Identifying SNP threshold from P2 sequences for investigating norovirus transmission.

Noroviruses are a group of non-enveloped single-stranded positive-sense RNA virus belonging to Caliciviridae family. They can be transmitted by the fecal-oral route from contaminated food and water and cause mainly acute gastroenteritis. Outbreaks of norovirus infections could be difficult to detect and investigate. In this study, we developed a simple threshold detection approach based on variations of the P2 domain of the capsid protein. We obtained sequences from the norovirus hypervariable P2 region using Sanger sequencing, including 582 pairs of epidemiologically-related strains from 35 norovirus outbreaks and 6402 pairs of epidemiologically-unrelated strains during the four epidemic seasons. Genetic distances were calculated and a threshold was performed by adopting ROC (Receiver Operating Characteristic) curve which identified transmission clusters in all tested outbreaks with 80 % sensitivity. In average, nucleotide diversity between outbreaks was 67.5 times greater than the diversity within outbreaks. Simple and accurate thresholds for detecting norovirus transmissions of three genotypes obtained here streamlines molecular investigation of norovirus outbreaks, thus enabling rapid and efficient responses for the control of norovirus.

The role of WSC domain-containing protein encoding gene AOL_s00043g401 in the growth and nematode trapping of Arthrobotrys oligospora.

Arthrobotrys oligospora is a model nematode-trapping fungus that has been extensively investigated to understand the interactions between fungi and nematodes. Nematode capture by A. oligospora is a complex process in which recognition of nematodes is generally believed to be mediated by lectins from the fungi. Lectins are a group of carbohydrate-binding proteins that widely exist in microorganisms and contain specific glycosylation recognition domains. In this work, we report the effect of a putative WSC domain-containing protein encoding gene AOL_s00043g401 (g401) on the growth and nematode-trapping of A. oligospora. The g401 gene was knocked out using the homologous recombination approach, and the △g401 mutant strain was then evaluated for its growth rate, conidial yield and germination rate, adaptation to environmental stresses, and nematocidal activity. Interestingly, the deletion of the putative lectin gene g401 had no significant effect on saprophytic growth, conidial yield and germination rate, responses to high salt, surfactant, and strong oxidative environments, as well as nematode-trapping efficiency of A. oligospora. We speculate that this phenomenon might have been caused by an intrinsic genetic compensation of the g401 in this fungus. For instance, more copies of WSC domain encoding genes or PQQ-DH domain encoding genes were found in the genome of A. oligospora. These findings provide further experimental evidence on the effect of lectin-related functional proteins in A. oligospora on nematode capture and will help further analyze their potential roles in the biological control of nematodes in the future.

The fucose-specific lectin gene AOL_s00054g276 affects trap formation and nematocidal activity of the nematophagous fungus Arthrobotrys oligospora.

Nematode-trapping fungi are natural enemies of nematodes in nature. Arthrobotrys oligospora, a typical nematode-trapping fungus with a clear genetic background, can capture and infect nematodes by forming adhesive three-dimensional networks. Lectins, a class of glycoproteins containing glycosyl-specific recognition domains, play an important role in biological recognition. However, the fucose-specific lectins have rarely been studied regarding the process of preying on nematodes. In this study, we characterized the biological role of the fucose-specific lectin-encoding gene AOL_s00054g276 (g276) in A. oligospora. The gene g276 was first deleted based on homologous recombination, then the phenotype and nematocidal activity of the Δg276 mutant was evaluated. The results showed that the deletion of gene g276 delayed trap formation and weakened its nematocidal activity; however, mycelial growth, conidia production, conidial germination rates and adaption to environmental stresses were not affected. Our results suggest that the fucose-specific lectin-encoding gene g276 might be associated with the morphogenesis of this fungus, and its deletion resulted in a significantly low density of three-dimensional traps (P < 0.05) and a significantly low nematode-trapping efficiency (P < 0.001). These findings provide a basis for further elucidating the mechanism of A. oligospora preying on nematodes and lay a foundation for the development and utilization of fungal-derived lectins for nematode control in the future.

The role of Jacalin-related lectin gene AOL_s00083g511 in the development and pathogenicity of the nematophagous fungus Arthrobotrys oligospora.

