Transcriptional induction of TGF-ß1 and endothelial-to-mesenchymal transition cell markers in human umbilical vein endothelial cells by Ebola virus infection.

BACKGROUND: Ebola virus (EBOV) causes a serious hemorrhagic disease in humans, with a mortality rate of up to 80%. Despite significant achievements in the past decades elucidating the pathogenesis of EBOV, there is still much to be elucidated about the cell type-specific host response and their functional roles during infection. OBJECTIVE: This study aimed to gain insight into cell type-specific host responses to EBOV infection. METHODS: Real-time RT-qPCR analysis was used to identify host transcriptional changes in epithelial Caco-2 cells and endothelial HUVECs by EBOV infection. RESULTS: EBOV efficiently infected to both Caco-2 cells and HUVECs, depending on the time of infection. However, changes in the transcriptional levels of several host cellular genes following viral infection showed significant differences between Caco-2 cells and HUVECs. EBOV infection increases the transcription of TGF-ß1, a key factor in epithelium-to-mesenchyme transition (EMT), only in HUVECs, but not in Caco-2 cells. This upregulation in turn induces the transcription of other EMT signaling molecules such as snail, slug and MMP9, ultimately leading to endothelial-to-mesenchymal transition (EndMT). Furthermore, this EndMT process appears to be associated with increased transcription of stem-cell markers such as Klf4, Sox2 and Oct4. However, most of these transcriptional changes due to EBOV infection did not occur in Caco-2 cells, suggesting that EMT or EndMT by EBOV infection is cell type-specific. CONCLUSION: We propose that EBOV infection induces the expression of TGF-ß1-mediated signals in endothelial HUVECs, resulting in EndMT. This could provide broader information to elucidate the pathogenesis of Ebola virus disease.

Nucleoporin 210 Serves a Key Scaffold for SMARCB1 in Liver Cancer.

The roles of chromatin remodelers and their underlying mechanisms of action in cancer remain unclear. In this study, SMARCB1, known initially as a bona fide tumor suppressor gene, was investigated in liver cancer. SMARCB1 was highly upregulated in patients with liver cancer and was associated with poor prognosis. Loss- and gain-of-function studies in liver cells revealed that SMARCB1 loss led to reduced cell proliferation, wound healing capacity, and tumor growth in vivo. Although upregulated SMARCB1 appeared to contribute to switch/sucrose nonfermentable (SWI/SNF) complex stability and integrity, it did not act using its known pathways antagonism with EZH2 or association between TP53 or AMPK. SMARCB1 knockdown induced a mild reduction in global H3K27 acetylation, and chromatin immunoprecipitation sequencing of SMARCB1 and acetylated histone H3K27 antibodies before and after SMARCB1 loss identified Nucleoporin210 (NUP210) as a critical target of SMARCB1, which bound its enhancer and changed H3K27Ac enrichment and downstream gene expression, particularly cholesterol homeostasis and xenobiotic metabolism. Notably, NUP210 was not only a putative tumor supporter involved in liver cancer but also acted as a key scaffold for SMARCB1 and P300 to chromatin. Furthermore, SMARCB1 deficiency conferred sensitivity to doxorubicin and P300 inhibitor in liver cancer cells. These findings provide insights into mechanisms underlying dysregulation of chromatin remodelers and show novel associations between nucleoporins and chromatin remodelers in cancer. SIGNIFICANCE: This study reveals a novel protumorigenic role for SMARCB1 and describes valuable links between nucleoporins and chromatin remodelers in cancer by identifying NUP210 as a critical coregulator of SMARCB1 chromatin remodeling activity.

Heterogeneity in liver histopathology is associated with GSK-3ß activity and mitochondrial dysfunction in end-stage diabetic rats on differential diets.

