Incorporation of aptamers in the terminal loop of shRNAs yields an effective and novel combinatorial targeting strategy.

Gene therapy by engineering patient's own blood cells to confer HIV resistance can potentially lead to a functional cure for AIDS. Toward this goal, we have previously developed an anti-HIV lentivirus vector that deploys a combination of shRNA, ribozyme and RNA decoy. To further improve this therapeutic vector against viral escape, we sought an additional reagent to target HIV integrase. Here, we report the development of a new strategy for selection and expression of aptamer for gene therapy. We developed a SELEX protocol (multi-tag SELEX) for selecting RNA aptamers against proteins with low solubility or stability, such as integrase. More importantly, we expressed these aptamers in vivo by incorporating them in the terminal loop of shRNAs. This novel strategy allowed efficient expression of the shRNA-aptamer fusions that targeted RNAs and proteins simultaneously. Expressed shRNA-aptamer fusions targeting HIV integrase or reverse transcriptase inhibited HIV replication in cell cultures. Viral inhibition was further enhanced by combining an anti-integrase aptamer with an anti-HIV Tat-Rev shRNA. This construct exhibited efficacy comparable to that of integrase inhibitor Raltegravir. Our strategy for the selection and expression of RNA aptamers can potentially extend to other gene therapy applications

An integrated web medicinal materials DNA database: MMDBD (Medicinal Materials DNA Barcode Database)

<p>Abstract</p> <p>Background</p> <p>Thousands of plants and animals possess pharmacological properties and there is an increased interest in using these materials for therapy and health maintenance. Efficacies of the application is critically dependent on the use of genuine materials. For time to time, life-threatening poisoning is found because toxic adulterant or substitute is administered. DNA barcoding provides a definitive means of authentication and for conducting molecular systematics studies. Owing to the reduced cost in DNA authentication, the volume of the DNA barcodes produced for medicinal materials is on the rise and necessitates the development of an integrated DNA database.</p> <p>Description</p> <p>We have developed an integrated DNA barcode multimedia information platform- Medicinal Materials DNA Barcode Database (MMDBD) for data retrieval and similarity search. MMDBD contains over 1000 species of medicinal materials listed in the Chinese Pharmacopoeia and American Herbal Pharmacopoeia. MMDBD also contains useful information of the medicinal material, including resources, adulterant information, medical parts, photographs, primers used for obtaining the barcodes and key references. MMDBD can be accessed at <url>http://www.cuhk.edu.hk/icm/mmdbd.htm</url>.</p> <p>Conclusions</p> <p>This work provides a centralized medicinal materials DNA barcode database and bioinformatics tools for data storage, analysis and exchange for promoting the identification of medicinal materials. MMDBD has the largest collection of DNA barcodes of medicinal materials and is a useful resource for researchers in conservation, systematic study, forensic and herbal industry.</p

The minibrain kinase homolog, mbk-2, is required for spindle positioning and asymmetric cell division in early C. elegans embryos

AbstractIn the newly fertilized Caenorhabditis elegans zygote, cytoplasmic determinants become localized asymmetrically along the anterior–posterior (A–P) axis of the embryo. The mitotic apparatus then orients so as to cleave the embryo into anterior and posterior blastomeres that differ in both size and developmental potential. Here we describe a role for MBK-2, a member of the Dyrk family of protein kinases, in asymmetric cell division in C. elegans. In mbk-2 mutants, the initial mitotic spindle is misplaced and cytoplasmic factors, including the germline-specific protein PIE-1, are mislocalized. Our findings support a model in which MBK-2 down-regulates the katanin-related protein MEI-1 to control spindle positioning and acts through distinct, as yet unknown factors, to control the localization of cytoplasmic determinants. These findings in conjunction with work from Schizosaccharomyces pombe indicate a possible conserved role for Dyrk family kinases in the regulation of spindle placement during cell division

EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma.

