Optimization of Zebrafish Larvae 6-OHDA Exposure for Neurotoxin Induced Dopaminergic Marker Reduction.

Parkinson's disease (PD) is a neurodegenerative disorder that is clinically assessed by motor symptoms associated with the loss of midbrain dopaminergic neurons affecting the quality of life for over 8.5 million people worldwide. The neurotoxin 6-hydroxydopamine (6-OHDA) has been used to chemically induce a PD-like state in zebrafish larvae by several laboratories; however, highly variable concentration, methodology, and reagents have resulted in conflicting results suggesting a need to investigate these issues of reproducibility. We propose a protocol that addresses the differences in methodology and induces changes in 6 days postfertilization (dpf) larvae utilizing a 24-h exposure at 3 dpf with 30 µM 6-OHDA. Despite ∼50% lethality, no morphological or development differences in surviving fish are observed. Definition of our model is defined by downregulation of the expression of th1 by reverse transcriptase-quantitative polymerase chain reaction, a marker for dopaminergic neurons and a reduction in movement. Additionally, we observed a downregulation of pink1 and an upregulation of sod1 and sod2, indicators of mitochondrial dysfunction and response to reactive oxygen species, respectively.

Exposure of Mice to Thirdhand Smoke Modulates In Vitro and In Vivo Platelet Responses.

Smoking is a risk factor for a variety of deleterious conditions, such as cancer, respiratory disease and cardiovascular disease. Thrombosis is an important and common aspect of several cardiovascular disease states, whose risk is known to be increased by both first- and secondhand smoke. More recently, the residual cigarette smoke that persists after someone has smoked (referred to as thirdhand smoke or THS) has been gaining more attention, since it has been shown that it also negatively affects health. Indeed, we have previously shown that 6-month exposure to THS increases the risk of thrombogenesis. However, neither the time-dependence of THS-induced thrombus formation, nor its sex dependence have been investigated. Thus, in the present study, we investigated these issues in the context of a shorter exposure to THS, specifically 3 months, in male and female mice. We show that the platelets from 3-month THS-exposed mice exhibited enhanced activation by agonists. Moreover, we also show that mice of both sexes exposed to THS have decreased tail bleeding as well as decreased thrombus occlusion time. In terms of the role of sex, intersex disparities in thrombus development and hemostasis as well as in platelet aggregation were, interestingly, observed. Together, our findings show that exposing mice to THS for 3 months is sufficient to predispose them to thrombosis; which seems to be driven, at least in part, by an increased activity in platelets, and that it does not manifest equally in both sexes.

The Antidepressant Duloxetine Inhibits Platelet Function and Protects against Thrombosis.

While cardiovascular disease (CVD) is the leading cause of death, major depressive disorder (MDD) is the primary cause of disability, affecting more than 300 million people worldwide. Interestingly, there is evidence that CVD is more prevalent in people with MDD. It is well established that neurotransmitters, namely serotonin and norepinephrine, are involved in the biochemical mechanisms of MDD, and consequently, drugs targeting serotonin-norepinephrine reuptake, such as duloxetine, are commonly prescribed for MDD. In this connection, serotonin and norepinephrine are also known to play critical roles in primary hemostasis. Based on these considerations, we investigated if duloxetine can be repurposed as an antiplatelet medication. Our results-using human and/or mouse platelets show that duloxetine dose-dependently inhibited agonist-induced platelet aggregation, compared to the vehicle control. Furthermore, it also blocked agonist-induced dense and α-granule secretion, integrin αIIbß3 activation, phosphatidylserine expression, and clot retraction. Moreover duloxetine-treated mice had a significantly prolonged occlusion time. Finally, duloxetine was also found to impair hemostasis. Collectively, our data indicate that the antidepressant duloxetine, which is a serotonin-norepinephrine antagonist, exerts antiplatelet and thromboprotective effects and inhibits hemostasis. Consequently, duloxetine, or a rationally designed derivative, presents potential benefits in the context of CVD, including that associated with MDD.

Analysis of the Zn-Binding Domains of TRIM32, the E3 Ubiquitin Ligase Mutated in Limb Girdle Muscular Dystrophy 2H.