While liver histopathology is heterogeneous in diabetes, the underlying mechanisms remain unclear. We investigated whether glycemic variation resulting from differential diets can induce heterogeneity in diabetic liver and the underlying molecular mechanisms. We generated end-stage non-obese diabetic model rats by subtotal-pancreatectomy in male Sprague- Dawley rats and ad libitum diet for 7 weeks (n = 33). The rats were then divided into three groups, and fed a standard- or a low-protein diet (18 or 6 kcal%, respectively), for another 7 weeks: to maintain hyperglycemia, 11 rats were fed ad libitum (18AL group); to achieve euglycemia, 11 were calorierestricted (18R group), and 11 were both calorie- and proteinrestricted with the low-protein diet (6R group). Overnightfasted liver samples were collected after the differential diets together with sham-control (18S group), and histology and molecular changes were compared. Hyperglycemic-18AL showed glycogenic hepatopathy (GH) without steatosis, with the highest GSK-3ß inactivation because of Akt activation during hyperglycemia; mitochondrial function was not impaired, compared to the 18S group. Euglycemic-18R showed neither GH nor steatosis, with intermediate GSK-3ß activation and mitochondrial dysfunction. However, euglycemic-6R showed both GH and steatosis despite the highest GSK-3ß activity and no molecular evidence of increased lipogenesis or decreased ApoB expression, where mitochondrial dysfunction was highest among the groups. In conclusion, heterogeneous liver histopathology developed in end-stage non-obese diabetic rats as the glycemic levels varied with differential diets, in which protein content in the diets as well as glycemic levels differentially influenced GSK-3ß activity and mitochondrial function in insulin-deficient state. [BMB Reports 2020; 53(2): 100-105].

Salmonella­induced miR­155 enhances necroptotic death in macrophage cells via targeting RIP1/3.

Salmonella enterica serovar Typhimurium (hereafter referred to as Salmonella), a virulent pathogen, is known to induce host­cell death. Using reverse transcription­quantitative polymerase chain reaction, a 28­fold increase of microRNA (miR)­155 expression in RAW 264.7 macrophages was observed following infection with Salmonella for 24 h. This miR­155 upregulation increased macrophage cell death by up to 40% in 48 h following infection. Western blot analysis revealed that receptor interacting protein 1 (RIP1) and 3 (RIP3) were increased at 18 h following miR­155 transfection to macrophages, similar to Salmonella infection. In addition, inhibition of RIP1 by pre­incubating macrophages with necrostatin­1, a RIP1 specific inhibitor, increased the viability of Salmonella­infected cells and miR­155­transfected cells by up to 20%. The cleavage of poly (adenosine diphosphate­ribose) polymerase­1 (PARP­1) was also enhanced by miR­155 induction upon Salmonella infection. Therefore, it was suggested that RIP1/3­induced necroptosis and PARP­1­mediated necrosis caused by miR­155 induction may represent distinct routes of programmed necrotic cell death of Salmonella­infected macrophages.

Rapid detection and quantification of Ebola Zaire virus by one-step real-time quantitative reverse transcription-polymerase chain reaction.

Given that Ebola virus causes severe hemorrhagic fever in humans with mortality rates as high as 90%, rapid and accurate detection of this virus is essential both for controlling infection and preventing further transmission. Here, a one-step qRT-PCR assay for rapid and quantitative detection of an Ebola Zaire strain using GP, VP24 or VP40 genes as a target is introduced. Routine assay conditions for hydrolysis probe detection were established from the manufacturer's protocol used in the assays. The analytical specificity and sensitivity of each assay was evaluated using in vitro synthesized viral RNA transcripts. The assays were highly specific for the RNA transcripts, no cross-reactivity being observed among them. The limits of detection of the assays ranged from 102 to 103 copies per reaction. The assays were also evaluated using viral RNAs extracted from cell culture-propagated viruses (Ebola Zaire, Sudan and Reston strains), confirming that they are gene- and strain-specific. The RT-PCR assays detected viral RNAs in blood samples from virus-infected animal, suggesting that they can be also a useful method for identifying Ebola virus in clinical samples.

Characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis isolates from Korea and analysis of the correlation between the mutations and pyrazinamidase activity.

To investigate the effect of natural pyrazinamidase (PncA) mutations on protein function, we analyzed expression and PncA activity of eight pncA point mutants identified in nineteen pyrazinamide-resistant Mycobacterium tuberculosis clinical isolates. Among them, two mutants (Y99D and T135P) showed high expression level and solubility comparable to those of the wild-type PncA protein, two (K48E and G97D) displayed low expression level and solubility, and four (C14R, H51P, W68S, and A146V) were insoluble. Interestingly, when possible structural effects of these mutations were predicted by the CUPSAT program based on the proposed three-dimensional structure of M. tuberculosis PncA, only two highly soluble mutant proteins (Y99D and T135P) were predicted to be stabilizing and have favorable torsion angles. However, the others exhibiting either low solubility or precipitation were foreseen to be destabilizing and/or have unfavorable torsion angles, suggesting that the alterations could interfere with proper protein folding, thereby decreasing or depleting protein solubility. A PncA activity assay demonstrated that two mutants (G97D and T135P) showed virtually no activity, but two other mutants (K48E and Y99D) exhibited wild-type activity, indicating that the PncA residues (Cys14, His51, Trp68, Gly97, Thr135, and Ala146) may be important for PncA activity and/or proper protein folding.

