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Supplemental material, sj-pdf-1-vdi-10.1177_1040638721994810 for Development and application of a real-time RT-PCR assay to rapidly detect H2 subtype avian influenza A viruses by Yixin Xiao, Fan Yang, Fumin Liu, Hangping Yao, Nanping Wu and Haibo Wu in Journal of Veterinary Diagnostic Investigation

AIDS Research and Human Retroviruses officially retracts the Instant Online/Just Accepted version of the article entitled, "Beijing as the hub of CRF07_BC transmission from the intravenous drug use...

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally with more than 33 million patients diagnosed, taking more than a million lives. Abundant mutations were observed but the functional consequences of these mutations are largely unknown. We report the mutation spectrum, replication dynamics, and infectivity of 11 patient-derived viral isolates in diverse cell lines, including the human lung cancer cell line Calu-3. We observed 46 mutations, including 9 different mutations in the spike gene. Importantly, these viral isolates show significant and consistent variations in replication dynamics and infectivity in tested cell lines, up to a 1500-fold difference in viral titers at 24 h after infecting Calu-3 cells. Moreover, we show that the variations in viral titers among viral isolates are positively correlated with blood clotting function but inversely correlated with the amount of red blood cell and hemoglobin in patients. Therefore, we provide direct evidence that naturally occurring mutations in SARS-CoV-2 can substantially change its replication dynamics and infectivity in diverse human cell lines, with clinical implications in vivo.

Structural principles underlying the composition and synergistic mechanisms of protective monoclonal antibody cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic antibody cocktail against SARS-CoV-2. On the basis of our previously identified humanized cross-neutralizing antibody H014, we systematically analyzed a fully human naive antibody library and rationally identified a potent neutralizing antibody partner, P17, which confers effective protection in animal model. Cryo-EM studies dissected the nature of the P17 epitope, which is SARS-CoV-2 specific and distinctly different from that of H014. High-resolution structure of the SARS-CoV-2 spike in complex with H014 and P17, together with functional investigations revealed that in a two-antibody cocktail, synergistic neutralization was achieved by S1 shielding and conformational locking, thereby blocking receptor attachment and viral membrane fusion, conferring high potency as well as robustness against viral mutation escape. Furthermore, cluster analysis identified a hypothetical 3rd antibody partner for further reinforcing the cocktail as pan-SARS-CoVs therapeutics.

SARS-CoV-2 is an enveloped virus responsible for the COVID-19 pandemic. Despite recent advances in the structural elucidation of SARS-CoV-2 proteins, the detailed architecture of the intact virus remains to be unveiled. Here we report the molecular assembly of the authentic SARS-CoV-2 virus using cryoelectron tomography (cryo-ET) and subtomogram averaging (STA). Native structures of the S proteins in pre- and postfusion conformations were determined to average resolutions of 8.7–11 Å. Compositions of the N-linked glycans from the native spikes were analyzed by mass spectrometry, which revealed overall processing states of the native glycans highly similar to that of the recombinant glycoprotein glycans. The native conformation of the ribonucleoproteins (RNPs) and their higher-order assemblies were revealed. Overall, these characterizations revealed the architecture of the SARS-CoV-2 virus in exceptional detail and shed light on how the virus packs its ∼30-kb-long single-segmented RNA in the ∼80-nm-diameter lumen., Graphical Abstract, Highlights • Molecular architecture of the authentic SARS-CoV-2 virus • Native structures of S in RBD down, one RBD up, and postfusion conformations • Compositions of the glycans from the native S are characterized by mass spectrometry • Structure and assembly of the RNPs are revealed in situ, Combined imaging analyses of 2,294 intact virions from the authentic SARS-CoV-2 virus resolve the S protein in pre- and postfusion conformations and characterize the molecular architecture of SARS-CoV-2 at high resolution.

The mutations in the SARS-CoV-2 virus genome during COVID-19 dissemination are unclear. In 788 COVID-19 patients from Zhejiang province, we observed decreased rate of severe/critical cases compared with patients in Wuhan. For mechanisms exploration, we isolated one strain of SARS-CoV-2 (ZJ01) from a mild COVID-19 patient. Thirty-five specific gene mutations were identified. Phylogenetic and relative synonymous codon usage analysis suggested that ZJ01 may be a potential evolutionary branch of SARS-CoV-2. We classified 54 global virus strains based on the base (C or T) at positions 8824 and 28247 while ZJ01 has T at both sites. The prediction of the Furin cleavage site (FCS) and sequence alignment indicated that the FCS may be an important site of coronavirus evolution. ZJ01 mutations identified near the FCS (F1-2) caused changes in the structure and electrostatic distribution of the S surface protein, further affecting the binding capacity of Furin. Single-cell sequencing and ACE2-Furin co-expression results confirmed that the Furin expression was especially higher in glands, liver, kidneys, and colon. The evolutionary pattern of SARS-CoV-2 towards FCS formation may result in its clinical symptom becoming closer to HKU-1 and OC43 caused mild flu-like symptoms, further showing its potential in differentiating into mild COVID-19 subtypes.

