Stable reference gene selection for Ophiocordyceps sinensis gene expression studies under different developmental stages and light-induced conditions.

The molecular mechanism of Chinese cordyceps formation has received a substantial amount of attention because of its usage as traditional Chinese medicine. The formation process of Chinese cordyceps includes two parts: asexual proliferation (Ophiocordyceps sinensis proliferates in the hemolymph of Thitarodes armoricanus larvae) and sexual development (formation and development of fruiting bodies). Therefore, validation of reference genes under different development stages and experimental conditions is crucial for RT-qPCR analysis. However, there is no report on stable reference genes at the development stage of O. sinensis fruiting body. In this study, 10 candidate reference genes, Actin, Cox5, Tef1, Ubi, 18s, Gpd, Rpb1, Try, Tub1 and Tub2, were selected and calculated their expression stability using four methods: geNorm, NormFinder, BestKeeper, and Comparative △Ct. After comprehensive analysis of the results of these four methods with RefFinder, we determined that the most stable reference genes during asexual reproduction of O. sinensis were Tef1 and Tub1, while the most stable reference genes during fruiting body development were Tyr and Cox5, and the most stable reference genes under light-induced conditions were Tyr and Tef1. Our study provides a guidance for reference genes selections at different proliferation processes with light stress of O. sinensis, and represents a foundation for studying the molecular mechanism of Chinese cordyceps formation.

Programmable multistage small-molecule nano-photosensitizer for multimodal imaging-guided photothermal therapy.

Photothermal therapy has become a promising approach as precision medicine to allow spatial control of therapeutic effect only in the site of interest. However, the full potential of PTT has not been realized due to the lack of simple photosensitizers (PSs) that can overcome multistage biological barriers and improve theranostic efficiency. Here, we develop a small molecule-based PS to enhance tumor-specific PTT by programming multistage transport and activation properties in molecular architecture. This PS can self-assemble into stable nanoparticles that accumulate passively in tumor, and then actively internalize through ligand-mediated endocytosis. Subsequently, the programmable degradable linkers are selectively cleaved, enabling size shrinkage for better tumor penetration, binding albumin to enhance the near-infrared fluorescence for low-background imaging, and activating photothermal conversion for tumor suppression. The self-delivery process can be programmed, representing the first multistage small-molecule nano-photosensitizer that overcomes multiple biological barriers and improves the PTT index of tumor. STATEMENT OF SIGNIFICANCE: Photothermal therapy has become a promising approach as precision medicine, but has not been realized due to the lack of simple photosensitizers that can overcome multistage biological barriers and improve theranostic efficiency. In this contribution, we solve this dilemma by developing a small molecule-based photosensitizer by programming multistage transport and activation properties in molecular architecture, which could self-assemble into stable nanoparticles that accumulate passively in tumor, and actively internalized through ligand-mediated endocytosis. Subsequently, the programmable activation by ROS triggered size reduction for tumor penetration and minimized the phototoxicity to normal tissue. The activatable fluorescence and photothermal properties made the photosensitizer intrinsically suitable for multimodal imaging-guided PTT, providing a promising supramolecular nanomedicine towards tumor precise diagnosis and therapy.

Lighting up Self-Quenching Nanoaggregates with Protein Corona for Simultaneous Intraoperative Imaging and Photothermal Theranostics of Metastatic Cancer.

Near-infrared (NIR) photothermal transduction agents (PTAs) with large rigid π-extended and planar structures are prone to aggregate in a physiological environment where their emission is often quenched due to the strong intermolecular dipole-dipole or π-π interactions. This aggregation-caused quenching effect greatly impedes their applications in image-guided photothermal theranostics. Herein, we made an interesting finding that engineering a bioinspired protein corona (PC), once thermodynamically stabilized in preferred orientations on PTA nanoaggregates, can produce brilliant NIR fluorescence with a high quantum yield (∼6.2%) without compromising their photothermal properties. Both experimental data and computational modeling suggest that the mechanism of fluorescence enhancement is due to the high-affinity binding of nano-sized PTA to albumin, which regulates the molecular conformation and aggregation state of PTA. High spatial and temporal resolution imaging of albumin PC-coated PTA aggregates enables image-guided photothermal therapy for cancer cells in sentinel lymph nodes to remarkably inhibit pulmonary metastasis. Such a treatment combined with the surgical removal of the primary tumor can prolong animal survival, which is a promising candidate for clinical applications in the treatment of advanced metastatic cancers.

Angelica sinensis polysaccharide (ASP) attenuates diosbulbin-B (DB)-induced hepatotoxicity through activating the MEK/ERK pathway.

