Inhibition of AKR1Cs by liquiritigenin and the structural basis.

In vivo and in vitro studies have confirmed that liquiritigenin (LQ), the primary active component of licorice, acts as an antitumor agent. However, how LQ diminishes or inhibits tumor growth is not fully understood. Here, we report the enzymatic inhibition of LQ and six other flavanone analogues towards AKR1Cs (AKR1C1, AKR1C2 and AKR1C3), which are involved in prostate cancer, breast cancer, and resistance of anticancer drugs. Crystallographic studies revealed AKR1C3 inhibition of LQ is related to its complementarity with the active site and the hydrogen bonds net in the catalytic site formed through C7-OH, aided by its nonplanar and compact structure due to the saturation of the C2C3 double bond. Comparison of the LQ conformations in the structures of AKR1C1 and AKR1C3 revealed the induced-fit conformation changes, which explains the lack of isoform selectivity of LQ. Our findings will be helpful for better understanding the antitumor effects of LQ on hormonally dependent cancers and the rational design of selective AKR1Cs inhibitors.

Corydalis decumbens Alleviates the Migration, Phagocytosis, and Inflammatory Response of Macrophages.

Background: The role of Corydalis decumbens (CD) in macrophage activation remains unclear, particularly in the Ras homolog family member A (RhoA) signaling pathway. Therefore, the present study aimed to investigate the effect of CD on the viability, proliferation, morphological changes, migration, phagocytosis, differentiation, and release of inflammatory factors and signaling pathways in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Methods: Cell counting kit-8 and water-soluble tetrazolium salt assays were used to evaluate the viability and proliferation of RAW264.7 macrophages. A transwell assay was examined to assess cell migration. The ingestion of lumisphere assay was employed to detect the phagocytic capacity of macrophages. Phalloidin staining was performed to observe morphological changes in the macrophages. An enzyme-linked immunosorbent assay was performed to quantify inflammation-related cytokines in cell culture supernatants. Cellular immunofluorescence and western blotting were adopted to show the expression of inflammation-related factors, biomarkers of M1/M2 subset macrophages, and factors of the RhoA signaling pathway. Results: We found that CD increased the viability and proliferation of RAW264.7 macrophages. CD also impaired the migration and phagocytic capacity of macrophages, induced anti-inflammatory M2 macrophage polarization, such as M2-like morphological changes, and upregulated M2 macrophage biomarkers and anti-inflammatory factors. We also observed that CD inactivated the RhoA signaling pathway. Conclusions: CD mediates the activation of LPS-stimulated macrophages, alleviates the inflammatory responses of macrophages, and activates related signaling pathways induced by LPS.

Five-lipoxygenase-activating protein-mediated CYLD attenuation is a candidate driver in hepatic malignant lesion.

Hepatocellular carcinoma (HCC) is an inflammation-associated cancer. However, the lipid pro-inflammatory mediators have only been seldom investigated in HCC pathogenesis. Cylindromatosis (CYLD) attenuation is involved in hepatocarcinogenesis. Here, we aimed to evaluate the significance of hepatic lipid pro-inflammatory metabolites of arachidonate-affected CYLD expression via the 5-lipoxygenase (5-LO) pathway. Resection liver tissues from HCC patients or donors were evaluated for the correlation of 5-LO/cysteinyl leukotrienes (CysLTs) signaling to the expression of CYLD. The impact of functional components in 5-LO/CysLTs cascade on survival of HCC patients was subsequently assessed. Both livers from canines, a preponderant animal for cancer research, and genetic-modified human HCC cells treated with hepatocarcinogen aristolochic acid I (AAI) were further used to reveal the possible relevance between 5-LO pathway activation and CYLD suppression. Five-LO-activating protein (FLAP), an essential partner of 5-LO, was significantly overexpressed and was parallel to CYLD depression, CD34 neovascular localization, and high Ki-67 expression in the resection tissues from HCC patients. Importantly, high hepatic FLAP transcription markedly shortened the median survival time of HCC patients after surgical resection. In the livers of AAI-treated canines, FLAP overexpression was parallel to enhanced CysLTs contents and the simultaneous attenuation of CYLD. Moreover, knock-in FLAP significantly diminished the expression of CYLD in AAI-treated human HCC cells. In summary, the hepatic FLAP/CysLTs axis is a crucial suppressor of CYLD in HCC pathogenesis, which highlights a novel mechanism in hepatocarcinogenesis and progression. FLAP therefore can be explored for the early HCC detection and a target of anti-HCC therapy.

