Proteomic analysis of the Heliothis virescens ascovirus 3i (HvAV-3i) virion.

Ascoviruses are enveloped, circular, double-stranded DNA viruses that can effectively control the appetite of lepidopteran larvae, thereby reducing the consequent damage and economic losses to crops. In this study, the virion of a sequenced Heliothis virescens ascovirus 3i (HvAV-3i) strain was used to perform proteomic analysis using both in-gel and in-solution digestion. A total of 81 viral proteins, of which 67 were associated with the virions, were identified in the proteome of HvAV-3i virions. Among these proteins, 23 with annotated functions were associated with DNA/RNA metabolism/transcription, virion assembly, sugar and lipid metabolism, signalling, cellular homoeostasis and cell lysis. Twenty-one viral membrane proteins were also identified. Some of the minor 'virion' proteins identified may be non-virion contaminants of viral proteins synthesized during replication, identified by more recent and highly sensitive methods. The extensive identification of the ascoviral proteome will establish a foundation for further investigation of ascoviral replication and infection.

Genomic analysis of a novel isolate Heliothis virescens ascovirus 3i (HvAV-3i) and identification of ascoviral repeat ORFs (aros).

Ascoviruses are circular double-stranded DNA viruses that infect insects. Herein we sequenced and analyzed the genome of the previously unrecorded ascovirus isolate Heliothis virescens ascovirus 3i (HvAV-3i). The genome size is 185,650 bp with 181 hypothetical open reading frames (ORFs). Additionally, definition based on ascovirus repeated ORFs (aros) is proposed; whereby the 29 aros from all sequenced Ascoviridae genomes are divided into six distinct groups. The topological relationship among the isolates of Heliothis virescens ascovirus 3a is (HvAV-3f, {HvAV-3h, [HvAV-3e, (HvAV-3g, HvAV-3i)]}) with every clade well supported by a Bayesian posterior probability of 1.00 and a Bootstrap value of 100%.

ICTV Virus Taxonomy Profile: Ascoviridae.

The family Ascoviridae includes viruses with circular dsDNA genomes of 100-200 kbp characterized by oblong enveloped virions of 200-400 nm in length. Ascoviruses mainly infect lepidopteran larvae and are mechanically transmitted by parasitoid wasps in which they may also replicate. Most known members belong to the genus Ascovirus, except one virus, that of the genus Toursvirus, which replicates in both its lepidopteran and parasitoid vector hosts. Ascoviruses cause high mortality among economically important insect pests, thereby controlling insect populations. This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Ascoviridae, which is available at www.ictv.global/report/ascoviridae.

Baculovirus FP25K Localization: Role of the Coiled-Coil Domain.