Diosbulbin-B (DB) is a promising therapeutic drug for cancer treatment; however, DB-induced hepatotoxicity seriously limits its clinical utilization. Based on this, the present study investigated whether the Angelica sinensis extract, angelica sinensis polysaccharide (ASP), was effective to attenuate DB-induced cytotoxicity in hepatocytes. The primary hepatocytes were isolated from rats and cultured in vitro, which were subsequently treated with high-dose DB (100 µM) and ASP (12 µg/ml) to establish the DB-induced hepatotoxicity models. MTT assay and flow cytometry (FCM) were performed to evaluate cell viability, and the results showed that high-dose DB-induced cell apoptosis and inhibition of proliferation were reversed by co-treating cells with ASP, which were supported by our Western Blot assay data that ASP upregulated Cyclin D1 and CDK2 to abrogate high-dose DB-induced cell cycle arrest. In addition, ASP exerted its regulating effects on cell autophagy, and we found that ASP increased LC3B-II/I ratio and Atg5, but decreased p62 to activate the autophagy flux. Of note, the MEK/ERK pathway could be activated by ASP in the DB-treated hepatocytes, and the protective effects of ASP on high-dose DB-induced hepatocyte death were abolished by co-treating cells with the autophagy inhibitor (3-methyladenine, 3-MA) and MEK/ERK selective inhibitor (SCH772984). Moreover, blockage of the MEK/ERK pathway suppressed cell autophagy in the hepatocytes co-treated with ASP and high-dose DB. Taken together, this in vitro study illustrated that ASP activated the MEK/ERK pathway mediated autophagy to suppress high-dose DB-induced hepatotoxicity.

The single-cell epigenomic and transcriptional landscape of immunity to influenza vaccination.

Emerging evidence indicates a fundamental role for the epigenome in immunity. Here, we mapped the epigenomic and transcriptional landscape of immunity to influenza vaccination in humans at the single-cell level. Vaccination against seasonal influenza induced persistently diminished H3K27ac in monocytes and myeloid dendritic cells (mDCs), which was associated with impaired cytokine responses to Toll-like receptor stimulation. Single-cell ATAC-seq analysis revealed an epigenomically distinct subcluster of monocytes with reduced chromatin accessibility at AP-1-targeted loci after vaccination. Similar effects were observed in response to vaccination with the AS03-adjuvanted H5N1 pandemic influenza vaccine. However, this vaccine also stimulated persistently increased chromatin accessibility at interferon response factor (IRF) loci in monocytes and mDCs. This was associated with elevated expression of antiviral genes and heightened resistance to the unrelated Zika and Dengue viruses. These results demonstrate that vaccination stimulates persistent epigenomic remodeling of the innate immune system and reveal AS03's potential as an epigenetic adjuvant.

Adjuvanting a subunit COVID-19 vaccine to induce protective immunity.

The development of a portfolio of COVID-19 vaccines to vaccinate the global population remains an urgent public health imperative1. Here we demonstrate the capacity of a subunit vaccine, comprising the SARS-CoV-2 spike protein receptor-binding domain displayed on an I53-50 protein nanoparticle scaffold (hereafter designated RBD-NP), to stimulate robust and durable neutralizing-antibody responses and protection against SARS-CoV-2 in rhesus macaques. We evaluated five adjuvants including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an α-tocopherol-containing oil-in-water emulsion; AS37, a Toll-like receptor 7 (TLR7) agonist adsorbed to alum; CpG1018-alum, a TLR9 agonist formulated in alum; and alum. RBD-NP immunization with AS03, CpG1018-alum, AS37 or alum induced substantial neutralizing-antibody and CD4 T cell responses, and conferred protection against SARS-CoV-2 infection in the pharynges, nares and bronchoalveolar lavage. The neutralizing-antibody response to live virus was maintained up to 180 days after vaccination with RBD-NP in AS03 (RBD-NP-AS03), and correlated with protection from infection. RBD-NP immunization cross-neutralized the B.1.1.7 SARS-CoV-2 variant efficiently but showed a reduced response against the B.1.351 variant. RBD-NP-AS03 produced a 4.5-fold reduction in neutralization of B.1.351 whereas the group immunized with RBD-NP-AS37 produced a 16-fold reduction in neutralization of B.1.351, suggesting differences in the breadth of the neutralizing-antibody response induced by these adjuvants. Furthermore, RBD-NP-AS03 was as immunogenic as a prefusion-stabilized spike immunogen (HexaPro) with AS03 adjuvant. These data highlight the efficacy of the adjuvanted RBD-NP vaccine in promoting protective immunity against SARS-CoV-2 and have led to phase I/II clinical trials of this vaccine (NCT04742738 and NCT04750343).