Simple and rapid discrimination of embB codon 306 mutations in Mycobacterium tuberculosis clinical isolates by a real-time PCR assay using an LNA-TaqMan probe.

Single nucleotide polymorphisms in the codon 306 of embB gene are most frequently reported in ethambutol-resistant Mycobacterium tuberculosis clinical isolates. Here, we report a simple and rapid real-time PCR assay using a locked nucleic acid (LNA)-TaqMan probe for discriminating the embB306 mutations. The use of a 15-bp chimeric LNA/DNA probe led to a relatively higher level of sensitivity and fluorescence signal in the wild-type embB306 ATG codon. Therefore, the mutant alleles were easily distinguishable from the wild-type allele by their distinctive amplification curve shapes without a melting analysis of the PCR product. This system was fast and less than 0.1 pg of genomic DNA per reaction was needed for detection. Because the results from this real-time assay were absolutely consistent with those from DNA sequencing, it can be effectively applied as a simple and rapid method for primary screening of embB306 mutations that occur frequently in ethambutol-resistant and/or multidrug-resistant M. tuberculosis isolates.

Molecular characterization of drug-resistant and -susceptible Mycobacterium tuberculosis isolated from patients with tuberculosis in Korea.

We investigated the causal relationship between genotype and phenotype of drug-resistant Mycobacterium tuberculosis isolates obtained from patients with pulmonary tuberculosis (TB) in Korea. Of 80 isolates tested, 17, 20, 1, and 7 isolates were mono-resistant to ethambutol (EMB), isoniazid (INH), pyrazinamide (PZA), and rifampicin (RFP), respectively, and 31 isolates (38.8%) were multidrug-resistant (MDR). Sequencing analysis showed that 78% (32/41) of RFP-resistant strains had mutations in the rifampicin resistance-determining region (RRDR) of rpoB, and the mutation at rpoB531 (59.4%) was most abundant. In 52 INH-resistant strains, mutations were found mostly at C-15T (n = 21, 40.4%) in the inhA promoter region as well as at katG315 (n = 12, 23.1%). Mutations at embB306 were mostly found in 26.7% (12/45) of EMB-resistant isolates. New mutations found here in MDR isolates include rpoB523 (Gly523Glu) and embB319 (Tyr319Ser). Consequently, mutations in the rpoB531, C-15T in the inhA promoter region, embB306, and katG315 would be a useful marker for rapid detection of MDR M. tuberculosis isolates in Korea.

A small nonhuman primate model for filovirus-induced disease.

Ebolavirus and Marburgvirus are members of the filovirus family and induce a fatal hemorrhagic disease in humans and nonhuman primates with 90% case fatality. To develop a small nonhuman primate model for filovirus disease, common marmosets (Callithrix jacchus) were intramuscularly inoculated with wild type Marburgvirus Musoke or Ebolavirus Zaire. The infection resulted in a systemic fatal disease with clinical and morphological features closely resembling human infection. Animals experienced weight loss, fever, high virus titers in tissue, thrombocytopenia, neutrophilia, high liver transaminases and phosphatases and disseminated intravascular coagulation. Evidence of a severe disseminated viral infection characterized principally by multifocal to coalescing hepatic necrosis was seen in EBOV animals. MARV-infected animals displayed only moderate fibrin deposition in the spleen. Lymphoid necrosis and lymphocytic depletion observed in spleen. These findings provide support for the use of the common marmoset as a small nonhuman primate model for filovirus induced hemorrhagic fever.

Molecular cloning, purification, and characterization of a superoxide dismutase from a fast-growing Mycobacterium sp. Strain JC1 DSM 3803.