Members of the tripartite motif family of E3 ubiquitin ligases are characterized by the presence of a conserved N-terminal module composed of a RING domain followed by one or two B-box domains, a coiled-coil and a variable C-terminal region. The RING and B-box are both Zn-binding domains but, while the RING is found in a large number of proteins, the B-box is exclusive to the tripartite motif (TRIM) family members in metazoans. Whereas the RING has been extensively characterized and shown to possess intrinsic E3 ligase catalytic activity, much less is known about the role of the B-box domains. In this study, we adopted an in vitro approach using recombinant point- and deletion-mutants to characterize the contribution of the TRIM32 Zn-binding domains to the activity of this E3 ligase that is altered in a genetic form of muscular dystrophy. We found that the RING domain is crucial for E3 ligase activity and E2 specificity, whereas a complete B-box domain is involved in chain assembly rate modulation. Further, in vitro, the RING domain is necessary to modulate TRIM32 oligomerization, whereas, in cells, both the RING and B-box cooperate to specify TRIM32 subcellular localization, which if altered may impact the pathogenesis of diseases.

Identification of circulating protein biomarkers in patients with hepatocellular carcinoma concomitantly infected with chronic hepatitis C virus.

CONTEXT: The incidence rate of hepatocellular carcinoma (HCC) is higher in developing countries, and most cases are associated with chronic hepatitis C virus (HCV) infection. OBJECTIVE: To evaluate the circulating proteins as liver biomarkers for the identification of HCC associated with HCV infection in Egyptian patients using LC-MS/MS analysis. METHODS: Blood sera were collected from 31 HCC patients and the fractionated proteins were subjected to LC-MS/MS analysis. Protein candidates were validated by enzyme-linked immunosorbent assay (ELISA). RESULTS: Thirty-three proteins were significantly identified in the sera of HCC patients with persistent HCV infection. These proteins are involved in several biological processes including acute phase response, complement activation, hemostasis process and lipid metabolism. The level of lectin galactoside-binding soluble 3 binding protein (LGALS3BP), Kininogen-1 (KNG1), serum amyloid A2 (SAA2) and paraoxonase 1 (PON1) and alpha-fetoprtoein (AFP) were elevated in serum. CONCLUSION: In HCC patients with chronic HCV infection, we identified a group of differentially expressed circulating proteins involved in regulating different cellular mechanisms.

Inflammatory and Non-inflammatory Breast Cancer: A Potential Role for Detection of Multiple Viral DNAs in Disease Progression.

BACKGROUND: Inflammatory breast cancer (IBC) is the most lethal form of breast cancer. Multiple viral infections in IBC tissues were found to be associated with disease pathogenesis. OBJECTIVE: The aim of the present study was to correlate the incidence of viral DNA with breast cancer progression. MATERIALS AND METHODS: Overall, 135 women diagnosed with breast cancer were enrolled in this study. Using polymerase chain reaction and sequencing assays, we determined the incidence of human papillomavirus types 16 and 18 (HPV-16 and -18), human cytomegalovirus (HCMV), Epstein-Barr virus, human herpes simplex virus type 1 and 2, and human herpes virus type 8 (HHV-8) in breast carcinoma tissue biopsies. We also assessed the expression of the cell proliferation marker Ki-67 by immunohistochemistry in association with the incidence of viral DNA. RESULTS: HCMV and HPV-16 were the most detected viral DNAs in breast carcinoma tissues; however, the frequency of HCMV and HHV-8 DNA were significantly higher in IBC than non-IBC tissues. Moreover, the prevalence of multiple viral DNAs was higher in IBC than non-IBC tissues. The incidence of multiple viral DNAs positively correlates with tumor size and number of metastatic lymph nodes in both non-IBC and IBC patients. The expression of Ki-67 was found to be significantly higher in both non-IBC and IBC tissues in which multiple viral DNAs were detected. CONCLUSIONS: The incidence of multiple viral DNAs in IBC tissues was higher compared with non-IBC tissues. The present results suggest the possibility of a functional relationship between the presence of multiple viral DNAs and disease pathogenesis.

Investigating the pretreatment miRNA expression patterns of advanced hepatocellular carcinoma patients in association with response to TACE treatment.