Seroprevalence of neutralizing antibodies against adenovirus type 26 and 35 in healthy populations from Guangdong and Shandong provinces, China.

Human adenoviruses type 26 (HAdV26) and type 35 (HAdV35) have increasingly become the choice of adenovirus vectors for vaccine application. However, the population pre-existing immunity to these two adenoviruses in China, which may reduce vaccine efficacy, remains largely unknown. Here, we established micro-neutralizing (MN) assays to investigate the seroprevalence of neutralizing antibodies (nAbs) against HAdV26 and HAdV35 in the general population of Guangdong and Shandong provinces, China. A total of 1184 serum samples were collected, 47.0% and 15.8% of which showed HAdV26 and HAdV35 nAb activity, respectively. HAdV26-seropositive individuals tended to have more moderate nAbs titers (201-1000), while HAdV35-seropositive individuals appeared to have more low nAbs titers (72-200). The seropositive rates of HAdV26 and HAdV35 in individuals younger than 20 years old were very low. The seropositive rates of HAdV26 increased with age before 70 years old and decreased thereafter, while HAdV35 seropositive rates did not show similar characteristics. Notably, the seropositive rates and nAb levels of both HAdV26 and HAdV35 were higher in Guangdong Province than in Shandong Province, but did not exert significant differences between males and females. The seroprevalence between HAdV26 and HAdV35 showed little correlation, and no significant cross-neutralizing activity was detected. These results clarified the characteristics of the herd immunity against HAdV26 and HAdV35, and provided information for the rational development and application of HAdV26 and HAdV35 as vaccine vectors in China.

[Effect of electroacupuncture on complement C1q and microglia phagocytosis in hippocampus of SAMP8 mice].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the expression of Iba-1, complement C1q and CD68 in hippocampus of SAMP8 mice, so as to explore its mechanisms underlying improvement of Alzheimer's disease (AD). METHODS: Twenty-four male SAMP8 mice were randomly and equally divided into model and EA groups, and 12 SAMR1 mice were used as the control group. EA (2 Hz, 1.5-2.0 mA) was applied to "Baihui" (GV20), "Dazhui"(GV14) and "Shen-shu"(BL23) for 20 min once daily in the EA group, each course of treatment was 8 days, with an interval of 2 days between two courses, and the mice were treated for 3 courses. Morris water maze test was performed to assess the learning-memory ability of mice. The positive expression levels of Iba-1 and CD68 proteins in the hippocampus CA1 region were detected by immunohistochemistry. The mRNA and protein expression levels of Iba-1,C1q and CD68 in the hippocampus were detected by real-time PCR and Western blot, separately. RESULTS: Compared with the control group, the average escape latency of Morris water maze test was prolonged in the model group (P<0.01), duration of swimming in the original platform quadrant and the number of original platform crossing were significantly shorter and decreased respectively (P<0.01). Compared with the model group, the average escape latency in the EA group was shortened (P<0.05, P<0.01), the duration of swimming in the original platform quadrant and the number of original platform crossing were significantly prolonged and increased (P<0.01). The immunoactivity of Iba-1 and CD68 in hippocampal CA1 region, and mRNA and protein expression levels of hippocampal Iba-1,C1q and CD68 were significantly up-regulated in the model group in contrast to the control group (P<0.01, P<0.05), and obviously down-regulated except the mRNA expression level of hippocampal Iba-1 in the EA group relevant to the model group (P<0.01, P<0.05). CONCLUSION: EA can improve the learning and memory ability of SAMP8 mice, which may be associated with its effect in inhibiting of complement C1q-dependent microglial phagocytosis in the hippocampus.

Free energy perturbation (FEP)-guided scaffold hopping.

Scaffold hopping refers to computer-aided screening for active compounds with different structures against the same receptor to enrich privileged scaffolds, which is a topic of high interest in organic and medicinal chemistry. However, most approaches cannot efficiently predict the potency level of candidates after scaffold hopping. Herein, we identified potent PDE5 inhibitors with a novel scaffold via a free energy perturbation (FEP)-guided scaffold-hopping strategy, and FEP shows great advantages to precisely predict the theoretical binding potencies ΔG FEP between ligands and their target, which were more consistent with the experimental binding potencies ΔG EXP (the mean absolute deviations | Δ G FEP - Δ G EXP |  < 2 kcal/mol) than those ΔG MM-PBSA or ΔG MM-GBSA predicted by the MM-PBSA or MM-GBSA method. Lead L12 had an IC50 of 8.7 nmol/L and exhibited a different binding pattern in its crystal structure with PDE5 from the famous starting drug tadalafil. Our work provides the first report via the FEP-guided scaffold hopping strategy for potent inhibitor discovery with a novel scaffold, implying that it will have a variety of future applications in rational molecular design and drug discovery.

