The development and technologies of RNA therapeutics.

Since it was discovered for over 20 years ago, the potentiality of siRNAs in gene silencing in vitro and in vivo models has been recognized. Several studies in the new generation, molecular mechanisms, target attachment, and purification of RNA have supported the development of RNA therapeutics for a variety of applications. RNA therapeutics are growing rapidly with various platforms contributing to the standard of personalized medicine and rare disease treatment. Therefore, understanding the development and technologies of RNA therapeutics becomes a crucial point for new drug generation. Here, the primary purpose of this review is to provide a general view of six therapeutic categories that make up RNA-based therapeutic approaches, including RNA-target therapeutics, protein-targeted therapeutics, cellular reprogramming and tissues engineering, RNA-based protein replacement therapeutics, RNA-based genome editing, and RNA-based immunotherapies based on non-coding RNAs and coding RNA. Furthermore, we present an overview of the RNA strategies regarding viral approaches and nonviral approaches in designing a new generation of RNA technologies. The advantages and challenges of using RNA therapeutics are also discussed along with various approaches for RNA delivery. Therefore, this review is designed to provide updated reference evidence of RNA therapeutics in the battle against rare or difficult-to-treat diseases for researchers in this field.

Machine Learning Heuristics on Gingivobuccal Cancer Gene Datasets Reveals Key Candidate Attributes for Prognosis.

Delayed cancer detection is one of the common causes of poor prognosis in the case of many cancers, including cancers of the oral cavity. Despite the improvement and development of new and efficient gene therapy treatments, very little has been carried out to algorithmically assess the impedance of these carcinomas. In this work, from attributes or NCBI's oral cancer datasets, viz. (i) name, (ii) gene(s), (iii) protein change, (iv) condition(s), clinical significance (last reviewed). We sought to train the number of instances emerging from them. Further, we attempt to annotate viable attributes in oral cancer gene datasets for the identification of gingivobuccal cancer (GBC). We further apply supervised and unsupervised machine learning methods to the gene datasets, revealing key candidate attributes for GBC prognosis. Our work highlights the importance of automated identification of key genes responsible for GBC that could perhaps be easily replicated in other forms of oral cancer detection.

Target Capture Reveals the Complex Origin of Vietnamese Ginseng.

The global market of the medicinal plant ginseng is worth billions of dollars. Many ginseng species are threatened in the wild and effective sustainable development initiatives are necessary to preserve biodiversity at species and genetic level whilst meeting the demand for medicinal produce. This is also the case of Panax vietnamensis Ha & Grushv., an endemic and threatened ginseng species in Vietnam that is locally cultivated at different scales and has been the object of national breeding programs. To investigate the genetic diversity within cultivated and wild populations of P. vietnamensis we captured 353 nuclear markers using the Angiosperm-353 probe set. Genetic diversity and population structure were evaluated for 319 individuals of Vietnamese ginseng across its area of distribution and from wild and a varying range of cultivated areas. In total, 319 individuals were sampled. After filtering, 1,181 SNPs were recovered. From the population statistics, we observe high genetic diversity and high genetic flow between populations. This is also supported by the STRUCTURE analysis. The intense gene flow between populations and very low genetic differentiation is observed regardless of the populations' wild or cultivated status. High levels of admixture from two ancestral populations exist in both wild and cultivated samples. The high gene flow between populations can be attributed to ancient and on-going practices of cultivation, which exist in a continuum from understorey, untended breeding to irrigated farm cultivation and to trade and exchange activities. These results highlight the importance of partnering with indigenous peoples and local communities and taking their knowledge into account for biodiversity conservation and sustainable development of plants of high cultural value.

Inappropriate supply of antibiotics for common viral infections by community pharmacies in Vietnam: A standardised patient survey.