Two types of viruses are produced during the baculovirus life cycle: budded virus (BV) and occlusion-derived virus (ODV). A particular baculovirus protein, FP25K, is involved in the switch from BV to ODV production. Previously, FP25K from the model alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was shown to traffic ODV envelope proteins. However, FP25K localization and the domains involved are inconclusive. Here we used a quantitative approach to study FP25K subcellular localization during infection using an AcMNPV bacmid virus that produces a functional AcMNPV FP25K-green fluorescent protein (GFP) fusion protein. During cell infection, FP25K-GFP localized primarily to the cytoplasm, particularly amorphous structures, with a small fraction being localized in the nucleus. To investigate the sequences involved in FP25K localization, an alignment of baculovirus FP25K sequences revealed that the N-terminal putative coiled-coil domain is present in all alphabaculoviruses but absent in betabaculoviruses. Structural prediction indicated a strong relatedness of AcMNPV FP25K to long interspersed element 1 (LINE-1) open reading frame 1 protein (ORF1p), which contains an N-terminal coiled-coil domain responsible for cytoplasmic retention. Point mutations and deletions of this domain lead to a change in AcMNPV FP25K localization from cytoplasmic to nuclear. The coiled-coil and C-terminal deletion viruses increased BV production. Furthermore, a betabaculovirus FP25K protein lacking this N-terminal coiled-coil domain localized predominantly to the nucleus and exhibited increased BV production. These data suggest that the acquisition of this N-terminal coiled-coil domain in FP25K is important for the evolution of alphabaculoviruses. Moreover, with the divergence of preocclusion nuclear membrane breakdown in betabaculoviruses and membrane integrity in alphabaculoviruses, this domain represents an alphabaculovirus adaptation for nuclear trafficking of occlusion-associated proteins. IMPORTANCE: Baculovirus infection produces two forms of viruses: BV and ODV. Manufacturing of ODV involves trafficking of envelope proteins to the inner nuclear membrane, mediated partly through the FP25K protein. Since FP25K is present in alpha-, beta-, and gammabaculoviruses, it is uncertain if this trafficking function is conserved. In this study, we looked at alpha- and betabaculovirus FP25K trafficking by its localization. Alphabaculovirus FP25K localized primarily to the cytoplasm, whereas betabaculovirus FP25K localized to the nucleus. We found that an N-terminal coiled-coil domain present in all alphabaculovirus FP25K proteins, but absent in betabaculovirus FP25K, was critical for alphabaculovirus FP25K cytoplasmic localization. We believe that this represents an evolutionary process that partly led to the gain of function of this N-terminal coiled-coil domain in alphabaculovirus FP25K to aid in nuclear trafficking of occlusion-associated proteins. Due to betabaculovirus breakdown of the nuclear membrane before occlusion, this function is not needed, and the domain was lost or never acquired.

Genome sequence and organization analysis of Heliothis virescens ascovirus 3f isolated from a Helicoverpa zea larva.

The complete genome sequence of Heliothis virescens ascovirus 3f (HvAV-3f) was obtained. The HvAV-3f genome has a circular genome of 198,157bp with a G+C content of 46.0%, and encodes 190 open reading frames (ORFs) longer than 69 amino acids. Two major homologous regions (hrs) and 29 'baculovirus repeat ORFs' (bro) were found in the genome. BLAST analyses revealed that three HvAV-3f genes were homologous to that of lepidopteran insects. Nine ORFs were unique to HvAV-3f, in which two ORFs showed significant levels of similarity to genes that have not been previously described for ascoviruses in the Genbank database.

Reduction of polyhedrin mRNA and protein expression levels in Sf9 and Hi5 cell lines, but not in Sf21 cells, infected with Autographa californica multiple nucleopolyhedrovirus fp25k mutants.

During cell infection, the fp25k gene of baculoviruses frequently mutates, producing the few polyhedra (FP) per cell phenotype with reduced polyhedrin (polh) expression levels compared with wild-type baculoviruses. Here we report that the fp25k gene of the model baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), contains two hypermutable seven-adenine (A7) mononucleotide repeats (MNRs) that were mutated to A8 MNRs and a TTAA site that had host DNA insertions, producing fp25k mutants during Sf21 cell infection. The FP phenotype in Sf9 and Hi5 cells was more pronounced than in Sf21 cells. AcMNPV fp25k mutants produced similar levels of polyhedra or enhanced GFP, which were both under the control of the AcMNPV polh promoter for expression, in Sf21 cells but lower levels in Sf9 and Hi5 cells compared with AcMNPV with an intact fp25k gene. This correlated with the polh mRNA levels detected in each cell line. The majority of Sf21 cells infected with fp25 mutants showed high polh promoter-mediated GFP expression levels. Two cell lines subcloned from Sf21 cells that were infected with fp25k mutants showed different GFP expression levels. Furthermore, a small proportion of Hi5 cells infected with fp25k mutants showed higher production of polyhedra and GFP expression than the rest, and the latter was not correlated with increased m.o.i. Therefore, these data suggest that AcMNPV polh promoter-mediated gene expression activities differ in the three cell lines and are influenced by different cells within the cell line.