The novel indomethacin derivative CZ-212-3 exerts antitumor effects on castration-resistant prostate cancer by degrading androgen receptor and its variants.

Androgen receptor (AR) serves as a main therapeutic target for prostate cancer (PCa). However, resistance to anti-androgen therapy (SAT) inevitably occurs. Indomethacin is a nonsteroidal anti-inflammatory drug that exhibits activity against prostate cancer. Recently, we designed and synthesized a series of new indomethacin derivatives (CZ compounds) via Pd (II)-catalyzed synthesis of substituted N-benzoylindole. In this study, we evaluated the antitumor effect of these novel indomethacin derivatives in castration-resistant prostate cancer (CRPC). Upon employing CCK-8 cell viability assays and colony formation assays, we found that these derivatives had high efficacy against CRPC tumor growth in vitro. Among these derivatives, CZ-212-3 exhibited the most potent efficacy against CRPC cell survival and on apoptosis induction. Mechanistically, CZ-212-3 significantly suppressed the expression of AR target gene networks by degrading AR and its variants. Consistently, CZ-212-3 significantly inhibited tumor growth in CRPC cell line-based xenograft and PDX models in vivo. Taken together, the data show that the indomethacin derivative CZ-212-3 significantly inhibited CRPC tumor growth by degrading AR and its variants and could be a promising agent for CRPC therapy.

Bioadhesive poly(methyl vinyl ether-co-maleic anhydride)-TPGS copolymer modified PLGA/lipid hybrid nanoparticles for improving intestinal absorption of cabazitaxel.

A bioadhesive nanocarrier, PTNP, was constructed by utilizing a novel poly(methyl vinyl ether-co-maleic anhydride)- D-α-Tocopheryl polyethylene glycol succinate (PVMMA-TPGS) copolymer in the PLGA/lipid hybrid nanoparticles (PLGA NPs) for improving oral delivery of cabazitaxel (CTX). The PVMMA-TPGS was synthesized by the ring-opening polymerization of the anhydride groups with the hydroxyl groups, combining the bioadhesive property of PVMMA with P-glycoprotein (P-gp) inhibitory effect of TPGS. The CTX-loaded PTNPs (CTX-PTNPs) were prepared by an emulsification-solvent evaporation method and performed a spherical appearance with a uniform particle size of 192.2 nm. The CTX-PTNPs were surface negatively charged, and exhibited good drug loading (10.2%) and encapsulation efficiency (92.1%). A sustained drug release and high stability in simulated gastrointestinal environment were confirmed in in vitro studies. The in vitro mucin adhesion and in vivo intestinal retention experiments indicated that the PTNPs had a stronger bioadhesive effect and a notably longer intestinal retention than the control PLGA NPs, due to the interaction of PVMMA on the PTNP surface with the intestinal mucosa. Moreover, an enhanced intestinal permeability of the PTNPs was also verified in in vivo and ex vivo intestinal permeation studies, which was probably attributed to the extended retention of PTNPs in intestinal mucosa and the P-gp inhibitory effect of TPGS. As respected, in in vivo pharmacokinetic study, the Tmax and oral bioavailability of CTX were dramatically improved to 1.08 h and 28.84% by the PTNPs, respectively, obviously superior to the CTX solution and the PLGA NPs, further demonstrating the high-efficiency in oral delivery of CTX. Hence, this bioadhesive carrier is proposed to be a potential and promising strategy for increasing oral absorption of small molecule insoluble drugs.

Mucosal Priming with a Recombinant Influenza A Virus-Vectored Vaccine Elicits T-Cell and Antibody Responses to HIV-1 in Mice.