A cytosolic superoxide dismutase (SOD) was purified and characterized from a fast-growing Mycobacterium sp. strain JC1 DSM 3803 grown on methanol. The native molecular weight of the purified SOD was estimated to be 48 kDa. SDS-PAGE revealed a subunit of 23 kDa, indicating that the enzyme is a homodimer. The enzyme activity was inhibited by H(2)O(2) and azide. The purified SOD contained 1.12 and 0.56 g-atom of Mn and Fe per mol of enzyme, respectively, suggesting that it may be a Fe/Mn cambialistic SOD. The apo-SOD reconstitution study revealed that Mn salts were more specific than Fe salts in the SOD activity. The gene encoding the SOD was identified from the JC1 cosmid genomic library by PCR screening protocol. The cloned gene, sodA, had an open reading frame (ORF) of 624 nt, encoding a protein with a calculated molecular weight of 22,930 Da and pi of 5.33. The deduced SodA sequence exhibited 97.6% identity with that of Mycobacterium fortuitum Mn-SOD and clustered with other mycobacterial Mn-SODs. A webtool analysis on the basis of SOD sequence and structure homologies predicted the SOD as a tetrameric Mn-SOD, suggesting that the protein is a dimeric Mn-SOD having tetramer-specific sequence and structure characteristics.

Akt regulates the expression of MafK, synaptotagmin I, and syntenin-1, which play roles in neuronal function.

BACKGROUND: Akt regulates various cellular processes, including cell growth, survival, and metabolism. Recently, Akt's role in neurite outgrowth has also emerged. We thus aimed to identify neuronal function-related genes that are regulated by Akt. METHODS: We performed suppression subtractive hybridization on two previously established PC12 sublines, one of which overexpresses the wild-type (WT) form and the other, the dominant-negative (DN) form of Akt. These sublines respond differently to NGF's neuronal differentiation effect. RESULTS: A variety of genes was identified and could be classified into several functional groups, one of which was developmental processes. Two genes involved in neuronal differentiation and function were found in this group. v-Maf musculoaponeurotic fibrosarcoma oncogene homolog K (MafK) induces the neuronal differentiation of PC12 cells and immature telencephalon neurons, and synaptotagmin I (SytI) is essential for neurotransmitter release. Another gene, syntenin-1 (Syn-1) was also recognized in the same functional group into which MafK and SytI were classified. Syn-1 has been reported to promote the formation of membrane varicosities in neurons. Quantitative reverse transcription polymerase chain reaction analyses show that the transcript levels of these three genes were lower in PC12 (WT-Akt) cells than in parental PC12 and PC12 (DN-Akt) cells. Furthermore, treatment of PC12 (WT-Akt) cells with an Akt inhibitor resulted in the increase of the expression of these genes and the improvement of neurite outgrowth. These results indicate that dominant-negative or pharmacological inhibition of Akt increases the expression of MafK, SytI, and Syn-1 genes. Using lentiviral shRNA to knock down endogenous Syn-1 expression, we demonstrated that Syn-1 promotes an increase in the numbers of neurites and branches. CONCLUSIONS: Taken together, these results indicate that Akt negatively regulates the expression of MafK, SytI, and Syn-1 genes that all participate in regulating neuronal integrity in some way or another.

Cloning, expression and characterization of the catalase-peroxidase (KatG) gene from a fast-growing Mycobacterium sp. strain JC1 DSM 3803.

The gene encoding a catalase-peroxidase (KatG) was cloned from chromosomal DNA of a fast-growing Mycobacterium sp. strain JC1 DSM 3803. The nucleotide sequence of a 5.7 kb EcoRI fragment containing the katG and its flanking regions was determined. The fragment (5,706 bps) contained two complete open reading frames (ORFs) encoding putative ferric uptake regulator A (FurA) and KatG proteins. The cloned gene, katG, had an ORF of 2241 nt, encoding a protein with calculated molecular mass of 81,748 Da. The furA was located in the upstream of the katG with the same transcriptional direction and there was a 38 bp gap space between them. The deduced KatG and FurA protein sequences showed significant homologies to KatG2 and Fur2 of Mycobacterium smegmatis and clustered with other mycobacterial KatG and Fur-like proteins in phylogenetic trees, respectively. The recombinant KatG overproduced in Escherichia coli was nearly indistinguishable from the native JC1 catalase-peroxidase in enzymatic properties and also possessed the resistance to organic solvents, indicating that the cloned katG truly encodes the Mycobacterium sp. JC1 catalase-peroxidase. Difference spectroscopy revealed Mn(II) binding near the haem of the KatG. Transcript analysis of the furA-katG using RT-PCR suggests that the katG is independently transcribed from the furA.

The ginsenoside-Rb2 lowers cholesterol and triacylglycerol levels in 3T3-L1 adipocytes cultured under high cholesterol or fatty acids conditions.