Cell-dependent production of polyhedra and virion occlusion of Autographa californica multiple nucleopolyhedrovirus fp25k mutants in vitro and in vivo.

Members of the family Baculoviridae are insect-specific dsDNA viruses that have been used for biological control of insect pests in agriculture and forestry, as well as in research and pharmaceutical protein expression in insect cells and larvae. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the type species of the family Baculoviridae. During infection of AcMNPV in permissive cells, fp25k mutants are positively selected, leading to the formation of the few polyhedra (FP) phenotype with reduced yield of polyhedra and reduced virion occlusion efficiency, which leads to decreased oral infectivity for insects. Here we report that polyhedra of AcMNPV fp25k mutants produced from different insect cell lines and insects have differences in larval per os infectivity, and that these variations are due to different virion occlusion efficiencies in these cell lines and insects. Polyhedra of AcMNPV fp25k mutants produced from Sf cells (Sf21 and Sf9, derived from Spodoptera frugiperda) and S. frugiperda larvae had poorer virion occlusion efficiency than those from Hi5 cells (derived from Trichoplusia ni) and T. ni larvae, based on immunoblots, DNA isolation and larval oral infection analysis. AcMNPV fp25k mutants formed clusters of FP and many polyhedra (MP) in the fat body cells of both T. ni and S. frugiperda larvae. Transmission electron microscopy revealed that the nature of virion occlusion of AcMNPV fp25k mutants was dependent on the different cells of the T. ni fat body tissue. Taken together, these results indicate that the FP phenotype and virion occlusion efficiency of fp25k mutants are influenced by the host insect cells.

Genome of a Bombyx mori nucleopolyhedrovirus strain isolated from India.

Bombyx mori nucleopolyhedrovirus (BmNPV), a member of the Baculoviridae, is a major pathogen of silkworm and has also been recently developed as an expression vector for heterologous gene expression in the silkworm larvae and pupae. To better understand the diversity of this important baculovirus, we sequenced the complete genome of the BmNPV strain isolated from India, where its host is available throughout the year due to its tropical climate. The genome of the Indian strain consists of 127,879 nucleotides, with a G+C content of 40.36%. There are 138 open reading frames (ORFs) encoding the predicted proteins of more than 50 amino acids. Genomic comparison of the Indian strain with 3 other reported BmNPV strains showed that the baculovirus repeat ORFs (bro) and homologous repeat regions (hr's) are highly variable. These results suggest that the BmNPV strain heterogeneity is mainly caused by single-nucleotide polymorphisms (SNPs) and changes in the hr's and bro genes.

Genomic sequence of Heliothis virescens ascovirus 3g isolated from Spodoptera exigua.

Heliothis virescens ascovirus 3a (HvAV-3a), a member of the family Ascoviridae, has the highest diversity among ascovirus species that have been reported in Australia, Indonesia, China, and the United States. To understand the diversity and origin of this important ascovirus, the complete genome of the HvAV Indonesia strain (HvAV-3g), isolated from Spodoptera exigua, was determined to be 199,721 bp, with a G+C content of 45.9%. Therefore, HvAV-3g has the largest genome among the reported ascovirus genomes to date. There are 194 predicted open reading frames (ORFs) encoding proteins of 50 or more amino acid residues. In comparison to HvAV-3e reported from Australia, HvAV-3g has all the ORFs in HvAV-3e with 6 additional ORFs unique to HvAV-3g, including 1 peptidase C26 gene with the highest identity to Drosophila spp. and 2 gas vesicle protein U (GvpU) genes with identities to Bacillus megaterium. The five unique homologous regions (hrs) and 25 baculovirus repeat ORFs (bro) of HvAV-3g are highly variable.

Genome of Thysanoplusia orichalcea multiple nucleopolyhedrovirus lacks the superoxide dismutase gene.