Background: This study aimed to evaluate the appropriateness of antibiotic dispensing of private pharmacies in Vietnam. Methods: Standardised patient surveys were conducted in randomly selected community pharmacies across 40 districts in Vietnam. Four clinical scenarios were represented by patient actors: (a) an adult requesting treatment for a sibling with a viral upper respiratory tract infection (URTI), (b) a parent requesting treatment for a child with acute diarrhoea, (c) an adult making a direct antibiotic request, and (d) an adult presenting with an antibiotic prescription. We calculated the proportion of interactions that resulted in inappropriate supply of antibiotics and patient advice. Predictors of inappropriate antibiotic supply were assessed. Findings: Patient actors attended 949 pharmacies, resulting in 1266 clinical interactions. Antibiotics were inappropriately supplied to 92% (291/316) of adults requesting treatment for URTI symptoms, 43% (135/316) for children with acute diarrhoea symptoms and to 84% (267/317) of direct request for antibiotics. Only 49% of pharmacies advised patients regarding their antibiotic use. Female actors were more likely to be given antibiotics than male actors for URTI (aOR 2·71, 1·12-6·60) but not for diarrhoeal disease. Pharmacies in northern Vietnam were more likely than those in southern Vietnam to supply antibiotics without a prescription: for adult URTI (aOR=5·8, 95% CI: 2·2-14·9) and childhood diarrhoea (aOR=3·5, 95% CI: 2·0-6·0) symptoms, but less likely to dispense for direct antibiotics request. Interpretation: Inappropriate antibiotic supply was common in Vietnamese private pharmacies. Multifaceted measures are urgently needed to achieve WHO's global action plan for the optimal use of antimicrobials. Funding: This study was funded by a grant from the Australian Department of Foreign Affairs and Trade.

P2Y1 agonist HIC in combination with androgen receptor inhibitor abiraterone acetate impairs cell growth of prostate cancer.

P2Y receptors belong to the large superfamily of G-protein-coupled receptors and play a crucial role in cell death and survival. P2Y1 receptor has been identified as a marker for prostate cancer (PCa). A previously unveiled selective P2Y1 receptor agonist, the indoline-derived HIC (1-(1-((2-hydroxy-5-nitrophenyl)(4-hydroxyphenyl)methyl)indoline-4-carbonitrile), induces a series of molecular and biological responses in PCa cells PC3 and DU145, but minimal toxicity to normal cells. Here, we evaluated the combinatorial effect of HIC with abiraterone acetate (AA) targeted on androgen receptor (AR) on the inhibition of PCa cells. Here, the presence of HIC and AA significantly inhibited cell proliferation of PC3 and DU145 cells with time-dependent manner as a synerfistic combination. Moreover, it was also shown that the anticancer and antimetastasis effects of the combinratorial drugs were noticed through a decrease in colony-forming ability, cell migration, and cell invasion. In addition, the HIC + AA induced apoptotic population of PCa cells as well as cell cycle arrest in G1 progression phase. In summary, these studies show that the combination of P2Y1 receptor agonist, HIC and AR inhibitor, AA, effectively improved the antitumor activity of each drug. Thus, the combinatorial model of HIC and AA should be a novel and promising therapeutic strategy for treating prostate cancer.

Molecular interaction of HIC, an agonist of P2Y1 receptor, and its role in prostate cancer apoptosis.

Prostate cancer is a heterogeneous, slow growing asymptomatic cancer that predominantly affects man. A purinergic G-protein coupled receptor, P2Y1R, is targeted for its therapeutic value since it plays a crucial role in many key molecular events of cancer progression and invasion. Our previous study demonstrated that indoline derivative, 1 ((1-(2-Hydroxy-5-nitrophenyl) (4-hydroxyphenyl) methyl)indoline-4­carbonitrile; HIC), stimulates prostate cancer cell (PCa) growth inhibition via P2Y1R. However, the mode of interaction of P2Y1R with HIC involved in this process remains unclear. Here, we have reported the molecular interactions of HIC with P2Y1R. Molecular dynamics simulation was performed that revealed the stable specific binding of the protein-ligand complex. In vitro analysis has shown increased apoptosis of PCa-cells, PC3, and DU145, upon specific interaction of P2Y1R-HIC. This was further validated using siRNA analysis that showed a higher percentage of apoptotic cells in PCa-cells transfected with P2Y-siRNA-MRS2365 than P2Y-siRNA-HIC treatment. Decreased mitochondrial membrane potential (MMP) activity and reduced glutathione (GSH) level show their role in P2Y1R-HIC mediated apoptosis. These in silico and in vitro results confirmed that HIC could induce mitochondrial apoptotic signaling through the P2Y1R activation. Thus, HIC being a potential ligand upon interaction with P2Y1R might have therapeutic value for the treatment of prostate cancer.

Sero-Prevalence of SARS-CoV-2 Antibodies in High-Risk Populations in Vietnam.

