Identification and classification of the genomes of novel microviruses in poultry slaughterhouse.

Microviridae is a family of phages with circular ssDNA genomes and they are widely found in various environments and organisms. In this study, virome techniques were employed to explore potential members of Microviridae in a poultry slaughterhouse, leading to the identification of 98 novel and complete microvirus genomes. Using a similarity clustering network classification approach, these viruses were found to belong to at least 6 new subfamilies within Microviridae and 3 higher-level taxonomic units. Genome size, GC content and genome structure of these new taxa showed evident regularities, validating the rationality of our classification method. Our method can divide microviruses into about 45 additional detailed clusters, which may serve as a new standard for classifying Microviridae members. Furthermore, by addressing the scarcity of host information for microviruses, the current study significantly broadened their host range and discovered over 20 possible new hosts, including important pathogenic bacteria such as Helicobacter pylori and Vibrio cholerae, as well as different taxa demonstrated different host specificities. The findings of this study effectively expand the diversity of the Microviridae family, providing new insights for their classification and identification. Additionally, it offers a novel perspective for monitoring and controlling pathogenic microorganisms in poultry slaughterhouse environments.

Unveiling CRESS DNA Virus Diversity in Oysters by Virome.

Oysters that filter feed can accumulate numerous pathogens, including viruses, which can serve as a valuable viral repository. As oyster farming becomes more prevalent, concerns are mounting about diseases that can harm both cultivated and wild oysters. Unfortunately, there is a lack of research on the viruses and other factors that can cause illness in shellfish. This means that it is harder to find ways to prevent these diseases and protect the oysters. This is part of a previously started project, the Dataset of Oyster Virome, in which we further study 30 almost complete genomes of oyster-associated CRESS DNA viruses. The replication-associated proteins and capsid proteins found in CRESS DNA viruses display varying evolutionary rates and frequently undergo recombination. Additionally, some CRESS DNA viruses have the capability for cross-species transmission. A plethora of unclassified CRESS DNA viruses are detectable in transcriptome libraries, exhibiting higher levels of transcriptional activity than those found in metagenome libraries. The study significantly enhances our understanding of the diversity of oyster-associated CRESS DNA viruses, emphasizing the widespread presence of CRESS DNA viruses in the natural environment and the substantial portion of CRESS DNA viruses that remain unidentified. This study's findings provide a basis for further research on the biological and ecological roles of viruses in oysters and their environment.

A discussion of RNA virus taxonomy based on the 2020 International Committee on Taxonomy of Viruses report.

RNA viruses have a higher mutation rate than DNA viruses; however, RNA viruses are insufficiently studied outside disease settings. The International Committee on Taxonomy of Viruses (ICTV) is an organization set up by virologists to standardize virus classification. To better understand ICTV taxonomy and the characteristics and rules of different RNA virus families, we analyzed the 3,529 RNA viruses included in the 2020 ICTV report using five widely used metrics: length, host, GC content, number of predicted ORFs, and sequence similarity. The results show that host type has a significant influence on viral genome length and GC content. The genome lengths of virus members within the same genus are quite similar: 98.28% of the genome length differences within any particular genus are less than 20%. The species within those genera containing segmented viruses also have a similar length and number of segments. The number of predicted ORFs in the RNA viral genomes also shows a strong, statistically significant correlation with genome length. We suggest that due to the high mutation rate of RNA virus genomes, current RNA virus classification should mainly rely on protein similarities rather than nucleic acid similarities.

Novel RNA viruses in oysters revealed by virome.

Eighteen novel RNA viruses were found in Crassostrea hongkongensis. Phylogenic analysis shows evidence of recombination between major genes of viruses. Picobirnaviruses are ubiquitous and abundant in oysters.

Differential expression profile analysis of PSTK-regulated mRNAs in podocytes.

