions of the plants and, in some cases, was impressive. In conclusion, environmental stress can shift Pistacia lentiscus' metabolism to synthesize different biogenic products, which can be considered as exploitable for the pharmaceutical or food industry.

Dengue Virus Replication Is Associated with Catecholamine Biosynthesis and Metabolism in Hepatocytes.

Previously, the association between the catecholamine biosynthetic enzyme L-Dopa decarboxylase (DDC) and Dengue virus (DV) replication was demonstrated in liver cells and was found to be mediated at least by the interaction between DDC and phosphoinositide 3-kinase (PI3K). Here, we show that biogenic amines production and uptake impede DV replication in hepatocytes and monocytes, while the virus reduces catecholamine biosynthesis, metabolism, and transport. To examine how catecholamine biosynthesis/metabolism influences DV, first, we verified the role of DDC by altering DDC expression. DDC silencing enhanced virus replication, but not translation, attenuated the negative effect of DDC substrates on the virus and reduced the infection related cell death. Then, the role of the downstream steps of the catecholamine biosynthesis/metabolism was analyzed by chemical inhibition of the respective enzymes, application of their substrates and/or their products; moreover, reserpine, the inhibitor of the vesicular monoamine transporter 2 (VMAT2), was used to examine the role of uptake/storage of catecholamines on DV. Apart from the role of each enzyme/transporter, these studies revealed that the dopamine uptake, and not the dopamine-signaling, is responsible for the negative effect on DV. Accordingly, all treatments expected to enhance the accumulation of catecholamines in the cell cytosol suppressed DV replication. This was verified by the use of chemical inducers of catecholamine biosynthesis. Last, the cellular redox alterations due to catecholamine oxidation were not related with the inhibition of DV replication. In turn, DV apart from its negative impact on DDC, inhibits tyrosine hydroxylase, dopamine beta-hydroxylase, monoamine oxidase, and VMAT2 expression.

Significance of Catecholamine Biosynthetic/Metabolic Pathway in SARS-CoV-2 Infection and COVID-19 Severity.

The SARS-CoV-2 infection was previously associated with the expression of the dopamine biosynthetic enzyme L-Dopa decarboxylase (DDC). Specifically, a negative correlation was detected between DDC mRNA and SARS-CoV-2 RNA levels in in vitro infected epithelial cells and the nasopharyngeal tissue of COVID-19 patients with mild/no symptoms. However, DDC, among other genes related to both DDC expression and SARS-CoV-2-infection (ACE2, dACE2, EPO), was upregulated in these patients, possibly attributed to an orchestrated host antiviral response. Herein, by comparing DDC expression in the nasopharyngeal swab samples of severe/critical to mild COVID-19 cases, we showed a 20 mean-fold reduction, highlighting the importance of the expression of this gene as a potential marker of COVID-19 severity. Moreover, we identified an association of SARS-CoV-2 infection with the expression of key catecholamine biosynthesis/metabolism-related genes, in whole blood samples from hospitalized patients and in cultured cells. Specifically, viral infection downregulated the biosynthetic part of the dopamine pathway (reduction in DDC expression up to 7.5 mean-fold), while enhanced the catabolizing part (increase in monoamine oxidases A and B expression up to 15 and 10 mean-fold, respectively) in vivo, irrespectively of the presence of comorbidities. In accordance, dopamine levels in the sera of severe cases were reduced (up to 3.8 mean-fold). Additionally, a moderate positive correlation between DDC and MAOA mRNA levels (r = 0.527, p < 00001) in the blood was identified upon SARS-CoV-2-infection. These observations were consistent to the gene expression data from SARS-CoV-2-infected Vero E6 and A549 epithelial cells. Furthermore, L-Dopa or dopamine treatment of infected cells attenuated the virus-derived cytopathic effect by 55% and 59%, respectively. The SARS-CoV-2 mediated suppression of dopamine biosynthesis in cell culture was, at least in part, attributed to hypoxia-like conditions triggered by viral infection. These findings suggest that L-Dopa/dopamine intake may have a preventive or therapeutic value for COVID-19 patients.

Association of Hepatitis C Virus Replication with the Catecholamine Biosynthetic Pathway.