The effects of the ginsenoside Rb2 (Rb2) on lipid metabolism were characterized in 3T3-L1 adipocytes to evaluate their utility for treating obesity. While the amounts of total cholesterol and triacylglycerol (TAG) were markedly increased in the adipocytes treated with high amounts of cholesterol and fetal bovine serum (FBS), the test groups treated with Rb2 showed levels that were close to normal. The effect of Rb2 on these cells was comparable to that of lovastatin. Rb2 enhanced the expression of the sterol regulated element binding protein (SREBP) mRNA whereas treatment with cholesterol and FBS led to a reduction in the abundance of this transcript. The activity of fatty acid synthetase (FAS) was lower in the cholesterol group compared to the Rb2 treatment group suggesting that the observed decrease in cholesterol levels and activated SREBP was mediated by Rb2. Treatment with Rb2 also resulted in a decrease in TAG levels in adipocytes cultured under high fatty acid conditions. This effect was mediated by stimulating the expression of SREBP and Leptin mRNA, suggesting that Rb2 might be a valuable component capable of lowering the levels of lipids.

Protection against lethal challenge by Ebola virus-like particles produced in insect cells.

Ebola virus-like particles (VLPs) were produced in insect cells using a recombinant baculovirus expression system and their efficacy for protection against Ebola virus infection was investigated. Two immunizations with 50 microg Ebola VLPs (high dose) induced a high level of antibodies against Ebola GP that exhibited strong neutralizing activity against GP-mediated virus infection and conferred complete protection of vaccinated mice against lethal challenge by a high dose of mouse-adapted Ebola virus. In contrast, two immunizations with 10 microg Ebola VLPs (low dose) induced 5-fold lower levels of antibodies against GP and these mice were not protected against lethal Ebola virus challenge, similar to control mice that were immunized with 50 microg SIV Gag VLPs. However, the antibody responses against GP were boosted significantly after a third immunization with 10 microg Ebola VLPs to similar levels as those induced by two immunizations with 50 microg Ebola VLPs, and vaccinated mice were also effectively protected against lethal Ebola virus challenge. Furthermore, serum viremia levels in protected mice were either below the level of detection or significantly lower compared to the viremia levels in control mice. These results show that effective protection can be achieved by immunization with Ebola VLPs produced in insect cells, which give high production yields, and lend further support to their development as an effective vaccine strategy against Ebola virus.

Presence of an inducible semicarbazide-sensitive amine oxidase in Mycobacterium sp. Strain JC1 DSM 3803 grown on benzylamine.

Mycobacterium sp. strain JC1 was capable of growth on benzylamine as a sole source of carbon and energy. The primary deamination of benzylamine was mediated by an inducible amine oxidase, which can also oxidize tyramine, histamine, and dopamine. Inhibitor study identified this enzyme as a copper-containing amine oxidase sensitive to semicarbazide.

Purification, identification, and biochemical characterization of a host-encoded cysteine protease that cleaves a leishmaniavirus gag-pol polyprotein.

Leishmania RNA virus (LRV) is a double-stranded RNA virus that infects some strains of the protozoan parasite leishmania As with other totiviruses, LRV presumably expresses its polymerase by a ribosomal frameshift, resulting in a capsid-polymerase fusion protein. We have demonstrated previously that an LRV capsid-polymerase polyprotein is specifically cleaved by a Leishmania-encoded cysteine protease. This study reports the purification of this protease through a strategy involving anion-exchange chromatography and affinity chromatography. By using a Sepharose-immobilized lectin, concanavalin A, we isolated a fraction enriched with LRV polyprotein-specific protease activity. Analysis of the active fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoreses and silver staining revealed a 50-kDa protein that, upon characterization by high-pressure liquid chromatography electrospray tandem mass spectrometry (electrospray ionization/MS/MS), was identified as a cysteine protease of trypanosomes. A partial amino acid sequence derived from the MS/MS data was compared with a protein database using BLAST software, revealing homology with several cysteine proteases of Leishmania and other trypanosomes. The protease exhibited remarkable temperature stability, while inhibitor studies characterized the protease as a trypsin-like cysteine protease-a novel finding for leishmania. To elucidate substrate preferences, a panel of deletion mutations and single-amino-acid mutations were engineered into a Gag-Pol fusion construct that was subsequently transcribed and translated in vitro and then used in cleavage assays. The data suggest that there are a number of cleavage sites located within a 153-amino-acid region spanning both the carboxy-terminal capsid region and the amino-terminal polymerase domain, with LRV capsid exhibiting the greatest susceptibility to proteolysis.