This study aimed to elucidate the precise mechanisms underlying the protective effects of phosphoseryl-tRNA kinase (PSTK) against cisplatin-induced podocyte injury. PSTK overexpression and knockdown vectors were generated and transfected into murine podocyte cells-5. PSTK levels were measured, and transcriptome sequencing was conducted. Differential expression analysis was performed to identify messenger RNAs (mRNAs) that were positively and negatively correlated with PSTK. We selected 10 candidate genes identified via real-time quantitative polymerase chain reaction and Western blot analysis for further analysis. As expected, PSTK levels were significantly higher in PSTK-overexpressing podocytes and significantly lower in PSTK-knockdown podocytes. PSTK overexpression resulted in the upregulation of 122 genes and downregulation of 372 genes in podocytes. On the other hand, PSTK knockdown resulted in the upregulation of 231 genes and downregulation of 445 genes. Furthermore, the analysis revealed that 11 genes were positively correlated with PSTK, whereas 20 genes were negatively correlated with PSTK. The obtained PSTK-regulated genes were primarily involved in molecular function, biological process, and cellular component, as well as the angiogenesis pathway. The Wnt family member 10A levels were significantly higher after PSTK overexpression, but were significantly lower after PSTK knockdown. In addition, Na+/K+ ATPase subunit α-2 and matrix metalloproteinase 9 levels were significantly downregulated after PSTK overexpression, but significantly upregulated upon PSTK knockdown. Cell proliferation was significantly increased upon PSTK overexpression, but significantly decreased upon PSTK suppression. The results of this study not only identified several significant PSTK-regulated genes for further validation, but also provided insights into the mechanisms underlying the protective effects of PSTK on podocytes.

Hepatic ALT isoenzymes are elevated in gluconeogenic conditions including diabetes and suppressed by insulin at the protein level.

BACKGROUND: Alanine transaminase (ALT) plays an important role in gluconeogenesis by converting alanine into pyruvate for glucose production. Early studies have shown that ALT activities are upregulated in gluconeogenic conditions and may be implicated in the development of diabetes. ALT consists of two isoforms, ALT1 and ALT2, with distinctive subcellular and tissue distributions. Whether and how they are regulated are largely unknown. METHODS: By using Western blotting analysis, we measured hepatic ALT isoforms at the protein level in obese and diabetic animals and in Fao hepatoma cells treated with dexamethasone and insulin. In addition, we measured glucose output in Fao cells over-expressing ALT1 and ALT2. RESULTS: Both ALT isoforms in the liver were increased in diabetic Goto-Kakizaki rats and during fasting. However, in ob/ob mice, only ALT2, but not ALT1, protein levels were elevated, and the increase of ALT2 was correlated with that of ALT activity. We further demonstrated that, in vitro, both ALT1 and ALT2 were induced by glucocorticoid dexamethasone, but suppressed by insulin in Fao cells. Finally, we showed that the over-expression of ALT1 and ALT2 in Fao cells directly increased glucose output. CONCLUSIONS: We have shown the similarity and difference in the regulation of ALT isoforms in gluconeogenic conditions at the protein level, supporting that ALT isoenzymes play an important role in glucose metabolism and may be implicated the development of insulin resistance and diabetes.

Estrogen inhibits LPS-induced IL-6 production in macrophages partially via the nongenomic pathway.

17ß-estradiol (E2)-signaling is widely considered to be mediated through the transcription-regulating intracellular estrogen receptor (iER). In this study, using the cell-impermeable E2-BSA, we investigated the nongenomic effects of E2 on the IL-6 production, MAPK and transcription factor activation following LPS stimulation in mouse bone marrow-derived macrophages (BMMs). It was found that E2 normalized LPS-induced IL-6 production in BMMs. Although the increase in IL-6 production induced by LPS was also attenuated by E2-BSA treatment, the capacity of BMMs to produce the IL-6 cytokine remained higher than the control. In addition, the iER blocker, ICI 182780, did not abolish the total effects of E2 on LPS-stimulated IL-6 production capacity in BMMs. Furthermore, E2 and E2-BSA attenuated the LPS activation of p38 but not that of ERK1/2 and JNK. The p38 inhibitor, SB 203580, significantly reduced the LPS-induced IL-6 production. Moreover, E2 and E2-BSA inhibited LPS-induced activation of NF-κB. This inhibitory effect was associated with decreases in nuclear p65 protein levels. Taken together, these results indicate that E2 has an inhibitory effect on LPS-induced IL-6 production in BMMs through inhibition of p38 MAPK phosphorylation, and blockade of NF-κB activation. These effects are mediated at least in part via a nongenomic pathway.