Thysanoplusia orichalcea multiple nucleopolyhedrovirus (ThorMNPV) has high virulence to Trichoplusia ni and Pseudoplusia includens larvae, with a potential for biological control of insect pests. The genome of ThorMNPV was sequenced and found to be 132,978 bp, with a G+C content of 37.9%. There are 145 predicted open reading frames (ORFs), encoding proteins of 50 or more amino acid residues with minimal overlap. Of the 145 ORFs, 141 appeared to be homologous to those of Autographa californica MNPV (AcMNPV). In comparison to AcMNPV, 9 ORFs of AcMNPV were absent in ThorMNPV, including the superoxide dismutase (sod) gene.

"Megavirales", a proposed new order for eukaryotic nucleocytoplasmic large DNA viruses.

The nucleocytoplasmic large DNA viruses (NCLDVs) comprise a monophyletic group of viruses that infect animals and diverse unicellular eukaryotes. The NCLDV group includes the families Poxviridae, Asfarviridae, Iridoviridae, Ascoviridae, Phycodnaviridae, Mimiviridae and the proposed family "Marseilleviridae". The family Mimiviridae includes the largest known viruses, with genomes in excess of one megabase, whereas the genome size in the other NCLDV families varies from 100 to 400 kilobase pairs. Most of the NCLDVs replicate in the cytoplasm of infected cells, within so-called virus factories. The NCLDVs share a common ancient origin, as demonstrated by evolutionary reconstructions that trace approximately 50 genes encoding key proteins involved in viral replication and virion formation to the last common ancestor of all these viruses. Taken together, these characteristics lead us to propose assigning an official taxonomic rank to the NCLDVs as the order "Megavirales", in reference to the large size of the virions and genomes of these viruses.

USP9X expression is functionally related to laryngeal cancer.

An increasing number of studies have shown that USP9X is closely related to cancer. However, its role in carcinogenesis and progression of laryngeal cancer has not yet been investigated. In this study, we found that USP9X was upregulated in laryngeal cancer tissues. The expression of USP9X was significantly correlated with degree of laryngeal cancer differentiation and lymphatic metastasis. USP9X knockdown led to a decrease in the ability of proliferation, migration, and invasion of FaDu cells. The proportion of FaDu apoptotic cells increased by interfering with the endogenous expression of USP9X. We speculated that inhibiting USP9X might induce apoptosis in FaDu cells by downregulating Mcl-1 and upregulating Bax protein expression. Our findings for the first time suggest the expression level and trend of USP9X in laryngeal cancer tissue and USP9X may plays an important role in promoting the occurrence and progression of laryngeal cancer. USP9X may be a potential target for intervention in treatment of laryngeal cancer.

AcMNPV enhances infection by ThorNPV in Sf21 cells and SeMNPV in Hi5 cells.

An expression cassette containing the DsRed2 gene, which encodes the red fluorescent protein (RFP), was inserted into the wide-host-range Autographa californica M nucleopolyhedrovirus (AcMNPV) at the polyhedrin locus (vAcDsRed2). An expression cassette containing the enhanced green fluorescent protein (EGFP) gene was inserted at the gp37 locus of the narrow-host-range Thysanoplusia orichalcea MNPV (ThorMNPV) and the p10 locus of Spodoptera exigua MNPV (SeMNPV) to produce vThGFP and vSeGFP, respectively. vThGFP and vSeGFP are poor at infecting Sf21 and Hi5 cells, respectively, whereas vAcDsRed2 is highly infectious to both cell lines. During co-infection, vAcDsRed2 enhanced vThGFP infection in Sf21 cells by approximately 20-fold, and it enhanced vSeGFP infection in Hi5 cells by more than 300-fold, as detected by fluorescence measurements. In contrast, vThGFP reduced vAcDsRed2 infection by 5.4-fold in Sf21 cells, while vSeGFP reduced vAcDsRed2 by 3.2-fold in Hi5 cells. Plaque assay data did not suggest viral recombination, but vThGFP plaques surrounded by vAcDsRed2 plaques were observed. A viral DNA replication assay performed by real-time quantitative PCR suggested that the detected fluorescence correlated with virus replication. Sf21 cells infected with vAcDsRed2 were resistant to superinfection by viruses of the same type expressing EGFP (vAcGFP). These results demonstrated that AcMNPV could enhance replication of ThorMNPV and SeMNPV in non-permissive cells without recombination.