Arthrobotrys oligospora is a model species of nematophagous fungi and has great potential for the biological control of nematode diseases. Lectin is a protein that binds to carbohydrates and their complexes with high specificity, which mediates recognition events in various physiological and pathological processes. This study aimed to investigate the role of the Jacalin-related lectin (JRL) gene, AOL_s00083g511, in A. oligospora development. Through a homology recombination approach, we obtained the AOL_s00083g511 knockout mutant strain (Ag511). Next, the biological characteristics of the Ag511 mutant strain, including growth rate, conidia germination rate, adaptation to environmental stresses, and nematocidal activity, were compared with those of the wild-type (WT) strain. The results showed that the JRL gene AOL_s00083g511 did not affect fungal growth, conidia germination, 3D-trap formation, and the ability of A. oligospora to prey on nematodes significantly. We speculate that this phenomenon may be caused by a loss of the key ß1-ß2 loops in the AOL_ s00083g511-encoded JRL domain and an intrinsic genetic compensation of AOL_s00083g511 in this fungus. The growth rates of both strains on high salt or surfactant media were similar; however, in the strong oxidation medium, the growth rate of the Ag511 mutant was significantly lower than that of the WT strain, indicating that AOL_s00083g511 might play a role in oxidative stress resistance. These findings provide a basis for further analysis of the related functions of the JRL gene in A. oligospora and their potential roles in the biological control of nematodes in the future.

Liquiritin from Glycyrrhiza uralensis Attenuating Rheumatoid Arthritis via Reducing Inflammation, Suppressing Angiogenesis, and Inhibiting MAPK Signaling Pathway.

Among the various treatments, induction of synoviocyte apoptosis by natural products during a rheumatoid arthritis (RA) pathological condition can be considered to have vast potential. However, it is unclear that liquiritin, a kind of natural flavonoid extracted from the roots of Glycyrrhiza uralensis, induced the apoptosis of the synovial membrane and its molecular mechanism. In this study, interleukin-1ß (IL-1ß)-RA-FLS cells were incubated with different concentrations of liquiritin. An MTT assay, Hoechst 33342 staining, JC-1 staining, and Western blot were used to check the viability, cell apoptosis, mitochondrial membrane potential changes, and the expression of related proteins, respectively. In vivo, a TUNEL assay and HE staining of tissue were used for histopathological evaluation. Our results showed that liquiritin significantly inhibited the proliferation of IL-1ß-induced-RA-FLS, promoted nuclear DNA fragmentation, and changed the mitochondrial membrane potential to accelerate cell apoptosis. Liquiritin downregulated the ratio of Bcl-2/Bax and inhibited the VEGF expression and phosphorylation of JNK and P38. Moreover, liquiritin improved the clinical score of rheumatism, inflammatory infiltration, and angiogenesis and induced apoptosis of the synovial tissue in vivo. Hence, liquiritin ameliorates RA by reducing inflammation, blocking MAPK signaling, and restraining angiogenesis.

Epigenetic synergy between decitabine and platinum derivatives.

BACKGROUND: Aberrant epigenetic silencing of tumor suppressor genes has been recognized as a driving force in cancer. Epigenetic drugs such as the DNA methylation inhibitor decitabine reactivate genes and are effective in myeloid leukemia, but resistance often develops and efficacy in solid tumors is limited. To improve their clinical efficacy, we searched among approved anti-cancer drugs for an epigenetic synergistic combination with decitabine. RESULTS: We used the YB5 cell line, a clonal derivative of the SW48 colon cancer cell line that contains a single copy of a hypermethylated cytomegalovirus (CMV) promoter driving green fluorescent protein (GFP) to screen for drug-induced gene reactivation and synergy with decitabine. None of the 16 anti-cancer drugs tested had effects on their own. However, in combination with decitabine, platinum compounds showed striking synergy in activating GFP. This was dose dependent, observed both in concurrent and sequential combinations, and also seen with other alkylating agents. Clinically achievable concentrations of carboplatin at (25 µM) and decitabine reactivated GFP in 28 % of the YB5 cells as compared to 15 % with decitabine alone. Epigenetic synergy was also seen at endogenously hypermethylated tumor suppressor genes such as MLH1 and PDLIM4. Genome-wide studies showed that reactivation of hypermethylated genes by the combination was significantly better than that induced by decitabine alone or carboplatin alone. Platinum compounds did not enhance decitabine-induced hypomethylation. Rather, we found significantly inhibited HP1α expression by carboplatin and the combination. This was accompanied by increased histone H3 lysine 4 (H3K4) trimethylation and histone H3 lysine 9 (H3K9) acetylation at reactivated genes (P < 0.0001) and reduced occupancy by methyl-binding proteins including MeCP2 and methyl-CpG-binding domain protein 2 (MBD2) (P < 0.0001). CONCLUSIONS: Our results suggest that the combination of decitabine with platinum analogs shows epigenetic synergy that might be exploited in the treatment of different cancers.