[NF-κ B mediates the effect of glucosylceramide synthase on P-glycoprotein modulation in a drug-resistance leukemia cell line].

OBJECTIVE: To investigate whether transcription factor-kappaB (NF-κ B) is involved in the modulation of P-glycoprotein (P-gp) by glucosylceramide synthase (GCS) in a multidrug resistance leukemia cell line K562/A02 and to explore the relationship between NF-κ B and extracelluar signal-regulated kinase (ERK). METHODS: K562/A02 cells were treated with GCSsiRNA, pyrrolidine dithiocarbamate (PDTC, a NF-κ B specific inhibitor) and U0126 (a MEK1/2 inhibitor), respectively. The expression of GCS and multidrug resistance protein 1 (MDR1) mRNA were analyzed with qRT-PCR. Various proteins of different groups were measured by Western blotting. RESULTS: After transfected with GCSsiRNA for 48 h, GCS mRNA were reduced by 62% (51%-73%) and MDR1 mRNA was reduced by 52% (43%-61%) in the K562/A02 cells. Compared with the negative control, relative expression of NF-κ B p65 in nuclear and P-ERK1/2 were both down-regulated, and P-gp was also inhibited significantly at 72 h after transfected with GCSsiRNA (P< 0.05). In addition, the expression of P-gp was decreased at 24 h with 80 µ mol/L PDTC and 48 h with 20 µ mol/L PDTC. P-ERK1/2 was inhibited significantly when the cells were treated with 20 µ mol/L U0126 for 48 h. The expression of NF-κ B p65 in nuclear and P-gp were also down-regulated. CONCLUSION: NF-κ B can modulate the effect of GCS on P-gp in K562/A02 cells. P-ERK1/2 can activate NF-κ B in above signal transduction pathway.

Estrogen-induced nongenomic calcium signaling inhibits lipopolysaccharide-stimulated tumor necrosis factor α production in macrophages.

Estrogen is traditionally thought to exert genomic actions through members of the nuclear receptor family. Here, we investigated the rapid nongenomic effects of 17ß-estradiol (E2) on tumor necrosis factor α (TNF-α) production following lipopolysaccharide (LPS) stimulation in mouse bone marrow-derived macrophages (BMMs). We found that LPS induced TNF-α production in BMMs via phosphorylation of p38 mitogen-activated protein kinase (MAPK). E2 itself did not affect the MAPK pathway, although it attenuated LPS-induced TNF-α production through suppression of p38 MAPK activation. Recently, G protein-coupled receptor 30 (GPR30) was suggested to be a membrane estrogen receptor (mER) that can mediate nongenomic estradiol signaling. We found that BMMs expressed both intracellular estrogen receptors (iER) and mER GPR30. The specific GPR30 antagonist G-15 significantly blocked effects of estradiol on LPS-induced TNF-α production, whereas an iER antagonist did not. Moreover, E2 induced a rapid rise in intracellular free Ca(2+) that was due to the influx of extracellular Ca(2+) and was not inhibited by an iER antagonist or silencing of iER. Ca(2+) influx was also induced by an impermeable E2 conjugated to BSA (E2-BSA), which has been used to investigate the nongenomic effects of estrogen. Consequently, Ca(2+), a pivotal factor in E2-stimulated nongenomic action, was identified as the key mediator. The inhibitory effects of E2 on LPS-induced TNF-α production and p38 MAPK phosphorylation were dependent on E2-triggered Ca(2+) influx because BAPTA, an intracellular Ca(2+) chelator, prevented these effects. Taken together, these data indicate that E2 can down-regulate LPS-induced TNF-α production via blockade of p38 MAPK phosphorylation through the mER-mediated nongenomic Ca(2+) signaling pathway in BMMs.

