A high-throughput single cell-based antibody discovery approach against the full-length SARS-CoV-2 spike protein suggests a lack of neutralizing antibodies targeting the highly conserved S2 domain.

Coronavirus disease 2019 pandemic continues globally with a growing number of infections, but there are currently no effective antibody drugs against the virus. In addition, 90% amino acid sequence identity between the S2 subunit of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-CoV S proteins attracts us to examine S2-targeted cross-neutralizing antibodies that are not yet well defined. We therefore immunized RenMab mice with the full-length S protein and constructed a high-throughput antibody discovery method based on single-cell sequencing technology to isolate SARS-CoV-2 S-targeted neutralizing antibodies and cross-neutralizing antibodies against the S2 region of SARS-CoV-2/SARS-CoV S. Diversity of antibody sequences in RenMab mice and consistency in B-cell immune responses between RenMab mice and humans enabled screening of fully human virus-neutralizing antibodies. From all the frequency >1 paired clonotypes obtained from single-cell V(D)J sequencing, 215 antibodies with binding affinities were identified and primarily bound S2. However, only two receptor-binding domain-targeted clonotypes had neutralizing activity against SARS-CoV-2. Moreover, 5' single-cell RNA sequencing indicated that these sorted splenic B cells are mainly plasmablasts, germinal center (GC)-dependent memory B-cells and GC B-cells. Among them, plasmablasts and GC-dependent memory B-cells were considered the most significant possibility of producing virus-specific antibodies. Altogether, using a high-throughput single cell-based antibody discovery approach, our study highlighted the challenges of developing S2-binding neutralizing antibodies against SARS-CoV-2 and provided a novel direction for the enrichment of antigen-specific B-cells.

HBB contributes to individualized aconitine-induced cardiotoxicity in mice via interfering with ABHD5/AMPK/HDAC4 axis.

The root of Aconitum carmichaelii Debx. (Fuzi) is an herbal medicine used in China that exerts significant efficacy in rescuing patients from severe diseases. A key toxic compound in Fuzi, aconitine (AC), could trigger unpredictable cardiotoxicities with high-individualization, thus hinders safe application of Fuzi. In this study we investigated the individual differences of AC-induced cardiotoxicities, the biomarkers and underlying mechanisms. Diversity Outbred (DO) mice were used as a genetically heterogeneous model for mimicking individualization clinically. The mice were orally administered AC (0.3, 0.6, 0.9 mg· kg-1 ·d-1) for 7 d. We found that AC-triggered cardiotoxicities in DO mice shared similar characteristics to those observed in clinic patients. Most importantly, significant individual differences were found in DO mice (variation coefficients: 34.08%-53.17%). RNA-sequencing in AC-tolerant and AC-sensitive mice revealed that hemoglobin subunit beta (HBB), a toxic-responsive protein in blood with 89% homology to human, was specifically enriched in AC-sensitive mice. Moreover, we found that HBB overexpression could significantly exacerbate AC-induced cardiotoxicity while HBB knockdown markedly attenuated cell death of cardiomyocytes. We revealed that AC could trigger hemolysis, and specifically bind to HBB in cell-free hemoglobin (cf-Hb), which could excessively promote NO scavenge and decrease cardioprotective S-nitrosylation. Meanwhile, AC bound to HBB enhanced the binding of HBB to ABHD5 and AMPK, which correspondingly decreased HDAC-NT generation and led to cardiomyocytes death. This study not only demonstrates HBB achievement a novel target of AC in blood, but provides the first clue for HBB as a novel biomarker in determining the individual differences of Fuzi-triggered cardiotoxicity.

[Discussion and Concern on Animal Study for Orthopaedic Implantable Devices].

In recent years, new orthopaedic implantable devices continue to emerge, which require higher requirements for technical evaluation. Animal study is an important part of the research and development process for the new orthopedic implantable devices, which provides relevant evidence for product design and stereotyping. By introducing the purpose of animal study, and the application of 3R principle (replacement, reduction, refinement) in this field, we summarize the concern on the animal study, in order to provide reference for the development and research of new orthopedic implantable devices and biomaterials. At the same time, the application of evidence-based research methods such as systematic review in the field is introduced, which provides new tools and approaches for the technical review and regulatory science.