Recombinant influenza A viral (IAV) vectors are potential to stimulate systemic and mucosal immunity, but the packaging capacity is limited and only one or a few epitopes can be carried. Here, we report the generation of a replication-competent IAV vector that carries a full-length HIV-1 p24 gene linked to the 5'-terminal coding region of the neuraminidase segment via a protease cleavage sequence (IAV-p24). IAV-p24 was successfully rescued and stably propagated, and P24 protein was efficiently expressed in infected mammalian cells. In BALB/c mice, IAV-p24 showed attenuated pathogenicity compared to that of the parental A/PR/8/34 (H1N1) virus. An intranasal inoculation with IAV-p24 elicited moderate HIV-specific cell-mediated immune (CMI) responses in the airway and vaginal tracts and in the spleen, and an intranasal boost with a replication-incompetent adenovirus type 2 vector expressing the HIV-1 gag gene (Ad2-gag) greatly improved these responses. Importantly, compared to an Ad2-gag prime plus IAV-p24 boost regimen, the IAV-p24 prime plus Ad2-gag boost regimen had a greater efficacy in eliciting HIV-specific CMI responses. P24-specific CD8+ T cells and antibodies were robustly provoked both systemically and in mucosal sites and showed long-term durability, revealing that IAV-p24 may be used as a mucosa-targeted priming vaccine. Our results illustrate that IAV-p24 is able to prime systemic and mucosal immunity against HIV-1 and warrants further evaluation in nonhuman primates.IMPORTANCE An effective HIV-1 vaccine remains elusive despite nearly 40 years of research. CD8+ T cells and protective antibodies may both be desirable for preventing HIV-1 infection in susceptible mucosal sites. Recombinant influenza A virus (IAV) vector has the potential to stimulate these immune responses, but the packaging capacity is extremely limited. Here, we describe a replication-competent IAV vector expressing the HIV-1 p24 gene (IAV-p24). Unlike most other IAV vectors that carried one or several antigenic epitopes, IAV-p24 stably expressed the full-length P24 protein, which contains multiple epitopes and is highly conserved among all known HIV-1 sequences. Compared to the parental A/PR/8/34 (H1N1) virus, IAV-p24 showed an attenuated pathogenicity in BALB/c mice. When combined with an adenovirus vector expressing the HIV-1 gag gene, IAV-p24 was able to prime P24-specific systemic and mucosal immune responses. IAV-p24 as an alternative priming vaccine against HIV-1 warrants further evaluation in nonhuman primates.

Utilization of PLGA nanoparticles in yeast cell wall particle system for oral targeted delivery of exenatide to improve its hypoglycemic efficacy.

Oral delivery of exenatide (EXE), a high-efficiency therapeutic peptide, is urgently needed for long-term treatment of diabetes. In this study, a polylactide-co-glycoside (PLGA) nanoparticles (NPs) in yeast cell wall particle (YCWP) system was built to improve the intestinal absorption of EXE by efficient protection of EXE against gastrointestinal degradation and intestinal phagocytic cell targeted delivery. The EXE-loaded PLGA NPs were prepared by a double emulsion solvent diffusion method and exhibited a uniformly spherical appearance, a nano size (92.4 ± 4.6 nm) and a positive surface charge (+32.3 ± 3.8 mV). And then, the NPs were successfully loaded into the YCWPs by a solvent hydration - lyophilization cycle method to obtain the EXE-PLGA NPs @YCWPs, which was verified by scanning electron microscope and confocal laser scanning microscopy. An obvious sustained drug release and a reduced burst release were achieved by this nano-in-micro carrier. Moreover, the gastrointestinal stability of EXE in PLGA NPs @YCWPs was significantly higher than that in PLGA NPs in the simulated gastrointestinal environment, which were useful in enhancing the intestinal absorption of EXE. In biodistribution study, the EXE-PLGA NPs @YCWPs could quickly reached the root of the villi, and even partly entered the inner of the villi, especially in ileum and Peyer's patches. In vitro cell evaluation demonstrated an efficient ß-glucan receptor mediated endocytosis and transport of EXE-PLGA NPs @YCWPs by the macrophage RAW 264.7 cells, suggesting a potential intestinal macrophage targeted absorptive pathway. The in vivo pharmacokinetic study showed a preferred hypoglycemic effect and an increased pharmacological availability (13.7 ± 4.1%) after oral administration of the EXE-PLGA NPs @YCWPs. It is believed that the PLGA nanoparticles in YCWP system could become an efficient strategy to orally deliver therapeutic peptide drugs.

A Live-Attenuated Zika Virus Vaccine with High Production Capacity Confers Effective Protection in Neonatal Mice.