Adjuvanting a subunit SARS-CoV-2 nanoparticle vaccine to induce protective immunity in non-human primates.

The development of a portfolio of SARS-CoV-2 vaccines to vaccinate the global population remains an urgent public health imperative. Here, we demonstrate the capacity of a subunit vaccine under clinical development, comprising the SARS-CoV-2 Spike protein receptor-binding domain displayed on a two-component protein nanoparticle (RBD-NP), to stimulate robust and durable neutralizing antibody (nAb) responses and protection against SARS-CoV-2 in non-human primates. We evaluated five different adjuvants combined with RBD-NP including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an alpha-tocopherol-containing squalene-based oil-in-water emulsion used in pandemic influenza vaccines; AS37, a TLR-7 agonist adsorbed to Alum; CpG 1018-Alum (CpG-Alum), a TLR-9 agonist formulated in Alum; or Alum, the most widely used adjuvant. All five adjuvants induced substantial nAb and CD4 T cell responses after two consecutive immunizations. Durable nAb responses were evaluated for RBD-NP/AS03 immunization and the live-virus nAb response was durably maintained up to 154 days post-vaccination. AS03, CpG-Alum, AS37 and Alum groups conferred significant protection against SARS-CoV-2 infection in the pharynges, nares and in the bronchoalveolar lavage. The nAb titers were highly correlated with protection against infection. Furthermore, RBD-NP when used in conjunction with AS03 was as potent as the prefusion stabilized Spike immunogen, HexaPro. Taken together, these data highlight the efficacy of the RBD-NP formulated with clinically relevant adjuvants in promoting robust immunity against SARS-CoV-2 in non-human primates.

GPC3-targeted and curcumin-loaded phospholipid microbubbles for sono-photodynamic therapy in liver cancer cells.

More effective strategies are needed to improve the treatment of liver cancer. Sono-photodynamic therapy (SPDT) has a more obvious antitumor effect than sonodynamic therapy (SDT) or photodynamic therapy (PDT). We aimed to investigate Glypican-3-targeted, curcumin-loaded microbubbles (GPC3-CUR-MBs)-mediated SPDT in liver cancer cells in vitro and in vivo. GPC3-CUR-MBs were prepared by streptavidin-biotin interactions and the immune ligation method. The characterization and toxicity of GPC3-CUR-MBs and the anti-liver cancer effects of GPC3-CUR-MB-mediated SPDT in vitro and in vivo were studied. We synthetized GPC3-CUR-MBs and found that GPC3-CUR-MBs had no significant toxicity to HepG2 liver cancer cells. In terms of the anti-liver cancer effects in vitro and in vivo, when we used CUR, CUR-MBs or GPC3-CUR-MBs as the sono/photosensitizers, the outcome of SPDT was superior to that of SDT or PDT alone. The outcomes with GPC3-CUR-MBs were better than those with CUR or CUR-MBs in the SDT, PDT or SPDT groups. During the treatment period, the weight of the HepG2 tumor-bearing mice did not decrease significantly, and no significant evidence of lung, heart, liver, spleen and kidney damage was found with H&E staining. Our results indicated that the anti-liver tumor effect of SPDT was better than that of SDT and PDT and that GPC3-CUR-MBs were promising sono/photosensitizers.

Combinatorial screening of a panel of FDA-approved drugs identifies several candidates with anti-Ebola activities.

Ebola virus (EBOV), pathogen of Ebola hemorrhagic fever (EHF), is an enveloped filamental RNA virus. Recently, the EHF crisis occurred in the Democratic Republic of the Congo again highlights the urgency for its clinical treatments. However, no Food and Drug Administration (FDA)-approved therapeutics are currently available. Drug repurposing screening is a time- and cost-effective approach for identifying anti-EBOV therapeutics. Here, by combinatorial screening using pseudovirion and minigenome replicon systems we have identified several FDA-approved drugs with significant anti-EBOV activities. These potential candidates include azithromycin, clomiphene, chloroquine, digitoxin, epigallocatechin-gallate, fluvastatin, tetrandrine and tamoxifen. Mechanistic studies revealed that fluvastatin inhibited EBOV pseudovirion entry by blocking the pathway of mevalonate biosynthesis, while the inhibitory effect of azithromycin on EBOV maybe due to its intrinsic cationic amphiphilic structure altering the homeostasis of later endosomal vesicle similar as tamoxifen. Moreover, based on structure and pathway analyses, the anti-EBOV activity has been extended to other family members of statins, such as simvastatin, and multiple other cardiac glycoside drugs, some of which exhibited even stronger activities. More importantly, in searching for drug interaction, we found various synergy between several anti-EBOV drug combinations, showing substantial and powerful synergistic against EBOV infection. In conclusion, our work illustrates a successful and productive approach to identify new mechanisms and targets for treating EBOV infection by combinatorial screening of FDA-approved drugs.