[Discussion on Key Attention in Technical Review for Additive Manufacturing Acetabular Reconstruction Implantable Device].

With the development of additive manufacturing, the advantages of this type implant devices in the treatment of acetabular defects and reconstruction are becoming more and more prominent. The number of registration and declaration of such products is increasing day by day. According to the relevant requirements of the National Medical Products Administration for registration and application documents, combined with the characteristics of acetabular reconstruction implant products made of additive manufacturing, this study analyzes and summarizes the relevant requirements on raw material control, product performance research, product manufacturing, clinical evaluation, et al. We should pay more attention to in the registration and application materials submitted by the applicant. Provide opinions and suggestions for the next registration applicant to standardize product R&D and registration application documents, in order to help them optimize product R&D process, improve product quality and registration application efficiency.

Fluorescence biosensor for inorganic pyrophosphatase activity.

A highly sensitive and selective fluorescence biosensor for inorganic pyrophosphatase (PPase) activity has been developed based on special click ligation trigger hyperbranched rolling circle amplification (CLT-HRCA). Pyrophosphate ion (PPi) can coordinate with Cu2+ to form stable PPi/Cu2+ complex and Cu2+ in the complex cannot be reduced to Cu+. The addition of PPase causes the hydrolysis of PPi into orthophosphate (Pi) and therefore induces the releasing of Cu2+ from the stable PPi/Cu2+ complex, and the free Cu2+ is easily reduced to Cu+ by sodium ascorbate. Then Cu+ catalyzes the cyclization reaction between the specially designed 5'-azide and 3'-alkyne tagged padlock probes through Cu+ catalyzed azide-alkyne cycloaddition (CuAAC), which in turn initiates the hyperbranched rolling circle amplification (HRCA). Given that the CLT-HRCA products contain large amounts of double-stranded DNAs (dsDNAs), the addition of SYBR Green I resulted in the enhanced fluorescence signal. There was a linear relationship between the enhanced fluorescence intensity and the logarithm PPase activity ranging from 0.05 to 25 mU with a detection limit of 0.02 mU. Such proposed biosensor has been successfully applied to screen the potential PPase inhibitors and has accessed the related inhibit ability with high efficiency.

Physical properties and hydration behavior of a fast-setting bioceramic endodontic material.

BACKGROUND: To investigate the physical properties and the hydration behaviour of the fast-setting bioceramic iRoot FS Fast Set Root Repair Material (iRoot FS) and three other endodontic cements. METHODS: iRoot FS, Endosequence Root Repair Material Putty (ERRM Putty), gray and white mineral trioxide aggregate (G-MTA & W-MTA), and intermediate restorative material (IRM) were evaluated. The setting time was measured using ANSI/ADA standards. Microhardness was evaluated using the Vickers indentation test. Compressive strength and porosity were investigated at 7 and 28 days. Differential scanning calorimetry (DSC) was employed for the hydration test. RESULTS: iRoot FS had the shortest setting time of the four bioceramic cements (p < .001). The microhardness values of iRoot FS, ERRM Putty and MTA increased at different rates over the 28 days period. At day one, ERRM Putty had the lowest microhardness of the bioceramic cements (p < .001), but reached the same level as MTA at 4, 7 and 28 days. The microhardness of iRoot FS was lower than that of W-MTA at 7 and 28 days (p < .05). The porosity of the materials did not change after 7 days (p < .05). The compressive strength values at 28 days were significantly greater for all bioceramic groups compared to those at 7 days (p < .01). ERRM Putty had the highest compressive strength and the lowest porosity of the evaluated bioceramic cements (p < .05), followed by iRoot FS, W-MTA, and G-MTA, respectively. DSC showed that iRoot FS hydrated fastest, inducing an intense exothermic reaction. The ERRM Putty did not demonstrate a clear exothermic peak during the isothermal calorimetry test. CONCLUSIONS: iRoot FS had a faster setting time and hydrating process than the other bioceramic cements tested. The mechanical properties of iRoot FS, G-MTA and W-MTA were relatively similar.