Zika virus (ZIKV) infection during pregnancy has been linked to congenital abnormalities, such as microcephaly in infants. An efficacious vaccine is desirable for preventing the potential recurrence of ZIKV epidemic. Here, we report the generation of an attenuated ZIKV (rGZ02a) that has sharply decreased virulence in mice but grows to high titers in Vero cells, a widely approved cell line for manufacturing human vaccines. Compared to the wild-type ZIKV (GZ02) and a plasmid-launched rGZ02p, rGZ02a has 3 unique amino acid alterations in the envelope (E, S304F), nonstructural protein 1 (NS1, R103K), and NS5 (W637R). rGZ02a is more sensitive to type I interferon than GZ02 and rGZ02p, and causes no severe neurological disorders in either wild-type neonatal C57BL/6 mice or type I interferon receptor knockout (Ifnar1-/-) C57BL/6 mice. Immunization with rGZ02a elicits robust inhibitory antibody responses with a certain long-term durability. Neonates born to the immunized dams are effectively protected against ZIKV-caused neurological disorders and brain damage. rGZ02a as a booster vaccine greatly improves the protective immunity primed by Ad2-prME, an adenovirus-vectored vaccine expressing ZIKV prM and E proteins. Our results illustrate that rGZ02a-induced maternal immunity can be transferred to the neonates and confer effective protection. Hence, rGZ02a may be developed as an alternative live-attenuated vaccine and warrants further evaluation. IMPORTANCE Zika virus (ZIKV), a mosquito-borne flavivirus that has caused global outbreaks since 2013, is associated with severe neurological disorders, such as Guillian-Barré syndrome in adults and microcephaly in infants. The ZIKV epidemic has gradually subsided, but a safe and effective vaccine is still desirable to prevent its potential recurrence, especially in countries of endemicity with competent mosquito vectors. Here, we describe a novel live-attenuated ZIKV, rGZ02a, that carries 3 unique amino acid alterations compared to the wild-type GZ02 and a plasmid-launched rGZ02p. The growth capacity of rGZ02a is comparable to GZ02 in Vero cells, but the pathogenicity is significantly attenuated in two mice models. Immunization with rGZ02a elicits robust inhibitory antibody responses in the dams and effectively protects their offspring against ZIKV disease. Importantly, in a heterologous prime-boost regimen, rGZ02a effectively boosts the protective immunity primed by an adenovirus-vectored vaccine. Thus, rGZ02a is a promising candidate for a live-attenuated ZIKV vaccine.

Emergence and characterization of a putative novel human adenovirus recombinant HAdV-C104 causing pneumonia in Southern China.

In 2017, a survey of the molecular epidemiology of human adenovirus (HAdV) infections in Southern China based on hexon and fiber genotype demonstrated that the most prevalent genotypes of HAdV were HAdV-3 (n = 62), HAdV-2 (n = 21), and HAdV-7 (n = 16). In addition, two patients were co-infected with two genotypes of HAdV. Interestingly, a novel human adenovirus C recombinant genotype strain was isolated from one of the pneumonia patients in this survey. Phylogenetic, recombination, and proteotyping analysis showed that this novel pathogen originated from the recombination of parental viruses harboring the HAdV-1 penton and hexon gene, and the HAdV-2 fiber gene. It was named 'P1H1F2' and was assigned as HAdV-C104 based on the nomenclature protocol of using three major capsid proteins for characterization. Subsequent in vitro experiments demonstrated that HAdV-C104 had comparable proliferation capacity to HAdV-1, HAdV-2, and another recombination genotype P1H2F2. In addition, the HAdV-C104 infected patient was diagnosed with pneumonia and recovered after antiviral therapy. This report strengthens the hypothesis of recombination as a major pathway for the molecular evolution of HAdV-C species.

Design, synthesis and antitumor evaluation of novel 5-methylpyrazolo[1,5-a]pyrimidine derivatives as potential c-Met inhibitors.

