Design, synthesis, and anti-tumor activity of derivatives of ring A and C-28 of asiatic acid.

Based on computer-aided drug design (CADD), the active groups of the known active small molecule compounds that can bind to EGFR target protein were analyzed through the molecular docking method. Then, 12 novel asiatic acid derivatives were synthesized by introducing active groups at ring A and C-28 positions of asiatic acid. The structures of these novel compounds were determined by NMR and MS. Furthermore, the anti-tumor activities of these derivatives on human lung cancer cells (A549) and human breast cancer cells (MCF-7) were evaluated by MTT assay. In conclusion, compounds I4 and II3 have stronger anti-cancer activity than parent compounds, the activities were stronger than gefitinib and comparable to afatinib, which may be potential candidate compounds for tumor therapy.

Virome Analysis of Aconitum carmichaelii Reveals Infection by Eleven Viruses, including Two Potentially New Species.

Aconitum carmichaelii is a herbaceous herb indigenous to China that has been cultivated for traditional medicine for centuries. Virus-like symptoms of A. carmichaelii plants were observed on leaves in some A. carmichaelii plantations in Zhanyi and Wuding Counties, Yunnan Province, southwest China. High-throughput sequencing (HTS) was performed on 28 symptomatic plants, and the results revealed infection with 11 viruses, including 2 novel viruses and 9 previously described viruses: Aconitum amalgavirus 1 (AcoAV-1), aconite virus A (AcVA), cucumber mosaic virus (CMV), currant latent virus (CuLV), apple stem grooving virus (ASGV), chilli veinal mottle virus (ChiVMV), tomato spotted wilt orthotospovirus (TSWV), tobacco vein distorting virus (TVDV), and potato leafroll virus (PLRV). Two novel viruses tentatively named Aconitum potyvirus 1 and Aconitum betapartitivirus 1, were supported by sequence and phylogenetic analysis results of their genomes. We proposed the names Potyvirus aconiti and Betapartitivirus aconiti. RT-PCR assays of 142 plants revealed the predominance and widespread distribution of CMV, AcVA, and AcoPV-1 in plantations. The detection of isolates of CuLV, ASGV, ChiVMV, TSWV, TVDV, and PLRV infections for the first time in A. carmichaelii expands their known host ranges.

Design, synthesis, and antitumor activity of novel oleanolic acid analogues targeting EGFR.

Based on the simulated docking of Epidermal growth factor receptor inhibitors with known active small molecule compounds, computer-aided drug design technology was used to analyze key amino acid fragments and determine the active groups binding with key sites. Then, twelve novel analogues of oleanolic acid (OA) were synthesized by introducing active groups at the C-3 and C-28 positions of OA. The structures of these novel analogues were confirmed by NMR and MS. Furthermore, the antitumor activities of these novel analogues were evaluated by MTT assay. As a result, compounds I3 and II3 showed stronger cytotoxicity on tumor cells than positive controls. In conclusion, our study synthesized twelve novel analogues of OA and determined compounds I3 and II3 had better antitumor effect, which may be potential candidate compounds for tumor therapy.

Synthesis and anti-tumor activity of asiatic acid derivatives targeting VEGFR.

To discovery novel VEGFR inhibitors, 12 novel asiatic acid derivatives were designed by computer-aided drug design (CADD) technology. Then, these novel asiatic acid derivatives were synthesized by introducing active groups at ring A and C-28 positions of asiatic acid. The structures of these novel analogues were confirmed by NMR and MS. Moreover, the anti-tumor activities of these novel asiatic acid derivatives on human hepatoma cells HepG2 and human gastric cancer cells SGC7901 were evaluated by MTT assay. As a result, compounds I2 and II4 showed stronger cytotoxicity on tumor cells than asiatic acid and positive control drugs such as gefitinib and paclitaxel. In conclusion, our study synthesized twelve novel asiatic acid derivatives and determined compounds I2 and II4 had better anti-tumor effect which may be potential candidate compounds for tumor therapy.

The N-Myc-responsive lncRNA MILIP promotes DNA double-strand break repair through non-homologous end joining.