AMPK phosphorylates GBF1 for mitotic Golgi disassembly.

In mammalian cells, the Golgi apparatus undergoes extensive fragmentation during mitosis; this is required not only for the partitioning of the complex but also for the process of mitosis. However, the molecular mechanism underlying the mitotic fragmentation of the Golgi is far from clear. Here, we show that AMP-activated protein kinase (AMPK) is phosphorylated and activated when cells enter mitosis. Activated AMPK phosphorylates GBF1, a guanine nucleotide exchange factor (GEF) for Arf-GTPases, disassociating GBF1 from the Golgi membrane and abolishing the action of GBF1 as an Arf1-GEF. We further demonstrate that the phosphorylation of AMPK and GBF1 is essential for Golgi disassembly and subsequent mitosis entry. These data suggest that AMPK-GBF1-Arf1 signaling is involved in the regulation of Golgi fragmentation during mitosis.

AP1 is essential for generation of autophagosomes from the trans-Golgi network.

Despite recent advances in understanding the functions of autophagy in developmental and pathological conditions, the underlying mechanism of where and how autophagosomal structures acquire membrane remains enigmatic. Here, we provide evidence that post-Golgi membrane traffic plays a crucial role in autophagosome formation. Increased secretion of constitutive cargo from the trans-Golgi network (TGN) to the plasma membrane induced the formation of microtubule-associated protein light chain 3 (LC3)-positive structures. At the early phase of autophagy, LC3 associated with and then budded off from a distinct TGN domain without constitutive TGN-to-plasma cargo and TGN-to-endosome proteins. The clathrin adaptor protein AP1 and clathrin localized to starvation- and rapamycin-induced autophagosomes. Dysfunction of the AP1-dependent clathrin coating at the TGN but not at the plasma membrane prevented autophagosome formation. Our results thus suggest an essential role of the TGN in autophagosome biogenesis, providing membrane to autophagosomes through an AP1-dependent pathway.

Effects of drying methods on physicochemical and immunomodulatory properties of polysaccharide-protein complexes from litchi pulp.

Dried litchi pulp has been used in traditional remedies in China for many years to treat various diseases, and the therapeutic activity has been, at least partly, attributed to the presence of bioactive polysaccharides. Polysaccharide-protein complexes from vacuum freeze-(VF), vacuum microwave-(VM) and heat pump (HP) dried litchi pulp, which were coded as LP-VF, LP-VM and LP-HP, were comparatively studied on the physicochemical and immunomodulatory properties. LP-HP had a predominance of galactose, while glucose was the major sugar component in LP-VF and LP-VM. Compared with LP-VF and LP-VM, LP-HP contained more aspartate and glutamic in binding protein. LP-HP also exhibited a stronger stimulatory effect on splenocyte proliferation at 200 µg/mL and triggered higher NO, TNF-α and IL-6 secretion from RAW264.7 macrophages. Different drying methods caused the difference in physicochemical properties of polysaccharide-protein complexes from dried litchi pulp, which resulted in significantly different immunomodulatory activity. HP drying appears to be the best method for preparing litchi pulp to improve its immunomodulatory properties.

Modeling and analysis of a stochastic giving-up-smoking model with quit smoking duration.

Smoking has gradually become a very common behavior, and the related situation in different groups also presents different forms. Due to the differences of individual smoking cessation time and the interference of environmental factors in the spread of smoking behavior, we establish a stochastic giving up smoking model with quit-smoking duration. We also consider the saturated incidence rate. The total population is composed of potential smokers, smokers, quitters and removed. By using Itô's formula and constructing appropriate Lyapunov functions, we first ensure the existence of a unique global positive solution of the stochastic model. In addition, a threshold condition for extinction and permanence of smoking behavior is deduced. If the intensity of white noise is small, and $ \widetilde{\mathcal{R}}_0 < 1 $, smokers will eventually become extinct. If $ \widetilde{\mathcal{R}}_0 > 1 $, smoking will last. Then, the sufficient condition for the existence of a unique stationary distribution of the smoking phenomenon is studied as $ R_0/ > 1 $. Finally, conclusions are explained by numerical simulations.