Downregulation of CircRNA CDR1as specifically triggered low-dose Diosbulbin-B induced gastric cancer cell death by regulating miR-7-5p/REGγ axis.

Diosbulbin-B (DB) was the main compound of Dioscorea bulbifera L, which was widely used for cancer treatment in Asia. However, the hepatotoxicity induced by high-dose DB seriously limited its possibility using for gastric cancer (GC) treatment in clinic. In this study, we found that DB inhibited GC cells and hepatocytes cell viability in a dose- and time-dependent manner. Specifically, high-dose DB (50µM) significantly inhibited cell proliferation and promoted cell apoptosis, while low dose DB (12.5µM) had little effects on cell viability. Besides, high-dose DB (50µM) significantly decreased CircRNA CDR1as levels in gastric cancer cells instead of hepatocytes. Notably, knock-down of CircRNA CDR1as triggered low-dose DB (12.5µM) induced GC cell death, but had little effects on hepatocytes proliferation and apoptosis. Further results showed that CircRNA CDR1as increased REGγ expressions in GC cells by sponging miR-7-5p, and high-dose DB (50µM) increased miR-7-5p levels and inhibited REGγ expressions in GC cells instead of hepatocytes. In addition, either downregulated miR-7-5p or overexpressed REGγ reversed the promoting effects of downregulated CircRNA CDR1as on low-dose DB-induced GC cell death. Taken together, we concluded that knock-down of CircRNA CDR1as specifically promoted the cytotoxic effects of low-dose DB on GC cells instead of hepatocytes by regulating miR-7-5p/REGγ axis.

Chloroquine, a FDA-approved Drug, Prevents Zika Virus Infection and its Associated Congenital Microcephaly in Mice.

Zika virus (ZIKV) has become a global public health emergency due to its rapidly expanding range and its ability to cause severe congenital defects such as microcephaly. However, there are no FDA-approved therapies or vaccines against ZIKV infection. Through our screening of viral entry inhibitors, we found that chloroquine (CQ), a commonly used antimalarial and a FDA-approved drug that has also been repurposed against other pathogens, could significantly inhibit ZIKV infection in vitro, by blocking virus internalization. We also demonstrated that CQ attenuates ZIKV-associated morbidity and mortality in mice. Finally, we proved that CQ protects fetal mice from microcephaly caused by ZIKV infection. Our methodology of focusing on previously identified antivirals in screens for effectiveness against ZIKV proved to be a rapid and efficient means of discovering new ZIKV therapeutics. Selecting drugs that were previously FDA-approved, such as CQ, also improves the likelihood that they may more quickly reach stages of clinical testing and use by the public.

Screening for Novel Small-Molecule Inhibitors Targeting the Assembly of Influenza Virus Polymerase Complex by a Bimolecular Luminescence Complementation-Based Reporter System.

Influenza virus RNA-dependent RNA polymerase consists of three viral protein subunits: PA, PB1, and PB2. Protein-protein interactions (PPIs) of these subunits play pivotal roles in assembling the functional polymerase complex, which is essential for the replication and transcription of influenza virus RNA. Here we developed a highly specific and robust bimolecular luminescence complementation (BiLC) reporter system to facilitate the investigation of influenza virus polymerase complex formation. Furthermore, by combining computational modeling and the BiLC reporter assay, we identified several novel small-molecule compounds that selectively inhibited PB1-PB2 interaction. Function of one such lead compound was confirmed by its activity in suppressing influenza virus replication. In addition, our studies also revealed that PA plays a critical role in enhancing interactions between PB1 and PB2, which could be important in targeting sites for anti-influenza intervention. Collectively, these findings not only aid the development of novel inhibitors targeting the formation of influenza virus polymerase complex but also present a new tool to investigate the exquisite mechanism of PPIs. IMPORTANCE Formation of the functional influenza virus polymerase involves complex protein-protein interactions (PPIs) of PA, PB1, and PB2 subunits. In this work, we developed a novel BiLC assay system which is sensitive and specific to quantify both strong and weak PPIs between influenza virus polymerase subunits. More importantly, by combining in silico modeling and our BiLC assay, we identified a small molecule that can suppress influenza virus replication by disrupting the polymerase assembly. Thus, we developed an innovative method to investigate PPIs of multisubunit complexes effectively and to identify new molecules inhibiting influenza virus polymerase assembly.