A series of novel 5-methylpyrazolo[1,5-a]pyrimidine derivatives (10a-10x) were designed, synthesized, and evaluated for their in vitro inhibitory activities against c-Met kinase and antiproliferative activities against the SH-SY5Y, MDA-MB-231, A549, and HepG2 cell lines. Most of the compounds remarkably inhibited c-Met kinase and showed moderate to good cytotoxicity and selectivity toward the four cancer cell lines. Among them, compounds 10b and 10f were the two most potent selective c-Met inhibitors with half-maximal inhibitory concentration (IC50) values of 5.17 ± 0.48 nM and 5.62 ± 0.78 nM, respectively, and suppression abilities comparable with the positive control cabozantinib. Cell proliferation assay further demonstrated that the two most promising compounds 10a and 10b also showed good cytotoxicity and selectivity toward MDA-MB-231 cells, with IC50 values of 26.67 ± 2.56 µM and 26.83 ± 2.41 µM, respectively. Compounds 10f and 10g showed cytotoxicity and selectivity toward A549 cells, with IC50 values of 20.20 ± 2.04 µM and 21.65 ± 1.58 µM, respectively. All antiproliferative activities were within the range of those of cabozantinib. Notably, these compounds presented relatively low hepatotoxicity compared with reference drugs. Moreover, the preliminary structure-activity relationship and docking studies revealed that replacement of a nitrogen-containing heterocycle on the R2 (block A) group might improve the c-Met kinase inhibitory and antiproliferative effects in MDA-MB-231 cells, whereas displacement by a substituted benzene ring, especially for the p-fluorophenyl or 4-fluoro-3-methoxyphenyl moiety, on the R2 group enhanced cytotoxicity toward A549 cells. Together, these results suggest that 10b and 10f are promising compounds and provide a basis for their development as new antitumor agents.

Design, Synthesis and Cytotoxicity Evaluation of Novel Indole Derivatives Containing Benzoic Acid Group as Potential AKR1C3 Inhibitors.

Castration-resistant prostate cancer (CRPC) is a fatal, metastatic form of prostate cancer, characterized by reactivation of the androgen axis. Aldo-keto reductase 1C3 (AKR1C3) converts androstenedione (AD) and 5α-androstanedione to testosterone (T) and 5α-dihydrotestosterone (DHT), respectively. In CRPC, AKR1C3 is upregulated and implicated in drug resistance and has been regarded as a potential therapeutic target. Here we examined a series of indole derivatives containing benzoic acid or phenylhydroxamic acid and found that 4-({3-[(3,4,5-trimethoxyphenyl)sulfanyl]-1H-indol-1-yl}methyl)benzoic acid (3e) and N-hydroxy-4-({3-[(3,4,5-trimethoxyphenyl)sulfanyl]-1H-indol-1-yl}methyl)benzamide (3q) inhibited 22Rv1 cell proliferation with IC50 values of 6.37 µM and 2.72 µM, respectively. In enzymatic assay, compounds 3e and 3q exhibited potent inhibitory effect against AKR1C3 (IC50 =0.26 and 2.39 µM, respectively). These results indicated that compounds 3e and 3q might be useful leads for further investigation of more potential AKR1C3 inhibitors used for CRPC.

An adenovirus-vectored COVID-19 vaccine confers protection from SARS-COV-2 challenge in rhesus macaques.

The rapid spread of coronavirus SARS-CoV-2 greatly threatens global public health but no prophylactic vaccine is available. Here, we report the generation of a replication-incompetent recombinant serotype 5 adenovirus, Ad5-S-nb2, carrying a codon-optimized gene encoding Spike protein (S). In mice and rhesus macaques, intramuscular injection with Ad5-S-nb2 elicits systemic S-specific antibody and cell-mediated immune (CMI) responses. Intranasal inoculation elicits both systemic and pulmonary antibody responses but weaker CMI response. At 30 days after a single vaccination with Ad5-S-nb2 either intramuscularly or intranasally, macaques are protected against SARS-CoV-2 challenge. A subsequent challenge reveals that macaques vaccinated with a 10-fold lower vaccine dosage (1 × 1010 viral particles) are also protected, demonstrating the effectiveness of Ad5-S-nb2 and the possibility of offering more vaccine dosages within a shorter timeframe. Thus, Ad5-S-nb2 is a promising candidate vaccine and warrants further clinical evaluation.

Human Desmoglein-2 and Human CD46 Mediate Human Adenovirus Type 55 Infection, but Human Desmoglein-2 Plays the Major Roles.