The protooncoprotein N-Myc, which is overexpressed in approximately 25% of neuroblastomas as the consequence of MYCN gene amplification, has long been postulated to regulate DNA double-strand break (DSB) repair in neuroblastoma cells, but experimental evidence of this function is presently scant. Here, we show that N-Myc transcriptionally activates the long noncoding RNA MILIP to promote nonhomologous end-joining (NHEJ) DNA repair through facilitating Ku70-Ku80 heterodimerization in neuroblastoma cells. High MILIP expression was associated with poor outcome and appeared as an independent prognostic factor in neuroblastoma patients. Knockdown of MILIP reduced neuroblastoma cell viability through the induction of apoptosis and inhibition of proliferation, retarded neuroblastoma xenograft growth, and sensitized neuroblastoma cells to DNA-damaging therapeutics. The effect of MILIP knockdown was associated with the accumulation of DNA DSBs in neuroblastoma cells largely due to decreased activity of the NHEJ DNA repair pathway. Mechanistical investigations revealed that binding of MILIP to Ku70 and Ku80 increased their heterodimerization, and this was required for MILIP-mediated promotion of NHEJ DNA repair. Disrupting the interaction between MILIP and Ku70 or Ku80 increased DNA DSBs and reduced cell viability with therapeutic potential revealed where targeting MILIP using Gapmers cooperated with the DNA-damaging drug cisplatin to inhibit neuroblastoma growth in vivo. Collectively, our findings identify MILIP as an N-Myc downstream effector critical for activation of the NHEJ DNA repair pathway in neuroblastoma cells, with practical implications of MILIP targeting, alone and in combination with DNA-damaging therapeutics, for neuroblastoma treatment.

Complete genome sequence of Aconitum amalgavirus 1, a distinct member of the genus Amalgavirus.

A novel virus named Aconitum amalgavirus 1 (AcoAV-1) was identified in Chinese aconite (Aconitum carmichaelii) plants. The complete genome of AcoAV-1 is 3,370 nucleotides long, containing two partially overlapping open reading frames encoding a putative coat protein and a RNA-dependent RNA polymerase, respectively. Its fusion protein shares 34.9%-50.7% amino acid sequence identity with other amalgaviruses. Phylogenetic analysis showed that this virus formed a clade with blueberry latent virus and four other related viruses, suggesting that it belongs to the genus Amalgavirus in the family Amalgaviridae.

Development of a Recombinase-aided Amplification Combined With Lateral Flow Dipstick Assay for the Rapid Detection of the African Swine Fever Virus.

OBJECTIVE: To establish a sensitive, simple and rapid detection method for African swine fever virus (ASFV) B646L gene. METHODS: A recombinase-aided amplification-lateral flow dipstick (RAA-LFD) assay was developed in this study. Recombinase-aided amplification (RAA) is used to amplify template DNA, and lateral flow dipstick (LFD) is used to interpret the results after the amplification is completed. The lower limits of detection and specificity of the RAA assay were verified using recombinant plasmid and pathogenic nucleic acid. In addition, 30 clinical samples were tested to evaluate the performance of the RAA assay. RESULTS: The RAA-LFD assay was completed within 15 min at 37 °C, including 10 min for nucleic acid amplification and 5 minutes for LFD reading results. The detection limit of this assay was found to be 200 copies per reaction. And there was no cross-reactivity with other swine viruses. CONCLUSION: A highly sensitive, specific, and simple RAA-LFD method was developed for the rapid detection of the ASFV.

The pan-cancer lncRNA PLANE regulates an alternative splicing program to promote cancer pathogenesis.

Genomic amplification of the distal portion of chromosome 3q, which encodes a number of oncogenic proteins, is one of the most frequent chromosomal abnormalities in malignancy. Here we functionally characterise a non-protein product of the 3q region, the long noncoding RNA (lncRNA) PLANE, which is upregulated in diverse cancer types through copy number gain as well as E2F1-mediated transcriptional activation. PLANE forms an RNA-RNA duplex with the nuclear receptor co-repressor 2 (NCOR2) pre-mRNA at intron 45, binds to heterogeneous ribonucleoprotein M (hnRNPM) and facilitates the association of hnRNPM with the intron, thus leading to repression of the alternative splicing (AS) event generating NCOR2-202, a major protein-coding NCOR2 AS variant. This is, at least in part, responsible for PLANE-mediated promotion of cancer cell proliferation and tumorigenicity. These results uncover the function and regulation of PLANE and suggest that PLANE may constitute a therapeutic target in the pan-cancer context.

c-Myc inactivation of p53 through the pan-cancer lncRNA MILIP drives cancer pathogenesis.