Plain metallic biomaterials: opportunities and challenges.

The 'plainification of materials' has been conceptualized to promote the sustainable development of materials. This perspective, for the first time in the field of biomaterials, proposes and defines 'plain metallic biomaterials (PMBs)' with demonstrated research and application case studies of pure titanium with high strength and toughness, and biodegradable, fine-grained and high-purity magnesium. Then, after discussing the features, benefits and opportunities of PMBs, the challenges are analyzed from both technical and regulatory aspects. Regulatory perspectives on PMB-based medical devices are also provided for the benefit of future research, development and commercialization.

Bile acids promote the development of HCC by activating inflammasome.

BACKGROUND: Hepatocellular carcinoma (HCC) is associated with chronic inflammation caused by different factors; especially, the interaction of inflammatory pathways and bile acids (BAs) can affect hepatocyte proliferation, death, and regeneration, but whether BAs promote HCC progression through inflammatory pathways and the mechanisms is still unclear. METHODS AND RESULTS: By examining cancer and tumor-adjacent tissue BA levels and genes associated with BA homeostasis in 37 HCC patients, we found that total bile acids (TBAs) were decreased by 36% and varying degrees of changes in factors regulating BA homeostasis (p < 0.05). In addition, we found that BA homeostasis was disturbed in diethylnitrosamine-induced HCC mouse models, and TBA was correlated with inflammasome activation during HCC progression (6-24 W) (p < 0.05). Similarly, the inflammasome and chenodeoxycholic acid (CDCA) content were suppressed in cholestasis model mice (Mrp2-deficient mice) (p < 0.05). In vitro, CDCA significantly promoted the malignant transformation of hepatocytes (p < 0.001), activated the inflammasome by triggering the release of mitochondrial reactive oxygen species and mitochondrial DNA, and ultimately induced pyroptosis. Furthermore, we found that CDCA has a targeted binding effect with HO-1 through molecular docking and Cellular Thermal Shift Assay experiments. CONCLUSIONS: In conclusion, we found that CDCA can trigger the excessive accumulation of mitochondrial reactive oxygen species by targeting HO-1 to promote the activation of the inflammasome and ultimately promote the progression of HCC. Our study provides a novel mechanism by which BAs promote HCC by activating the inflammasome and establishes the important role of BA homeostasis imbalance in the progression of HCC from the aspect of inflammation.

Functionalization of biomedical materials using fusion peptides for tissue regeneration.

Tissue development deformity or tissue defect is a major clinical challenge. Tissue engineering technology provides a promising solution to these problems. Among them, functional biomaterials with regenerative abilities are one of the development trends. Polypeptide is a small molecule that can be used to modify tissue engineering materials. However, the function of a single polypeptide molecule is limited and insufficient to construct comprehensive microenvironment for tissue regeneration. Fusion peptides combining two or more polypeptide molecules with different functions were expected to achieve multiple efficaciesin vivo, providing a novel solution for clinical tissue regeneration engineering applications. This paper reviews the construction methods, degradation process, and biological activities of fusion peptides, and presents recent global research progress and prospects concerning fusion peptides. It provides a reference helping to guide the future exploration and development of fusion peptide-based functional biomaterials for tissue engineering.

c-Myc-PD-L1 Axis Sustained Gemcitabine-Resistance in Pancreatic Cancer.