Human adenovirus type 55 (HAdV55) represents an emerging respiratory pathogen and causes severe pneumonia with high fatality in humans. The cellular receptors, which are essential for understanding the infection and pathogenesis of HAdV55, remain unclear. In this study, we found that HAdV55 binding and infection were sharply reduced by disrupting the interaction of viral fiber protein with human desmoglein-2 (hDSG2) but only slightly reduced by disrupting the interaction of viral fiber protein with human CD46 (hCD46). Loss-of-function studies using soluble receptors, blocking antibodies, RNA interference, and gene knockout demonstrated that hDSG2 predominantly mediated HAdV55 infection. Nonpermissive rodent cells became susceptible to HAdV55 infection when hDSG2 or hCD46 was expressed, but hDSG2 mediated more efficient HAd55 infection than hCD46. We generated two transgenic mouse lines that constitutively express either hDSG2 or hCD46. Although nontransgenic mice were resistant to HAdV55 infection, infection with HAdV55 was significantly increased in hDSG2+/+ mice but was much less increased in hCD46+/+ mice. Our findings demonstrate that both hDSG2 and hCD46 are able to mediate HAdV55 infection but hDSG2 plays the major roles. The hDSG2 transgenic mouse can be used as a rodent model for evaluation of HAdV55 vaccine and therapeutics.IMPORTANCE Human adenovirus type 55 (HAdV55) has recently emerged as a highly virulent respiratory pathogen and has been linked to severe and even fatal pneumonia in immunocompetent adults. However, the cellular receptors mediating the entry of HAdV55 into host cells remain unclear, which hinders the establishment of HAdV55-infected animal models and the development of antiviral approaches. In this study, we demonstrated that human desmoglein-2 (hDSG2) plays the major roles during HAdV55 infection. Human CD46 (hCD46) could also mediate the infection of HAdV55, but the efficiency was much lower than for hDSG2. We generated two transgenic mouse lines that express either hDSG2 or hCD46, both of which enabled HAd55 infection in otherwise nontransgenic mice. hDSG2 transgenic mice enabled more efficient HAdV55 infection than hCD46 transgenic mice. Our study adds to our understanding of HAdV55 infection and provides an animal model for evaluating HAdV55 vaccines and therapeutics.

Molecular typing and genomic characteristic of human adenoviruses in Datong, Northern China.

The molecular prevalence of human adenoviruses (HAdVs) in Datong city and molecular evolution of HAdV-C species is still obscure. Here, we explored the molecular prevalence of HAdVs by simultaneous sequencing of hexon and fiber. Then, the penton gene fragments of HAdV-C species were determined by sequencing. Finally, genomic and proteotyping analysis were performed for exploration of molecular evolution of unique HAdV-6. Our results showed that dominant molecular types of HAdVs were HAdV-3, HAdV-2, and HAdV-1 based on the hexon and fiber genotype. Among H2F2 isolates, P1H2F2 was most common, followed by P2H2F2 and HAdV-89. The clinical symptoms of HAdV-1 or HAdV-2 infected patients were more severe than HAdV-3 infected patients, the prognosis of HAdV-1, HAdV-2, and HAdV-3 infected patients was indifference. Genomic and proteotyping analysis demonstrated that DT15 was different from HAdV-6 prototype, with high-discrepant sequences localized in the E3 region. In conclusion, HAdV-1 and HAdV-2 have a high affinity to infect younger children and cause more severe symptoms than HAdV-3. The E3 gene of HAdV-C species was considered as highly recombination and mutation region.

[Effect of early intervention of electroacupuncture on learning-memory ability and level of hippocampal phosphorylated Tau protein in SAMP8 mice].

OBJECTIVE: To explore the effect of early intervention electroacupuncture (EA) at "Baihui" (GV 20), "Dazhui" (GV 14) and "Shenshu" (BL 23) on the learning-memory ability and the expression of phosphorylated Tau protein in the hippocampus of SAMP8 mice, so as to provide reference for the intervening period of EA for Alzheimer's disease (AD). METHODS: A total of 36 3-month old SAMP8 mice were randomly divided into a model group, a 3-month-old EA group and a 9-month-old EA group, 12 mice in each group. Twelve normal SAMR1 mice with the same age were taken as the control group. The mice in the 3-month-old EA group and 9-month-old EA group were treated with EA at "Baihui" (GV 20), "Dazhui" (GV 14) and "Shenshu" (BL 23) separately 3 months old and 9 months old (continuous wave, 2 Hz, 1.5-2 mA), 20 min each time, once a day, 8 days as a course of treatment, with an interval of 2 days between courses, totally 3 courses of treatment were given. The mice sample in each group was collected at the age of 10 months after the learning-memory ability tested by Morris water maze. The expression of phosphorylated Tau protein in the hippocampus was detected by immunohistochemistry and Western blot, and the expression of Tau mRNA was detected by real-time PCR. RESULTS: Compared with the control group, in the model group, the escape latency was significantly increased (P<0.01), the time of stay in the original platform quadrant and the number of crossing the platform quadrant were reduced (P<0.01), and the expressions of phosphorylated Tau protein and Tau mRNA in hippocampus were increased (P<0.01). Compared with the model group, in the 3-month-old EA group and 9-month-old EA group, the escape latency was significantly reduced (P<0.05), the time of stay in the original platform quadrant and the number of crossing the platform quadrant were increased (P<0.05), and the expressions of phosphorylated Tau protein and Tau mRNA in hippocampus were reduced (P<0.05). Compared with the 9-month-old EA group, in the 3-month-old EA group, the escape latency was significantly reduced (P<0.05), the time of stay in the original platform quadrant and the number of crossing the platform quadrant were increased (P<0.05), and the expressions of phosphorylated Tau protein and Tau mRNA were reduced (P<0.01). CONCLUSION: The early EA intervention could more effectively improve the learning-memory ability and inhibit phosphorylation of Tau protein in the hippocampus of SAMP8 mice.