Comparative analysis of a highly variable region within the genomes of Spodoptera frugiperda ascovirus 1d (SfAV-1d) and SfAV-1a.

The recently discovered ascoviruses have a worldwide distribution. Here we report a new member of the family Ascoviridae, Spodoptera frugiperda ascovirus 1d (SfAV-1d) with a variable region in the genome. Restriction fragment length polymorphism, Southern hybridization and genome sequencing analyses confirmed that SfAV-1d and the earlier reported SfAV-1a are closely related but are not identical. The genome size of SfAV-1d is approximately 100 kbp, which is about 57 kbp smaller than SfAV-1a. The SfAV-1d genome has a major deletion of 14 kbp that corresponds to one of the inverted repeat (IR) regions of SfAV-1a. Cloning and sequencing revealed that the region flanking the deletion within the SfAV-1d genome is highly variable. In all the variants of this region, the whole IR region is missing, with 88.2 % of the variants missing part of or the whole adjacent SfAV-1a ORF71, 94.1 % missing part of or the whole of adjacent ORF72 and 64.6 % missing part of or the whole of ORF73.

[Construction and identification of nemo-like kinase gene recombinant adenovirus vector].

OBJECTIVE: To construct the nemo-like kinase (NLK) gene recombinant adenovirus vector. METHODS: The AdEasy system was used to construct the recombinant adenovirus vector. Using reverse transcriptase polymerase chain reaction (RT-PCR), the full-length gene of NLK and its mutants (K155M, T286V, and C425Y) were amplified from HEK293 cells. The FLAG tag was appended at the C-terminal of NLK. After ligation and transformation, the NLK gene and its mutants were cloned into the pAdTrack-CMV vector. It was detected by PCR, sequencing, and Western blot analysis. Using DNA recombination and homogenous recombination, the normally expressed plasmids were linearized by the restriction enzyme-PmeI and PacI, then the enzyme-digested products were recycled by using ethanol precipitation. The purified product was transfected to HEK293A packaging cells with FuGENE HD transfection reagent. After amplification of the recombinant adenovirus, Western blot analysis was performed to detect the expression of NLK gene and its mutants. RESULTS: The successful construction of pAdtrack-CMV-NLK (and mutants) was confirmed by PCR and sequencing. Western blot analysis showed that the target genes and the recombinant adenovirus were obtained. This recombinant virus was able to express NLK protein and its mutants correctly in HCT 116 cells. CONCLUSION: The NLK gene recombinant adenovirus vector was successfully constructed and identified.

Identification of SARS-CoV-2 Variants and Their Clinical Significance in Hefei, China.

The ongoing coronavirus disease 2019 (COVID-19) pandemic represents one of the most exigent threats of our lifetime to global public health and economy. As part of the pandemic, from January 10 to March 10, 2020, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) began to spread in Hefei (Anhui Province, China) with a total of 174 confirmed cases of COVID-19. During this period, we were able to gather critical information on the transmission and evolution of pathogens through genomic surveillance. Particularly, the objective of our study was to track putative variants of SARS-CoV-2 circulating in Hefei for the first time and contribute to the global effort toward elucidating the molecular epidemic profile of the virus. Patients who showed symptoms of COVID-19 were routinely tested for SARS-CoV-2 infections via RT-PCR at the First Affiliated Hospital of Anhui Medical University. Whole-genome sequencing was performed on 97 clinical samples collected from 29 confirmed COVID-19 patients. As a result, we identified a local novel single-nucleotide polymorphism site (10,380) harboring a G → T mutation (Gly → Val) in Hefei. Further phylogenetic network analysis with all the sequences of SARS-CoV-2 deposited in GenBank collected in East and Southeast Asia revealed a local subtype of S-type SARS-CoV-2 (a1) harboring a C → T synonymous mutation (Leu) at position 18,060 of ORF1b, likely representing a local SARS-CoV-2 mutation site that is obviously concentrated in Hefei and the Yangtze River Delta region. Moreover, clinical investigation on the inflammatory cytokine profile of the patients suggested that mutations at positions 18,060 (the shared variable site of subtype a1) and 28,253(harboring a C → T synonymous mutation, Phe) were associated with milder immune responses in the patients.