Pancreatic cancer ranks fourth among cancer-related deaths, with a 5-years overall survival rate being below 10%. Gemcitabine (dFdC) has been considered the first-line drug for patients with pancreatic cancer. However, the clinical effectiveness is less than 20% due to drug resistance. Most importantly, overwhelming evidence suggested c-Myc and PD-L1 were generally highly expressed in pancreatic cancer patients. However, whether dFdC-resistant pancreatic cancer is associated with c-Myc and PD-L1 has not been elucidated. In our present study, we found that the expression of c-Myc and PD-L1 was markedly increased in pancreatic tumor tissues compared with adjacent tissues. Similarly, c-Myc and PD-L1 expression were also remarkably elevated in dFdC-resistant Panc-1 cells compared with parental cells. In addition, dFdC sensitivity was enhanced by the combination of dFdC and c-Myc inhibitors in Panc-1 cells. Interestingly, its sensitivity was reduced when c-Myc was overexpressed. Moreover, PD-L1 protein expression was dramatically down-regulated when treated with c-Myc inhibitors. Furthermore, artesunate (ARTS) screened from 18 compounds could reverse dFdC resistance in combination with dFdC in dFdC-resistant Panc-1 cells in vitro and suppressed DMBA-induced pancreatic cancer in vivo. In summary, our data revealed that the mechanism of dFdC resistance may be that c-Myc overexpression contributed to increased PD-L1 expression, and ARTS could overcome dFdC-resistant pancreatic cancer by inhibiting c-Myc and PD-L1. Our findings not only suggest c-Myc and PD-L1 as novel prognostic biomarkers in dFdC-resistant pancreatic cancer, but also provide ARTS as a promising candidate for overcoming dFdC resistance.

Alcohol triggered bile acid disequilibrium by suppressing BSEP to sustain hepatocellular carcinoma progression.

Bile acids (BAs), the most important components of bile, attribute predominately to maintain metabolic homeostasis. In hepatocellular carcinoma (HCC) patients, the BAs homeostasis was seriously disturbed, especially in those patients with alcohol-intake history. However, whether alcohol consumption could promote HCC progression via influencing BAs homeostasis and the precise mechanism underlying are still unclear. In our study, by collecting HCC specimens from both alcohol-drinkers (n = 15) and non-alcohol drinkers (n = 22), we found that compared to non-alcohol intake HCC patients, BAs homeostasis was disturbed in HCC patients who drank alcohol. Furthermore, ethanol treatment was also found to promote HCC progression by markedly activating oncogenes (RAS, MYC, MET, and HER2), while remarkably suppressing tumor suppressor genes (BRCA2 and APC). We evaluated 14 key functional genes that maintain the homeostasis of BAs and found that either in alcohol-intake HCC patients (n = 15), or in ethanol-treated mice, BSEP, rate-limiting transporter governing excreting BAs from liver into bile duct, was remarkably decreased when exposed to alcohol. Moreover, by screening for changes in the epigenetic landscape of liver cancer cells exposed to alcohol, we strikingly found that histone methyltransferases (RBBP-5, Suv39h1, ASH2L, and SET7/9) were increased, and KMT3B, KMT4, and KMT7 gene expression was also elevated, while histone demethyltransferases (JARID1a, JARID1b, JARID1c) were decreased. In summary, we found that alcohol could trigger BAs disequilibrium to initiate and promote HCC progression. Our study provided a novel and supplementary mechanism to determine the important role of alcohol-intake in HCC development regarding from the perspective of BAs homeostasis.

Tumor Microenvironment Acidity Triggers Lipid Accumulation in Liver Cancer via SCD1 Activation.

Acidification is recognized as the predominant characteristic of the tumor microenvironment (TME) and contributes to tumor progression. However, the mechanism of extracellular acidic TME directly influences intercellular pathologic responses remains unclear. Meanwhile, acidic TME is mainly ascribed to aberrant metabolism of lipids and glucose, but whether and how acidity affects metabolic reprogramming, especially for lipid metabolism, is still unknown. We found that lipid was significantly accumulated in liver cancer cells when exposed to acidic TME. Moreover, proteomic analysis showed that differentially expressed proteins were mainly clustered into fatty acid pathways. Subsequently, we found that acidification increased the expression of SCD1 by activating PI3K/AKT signaling pathway. Interestingly, we found that SCD1 directly bound to PPARα in the acidic TME, which vanished after 2-day reverse incubation in pH 7.4 medium, implying extracellular acidosis might influence intercellular function by mediating the binding affinity between SCD1 and PPARα under different pH gradients. In summary, our data revealed that acidosis could significantly trigger fatty acid synthesis to promote liver tumorigenesis by upregulating SCD1 in a PI3K/AKT activation dependent manner and simultaneously promote SCD1 binding to PPARα. Our study not only provides direct mechanistic evidence to support the vital role of acidosis in lipid metabolic reprogramming, but also provides novel insights for determining the binding affinity of functional proteins as a molecular mechanism to better understand the role of the acidic TME in tumor development. IMPLICATIONS: The acidic TME contributes to lipid accumulation in liver cancer by activating the PI3K/AKT signaling pathway and promoting SCD1-PPARα binding.