A bidirectional negative relationship between Hepatitis C virus (HCV) replication and gene expression of the catecholamine biosynthetic enzyme L-Dopa decarboxylase (DDC) was previously shown in the liver and attributed at least to an association of DDC with phosphatidylinositol 3-kinase (PI3K). Here, we report that the biosynthesis and uptake of catecholamines restrict HCV replication in hepatocytes, while HCV has developed ways to reduce catecholamine production. By employing gene silencing, chemical inhibition or induction of the catecholamine biosynthetic and metabolic enzymes and transporters, and by applying the substrates or the products of the respective enzymes, we unravel the role of the different steps of the pathway in viral infection. We also provide evidence that the effect of catecholamines on HCV is strongly related with oxidative stress that is generated by their autoxidation in the cytosol, while antioxidants or treatments that lower cytosolic catecholamine levels positively affect the virus. To counteract the effect of catecholamines, HCV, apart from the already reported effects on DDC, causes the down-regulation of tyrosine hydroxylase that encodes the rate-limiting enzyme of catecholamine biosynthesis and suppresses dopamine beta-hydroxylase mRNA and protein amounts, while increasing the catecholamine degradation enzyme monoamine oxidase. Moreover, the NS4B viral protein is implicated in the effect of HCV on the ratio of the ~50 kDa DDC monomer and a ~120 kDa DDC complex, while the NS5A protein has a negative effect on total DDC protein levels.

Alteration of L-Dopa decarboxylase expression in SARS-CoV-2 infection and its association with the interferon-inducible ACE2 isoform.

L-Dopa decarboxylase (DDC) is the most significantly co-expressed gene with ACE2, which encodes for the SARS-CoV-2 receptor angiotensin-converting enzyme 2 and the interferon-inducible truncated isoform dACE2. Our group previously showed the importance of DDC in viral infections. We hereby aimed to investigate DDC expression in COVID-19 patients and cultured SARS-CoV-2-infected cells, also in association with ACE2 and dACE2. We concurrently evaluated the expression of the viral infection- and interferon-stimulated gene ISG56 and the immune-modulatory, hypoxia-regulated gene EPO. Viral load and mRNA levels of DDC, ACE2, dACE2, ISG56 and EPO were quantified by RT-qPCR in nasopharyngeal swab samples from COVID-19 patients, showing no or mild symptoms, and from non-infected individuals. Samples from influenza-infected patients were analyzed in comparison. SARS-CoV-2-mediated effects in host gene expression were validated in cultured virus-permissive epithelial cells. We found substantially higher gene expression of DDC in COVID-19 patients (7.6-fold; p = 1.2e-13) but not in influenza-infected ones, compared to non-infected subjects. dACE2 was more elevated (2.9-fold; p = 1.02e-16) than ACE2 (1.7-fold; p = 0.0005) in SARS-CoV-2-infected individuals. ISG56 (2.5-fold; p = 3.01e-6) and EPO (2.6-fold; p = 2.1e-13) were also increased. Detected differences were not attributed to enrichment of specific cell populations in nasopharyngeal tissue. While SARS-CoV-2 virus load was positively associated with ACE2 expression (r≥0.8, p<0.001), it negatively correlated with DDC, dACE2 (r≤-0.7, p<0.001) and EPO (r≤-0.5, p<0.05). Moreover, a statistically significant correlation between DDC and dACE2 expression was observed in nasopharyngeal swab and whole blood samples of both COVID-19 and non-infected individuals (r≥0.7). In VeroE6 cells, SARS-CoV-2 negatively affected DDC, ACE2, dACE2 and EPO mRNA levels, and induced cell death, while ISG56 was enhanced at early hours post-infection. Thus, the regulation of DDC, dACE2 and EPO expression in the SARS-CoV-2-infected nasopharyngeal tissue is possibly related with an orchestrated antiviral response of the infected host as the virus suppresses these genes to favor its propagation.

Structure and organization of the secretion apparatus of the mastic tree (Pistacia lentiscus L.) and LC-HRMS analysis of leaf extracts.