Baculovirus interactions in vitro and in vivo.

Baculoviruses are promising viral insecticides and are safe for the environment. Interaction of baculoviruses in vitro and in vivo is a basic molecular and ecological question that has practical applications in agriculture. Cellular secretion is also a fundamental property in cell-cell communication. Here, we review recent investigations on how baculoviruses interact with insect cells and insect hosts. We focus particularly on a new interaction mechanism in which a secretion from cells infected with one virus enhances infection by a second virus. We also discuss a hypothesis that the secreted signals may serve as ligands that bind to the receptors on the surface of the cells that harbor the suppressed genomes of Thysanoplusia orichalcea MNPV (ThorMNPV) in Sf21 and Spodoptera exigua MNPV (SeMNPV) in High 5 to initiate signal transduction leading to the activation of genome replication of ThorMNPV in Sf21 and SeMNPV in High 5. We also discuss how the enhanced replication of SeMNPV replication by Autographa californica MNPV (AcMNPV) in nonpermissive insect cells depends on the types of cells. Interaction of baculoviruses in insects focused on mutualism and antagonism, even though the mechanism is not clear on mutualism. The antagonism of a Nucleopolyhedrovirus (NPV) with a Granulovirus (GV) has been extensively studied by a metalloprotein in the capsule of GV that disrupts the peritrophic membrane, a physical barrier to NPV entry to the midgut of larvae, to facilitate NPV infection.

[Epidemiological and molecular characterization of seasonal influenza A/H3N2 viruses circulating in Shenzhen, 2005 - 2007].

OBJECTIVE: To determine the epidemiological characteristics of seasonal influenza in Shenzhen from 2005 to 2007 and the molecular variation of HA1 domain of influenza H3N2 viruses. METHODS: The consultation rate for influenza-like illness (ILI) were calculated weekly for indicating the influenza activities (the Shenzhen Influenza Surveillance System mainly consisted of 16 institutions with 9 hospitals, 6 districts and one municipal centers of disease control and prevention). Pharyngeal swabs from the cases of ILI, which were collected during 2005 to 2007 from the city-wide and quality-controlled surveillance network, were used to propagate the viruses. The HA1 region of the influenza A/H3N2 viruses were detected by RT-PCR and sequenced subsequently. The analyses of pairwise amino acid variations, genetic clustering and phylogenetics was performed. RESULTS: The activity levels of influenza showed certain changes during each year from 2005 to 2007, and there were summer peaks from May to July in 2006 and 2007. The positive rates of influenza virus were 4.78% (114/2385), 5.77% (212/3674) and 12.12% (343/2831) from 2005 to 2007 respectively. The weekly isolating rates changed accordingly with the trend of the percentages of ILI. The proportions of influenza H3N2 virus were 25.46% (28/114) and 2.83% (6/212) in 2005 and in 2006 respectively, but the proportion increased to 62.68% (215/343), which indicated that H3N2 virus became the predominant strain in 2007. Phylogenetic clustering analysis of influenza H3N2 virus revealed that there were 5 clades. The viruses which were isolated in 2005 contained in the clade I and II, the viruses in 2006 were comprised in clade III, and clade IV and V included the viruses isolated in 2007. Although the stem of cladogram developed with one accord of the time isolated viruses, the viruses which were similar to vaccine strains had circulated in Shenzhen before a given strain was determined as vaccine strain by WHO. It was also noticed that more amino acid changes at antigenic sites, especially at sites A and B in the H3N2 viruses, were found in 2007 than that in 2005 and in 2006. But the sequences at the receptor-binding sites and disulphide bond sites were conserved and no new circulating strain for genetic reassortment had been found in the period. CONCLUSION: Shenzhen might be one of areas where the ongoing genetic drift of influenza H3N2 viruses appeared earlier in China. The changes of influenza H3N2 virus showed the active status in the population. The results suggested that monitoring seasonal influenza viruses by sequence analysis could provide important and timely information on the appearance of strains with epidemiologic significance.