Screening tumor specificity targeted by arnicolide D, the active compound of Centipeda minima and molecular mechanism underlying by integrative pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Herb-derived anti-tumor agents, such as paclitaxel and vincristine, exert significant but varied effectivenesses towards different cancer types. Similarly, Centipeda minima (CM) is a well-known traditional Chinese medicine that has been used to treat rhinitis, relieve pain and reduce swelling, and recently found to exert overwhelming anti-tumor effects against breast cancer, colon cancer, and nasopharyngeal carcinoma with different response rates. However, what is the optimizing cancer model that benefits most from CM, and what is the specific target underlying still require more exclusive and profound investigations. AIMS OF THE STUDY: This study aimed to explore the dominant tumor model and specific target of CM by integrative pharmacology and biological experiments. MATERIALS AND METHODS: The most predominant and specific cancer types that are sensitive to CM were screened and identified based on a combination network pharmacology and bioinformatics analysis. Compound-target network and protein-protein interaction of CM-related cancer targets were carried out to determine the most abundant active compound. Simultaneously, the priority target responsible for CM-related anti-tumor efficacy was further validated by molecular docking and in vitro experiments. RESULTS: In total, approximately 42% (8/19) of the targets were enriched in prostate cancer (p = 1.25E-09), suggesting prostate cancer would be the most sensitive tumor response to CM-related efficacy. Furthermore, we found that arnicolide D (ARD), the most abundant and representative active compound of CM, could directly bind to Src with binding energy of -7.3 kcal/mol, implying Src would be the priority target responsible for CM-related anti-tumor efficacy. Meanwhile, the results were further validated by solvent-induced protein precipitation (SIP) assay. In addition, PCR and WB results also revealed that either CM or ARD could not influence the gene expression of Src, while significantly decreased its protein expression instead, which further suggested that ARD might markedly shortene the Src protein half-life to promote Src protein degradation, thereby achieving significant anti-prostate cancer efficacy. CONCLUSION: Our findings not only suggest CM as a promising Src-targeting candidate for prostate cancer treatment, but also bring up a strategy for understanding the personalization of herbal medicines by using integrative pharmacology.

miR-7/TGF-ß2 axis sustains acidic tumor microenvironment-induced lung cancer metastasis.

Acidosis, regardless of hypoxia involvement, is recognized as a chronic and harsh tumor microenvironment (TME) that educates malignant cells to thrive and metastasize. Although overwhelming evidence supports an acidic environment as a driver or ubiquitous hallmark of cancer progression, the unrevealed core mechanisms underlying the direct effect of acidification on tumorigenesis have hindered the discovery of novel therapeutic targets and clinical therapy. Here, chemical-induced and transgenic mouse models for colon, liver and lung cancer were established, respectively. miR-7 and TGF-ß2 expressions were examined in clinical tissues (n = 184). RNA-seq, miRNA-seq, proteomics, biosynthesis analyses and functional studies were performed to validate the mechanisms involved in the acidic TME-induced lung cancer metastasis. Our data show that lung cancer is sensitive to the increased acidification of TME, and acidic TME-induced lung cancer metastasis via inhibition of miR-7-5p. TGF-ß2 is a direct target of miR-7-5p. The reduced expression of miR-7-5p subsequently increases the expression of TGF-ß2 which enhances the metastatic potential of the lung cancer. Indeed, overexpression of miR-7-5p reduces the acidic pH-enhanced lung cancer metastasis. Furthermore, the human lung tumor samples also show a reduced miR-7-5p expression but an elevated level of activated TGF-ß2; the expressions of both miR-7-5p and TGF-ß2 are correlated with patients' survival. We are the first to identify the role of the miR-7/TGF-ß2 axis in acidic pH-enhanced lung cancer metastasis. Our study not only delineates how acidification directly affects tumorigenesis, but also suggests miR-7 is a novel reliable biomarker for acidic TME and a novel therapeutic target for non-small cell lung cancer (NSCLC) treatment. Our study opens an avenue to explore the pH-sensitive subcellular components as novel therapeutic targets for cancer treatment.