MAIN CONCLUSION: The quantitative profile of the biochemicals secreted by summer and winter leaves, present noticeable differences and appear to be qualitatively different from the biochemical profile of the commercially valuable mastic. The anatomy of the root and the primary and secondary shoot as well as that of the summer and winter leaves of P. lentiscus was thoroughly investigated. The secreting network was tracked throughout the plant axis, from the root to the leaves, and the active secreting cells of the duct epithelium were localized, while the secondary metabolites produced within the cells of the summer and winter leaf tissues were identified histochemically. Numerous phytochemicals were identified in the leaf extracts with UHPLC-qTOF MS analysis. The analyzed extracts from summer and winter leaves displayed similar qualitative profile, although quantitative differences were evident, since, during the summer, the leaves tend to synthesize the more complex amongst the identified compounds. The phytochemical profile of the leaf extracts turns to be completely different compared to that of the valuable mastic harvested from the injured trunks. Many of the compounds common in mastic were not detected in the analyzed leaves samples. The numerous secreting ducts either fail to form a unified network, so composition of the secreted material varies in the different organs of the plant or they compose a continuous network, but the biochemical profile of the secreted material differs along the plant axis. Such a detailed investigation of the secretion network of the mastic tree may assist the improvement of the yield and promote the production of valuable phytochemicals through in vitro cultures.

Next-generation sequencing reveals alternative L-DOPA decarboxylase (DDC) splice variants bearing novel exons, in human hepatocellular and lung cancer cells.

The human L-DOPA decarboxylase (DDC) is an enzyme that displays a pivotal role in metabolic processes. It is implicated in various human disorders, including hepatocellular and lung cancer. Several splice variants of DDC have previously been described, most of which encode for protein isoforms of this enzyme. In the present study, we used next-generation sequencing (NGS) technology along with nested touchdown PCR and Sanger sequencing to identify new splice variants bearing novel exons of the DDC gene, in hepatocellular and lung cancer cell lines. Using an in-house-developed algorithm, we discovered seven novel DDC exons. Next, we determined the structure of ten novel DDC transcripts, three of which contain an open reading frame (ORF) and probably encode for three previously unknown protein isoforms of this enzyme. Future studies should focus on the elucidation of their role in cellular physiology and cancer pathobiology.

Revised Exon Structure of l-DOPA Decarboxylase (DDC) Reveals Novel Splice Variants Associated with Colorectal Cancer Progression.

Colorectal cancer (CRC) is a highly heterogenous malignancy with an increased mortality rate. Aberrant splicing is a typical characteristic of CRC, and several studies support the prognostic value of particular transcripts in this malignancy. l-DOPA decarboxylase (DDC) and its derivative neurotransmitters play a multifaceted role in physiological and pathological states. Our recent data support the existence of 6 DDC novel exons. In this study, we investigated the existence of additional DDC novel exons and transcripts, and their potential value as biomarkers in CRC. Next-generation sequencing (NGS) in 55 human cell lines coupled with Sanger sequencing uncovered 3 additional DDC novel exons and 20 splice variants, 7 of which likely encode new protein isoforms. Eight of these transcripts were detected in CRC. An in-house qPCR assay was developed and performed in TNM II and III CRC samples for the quantification of transcripts bearing novel exons. Extensive biostatistical analysis uncovered the prognostic value of specific DDC novel exons for patients' disease-free and overall survival. The revised DDC exon structure, the putative protein isoforms with distinct functions, and the prognostic value of novel exons highlight the pivotal role of DDC in CRC progression, indicating its potential utility as a molecular biomarker in CRC.

Human L-Dopa decarboxylase interaction with annexin V and expression during apoptosis.

l-Dopa Decarboxylase (DDC) is a pyridoxal requiring enzyme that catalyzes the decarboxylation of L-3,4-dihydroxyphenylalanine (l-Dopa) to Dopamine (DA). The function of DDC in physiological and pathological biochemical pathways remains poorly understood, while the function and regulation of human DDC isoforms is almost completely elusive. We have shown that Annexin V, a fundamental apoptosis marker, is an inhibitor of l-Dopa decarboxylase activity. Here we show the interaction of both the full-length DDC and the truncated isoform alternative DDC (Alt-DDC) with Annexin V in human tissue and cell lines. Interestingly, DDC isoform expression is enhanced or remains unaffected following staurosporine (STS) treatment, despite increased levels of cytotoxicity and apoptosis. The findings presented here provide novel insights concerning the involvement of DDC in programmed cell death.

Response of young Nerium oleander plants to long-term non-ionizing radiation.