Repetitive elements and enforced transcriptional repression co-operate to enhance DNA methylation spreading into a promoter CpG-island.

Repression of many tumor suppressor genes in cancer is concurrent with aberrantly increased DNA methylation levels at promoter CpG islands (CGIs). About one-fourth of empirically defined human promoters are surrounded by or contain clustered repetitive elements. It was previously observed that a sharp transition of methylation exists between highly methylated repetitive elements and unmethylated promoter-CGIs in normal tissues. The factors that lead to aberrant CGI hypermethylation in cancer remain poorly understood. Here, we established a site-specific integration system with enforced local transcriptional repression in colorectal cancer cells and monitored the occurrence of initial de novo methylation at specific CG sites adjacent to the CGI of the INSL6 promoter, which could be accelerated by binding a KRAB-containing transcriptional factor. Additional repetitive elements from P16 and RIL (PDLIM4), if situated adjacent to the promoter of INSL6, could confer DNA methylation spreading into the CGI particularly in the setting of KRAB-factor binding. However, a repressive chromatin alone was not sufficient to initiate DNA methylation, which required specific DNA sequences and was integration-site (and/or cell-line) specific. Overall, these results demonstrate a requirement for specific DNA sequences to trigger de novo DNA methylation, and repetitive elements as cis-regulatory factors to cooperate with advanced transcriptional repression in promoting methylation spreading.

DNA methylation does not stably lock gene expression but instead serves as a molecular mark for gene silencing memory.

DNA methylation is commonly thought of as a "molecular lock" that leads to permanent gene silencing. To investigate this notion, we tested 24 different histone deacetylase inhibitors (HDACi) on colon cancer cells that harbor a GFP locus stably integrated and silenced by a hypermethylated cytomegalovirus (CMV) promoter. We found that HDACi efficiently reactivated expression of GFP and many other endogenous genes silenced by DNA hypermethylation. After treatment, all promoters were marked with active chromatin, yet DNA hypermethylation did not change. Thus, DNA methylation could not prevent gene reactivation by drug-induced resetting of the chromatin state. In evaluating the relative contribution of DNA methylation and histone modifications to stable gene silencing, we followed expression levels of GFP and other genes silenced by DNA hypermethylation over time after treatment with HDACi or DNA-demethylating drugs. Reactivation of methylated loci by HDACi was detectable for only 2 weeks, whereas DNA-demethylating drugs induced permanent epigenetic reprogramming. Therefore, DNA methylation cannot be considered as a lock for gene expression but rather as a memory signal for long-term maintenance of gene silencing. These findings define chromatin as an important druggable target for cancer epigenetic therapy and suggest that removal of DNA methylation signals is required to achieve long-term gene reactivation.

Mechanisms of resistance to decitabine in the myelodysplastic syndrome.

PURPOSE: The DNA methylation inhibitor 5-aza-2'-deoxycytidine (DAC) is approved for the treatment of myelodysplastic syndromes (MDS), but resistance to DAC develops during treatment and mechanisms of resistance remain unknown. Therefore, we investigated mechanisms of primary and secondary resistance to DAC in MDS. PATIENTS AND METHODS: We performed Quantitative Real-Time PCR to examine expression of genes related to DAC metabolism prior to therapy in 32 responders and non-responders with MDS as well as 14 patients who achieved a complete remission and subsequently relapsed while on therapy (secondary resistance). We then performed quantitative methylation analyses by bisulfite pyrosequencing of 10 genes as well as Methylated CpG Island Amplification Microarray (MCAM) analysis of global methylation in secondary resistance. RESULTS: Most genes showed no differences by response, but the CDA/DCK ratio was 3 fold higher in non-responders than responders (P<.05), suggesting that this could be a mechanism of primary resistance. There were no significant differences at relapse in DAC metabolism genes, and no DCK mutations were detected. Global methylation measured by the LINE1 assay was lower at relapse than at diagnosis (P<.05). On average, the methylation of 10 genes was lower at relapse (16.1%) compared to diagnosis (18.1%) (P<.05). MCAM analysis showed decreased methylation of an average of 4.5% (range 0.6%-9.7%) of the genes at relapse. By contrast, new cytogenetic changes were found in 20% of patients. CONCLUSION: Pharmacological mechanisms are involved in primary resistance to DAC, whereas hypomethylation does not prevent a relapse for patients with DAC treatment.

Decoding the intrinsic mechanism that prohibits ALIX interaction with ESCRT and viral proteins.