The functions of the proto-oncoprotein c-Myc and the tumor suppressor p53 in controlling cell survival and proliferation are inextricably linked as "Yin and Yang" partners in normal cells to maintain tissue homeostasis: c-Myc induces the expression of ARF tumor suppressor (p14ARF in human and p19ARF in mouse) that binds to and inhibits mouse double minute 2 homolog (MDM2) leading to p53 activation, whereas p53 suppresses c-Myc through a combination of mechanisms involving transcriptional inactivation and microRNA-mediated repression. Nonetheless, the regulatory interactions between c-Myc and p53 are not retained by cancer cells as is evident from the often-imbalanced expression of c-Myc over wildtype p53. Although p53 repression in cancer cells is frequently associated with the loss of ARF, we disclose here an alternate mechanism whereby c-Myc inactivates p53 through the actions of the c-Myc-Inducible Long noncoding RNA Inactivating P53 (MILIP). MILIP functions to promote p53 polyubiquitination and turnover by reducing p53 SUMOylation through suppressing tripartite-motif family-like 2 (TRIML2). MILIP upregulation is observed amongst diverse cancer types and is shown to support cell survival, division and tumourigenicity. Thus our results uncover an inhibitory axis targeting p53 through a pan-cancer expressed RNA accomplice that links c-Myc to suppression of p53.

SENEBLOC, a long non-coding RNA suppresses senescence via p53-dependent and independent mechanisms.

Long non-coding RNAs (lncRNAs) have emerged as important biological tuners. Here, we reveal the role of an uncharacterized lncRNA we call SENEBLOC that is expressed by both normal and transformed cells under homeostatic conditions. SENEBLOC was shown to block the induction of cellular senescence through dual mechanisms that converge to repress the expression of p21. SENEBLOC facilitates the association of p53 with MDM2 by acting as a scaffold to promote p53 turnover and decrease p21 transactivation. Alternatively, SENEBLOC was shown to affect epigenetic silencing of the p21 gene promoter through regulation of HDAC5. Thus SENEBLOC drives both p53-dependent and p53-independent mechanisms that contribute to p21 repression. Moreover, SENEBLOC was shown to be involved in both oncogenic and replicative senescence, and from the perspective of senolytic agents we show that the antagonistic actions of rapamycin on senescence are dependent on SENEBLOC expression.

Construction of gold-siRNA NPR1 nanoparticles for effective and quick silencing of NPR1 in Arabidopsis thaliana.

In recent years, gold nanoparticles (AuNPs) have been widely used as gene silencing agents and therapeutics for treatment of cancers due to their high transfection efficiency and lack of cytotoxicity, but their roles in gene silencing in plants have not yet been reported. Here, we report synthesis of AuNPs-branched polyethylenimine and its integration with the small interfering RNAs (siRNA) of NPR1 to form a AuNPs-siRNA NPR1 compound. Our results showed that AuNPs-siRNA NPR1 was capable of infiltrating into Arabidopsis cells. AuNPs-siRNA NPR1 silenced 80% of the NPR1 gene in Arabidopsis. Bacteriostatic and ion leakage experiments suggest that the NPR1 gene in Arabidopsis leaves was silenced by AuNPs-siRNA NPR1 . In Columbia-0 plants, compared with the control group treated with buffer solution, the AuNPs-siRNA NPR1 treatment significantly increased the number of colonies and cell death, and the leaves turned yellow, similar to the phenotype of the npr1 leaves. These results indicated this AuNPs-siRNA NPR1 silencing the NPR1 gene method is simple, effective and quick (3 days), and a powerful tool to study gene functions in plants.

Standardization of BCR-ABL1 quantification on the international scale in China using locally developed secondary reference panels.

For patients with chronic myeloid leukemia, reverse transcription quantitative polymerase chain reaction is widely used in laboratories to quantify BCR-ABL1 fusion gene transcripts for disease management. Many efforts have been made to standardize the BCR-ABL1 testing assay, including the primary and secondary reference reagents, but the secondary standards have not been developed and used in the standardization program in China. With the use of armored RNA technology, armored RNA of BCR-ABL1 and control genes was manufactured to prepare the secondary reference material anchored to the World Health Organization primary reference calibrators for standardization of BCR-ABL1 testing assays. The secondary reference was sent to 30 laboratories in China for validation. Data from an external quality assessment after the standardization process were collected and analyzed as well. The assigned %BCR-ABL1/ABL1IS values of the four levels of the secondary material panels were 0.0118, 0.1345, 1.3808, and 19.4266, respectively. In validation trials, 70.0% (21/30) of laboratories obtained valid conversion factors for the BCR-ABL1 assay. All valid conversion factors from 11 international scale laboratories were equivalent to their respective previous values. External quality assessment data indicated that the accuracy and precision between laboratories were improved. Moreover, the quantity of the panels is abundant to be used as quality control samples for monitoring the shift of data. In this study, we established a secondary genetic reference panel for BCR-ABL1 quantification. This study will play a role in facilitating the worldwide dissemination of the international scale, especially in promoting the standardization of molecular monitoring in China.