Correlation of circulating MMP-9 with white blood cell count in humans: effect of smoking.

BACKGROUND: Matrix metalloproteinase-9 (MMP-9) is an emerging biomarker for several disease conditions, where white blood cell (WBC) count is also elevated. In this study, we examined the relationship between MMP-9 and WBC levels in apparently healthy smoking and non-smoking human subjects. METHODS: We conducted a cross-sectional study to assess the relationship of serum MMP-9 with WBC in 383 men and 356 women. Next, we divided the male population (women do not smoke in this population) into three groups: never (n = 243), current (n = 76) and former (n = 64) smokers and compared the group differences in MMP-9 and WBC levels and their correlations within each group. RESULTS: Circulating MMP-9 and WBC count are significantly correlated in men (R(2) = 0.13, p<0.001) and women (R(2) = 0.19, p<0.001). After stratification by smoking status, MMP-9 level was significantly higher in current smokers (mean ± SE; 663.3±43.4 ng/ml), compared to never (529.7±20.6) and former smokers (568±39.3). WBC count was changed in a similar pattern. Meanwhile, the relationship became stronger in current smokers with increased correlation coefficient of r = 0.45 or R(2) = 0.21 (p<0.001) and steeper slope of ß = 1.16±0.30 (p<0.001) in current smokers, compared to r = 0.26 or R(2) = 0.07 (p<0.001) and ß = 0.34±0.10 (p<0.001) in never smokers. CONCLUSIONS: WBC count accounts for 13% and 19% of MMP-9 variance in men and women, respectively. In non-smoking men, WBC count accounts for 7% of MMP-9 variance, but in smoking subjects, it accounts for up to 21% of MMP-9 variance. Thus, we have discovered a previously unrecognized correlation between the circulating MMP-9 and WBC levels in humans.

[Effect of glucosylceramide synthase on P-gp expression by ERK signal transduction pathway in leukemia multi-drug resistance cell line].

OBJECTIVE: To investigate the effect of glucosylceramide synthase (GCS) on P-glycoprotein (P-gp) expression via extracellular signal-regulated kinase (ERK) pathways in leukemia K562/A02 cell line. METHODS: K562/A02 multidrug resistance cells were treated with GCS siRNA and U0126, respectively. Expression of multidrug resistance protein 1 (MDR1) mRNA was analyzed with qRT-PCR. Phosphorylated ERK1/2, total ERK1/2 protein and P-gp in different groups were measured with Western blotting. RESULTS: After treated with U0126, P-ERK1/2 was decreased along with the increased U0126 concentration. P-ERK1/2 and P-gp were apparently down-regulated by U0126 at the concentrations of 20 µmol/L, 40 µmol/L and 60 µmol/L. After being transfected with GCS siRNA, GCS mRNA was inhibited by 70.50% (58.00%-76.00%) in K562/A02 cells. Compared with the negative control, both P-ERK1/2 and P-gp were inhibited significantly after RNAi for 72 hours (P<0.01 and P<0.05, respectively. CONCLUSION: GCS may affect the expression of P-gp by ERK signal transduction pathway in leukemia cells.

The effect of glucosylceramide synthase on P-glycoprotein function in K562/AO2 leukemia drug-resistance cell line.