MAIN CONCLUSION: Although exposure to low frequency electromagnetic radiation is harmful to plants, LF-EM irradiated Nerium oleander seedlings exhibited enhanced development and growth, probably taking advantage of defined structural leaf deformations. Currently, evidence supports the undesirable, often destructive impact of low frequency electromagnetic (LF-EM) radiation on plants. The response of plants to LF-EM radiation often entails induction in the biosynthesis of secondary metabolites, a subject matter that is well documented. Nerium oleander is a Mediterranean plant species, which evolved remarkable resistance to various environmental stress conditions. In the current investigation, cultivated N. oleander plants, following their long-term exposure to LF-EM radiation, exhibited major structural modifications as the flattening of crypts, the elimination of trichomes and the reduction of the layers of the epidermal cells. These changes co-existed with an oxidative stress response manifested by a significant increase in reactive oxygen species at both the roots and the above ground parts, a decline in the absorbance of light by photosynthetic pigments and the substantially increased biosynthesis of L-Dopa decarboxylase (DDC), an enzyme catalyzing the production of secondary metabolites that alleviate stress. The exposed plants exhibited greater primary plant productivity, despite a manifested photosynthetic pigment limitation and the severe oxidative stress. This unique response of N. oleander to severe abiotic stress conditions may be owed to the advantage offered by a structural change consistent to an easier diffusion of CO2 within the leaves. A major plant response to an emerging "pollutant" was documented.

Circulating miR-146a and miR-134 in predicting drug-resistant epilepsy in patients with focal impaired awareness seizures.

OBJECTIVE: Epilepsy is one of the most prevalent neurologic disorders, causing serious psychological problems and reducing quality of life. Although 20 different antiepileptic drugs (AEDs) have been approved by the US Food and Drug Administration (FDA), 30% of patients have drug-resistant epilepsy (DRE). Considering the role of miR-146a and miR-134 in neuroinflammation and dendritic functionality, respectively, the aim of this study was the clinical evaluation of circulating miR-146a and miR-134 as novel noninvasive molecular markers for the prognosis of refractory epilepsy. METHODS: The study included 162 patients with focal impaired awareness seizures. Total RNA was extracted from serum samples spiked with synthetic cel-miR-39-3p for normalization purposes. First-strand complementary DNA (cDNA) synthesis was performed using microRNA-specific stem-loop primers, and hsa-miR-134/146a levels were quantified by quantitative polymerase chain reaction (qPCR). DRE was used as clinical end point event. Internal validation was performed by bootstrap analysis, and decision curve analysis was used to evaluate the clinical benefit on disease prognosis. RESULTS: The circulating levels of both miR-134 and miR-146a were elevated in patients with drug-resistant seizures. The receiver-operating characteristic (ROC) curve and logistic regression analysis demonstrated that patients with increased circulating miR-134/146a levels are at significantly higher risk for developing DRE, independently of temporal lobe sclerosis, epilepsy duration, familial history, age at first seizure, age, body mass index (BMI), smoking behavior, and gender. Finally, decision curve analysis highlighted that the evaluation of circulating miR-134/146a led to superior clinical benefit for DRE prognosis and patients' risk stratification. SIGNIFICANCE: Elevated serum miR-134/146a levels are associated with a higher risk for AED-resistant epilepsy and could constitute novel noninvasive molecular markers to improve disease early prognosis and support precision medicine.

Emerging Role of l-Dopa Decarboxylase in Flaviviridae Virus Infections.

l-dopa decarboxylase (DDC) that catalyzes the biosynthesis of bioactive amines, such as dopamine and serotonin, is expressed in the nervous system and peripheral tissues, including the liver, where its physiological role remains unknown. Recently, we reported a physical and functional interaction of DDC with the major signaling regulator phosphoinosite-3-kinase (PI3K). Here, we provide compelling evidence for the involvement of DDC in viral infections. Studying dengue (DENV) and hepatitis C (HCV) virus infection in hepatocytes and HCV replication in liver samples of infected patients, we observed a negative association between DDC and viral replication. Specifically, replication of both viruses reduced the levels of DDC mRNA and the ~120 kDa SDS-resistant DDC immunoreactive functional complex, concomitant with a PI3K-dependent accumulation of the ~50 kDa DDC monomer. Moreover, viral infection inhibited PI3K-DDC association, while DDC did not colocalize with viral replication sites. DDC overexpression suppressed DENV and HCV RNA replication, while DDC enzymatic inhibition enhanced viral replication and infectivity and affected DENV-induced cell death. Consistently, we observed an inverse correlation between DDC mRNA and HCV RNA levels in liver biopsies from chronically infected patients. These data reveal a novel relationship between DDC and Flaviviridae replication cycle and the role of PI3K in this process.

Identification of novel alternative splice variants of the human L-DOPA decarboxylase (DDC) gene in human cancer cells, using high-throughput sequencing approaches.