Yunvjian-Medicated Serum Protects INS-1 Cells against Glucolipotoxicity-Induced Apoptosis through Autophagic Flux Modulation.

Yunvjian (YNJ) is a traditional Chinese medicine formula adopted to prevent and treat diabetes. Our previous results from animal experiments showed that YNJ decreased blood glucose. This study aimed to examine the effect of high glucose and high lipid (HG/HL) conditions on the proliferation and apoptosis of INS-1 cells and the possible protective mechanism of YNJ-medicated serum on INS-1 cells exposed to HG/HL conditions. INS-1 cells were cultured in RPMI 1640 medium after being passaged. Then, INS-1 cells in the logarithmic growth phase were collected and divided into five groups: control, HG/HL, HG/HL+5% YNJ-medicated serum, HG/HL+10% YNJ-medicated serum, and HG/HL+20% YNJ-medicated serum. MTT assay and flow cytometry were used to detect proliferation and apoptosis of INS-1 cells, respectively. Protein profiles of INS-1 cells were analyzed using a tandem mass tag (TMT) label-based quantitative proteomic approach. Western blotting was performed to verify the proteomic results. YNJ-medicated serum significantly promoted INS-1 cell proliferation and inhibited apoptosis. Proteomic results from the INS-1 cells in the control, HG/HL, and HG/HL+10% YNJ-medicated serum groups showed that 7,468 proteins were identified, of which 6,423 proteins were quantified. Compared with the HG/HL group,430 differential proteins were upregulated, and 671 were downregulated in the HG/HL+10% YNJ-medicated serum group. Compared with the control group, 711 differential proteins were upregulated and 455 were downregulated in the HG/HL group, whereas 10 differential proteins were upregulated and 9 were downregulated in the HG/HL+10% YNJ-medicated serum group. Furthermore, several proteins related to autophagy, including ATG3, ATG2B, GABARAP, WIPI2, and p62/SQSTM1, were verified by western blotting, and these results were consistent with the results obtained from the proteomics analysis. These results confirmed that the autophagy pathway is critical to glucolipotoxicity in INS-1 cells. YNJ-medicated serum exhibited a protective effect on INS-1 cells cultured under HG/HL conditions by regulating autophagy genes' expression and restoring the autophagic flux.

Screening, synthesis, crystal structure, and molecular basis of 6-amino-4-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitriles as novel AKR1C3 inhibitors.

AKR1C3 is a promising therapeutic target for castration-resistant prostate cancer. Herein, an evaluation of in-house library discovered substituted pyranopyrazole as a novel scaffold for AKR1C3 inhibitors. Preliminary SAR exploration identified its derivative 19d as the most promising compound with an IC50 of 0.160 µM among the 23 synthesized molecules. Crystal structure studies revealed that the binding mode of the pyranopyrazole scaffold is different from the current inhibitors. Hydroxyl, methoxy and nitro group at the C4-phenyl substituent together anchor the inhibitor to the oxyanion site, while the core of the scaffold dramatically enlarges but partially occupies the SP pockets with abundant hydrogen bond interactions. Strikingly, the inhibitor undergoes a conformational change to fit AKR1C3 and its homologous protein AKR1C1. Our results suggested that conformational changes of the receptor and the inhibitor should both be considered during the rational design of selective AKR1C3 inhibitors. Detailed binding features obtained from molecular dynamics simulations helped to finally elucidate the molecular basis of 6-amino-4-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitriles as AKR1C3 inhibitors, which would facilitate the future rational inhibitor design and structural optimization.