As a response to the coronavirus disease 2019 (COVID-19) pandemic, Vietnam enforced strict quarantine, contact tracing and physical distancing policies resulting in one of the lowest numbers of individuals infected with severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) globally. This study aimed to determine the prevalence of SARS-CoV-2 antibody positivity among high-risk populations in Vietnam. A prevalence survey was undertaken within four communities in Vietnam, where at least two COVID-19 cases had been confirmed. Participants were classified according to the location of exposure: household contacts, close contacts, community members, and healthcare workers (HCWs) responsible for treating COVID-19 cases. Participants completed a baseline questionnaire and SARS-CoV-2 IgG antibodies were quantified using a commercial assay. A total of 3049 community members and 149 health care workers consented to the study. Among 13 individuals who were seropositive (0.4%), five household contacts (5/27, 18.5%), one close contact (1/53, 1.9%), and seven community members (7/2954, 0.2%) had detectable SARS-CoV-2 antibodies. All HCWs were negative for SARS-CoV-2 antibodies. Participants were tested a median of 15.1 (interquartile range from 14.9 to 15.2) weeks after exposure. Our study found a low prevalence of SARS-CoV-2 antibodies in high-risk communities and healthcare workers in communities in Vietnam with known COVID-19 cases.

Synedrella nodiflora (Linn.) Gaertn. inhibits inflammatory responses through the regulation of Syk in RAW 264.7 macrophages.

Synedrella nodiflora (Linn.) Gaertn. (S. nodiflora) has long been used for the treatment of inflammatory diseases, including liver disease, asthma, rheumatism and earache, in tropical countries throughout America, Asia and Africa. However, the biological effects of S. nodiflora have not been extensively studied at the molecular level. Notably, it remains unclear how S. nodiflora exerts anti-inflammatory activity. In the present study, the anti-inflammatory mechanism of a methanol extract of S. nodiflora (MSN) in RAW 264.7 macrophages activated by lipopolysaccharide (LPS) was investigated. Non-cytotoxic concentrations of MSN (≤400 µg/ml) decreased the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), which resulted in a decrease in nitric oxide and prostaglandin E2 (PGE2) production. The mRNA expression of pro-inflammatory cytokines such as interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α was reduced upon MSN treatment. In addition, the activation of spleen tyrosine kinase (Syk) and Akt was suppressed by MSN. Taken together, these findings recommend the traditional medicinal application of S. nodiflora in the treatment of several inflammation-associated diseases and indicate the possibility of MSN as a novel therapeutic reagent of inflammation-related diseases.

Synthesis and preclinical validation of novel P2Y1 receptor ligands as a potent anti-prostate cancer agent.

Purinergic receptor is a potential drug target for neuropathic pain, Alzheimer disease, and prostate cancer. Focusing on the structure-based ligand discovery, docking analysis on the crystal structure of P2Y1 receptor (P2Y1R) with 923 derivatives of 1-indolinoalkyl 2-phenolic compound is performed to understand the molecular insights of the receptor. The structural model identified the top novel ligands, 426 (compound 1) and 636 (compound 2) having highest binding affinity with the docking score of -7.38 and -6.92. We have reported the interaction efficacy and the dynamics of P2Y1R protein with the ligands. The best hits synthesized were experimentally optimized as a potent P2Y1 agonists. These ligands exhibits anti-proliferative effect against the PC-3 and DU-145 cells (IC50 = 15 µM - 33 µM) with significant increase in the calcium level in dose- and time-dependent manner. Moreover, the activation of P2Y1R induced the apoptosis via Capase3/7 and ROS signaling pathway. Thus it is evidenced that the newly synthesized ligands, as a P2Y1R agonists could potentially act as a therapeutic drug for treating prostate cancer.

[Corrigendum] Methanol extract of Guettarda speciosa Linn. inhibits the production of inflammatory mediators through the inactivation of Syk and JNK in macrophages.

After the acceptance of the article and during the pre­publication stages, the author listed second on the paper, Young­Chang Cho, moved to a new affiliation (College of Pharmacy, Chonnam National University, Gwangju 61186, Korea). Therefore, the published paper should also have included details of the new affiliation as a 'Present address'. The author and affiliation information should have been shown as follows: HIEN THI THU LE1*, YOUNG­CHANG CHO1,2* and SAYEON CHO1. 1Laboratory of Molecular Pharmacological Cell Biology, College of Pharmacy, Chung­Ang University, Seoul 06974, Republic of Korea. *Contributed equally. 2Present address: College of Pharmacy, Chonnam National University, Gwangju 61186, Republic of Korea. The authors regret that this change was not made prior to the publication of their paper, and apologize for any inconvenience caused. [the original article was published in International Journal of Molecular Medicine 41: 1783­1791, 2018; DOI: 10.3892/ijmm.2018.3377].