Inhibition of hepatitis B virus (HBV) by LNA-mediated nuclear interference with HBV DNA transcription.

Silencing target genes with small regulatory RNAs is widely used to investigate gene function and therapeutic drug development. Recently, triplex-based approaches have provided another attractive means to achieve targeted gene regulation and gene manipulation at the molecular and cellular levels. Nuclear entry of oligonucleotides and enhancement of their affinity to the DNA targets are key points of such approaches. In this study, we developed lipid-based transport of a locked-nucleic-acid (LNA)-modified oligonucleotide for hepatitis B virus (HBV) DNA interference in human hepatocytes expressing HBV genomic DNA. In these cells, the LNA-modified oligonucleotides passed efficiently across the cell membrane, and lipid-coating facilitated translocation from the cytoplasm to the nucleus. The oligonucleotide specifically targeting HBV DNA clearly interfered with HBV DNA transcription as shown by a block in pregenomic RNA (pgRNA) production. The HBV DNA-targeted oligonucleotide suppressed HBV DNA replication and HBV protein production more efficiently than small interfering RNAs directed to the pgRNA. These results demonstrate that fusion with lipid can carry LNA-modified oligonucleotides to the nucleus where they regulate gene expression. Interfering with HBV DNA transcription by LNA-modified oligonucleotides has strong potential as a new strategy for HBV inhibition.

[Preliminary study on the efficacy and safety of oral sildenafil in pulmonary arterial hypertension in China].

OBJECTIVE: Sildenafil has been shown to be effective in pulmonary arterial hypertension (PAH). However, the impact of sildenafil on PAH has been under-investigated in China. The aim of the present study was to evaluate the efficacy and safety of oral sildenafil in PAH patients in China. METHODS: In this prospective, open-label and multi-center study, 90 patients were recruited from 14 centers to receive oral sildenafil (75 mg/d) for 12 weeks. They underwent a six-minute walk test (SMWT) and cardiac catheterization at the beginning and the end of 12 weeks. The primary endpoint was the changes in exercise capacity as assessed by SMWT. And the secondary endpoints included assessment of functional class, evaluation of cardiopulmonary hemodynamics and clinical deterioration (defined as death, transplantation and re-hospitalization for PAH). Drug safety and tolerability were also examined. RESULTS: There were 19 males and 71 females with an average age of 32.5 ± 12.1 years old (range: 18 - 61). Their etiologies were idiopathic (n = 15), related with congenital heart disease (n = 60), or related with connective tissue disease (n = 9) and chronic thromboembolic pulmonary hypertension (n = 6). Oral sildenafil significantly increased the SMWT distances [(342 ± 93) m vs. (403 ± 88) m, P < 0.001]. There was also remarkable improvement in Borg dyspnea score (2.9 ± 2.6 vs. 2.4 ± 2.0, P = 0.005). Furthermore, significant improvements in World Healthy Organization (WHO) functional class and cardiopulmonary hemodynamics were also found (mean pulmonary artery pressure, P < 0.001; cardiac index, P < 0.001; pulmonary vascular resistance, P < 0.001). Side effects were mild and consistent with other reports. CONCLUSION: This study confirms and extends previous studies. Oral sildenafil is both safe and effective for the treatment of adult PAH patients in China.