Quantitative (stereological) study of the epididymis and seminal vesicle in the rat from young to old.

There is a scarcity of morphometric data on the developmental and ageing changes in the epididymis and seminal vesicle in young and old rats. Eighty-six normal male Sprague-Dawley rats were randomly sampled from a cohort of animals aged 1-36 months (7-9 animals each age group). The epididymis and seminal vesicle (with the closely attached coagulating gland) were removed, and methacrylate resin-embedded sections were prepared for quantitative study of key histological structures by light microscopy. Stereological methods (point counting and optical disector) were used to estimate the total volumes of sperm mass, secretion (glandular lumen) and other structures and the number of spermatozoa. The results showed that the rapid growth of the reproductive organs was between 1 and 4 months of age. The epididymis stored the largest volume of sperm mass or number of spermatozoa at 12 months of age, but thereafter until 36 months of age, the sperm storage did not markedly diminish. The volume of secretion stored in the seminal vesicular gland declined by more than 35% from a plateau at 12-18 months until 36 months of age while that in the coagulating gland declined by more than 30% from a plateau at 18-24 months until 36 months of age.

An Integrated Approach for Identifying Molecular Subtypes in Human Colon Cancer Using Gene Expression Data.

Identifying molecular subtypes of colorectal cancer (CRC) may allow for more rational, patient-specific treatment. Various studies have identified molecular subtypes for CRC using gene expression data, but they are inconsistent and further research is necessary. From a methodological point of view, a progressive approach is needed to identify molecular subtypes in human colon cancer using gene expression data. We propose an approach to identify the molecular subtypes of colon cancer that integrates denoising by the Bayesian robust principal component analysis (BRPCA) algorithm, hierarchical clustering by the directed bubble hierarchical tree (DBHT) algorithm, and feature gene selection by an improved differential evolution based feature selection method (DEFSW) algorithm. In this approach, the normal samples being completely and exclusively clustered into one class is considered to be the standard of reasonable clustering subtypes, and the feature selection pays attention to imbalances of samples among subtypes. With this approach, we identified the molecular subtypes of colon cancer on the mRNA gene expression dataset of 153 colon cancer samples and 19 normal control samples of the Cancer Genome Atlas (TCGA) project. The colon cancer was clustered into 7 subtypes with 44 feature genes. Our approach could identify finer subtypes of colon cancer with fewer feature genes than the other two recent studies and exhibits a generic methodology that might be applied to identify the subtypes of other cancers.

[Study of Kinase Inhibitor WP1066 on the STAT3/JAK2 of Prostate Stromal Cells].

OBJECTIVE: To study the role of JAK2 signaling pathway in prostate stromal cells and the effect of inhibitor WP1066 on its expression. METHODS: The phosphorylation of JAK2 and STAT3 in prostate tissues of patients with benign prostatic hyperplasia (BHP) (n=4) and severe histological prostatitis (HP) plus BPH (n=4) was tested by using Western blot to verify the activation of their mediated signaling pathway. Kinase inhibitor WP1066 was added to prostate stromal cells to detect inhibition of the JAK2 and STAT3 activation launched by IL-6. RESULTS: JAK2 phosphorylation level (pJAK2) was significantly increased in the patients with severe HP plus BPH,and the expression of JAK2 or STAT3 was not decreased in WP1066 treatment cells. However,neither phosphorylation in JAK2 nor STAT3 was able to be detected in the cells treated with WP1066 or WP1066+IL-6,indicating that the signaling pathway of JAK2-STAT3 was inhibited. CONCLUSION: JAK/STAT signaling pathway is activated in patients with severe HP plus BPH , but could be inhibited by WP1066.

Viral metagenomics of six bat species in close contact with humans in southern China.