Mutational screening of germline RB1 gene in Vietnamese patients with retinoblastoma reveals three novel mutations.

Purpose: Retinoblastoma (Rb) is a rare and unique eye cancer that usually develops in the retinas of children less than 5 years old due to mutations in the RB1 gene. About 40% of affected individuals have the heritable form making genetics testing of the RB1 gene important for disease management. This study aims to identify germline mutations in RB1 in a cohort of patients with Rb from northern Vietnam. Methods: Genomic DNA was extracted from peripheral blood of 34 patients with Rb (nine unilateral and 25 bilateral cases) and their available parents. Twenty-seven exons, flanking sequences, and the promoter region of RB1 gene were screened for mutations with direct PCR sequencing. Multiplex ligation-dependent probe amplification (MLPA) was applied for patients with negative sequencing results. In the mutation-positive patients, their available parental DNA was analyzed to determine the parental origin of the mutation. Results: Germline mutations in RB1 were identified in 25 (73.53%) of 34 patients (four unilateral and 21 bilateral cases). Of these mutations, 19 were detected, including seven nonsense, six frameshift, four splice-site (one was identified in two siblings), and one missense, with Sanger sequencing. Three novel frameshift mutations were discovered in one unilateral and two bilateral patients. MLPA detected mutations in the RB1 gene in six bilateral cases, of whom five had a whole gene deletion (three familial cases) and one had a partial gene deletion (from exon 4 to exon 27) in one allele of the RB1 gene. Parental testing showed five mutations originated from the fathers and one was inherited from a mother who was mosaic for the mutation. Conclusions: This study provides a data set of germline mutations in the RB1 gene in Vietnamese patients with retinoblastoma. Screening of mutations in the RB1 gene can help to identify heritable Rb and contribute to clinical management and genetic counseling for affected families.

Methanol extract of Guettarda speciosa Linn. inhibits the production of inflammatory mediators through the inactivation of Syk and JNK in macrophages.

Guettarda speciosa Linn. (G. speciosa, Rubiaceae) has been used as a traditional medicinal plant in Asia for the treatment of various inflammatory conditions, including cough, fever and maternal postpartum infection. However, the mechanisms underlying the anti­inflammatory action of G. speciosa extracts have remained elusive. In the present study, the anti­inflammatory effects of the methanol extract of G. speciosa (MGS) were investigated in murine macrophages by measuring the production of inflammatory mediators and the underlying mechanisms of action by performing immunoblotting analysis of proteins that are potentially involved. MGS reduced nitric oxide (NO) production through regulation of the expression of inducible NO synthase (iNOS) in lipopolysaccharide­activated RAW 264.7 cells; however, cyclooxygenase­2, the enzyme responsible for prostaglandin E2 production, was not affected at the mRNA or protein level. MGS reduced interleukin­6 (IL­6) production, but had no effect on tumor necrosis factor (TNF)­α production. In addition, MGS suppressed the transcription of IL­6, but not that of IL­1ß and TNF­α. The effect of MGS on proinflammatory mediators resulted from the inhibition of the activation of spleen tyrosine kinase and c­Jun N­terminal kinase. In conclusion, the present study suggested that MGS may be a potential candidate for development as a therapeutic for alleviating inflammation.

Anticancer effects of methanol extract of Myrmecodia platytyrea Becc. leaves against human hepatocellular carcinoma cells via inhibition of ERK and STAT3 signaling pathways.

Myrmecodia platytyrea Becc., a member of the Rubiaceae family, is found throughout Southeast Asia and has been traditionally used to treat cancer. However, there is limited pharmacological information on this plant. We investigated the anticancer effects of the methanol extract of Myrmecodia platytyrea Becc. leaves (MMPL) and determined the molecular mechanisms underlying the effects of MMPL on metastasis in human hepatocellular carcinoma (HCC) cells. MMPL dose-dependently inhibited cell migration and invasion in SK­Hep1 and Huh7 cells. In addition, MMPL strongly suppressed the enzymatic activity of matrix metalloproteinases (MMP­2 and MMP­9). Diminished telomerase activity by MMPL resulted in the suppression of both telomerase activity and telomerase-associated gene expression. The levels of urokinase-type plasminogen activator receptor (uPAR) expression as well as the phosphorylation levels of signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase (ERK) were also attenuated by MMPL. The above results collectively suggest that MMPL has anticancer effects in HCC and that MMPL can serve as an effective therapeutic agent for treating human liver cancer.