Effect of Electroacupuncture on Transcutaneous Oxygen Partial Pressure During Hyperbaric Oxygen Therapy in Healthy Individuals.

CONTEXT: The goal of hyperbaric oxygen therapy (HBOT) is to increase the oxygen (O2) supply to the body significantly. Because of the toxic side effects and complications of hyperbaric oxygen (HBO2), the environmental pressure and treatment time must be restricted. The research team hypothesized that other therapies administered during HBOT could safely improve the value of the arterial oxygen partial pressure (PaO2) during HBOT and improve its therapeutic effect. OBJECTIVE: The study intended to investigate whether electroacupuncture (EA) while receiving HBOT had a greater effect for healthy individuals than HBOT or EA alone or EA combined with normobaric pure oxygen (pure O2). DESIGN: The research team designed a randomized, controlled trial. SETTING: The study was performed in the Department of Hyperbaric Medicine at the No. 401 Hospital of the People's Liberation Army in Qingdao, China. PARTICIPANTS: A total of 81 volunteers were recruited. After thorough physical examination and laboratory testing, 21 volunteers were excluded from the study. Participants included 60 healthy volunteers. INTERVENTION: Participants were randomly assigned to 1 of 4 groups of 15 participants each: (1) an HBOT group, (2) an EA group, (3) an EA During HBOT group, and (4) an EA Combined With Pure O2group. OUTCOME MEASURES: Because at the current technology level a blood gas analyzer cannot test PaO2during HBOT, transcutaneous oxygen partial pressure (PtcO2) of the participants was tested instead. Before, during, and after EA, variations in PtcO2were monitored in each group. RESULTS: For the EA During HBOT group, (1) the increase in PtcO2during EA was significantly greater than that observed for the other 3 groups (P > .05). CONCLUSIONS: The EA During HBOT method provided improvements in the efficacy, safety, and tolerability of HBOT, and the study's results partially demonstrated the accuracy of the research team's hypothesis that EA therapy applied during HBOT could safely improve the value of PtcO2(PaO2) during HBOT and produce a greater therapeutic effect.

Analysis of medicine consumption in peacekeeping level II hospitals.

BACKGROUND: The peacekeeping military units of contributing countries are unfamiliar with the conditions prevailing in foreign mission areas and therefore have difficulties with medical supplies storage. AIM: The aim of this study is to provide reasonable and practical guidance on the maintenance of medical supplies in the peacekeeping military units of contributing countries. MATERIALS AND METHODS: A total of 1,972 prescriptions were received by the pharmacy in the peacekeeping level II hospital in the Republic of Sudan from February to July of 2009 including a total of 186 drug categories and 17,713 minimum packing units. Pairwise comparison was performed using the c2 test. When the total number of samples was smaller than 40, the Fisher's exact test was adopted for pairwise comparison. RESULTS: The majority of the consumed medicines mainly belonged to 6 categories, including specialty drugs, anti-microbial drugs, Chinese patent medicines, gastrointestinal drugs, central nervous system drugs, and drugs regulating fluids, electrolytes, and acid-base balance. Altogether, the drugs in the 6 categories accounted for 74% of all consumed medicines that were divided into a total of 20 categories. CONCLUSIONS: Medicine consumption in peacekeeping level II hospitals is unique, therefore the drugs used in military medical facilities should be prepared according to their actual needs in the area of peacekeeping operations.

Antiangiogenetic effects of 4 varieties of grapes in vitro.

The purpose of this study was to investigate the inhibitory effects of grapes on the human umbilical vein endothelial (HUVE) cells' capillary tube formation and matrix metalloproteinase-2 (MMP-2) expression secreted into the medium. Four different grape varieties (Concord, Niagara, Chardonnay, and Pinot Noir) were extracted using 80% acetone and the extracts were stored at -80 degrees C. The total amount of phenolics and flavonoids for each of the 4 grape varieties were determined by spectrophotometry. Grape extracts were co-cultured with HUVE cells on Matrigel and inhibitory effects on tube formation were observed under a microscope. The inhibitory effects of grape extracts on MMP-2 expression were examined by zymogram. All 4 grape varieties inhibited the tube formation of HUVE cells in a dose-dependent manner on Matrigel. Except for Chardonnay, the other 3 grape varieties completely inhibited secretion of MMP-2 at 20 mg/mL. There was a significant positive relationship between the total phenolics and flavonoids and antiangiogenetic activities. The grapes tested have the potential to inhibit angiogenesis mainly by their phenolics and flavonoids contents, which partly contribute to their cancer chemopreventive efficacy.