Accumulating studies have shown that bats could harbor various important pathogenic viruses that could be transmitted to humans and other animals. Extensive metagenomic studies of different organs/tissues from bats have revealed a large number of novel or divergent viruses. To elucidate viral diversity and epidemiological and phylogenetic characteristics, six pooled fecal samples from bats were generated (based on bat species and geographic regions characteristic for virome analysis). These contained 500 fecal samples from six bat species, collected in four geographic regions. Metagenomic analysis revealed a plethora of divergent viruses originally found in bats. Multiple contigs from influenza A virus and coronaviruses in bats shared high identity with those from humans, suggesting possible cross-species transmission, whereas a number of contigs, whose sequences were taxonomically classifiable within Alphapapillomavirus, Betaretrovirus, Alpharetrovirus, Varicellovirus, Cyprinivirus, Chlorovirus and Cucumovirus had low identity to viruses in existing databases, which indicated possible evolution of novel viral species. None of the established caliciviruses and picornaviruses were found in the 500 fecal specimens. Papillomaviruses with high amino acid identity were found in Scotophilus kuhlii and Rhinolophus blythi, challenging the hypotheses regarding the strict host specificity and co-evolution of papillomaviruses. Phylogenetic analysis showed that four bat rotavirus A strains might be tentative G3 strains, according to the Rotavirus Classification Working Group classification.

Serum glycated albumin, glycated hemoglobin, and arterial stiffness in a general Chinese population.

BACKGROUND: Both glycated albumin (GA) and glycated hemoglobin (HbA1c) reflect the mean glucose levels. This study was conducted to investigate the relationships among GA, HbA1c, and arterial stiffness in the general population. METHODS: A total of 11,014 participants were included. Serum GA; HbA1c; and arterial stiffness indices, including brachial-ankle pulse wave velocity (baPWV) and central systolic blood pressure (cSBP), were measured. Single-factor and multivariate regression analyses were performed. Receiver operating characteristic (ROC) analysis was performed to compare the predictive value of GA, HbA1c, and their combination for arterial stiffness. All analyses were stratified by sex. RESULTS: Men had a lower GA level than women. GA, HbA1c, and plasma glucose levels were correlated. The levels of baPWV and cSBP increased across sex-specific quartiles of GA and HbA1c (P for trend<0.001 for all). Both GA and HbA1c were positively related to elevated baPWV and cSBP after adjusting for conventional factors (P<0.05 for all). These relationships remained significant when participants were divided into groups with normal glucose tolerance, prediabetes, or diabetes. Regarding screening for elevated baPWV and cSBP, the values of the area under the ROC curve (AUC) for GA were similar to those for HbA1c in men but were lower than those for HbA1c in women. The combination of GA and HbA1c did not improve the AUC compared with HbA1c alone. CONCLUSIONS: Both GA and HbA1c were associated with arterial stiffness. The predictive value of GA for arterial stiffness was similar in men but lower in women compared with that of HbA1c.

Obesity and novel cardiovascular markers in a population without diabetes and cardiovascular disease in China.

OBJECTIVE: To investigate associations of novel cardiovascular markers with obesity in a general population. METHODS: A total of 9361 individuals without diabetes or cardiovascular disease were studied between 2009 and 2012 in China. High-sensitivity cardiac troponin T (hs-cTnT), N-terminal pro-B-type natriuretic peptide (NT-proBNP), brachial-ankle pulse wave velocity (baPWV), pulse pressure, and central systolic blood pressure (cSBP) were assessed according to body mass index (BMI) levels and different BMI/metabolic syndrome (MetS) combinations. RESULTS: 'Levels of hs-cTnT, baPWV, pulse pressure, and cSBP increased across BMI levels. Obesity was positively associated with these markers in multivariate models (P<0.05 for all). When stratified by MetS, these associations remained significant in the non-MetS group, and compared with normal weight participants, the obese participants had 1.87 (95% confidence interval: 1.48, 2.36), 1.27 (1.02, 1.57), 1.89 (1.39, 2.57), and 2.71 (2.11, 3.47) fold risks for having elevated hs-cTnT, baPWV, pulse pressure, and cSBP, respectively, and had 1.61 (1.26, 2.05), 1.75 (1.27, 2.42), 2.45 (1.46, 4.11), and 3.14 (2.13, 4.62) fold risks for having 1, 2, 3, and 4 elevated cardiovascular markers, respectively; while no relationship was observed between obesity and these novel markers in the MetS group, after multivariate adjustment. These results were unchanged when using a waist-hip ratio, body fat per cent, and visceral adiposity index to redefine obesity. CONCLUSIONS: Obesity was positively associated with novel cardiovascular markers (except NT-proBNP) in participants without MetS rather than in participants with MetS. Obese participants without MetS also had higher odds of having more number of elevated cardiovascular markers.