Squamous cell carcinoma (SCC) and malignant pleural mesothelioma (MPM) are thoracic malignancies with very poor prognosis and limited treatment options. It is an established fact that most of the solid tumors have overexpression of EPHA2 receptor tyrosine kinase. EPHA2 is known to exhibit opposing roles towards cancer progression. It functions in inhibiting cancer survival and migration via a ligand and tyrosine kinase dependent signaling (Y772). Whereas it is known to promote tumor progression and cell migration through a ligand-independent signaling (S897). We analyzed the expression profile and mutational status of the ephrin receptor A2 (EPHA2) in SCC and MPM cell lines and primary patient specimens. The EPHA2 receptor was found to be either overexpressed, mutated or amplified in SCC and MPM. In particular, the EPHA2 mutants A859D and T647M were interesting to explore, A859D Y772 dead mutant exhibited lower levels of phosphorylation at Y772 compared to T647M mutant. Molecular Dynamics simulations studies suggested that differential changes in conformation might form the structural basis for differences in the level of EPHA2 activation. Consequently, A859D mutant cells exhibited increased proliferation as well as cell migration compared to controls and T647M mutant. Kinomics analysis demonstrated that the STAT3 and PDGF pathways were upregulated whereas signaling through CBL was suppressed. Considered together, the present work has uncovered the oncogenic characteristics of EPHA2 mutations in SSC and MPM reinstating the dynamics of different roles of EPHA2 in cancer. This study also suggests that a combination of doxazosin and other EPHA2 inhibitors directed to inhibit the pertinent signaling components may be a novel therapeutic strategy for MPM and Non-small cell lung cancer patients who have either EPHA2 or CBL alterations

The Enhanced metastatic potential of hepatocellular carcinoma (HCC) cells with sorafenib resistance

Acquired resistance towards sorafenib treatment was found in HCC patients, which results in poor prognosis. To investigate the enhanced metastatic potential of sorafenib resistance cells, sorafenib-resistant (SorR) cell lines were established by long-term exposure of the HCC cells to the maximum tolerated dose of sorafenib. Cell proliferation assay and qPCR of ABC transporter genes (ABCC1-3) were first performed to confirm the resistance of cells. Migration and invasion assays, and immunoblotting analysis on the expression of epithelial to mesenchymal transition (EMT) regulatory proteins were performed to study the metastatic potential of SorR cells. The expression of CD44 and CD133 were studied by flow cytometry and the gene expressions of pluripotency factors were studied by qPCR to demonstrate the enrichment of cancer stem cells (CSCs) in SorR cells. Control (CTL) and SorR cells were also injected orthotopically to the livers of NOD-SCID mice to investigate the development of lung metastasis. Increased expressions of ABCC1-3 were found in SorR cells. Enhanced migratory and invasive abilities of SorR cells were observed. The changes in expression of EMT regulatory proteins demonstrated an activation of the EMT process in SorR cells. Enriched proportion of CD44+ and CD44+CD133 + cells were also observed in SorR cells. All (8/8) mice injected with SorR cells demonstrated lung metastasis whereas only 1/8 mouse injected with CTL cells showed lung metastasis. HCC cells with sorafenib resistance demonstrated a higher metastatic potential, which may be due to the activated EMT process. Enriched CSCs were also demonstrated in the sorafenib resistant cells. This study suggests that advanced HCC patients with acquired sorafenib resistance may have enhanced tumor growth or distant metastasis, which raises the concern of long-term sorafenib treatment in advanced HCC patients who have developed resistance of sorafenib. © 2013 Chow et al.published_or_final_versio

Comparison of depressive symptoms among healthcare workers in high-risk versus low-risk areas during the first month of the COVID-19 pandemic in China