The adaptor protein ALIX [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] links retroviruses to ESCRT (endosomal sorting complex required for transport) machinery during retroviral budding. This function of ALIX requires its interaction with the ESCRT-III component CHMP4 (charged multivesicular body protein 4) at the N-terminal Bro1 domain and retroviral Gag proteins at the middle V domain. Since cytoplasmic or recombinant ALIX is unable to interact with CHMP4 or retroviral Gag proteins under non-denaturing conditions, we constructed ALIX truncations and mutations to define the intrinsic mechanism through which ALIX interactions with these partner proteins are prohibited. Our results demonstrate that an intramolecular interaction between Patch 2 in the Bro1 domain and the TSG101 (tumour susceptibility gene 101 protein)-docking site in the proline-rich domain locks ALIX into a closed conformation that renders ALIX unable to interact with CHMP4 and retroviral Gag proteins. Relieving the intramolecular interaction of ALIX, by ectopically expressing a binding partner for one of the intramolecular interaction sites or by deleting one of these sites, promotes ALIX interaction with these partner proteins and facilitates ALIX association with the membrane. Ectopic expression of a GFP (green fluorescent protein)-ALIX mutant with a constitutively open conformation, but not the wild-type protein, increases EIAV (equine infectious anaemia virus) budding from HEK (human embryonic kidney)-293 cells. These findings predict that relieving the autoinhibitory intramolecular interaction of ALIX is a critical step for ALIX to participate in retroviral budding.

Chromatin remodeling is required for gene reactivation after decitabine-mediated DNA hypomethylation.

The DNA hypomethylating drug decitabine (DAC) reactivates silenced gene expression in cancer and is approved for the treatment of the myelodysplastic syndrome. Gene reactivation after DAC is variable and incompletely understood. Here, we established a cell line system (YB5) derived from the SW48 colon cancer cell line to study DAC-induced reactivation. YB5 contains a hypermethylated cytomegalovirus promoter driving green fluorescent protein (GFP), and the locus is transcriptionally silent. GFP reexpression can be achieved by DAC treatment, but the expression level of individual cells is heterogeneous. DAC-treated YB5 cells were separated into GFP-positive and GFP-negative subpopulations. By comparing DAC-treated sorted GFP-positive and GFP-negative cells, we found that their methylation levels were similarly decreased but that histone modifications and histone H3 densities were remarkably different. Despite a similar degree of (incomplete) DNA hypomethylation, GFP-positive cells reverted to an active chromatin structure marked by higher H3K9 acetylation, lower H3K27 trimethylation, and lower promoter nucleosome density. GFP-negative cells had histone modifications and promoter nucleosome density, similar to parental cells. On DAC withdrawal, gradual resilencing and remethylation occurred in both GFP-positive and GFP-negative cells, and the resilencing correlated with a gradual increase in nucleosome occupancy in GFP-positive cells. These data show that hypomethylation alone after DAC is insufficient for gene expression induction, and that chromatin resetting to an active state including nucleosome eviction is required for activation of protein expression. Our findings suggest that gene expression is the key in optimizing DAC treatment strategies in the clinic.

Mechanisms of resistance to 5-aza-2'-deoxycytidine in human cancer cell lines.

5-aza-2'-deoxycytidine (DAC) is approved for the treatment of myelodysplastic syndromes, but resistance to this agent is common. In search for mechanisms of resistance, we measured the half maximal (50%) inhibitory concentration (IC(50)) of DAC and found it differed 1000-fold among a panel of cancer cell lines. The IC(50) was correlated with the doses of DAC that induced the most hypomethylation of long interspersed nuclear elements (LINE; R = 0.94, P < .001), but not with LINE methylation or DNA methyltransferase 1 (DNMT1), 3a, and 3b expression at baseline. Sensitivity to DAC showed a low correlation (R = 0.44, P = .11) to that of 5-azacytidine (AZA), but a good correlation to that of cytarabine (Ara-C; R = 0.89, P < .001). The 5 cell lines most resistant to DAC had a combination of low dCK, hENT1, and 2 transporters, and high cytosine deaminase. In an HL60 clone, resistance to DAC could be rapidly induced by drug exposure and was related to a switch from heterozygous to homozygous mutation of DCK. Transfection of wild-type DCK restored DAC sensitivity. DAC induced DNA breaks as evidenced by H2AX phosphorylation and increased homologous recombination rates by 7- to 10-fold. These results suggest that in vitro resistance to DAC can be explained by insufficient incorporation into DNA.