Hepatocellular carcinoma (HCC) is a lethal malignancy with poor prognosis and limited treatment options. Transarterial chemoembolization (TACE) using chemotherapy agents-doxorubicin and cisplatin-is an accepted treatment option for locally advanced hepatocellular carcinoma. In the current study, we analyzed the expression pattern of a selected panel of 94 miRNAs in archival samples that were collected prior to treatment from 15 Egyptian patients diagnosed with advanced hepatocelleular carcinoma. We observed an overall increase in miRNA expression in HCC samples compared with normal subjects. Out of 94 examined miRNAs, 53 were significantly upregulated while 3 miRNAs were downregulated in HCC samples compared to normal liver samples. Comparing the pretreatment miRNA expression profiles in HCC patients and the patients response to TACE treatment resulted in the identification of a set of 12 miRNAs that are significantly upregulated in nonresponders group. This miRNA panel includes miR-10a-1, miR-23a-1, miR-24, miR-26a, miR-27a, miR-30c, miR-30e, miR-106b, miR-133b, miR-199a, miR-199-3p, and miR-200b. Furthermore, we observed that a panel of 10 miRNAs was significantly associated with patients' survival status at 1 year. These results highlight the potential implications of pretreatment miRNAs expression profiling in prediction of the patients' response to TACE treatment in liver cancer.

Reevaluation of the predicted gene structure of Dictyostelium cystatin A3 (cpiC) by nucleotide sequence determination of its cDNA* and its phylogenetic position in the cystatin superfamily.

Cystatins, cysteine protease inhibitors, are widely distributed among eukaryotes. We reevaluated the structure of the gene cpiC, a gene encoding the third identified member of cystatin family (cystatin A3) that was predicted in the genome database of the social amoeba Dictyostelium discoidium (dictyBase) but remained controversial. We determined the sequences of cDNA and PCR-amplified genomic DNA fragment and found a critical error in the registered nucleotide sequence. The corrected cystatin A3 gene has an open reading frame (ORF) without intron sequence interruption and encodes 94 amino acids (aa), in contrast to the previously predicted sequence of either 80, 82 or 118 aa. The cDNA has an unusual internal poly(A) sequence of 31 adenines, which immediately follows the translation termination codon (TAA) located 146 nucleotides upstream of the post-transcriptional polyadenylation site. The amino acid sequence of Dictyostelium cystatin A3 shows a high similarity to those of previously reported Dictyostelium cystatins as well as Family I cystatins of higher eukaryotes.

DdAlix, an Alix/AIP1 homolog in Dictyostelium discoideum, is required for multicellular development under low Ca2+ conditions.

Apoptosis-linked gene 2 (ALG-2) interacting protein X (Alix), also called AIP1, is a widely conserved protein in eukaryotes. Alix and its homologs are involved in various phenomena such as apoptosis, regulation of cell adhesion, protein sorting, adaptation to stress conditions, and budding of human immunodeficiency virus (HIV). To investigate the role of Alix in development, we identified an Alix homolog in the cellular slime mold Dictyostelium discoideum and disrupted the gene by homologous recombination. The growth of DdAlix deletion mutant (alx-) cells was significantly impaired in the presence of 5 mM Li+. On an agar plate, alx- cells underwent normal development and formed fruiting bodies indistinguishable from those formed by wild-type cells. However, alx- cells could not form fruiting bodies in the presence of 5 mM Li+. Similar results were obtained when cells were developed in the presence of 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), which is an antagonist of intracellular Ca2+ store. Furthermore, when the extracellular free Ca2+ was reduced to 10 nM, the ability of alx- cells, but not that of wild-type cells, to form fruiting bodies was impaired. The results indicate that DdAlix is essential for development under low Ca2+ conditions and suggest that DdAlix is involved in Ca2+ signaling during development.

Identification of cysteine protease inhibitors that belong to cystatin family 1 in the cellular slime mold Dictyostelium discoideum.

Family 1 cystatins are cytosolic inhibitors of cysteine proteases, and they are conserved in higher eukaryotes. We characterized two newly identified family 1 cystatins of the cellular slime mold Dictyostelium discoideum, cystatin A1 and A2. Their recombinant proteins showed specific inhibitory activity against papain and cathepsin B, respectively. Using specific polyclonal antibodies, we found that cystatin A1 is stably expressed throughout the life cycle of Dictyostelium, whereas cystatin A2 expression is up-regulated during the course of development.