IntroductionThe psychological health of healthcare workers (HCWs) has become a significant concern, particularly during the initial stage of a pandemic. This study compared the depressive symptoms among HCWs in high-risk areas (HRAs) and low-risk areas (LRAs) with matching demographics.MethodsA cross-sectional study was employed to compare the depressive symptoms (Patient Health Questionnaire score ≥ 10), workplace environment characteristics, the Health Belief Model (HBM) and socio-demographics of the HCWs working in HRAs and LRAs in several accessible regions (mainly Hubei Province and Guangdong–Hong Kong–Macao Greater–Bay–Area) in China. Eight hundred eighty-five HCWs were recruited for unmatched analysis between March 6 and April 2, 2020. After matching with occupation and years of service using a 1:2 ratio, 146 HCWs in HRAs and 290 HCWs in LRAs were selected for matched analysis. Subgroup analyzes were performed using two individual logistic regressions to delineate the associated factors in LRAs and HRAs, respectively.ResultsHCWs in LRAs (Prevalence = 23.7%) had 1.96 times higher odds of depressive symptoms than those in HRAs (Prevalence = 15.1%) after adjusting for occupation and years of service (p &lt; 0.001). Significant differences in workplace environment characteristics (p &lt; 0.001) and the 5-dimension of the HBM of HCWs (p &lt; 0.001 to p = 0.025) were found between HRAs and LRAs.Logistic regression showed that workers with years of service between 10 and 20  years (OR:6.27), ever had contact with COVID-19 patients (OR:14.33) and had higher scores of “perceived barrier” of HBM (OR:4.48) predicted depressive symptoms in HRAs while working in pneumology departments and infectious disease units (OR:0.06), and high “self-efficacy” in the HBM (OR:0.13) was a protective factor against depressive symptoms.Contrarily, in LRAs, those HCWs who worked in ICUs (OR:2.59), had higher scores of “perceived susceptibility toward the COVID-19 outbreak” (OR:1.41), “perceived severity of the pandemic” (OR:1.25), and “perceived barriers of wearing masks” (OR:1.43) in the HBM predicted depressive symptoms. High “cues to action” (OR:0.79), and better “knowledge” (OR:0.79) in the HBM were protective factors against depressive symptoms.ConclusionThe risk of depressive symptoms of HCWS was double in LRAs than in HRAs in the first month of the COVID-19 pandemic. Furthermore, salient predictors for depressive symptoms among HCWs in HRAs and LRAs were very different

Population genomic analyses of protected incense trees Aquilaria sinensis reveal the existence of genetically distinct subpopulations

The incense tree Aquilaria sinensis (Thymelaeaceae) can produce agarwood with commercial values and is now under threat from illegal exploitation in Hong Kong, impairing the local population and biodiversity. Together with other species of Aquilaria, it is listed in the CITES Appendix II, which strictly regulates its international trade. To understand the population structure of A. sinensis and to make relevant conservation measures, we have sequenced 346 individuals collected in Hong Kong and southern mainland China. Population genomic analyses including principal component analysis, neighbor-joining tree construction, ADMIXTURE, and hierarchical pairwise-FST analyses suggested that genetically distinct populations are contained in certain areas. Genomic scan analyses further detected single-nucleotide polymorphism (SNP) outliers related to plant defense, including the CYP71BE gene cluster. In addition to the population analyses, we have developed a modified hexadecyltrimethyl-ammonium bromide (CTAB) DNA extraction protocol for obtaining DNA from agarwood samples in this study, and resequencing of DNA extracted from two agarwood samples using this method allows us to successfully map to the sample corresponding localities in the phylogenetic tree. To sum up, this study suggested that there is a genetically distinct subpopulation of incense tree in Hong Kong that would require special conservation measures and established a foundation for future conservation measures

Solution structure of the dimerization domain of ribosomal protein P2 provides insights for the structural organization of eukaryotic stalk

The lateral stalk of ribosome is responsible for kingdom-specific binding of translation factors and activation of GTP hydrolysis that drives protein synthesis. In eukaryotes, the stalk is composed of acidic ribosomal proteins P0, P1 and P2 that constitute a pentameric P-complex in 1: 2: 2 ratio. We have determined the solution structure of the N-terminal dimerization domain of human P2 (NTD-P2), which provides insights into the structural organization of the eukaryotic stalk. Our structure revealed that eukaryotic stalk protein P2 forms a symmetric homodimer in solution, and is structurally distinct from the bacterial counterpart L12 homodimer. The two subunits of NTD-P2 form extensive hydrophobic interactions in the dimeric interface that buries 2400 Å2 of solvent accessible surface area. We have showed that P1 can dissociate P2 homodimer spontaneously to form a more stable P1/P2 1 : 1 heterodimer. By homology modelling, we identified three exposed polar residues on helix-3 of P2 are substituted by conserved hydrophobic residues in P1. Confirmed by mutagenesis, we showed that these residues on helix-3 of P1 are not involved in the dimerization of P1/P2, but instead play a vital role in anchoring P1/P2 heterodimer to P0. Based on our results, models of the eukaryotic stalk complex were proposed