Long Non-coding RNA H19 Suppression Protects the Endothelium Against Hyperglycemic-Induced Inflammation via Inhibiting Expression of miR-29b Target Gene Vascular Endothelial Growth Factor a Through Activation of the Protein Kinase B/Endothelial Nitric Oxide Synthase Pathway.

It has been shown that non-coding RNAs (ncRNAs) play an important regulatory role in pathophysiological processes involving inflammation. The vascular endothelial growth factor A (VEGFA) gene also participates in the inflammatory process. However, the relationships between ncRNAs and VEGFA are currently unclear. Here, this study was designed to determine the relationship between long non-coding RNA (lncRNA) H19, mircoRNA29b (miR-29b), and VEGFA in the development of diabetes mellitus (DM). We demonstrate that H19 is upregulated and miR-29b downregulated in individuals with DM and directly binds miR-29b. VEGFA is the target of miR-29b in the vascular endothelium of individuals with DM. We found that positive modulation of miR29b and inhibition of H19 and VEGFA significantly attenuates high glucose-induced endothelial inflammation and oxidative stress. We also found that the protein kinase B/endothelial nitric oxide synthase (AKT/eNOS) signal pathway in endothelial cells is activated through regulation of miR29b and H19 endogenous RNAs. We conclude that H19 suppression protects the endothelium against high glucose-induced inflammation and oxidative stress in endothelial cells by upregulation of miR-29b and downregulation of VEGFA through AKT/eNOS signal pathway activation. These results suggest a novel link between dysregulated ncRNA expression, inflammation, and the signaling pathway in the vascular endothelium of individuals with DM, indicating a promising strategy for preventing cardiovascular disease in such individuals.

Melatonin prevents oxidized low-density lipoprotein-induced increase of myosin light chain kinase activation and expression in HUVEC through ERK/MAPK signal transduction.

Melatonin, the main secretary product of the pineal gland, is potentially effective in the prevention of a number of diseases in which free radical processes are involved. The development of hypercholesterolemia is a multifactorial process in which elevated oxidized low-density lipoprotein (ox-LDL) levels play a central role. The purpose of this study was to test whether melatonin prevents ox-LDL-induced increase of myosin light chain kinase (MLCK) activation and expression in human umbilical vein endothelial cells (HUVECs) through extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signal transduction. HUVEC were cultured in vitro and treated with ox-LDL, melatonin, and PD98059 (a selective inhibitor of ERK), respectively. The expression, transcription, and activity of MLCK were measured by western blot, immunohistochemistry, reverse transcription-polymerase chain reaction and gamma-(32)P-adenosine triphosphate (ATP) incorporation, respectively. The results showed that the expression and activity of MLCK were increased in ox-LDL-treated HUVECs and this was decreased by melatonin and PD98059. The expression and activity of MLCK induced by ox-LDL was associated with the phosphorylation of ERK. These results indicate for the first time that hypercholesterolemia may be associated with MLCK expression and the activity which can be reduced by melatonin through ERK/MAPK signal transduction.