Previous work from our laboratory demonstrated that glucosylceramide synthase (GCS) and multidrug resistance 1 gene (MDR1) are co-overexpressed in drug-resistant leukemia cells. We hypothesized that GCS and MDR1 may interact. In this study, we used RNA interference (RNAi) to silence the GCS or MDR1 gene in K562/AO2 drug-resistant cells. The sensitivity of cells to different treatments with doxorubicin was evaluated. We used Taqman probe fluorescence real-time quantitative PCR, and detected expression of GCS and MDR1 mRNAs in different interfering groups. Intracellular mean fluorescence intensity (MFI), which represents rhodamine123 (rh123) retention, was determined by flow cytometry (FCM). An MTT cytotoxicity assay showed that the 50% inhibition concentration (IC50) of doxorubicin of K562/AO2 cells (138.25 ± 3.75 µg/ml) was significantly higher than that of K562 drug-sensitive cells (2.125 ± 0.125 µg/ml), and that IC50 was evidently lower in K562/AO2 cells, whether it was transfected with a small interfering RNA (siRNA) targeting GCS (GCSsiRNA) or one targeting MDR1 (MDR1siRNA). Compared with untreated K562/AO2 cells, the inhibition rates of GCS mRNA in the cells transfected with GCSsiRNA for 9 and 36 h were 56.67 ± 9.29% (p < 0.05) and 74 ± 6.38% (p < 0.05), respectively. Interestingly, the expression of MDR1 mRNA was also inhibited to 51.7 ± 4.5% (p < 0.05) 36 h after transfection with GCSsiRNA, but there was no significant difference in MDR1 expression at 9 h post-transfection in cells treated with GCSsiRNA and a negative control. It is well known that rh123 retention in cells results from an efflux function of P-glycoprotein (P-gp). In K562 cells, rh123 retention was much higher than in K562/AO2 cells (p < 0.01). We also noted that rh123 retention in the K562/AO2 cells transfected with GCSsiRNA for 48 h was significantly higher than in the negative control group. In conclusion, we show in the present study that inhibition of the GCS gene affects the expression of MDR1 mRNA and P-gp function.

[Study on the correlation of GCS and MDR1 genes in inducing multidrug resistance in human K562/A02 cell line].

OBJECTIVE: To investigate the correlation of glucosylceramide synthase (GCS) gene and multidrug resistance 1 (MDR1) gene in inducing multidrug resistance in human multidrug-resistant K562/A02 cell line, and search for a novel strategy for reversing multidrug resistance of leukemia cells. METHODS: The expression levels of GCS and MDR1 mRNA in K562 and K562/A02 cells were assayed by RT-PCR. siRNAs targeting the GCS and MDR1 gene were transfected into K562/A02 cells with liposome, respectively. The differential expression of GCS and MDR1 mRNAs, as well as their correlation, were detected by RT-PCR and real time quantitative-PCR(QPCR). RESULTS: The expression level of GCS and MDR1 mRNA was dramatically lower in drug-sensitive K562 cells compared with the K562/A02 cells. The GCS mRNA was inhibited by 73%(59%-82%) and MDR1 mRNA expression was down regulated by 67% (38%-82%) in K562/A02 cells after being transfected with GCS siRNA. The expression level of MDR1 mRNA was inhibited by 81%(63%-91%) and GCS mRNA expression had no apparent change in K562/A02 cells treated with MDR1 small interference RNA(siRNA). CONCLUSION: Positive correlation was detected between the expression of GCS and MDR1 mRNA in K562/A02 cells and MDR1 mRNA expression was down regulated after silencing the GCS gene expression.

NME1 at the human maternal-fetal interface downregulates titin expression and invasiveness of trophoblast cells via MAPK pathway in early pregnancy.

Nometastatic gene 23-H1 (NME1, also known as nm23-H1) is a wide-spectrum tumor metastasis suppressor gene that plays an important role in suppressing the invasion and metastasis of tumor cells. It has been demonstrated that NME1 is also expressed in human first-trimester placenta, but its function at maternal-fetal interface is not clear. The present study aimed to elucidate the biological function of NME1 at the maternal-fetal interface, especially on invasion of the human extravillous cytotrophoblasts (EVCTs). NME1 has been identified in both human trophoblast cells and decidual stromal cells (DSCs) in early pregnancy. We have proved that NME1 silencing in vitro increases the titin protein translation in the invasive EVCTs. Moreover, NME1 can inactivate the phospho-extracellular signal-regulated kinase 1/2 (P-ERK1/2) in trophoblasts in a time-dependent manner, and U0126, an inhibitor of MAPK/ERK, can inhibit partly the enhanced invasiveness and titin expression in trophoblasts induced by NME1 silencing. Interestingly, the expression of NME1 in either villi or decidua is higher significantly in miscarriage than that of the normal early pregnancy. These findings first reveal that the NME1 expressed in trophoblasts and DSCs controls the inappropriate invasion of human first-trimester trophoblast cells via MAPK/ERK1/2 signal pathway, and the overexpression of NME1 at maternal-fetal interface leads to pregnancy wastage.