Accuracy of detecting residual disease after neoadjuvant chemoradiotherapy for esophageal squamous cell carcinoma (preSINO trial): A prospective multicenter diagnostic cohort study

Background: After neoadjuvant chemoradiotherapy (nCRT) for esophageal cancer, high pathologically complete response (pCR) rates are being achieved especially in patients with squamous cell carcinoma (SCC). An active surveillance strategy has been proposed for SCC patients with clinically complete response (cCR) after nCRT. To justify omitting surgical resection, patients with residual disease should be accurately identified. The aim of this study is to assess the accuracy of response evaluations after nCRT based on the preSANO trial, including positron emission tomography with computed tomography (PET-CT), endoscopy with bite-on-bite biopsies and endoscopic ultrasonography (EUS) with fine-needle aspiration (FNA) in patients with potentially curable esophageal SCC. Methods: Operable esophageal SCC patients who are planned to undergo nCRT according to the CROSS regimen and are planned to undergo surgery will be recruited from four Asian centers. Four to 6 weeks after completion of nCRT, patients will undergo a first clinical response evaluation (CRE-1) consisting of endoscopy with bite-on-bite biopsies. In patients without histological evidence of residual tumor (i.e. without positive biopsies), surgery will be postponed another 6 weeks. A second clinical response evaluation (CRE-2) will be performed 10-12 weeks after completion of nCRT, consisting of PET-CT, endoscopy with bite-on-bite biopsies and EUS with FNA. Immediately after CRE-2 all patients without evidence of distant metastases will undergo esophagectomy. Results of CRE-1 and CRE-2 as well as results of the three single diagnostic modalities will be correlated to pathological response in the resection specimen (gold standard) for calculation of sensitivity, specificity, negative predictive value and positive predictive value. Discussion: If the current study shows that major locoregional residual disease (> 10% residual carcinoma or any residual nodal disease) can be accurately (i.e. with sensitivity of 80.5%) detected in patients with esophageal SCC, a prospective trial will be conducted comparing active surveillance with standard esophagectomy in patients with a clinically complete response after nCRT (SINO trial). Trial registration: The preSINO trial has been registered at ClinicalTrials.gov as NCT03937362 (May 3, 2019)

Solution structure of the dimerization domain of ribosomal protein P2 provides insights for the structural organization of eukaryotic stalk

The lateral stalk of ribosome is responsible for kingdom-specific binding of translation factors and activation of GTP hydrolysis that drives protein synthesis. In eukaryotes, the stalk is composed of acidic ribosomal proteins P0, P1 and P2 that constitute a pentameric P-complex in 1: 2: 2 ratio. We have determined the solution structure of the N-terminal dimerization domain of human P2 (NTD-P2), which provides insights into the structural organization of the eukaryotic stalk. Our structure revealed that eukaryotic stalk protein P2 forms a symmetric homodimer in solution, and is structurally distinct from the bacterial counterpart L12 homodimer. The two subunits of NTD-P2 form extensive hydrophobic interactions in the dimeric interface that buries 2400 Å2 of solvent accessible surface area. We have showed that P1 can dissociate P2 homodimer spontaneously to form a more stable P1/P2 1 : 1 heterodimer. By homology modelling, we identified three exposed polar residues on helix-3 of P2 are substituted by conserved hydrophobic residues in P1. Confirmed by mutagenesis, we showed that these residues on helix-3 of P1 are not involved in the dimerization of P1/P2, but instead play a vital role in anchoring P1/P2 heterodimer to P0. Based on our results, models of the eukaryotic stalk complex were proposed