Anti­inflammatory effects on murine macrophages of ethanol extracts of Lygodium japonicum spores via inhibition of NF­κB and p38.

The spores of Lygodium japonicum (Thunb.) Sw. (L. japonicum) have been used in traditional Chinese medicine for the treatment of various inflammatory diseases. However, the molecular mechanisms underlying their anti­inflammatory effects have yet to be elucidated. In the present study, we investigated the anti­inflammatory effects of ethanol extracts of L. japonicum spores (ELJ) by measuring the production of inflammatory mediators, and explored the molecular mechanisms underlying the effects of ELJ in murine macrophages in vitro using immunoblotting analyses. At non­cytotoxic concentrations of (50­300 µg/ml), ELJ was revealed to significantly suppress the production of nitric oxide (NO) and tumor necrosis factor (TNF)­α in lipopolysaccharide (LPS)­stimulated murine RAW 264.7 macrophages; ELJ repressed the production of interleukin (IL)­6 only at high concentrations (≥200 µg/ml). The ELJ­mediated decrease in NO production was demonstrated to depend on the downregulation of inducible NO synthase mRNA and protein expression. Conversely, the mRNA and protein expression of cyclooxygenase­2 were not affected by ELJ. In addition, ELJ was revealed to inhibit the mRNA expression of IL­6, IL­1ß, and TNF­α in LPS­stimulated RAW 264.7 macrophages. The effects of ELJ on proinflammatory mediators may have been due to the stabilization of inhibitor of κBα and the inhibition of p38 mitogen­activated protein kinase (MAPK). These results suggested that ELJ may suppress LPS­induced inflammatory responses in murine macrophages in vitro, through the negative regulation of p38 MAPK and nuclear factor (NF)­κB. Therefore, ELJ may have potential as a novel candidate for the development of therapeutic strategies aimed at alleviating inflammation.

Suppression of inflammation by the rhizome of Anemarrhena asphodeloides via regulation of nuclear factor-κB and p38 signal transduction pathways in macrophages.

The rhizome of Anemarrhena asphodeloides Bunge (A. asphodeloides) has been used as a traditional East Asian medicine for the treatment of various types of inflammatory disease. However, to the best of our knowledge, there have been no systemic studies regarding the molecular mechanisms of action of the A. asphodeloides rhizome anti-inflammatory effects. The aim of the present study was to elucidate the anti-inflammatory effects and underlying mechanism of action of ethanol extracts of the rhizome of A. asphodeloides (EAA) in murine macrophages. Non-cytotoxic concentrations of EAA (10-100 µg/ml) significantly decreased the production of NO and interleukin (IL)-6 in lipopolysaccharide (LPS)-stimulated macrophages, while the production of tumor necrosis factor-α was not regulated by EAA. EAA-mediated reduction of nitric oxide (NO) was due to reduced expression levels of inducible NO synthase (iNOS). Furthermore, protein expression levels of LPS-induced cyclooxygenase-2, another inflammatory enzyme, were alleviated in the presence of EAA. EAA-mediated reduction of those proinflammatory mediators was due to inhibition of nuclear factor-κB (NF-κB) and activator protein 1 transcriptional activities followed by the stabilization of inhibitor of κ Bα and inhibition of p38, respectively. These results indicate that EAA suppresses LPS-induced inflammatory responses by negatively regulating p38 and NF-κB, indicating that EAA is a candidate treatment for alleviating inflammation.

Inhibition of protein tyrosine phosphatase non-receptor type 2 by PTP inhibitor XIX: Its role as a multiphosphatase inhibitor.

Protein tyrosine phosphatases (PTPs) play crucial roles in signal transduction and their functional alteration has been detected in many diseases. PTP inhibitors have been developed as therapeutic drugs for diseases that are related to the activity of PTPs. In this study, PTP inhibitor XIX, an inhibitor of CD45 and PTEN, was investigated whether it inhibits other PTPs. Protein tyrosine phosphatase non-receptor type 2 (PTPN2) was selectively inhibited by the inhibitor in a competitive manner. Drug affinity responsive target stability (DARTS) analysis showed that the inhibitor induces conformational changes in PTPN2. Phosphorylation levels of signal transducer and activator of transcription 3 (STAT3) at Tyr-705, a crucial site for STAT3 activation and target site of PTPN2, decreased upon exposure to the inhibitor. Our results suggest that PTP inhibitor XIX might be considered as an effective regulator of PTPN2 for treating diseases related to PTPN2. [BMB Reports 2017; 50(6): 329-334].