GCS induces multidrug resistance by regulating apoptosis-related genes in K562/AO2 cell line.

We have previously shown that the expression of glucosylceramide synthase (GCS) gene in drug-resistant K562/AO2 human leukemia cell was higher than that in drug-sensitive K562 cell, and the sensitivity to adriamycin of K562/AO2 cell was enhanced by inhibiting GCS. It is concluded that the overexpression of GCS gene is one of the reasons which lead to multidrug resistance (MDR) of leukemia cell. Meanwhile, we also found that higher expression of Bcl-2 gene and protein were exhibited in K562/AO2 cell compared with K562 cell. Basing on this, we hypothesized that the high expression of GCS gene which results in MDR of leukemia cell is correlated with Bcl-2 signal transduction. In order to validate the hypothesis, the inhibition of GCS gene in K562/AO2 cell was observed by using chemical suppressor PPMP and siRNA targeted at GCS, and applying RT-PCR and flow cytometry, the expression levels of apoptosis-related gene Bcl-2 and Bax were analyzed before and after inhibiting GCS gene in K562/AO2 cell. The results demonstrated that the gene and protein of Bcl-2 in K562/AO2 cell were both down-regulated significantly after GCS gene being inhibited; however, the Bax mRNA expression had no apparent change in different groups. This suggested that GCS gene may contributed to MDR of human leukemia cell K562/AO2 by Bcl-2 signal transduction.

Overexpression of glucosylceramide synthase in associated with multidrug resistance of leukemia cells.

Ceramide, as a second messenger, initiates one of the major signal transduction pathways in tumor apoptosis. Glucosylceramide synthase (GCS) catalyzes glycosylation of ceramide and produces glucosylceramide. Through GCS, ceramide glycosylation allows cellular escape from ceramide-induced programmed cell death. Here we investigated the expression of GCS in human leukemia cells and an association between GCS and multidrug resistance of leukemia cells. Using RT-PCR technique the level of GCS gene was detected in 65 clinical multidrug resistance/non-resistance cases with leukemia, and in K562 and K562/A02 cell lines. AlamarBlue Assay was applied to confirm the multidrug resistant of K562/A02 cells. PPMP, which is a chemical inhibitor for GCS, was used to determine the relationship between GCS and drug-resistance in K562/A02 cells. In addition, multidrug resistance gene (mdr1), Bcl-2 and Bax mRNA was also analyzed by RT-PCR. The expression of GCS and mdr1 mRNA in clinic multidrug resistance samples exhibited significantly increased compared with clinic drug sensitive group (P<0.05). There was the positive correlation both the expression of GCS and mdr1 genes in leukemia samples (P<0.01, gamma=0.7). AlamarBlue Assay showed that the K562/A02 cell line was 115-fold more resistant to adriamycin and 36-fold more resistant to vincristine compared with drug-sensitive K562 cell line. There also was significant expression difference of GCS and mdr1 genes between K562 and K562/A02 cells. Bcl-2 gene exhibited higher expressions whatever in clinic drug-resistance samples or K562/A02 cells, whereas the expressions of Bax gene were higher in drug-sensitive samples and K562 cells. PPMP increased sensitivity to adriamycin toxicity by inhibiting GCS in K562/A02 cells. Therefore, it is suggested that a high level of GCS in leukemia is possible contributed to multidrug resistance of leukemia cells. Abnormally expressions of the genes in associated with cell apoptosis might be one of the main molecular pathology mechanisms of multidrug resistance caused by GCS gene.