The human L-DOPA decarboxylase (DDC) is a gene that has been in the center of research attention in many laboratories the last decades, due to its major implication in various disorders, including many types of cancer. In the current work, we used in-house developed RACE and high-throughput sequencing approaches, in order to detect and identify novel DDC transcripts. Bioinformatic analysis revealed new alternative splicing events that support the existence of novel DDC transcripts. As a result, a total of 14 DDC splice variants were identified and their expression profile was investigated in a wide panel of human cancer cell lines. From all 14 novel DDC transcripts that were identified, 9 transcripts are predicted to encode new protein isoforms, while the remaining 5 are nonsense-mediated mRNA decay (NMD) candidates. Our results demonstrate that the human DDC gene undergoes complex processing leading to the figuration of multiple mRNA isoforms in cancer cells.

Mobile phone electromagnetic radiation affects Amyloid Precursor Protein and α-synuclein metabolism in SH-SY5Y cells.

In this study, the effects of low-level, GSM emitted ElectroMagnetic Field (EMF) on Amyloid Precursor Protein (APP) and alpha-synuclein (α-syn) in human neuroblastoma cells was investigated. Our data indicated alterations on APP processing and cellular topology, following EMF exposure (ℇ = 10.51 V/m, SAR = 0.23 W/kg, exposure time: 3 times, for 10 min, for 2 days). Furthermore, changes in monomeric α-syn accumulation and multimerization, as well as induction of oxidative stress and cell death, were documented. The results presented here require further investigation to determine potential links of EMF with the molecular pathogenic mechanisms in Alzheimer's and Parkinson's Diseases.

L-Dopa decarboxylase interaction with the major signaling regulator ΡΙ3Κ in tissues and cells of neural and peripheral origin.

L-Dopa decarboxylase (DDC) catalyzes the decarboxylation of L-Dopa to dopamine and 5-hydroxytryptophan (5-HTP) to serotonin. Although DDC has been purified from a variety of peripheral organs, including the liver, kidney and pancreas, the physiological significance of the peripherally expressed enzyme is not yet fully understood. DDC has been considered as a potential novel biomarker for various types of cancer, however, the role of DDC in the development of hepatocellular carcinoma (HCC) remains to be evaluated. Phosphatidylinositol 3-kinase (PI3K), on the other hand, has been shown to play a key role in the tumorigenesis, proliferation, metastasis, apoptosis, and angiogenesis of HCC by regulating gene expression. We initially identified the interaction of DDC with PI3K by means of the phage display methodology. This association was further confirmed in human hepatocellular carcinoma cell lines, human embryonic kidney cells, human neuroblastoma cells, as well as mouse brain, by the use of specific antibodies raised against DDC and PI3K. Functional aspects of the above interaction were studied upon treatment with the DDC inhibitor carbidopa and the PI3K inhibitor LY294002. Interestingly, our data demonstrate the expression of the neuronal type DDC mRNA in HCC cells. The present investigation provides new evidence on the possible link of DDC with the PI3K pathway, underlining the biological significance of this complex enzyme.

The Role of Tissue Oxygen Tension in Dengue Virus Replication.

Low oxygen tension exerts a profound effect on the replication of several DNA and RNA viruses. In vitro propagation of Dengue virus (DENV) has been conventionally studied under atmospheric oxygen levels despite that in vivo, the tissue microenvironment is hypoxic. Here, we compared the efficiency of DENV replication in liver cells, monocytes, and epithelial cells under hypoxic and normoxic conditions, investigated the ability of DENV to induce a hypoxia response and metabolic reprogramming and determined the underlying molecular mechanism. In DENV-infected cells, hypoxia had no effect on virus entry and RNA translation, but enhanced RNA replication. Overexpression and silencing approaches as well as chemical inhibition and energy substrate exchanging experiments showed that hypoxia-mediated enhancement of DENV replication depends on the activation of the key metabolic regulators hypoxia-inducible factors 1α/2α (HIF-1α/2α) and the serine/threonine kinase AKT. Enhanced RNA replication correlates directly with an increase in anaerobic glycolysis producing elevated ATP levels. Additionally, DENV activates HIF and anaerobic glycolysis markers. Finally, reactive oxygen species were shown to contribute, at least in part through HIF, both to the hypoxia-mediated increase of DENV replication and to virus-induced hypoxic reprogramming. These suggest that DENV manipulates hypoxia response and oxygen-dependent metabolic reprogramming for efficient viral replication.