Background Highly pathogenic influenza A (H5N8) viruses have caused several worldwide outbreaks in birds and are of potential risk to humans. Thus, a specific, rapid and sensitive method for detection is urgently needed. Methods In the present study, TaqMan minor groove binder probes and multiplex real-time RT-PCR primers were designed to target the H5 hemagglutinin and N8 neuraminidase genes. A total of 38 strains of avian influenza viruses and other viruses were selected to test the performance of the assay. Results The results showed that only H5 and N8 avian influenza viruses yielded a positive signal, while all other subtypes avian influenza viruses and other viruses were negative. High specificity, repeatability, and sensitivity were achieved, with a detection limit of 10 copies per reaction. Conclusions The developed assay could be a powerful tool for rapid detection of H5N8 influenza viruses in the future.

Ebola virus (EBOV) can induce autophagy to benefit the virus life cycle, but detailed mechanisms remain to be elucidated. We previously found that EBOV GP and VP40 proteins interact with HSC70 (HSPA8), HSPA1A, and HSP90AB1. Thus, we presumed that EBOV likely induced autophagy by virus protein-host HSC70 or co-chaperon interactions via chaperone-mediated autophagy (CMA). We developed EBOV-trVLPs to model the EBOV life cycle, infected 293T cells with trVLPs, evaluated CMA by GFP-LC3 and RFP-LAMP1 co-localization, transmission electron microscopy (TEM) observation, and immunoblot analysis. The results demonstrated that EBOV-trVLPs induce autophagy which could not be inhibited by 3-MA significantly; autophagosomes and autolysosomes are obviously in the cytoplasm confirming CMA in cells infected with trVLPs. Meanwhile, a knockdown of HSC70 and relevant co-chaperones could inhibit trVLPs-associated autophagy, but no effort to Akt/mTOR/PHLPP1 pathway. These data indicate that EBOV-trVLPs could induce autophagy by CMA but was not limited by the CMA pathway. HSC70, HSPA1A, and HSP90AB1 participate and regulate CMA induced by EBOV-trVLPs. This was the first study about EBOV-trVLPs-induction of CMA and provides insight into the viral protein-host protein interaction, which is probably associated with CMA.HighlightsEBOV-trVLPs induce chaperone-mediated autophagy (CMA) but are not limit by CMA.HSC70, HSPA1A, and HSP90AB1 facilitate EBOV-trVLPs to induce autophagyKnockdown of HSC70, HSPA1A, and HSP90AB1 inhibit EBOV-trVLPs-induced CMA.

To establish a rapid detection method for H7N9 avian influenza virus (AIV), monoclonal antibodies (mAbs) against hemagglutinin (HA) of H7N9 were developed to establish an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA). AC-ELISA achieved high specificity and sensitivity, with a detection limit of 3.9 ng/mL for H7N9 HA protein (A/Zhejiang/DTID-ZJU01/2013), and 2-2 HA unit/100 μL for live H7N9 AIV. The inter- and intra-assay coefficient of variation was less than 10%. Compared with conventional virus isolation detection, the sensitivity and specificity were 94.96% and 88.24%, respectively. AC-ELISA proved to be a rapid and practical technique for the detection of H7N9 AIV.

The HIV-1 CRF07_BC strain is now the major recombinant form in China among the population of men who have sex with men (MSM), and has critically contributed to the HIV-1 epidemic in recent years. The phylodynamic and virological differences among CRF07_BC clusters circulating in MSM, and the factors that could be driving their evolution, remains unclear. Using the HIV-1 CRF07_BC strains obtained from the Los Alamos HIV database, we undertook a large-scale phylogenetic analysis using the maximum likelihood method of partial gag, pol, and env segments to infer their evolutionary relationships. The demographic histories of clusters were determined using the Bayesian Markov chain Monte Carlo (MCMC) method. For four pol clusters we analyzed the non-synonymous (dN) to synonymous (dS) substitution rates and performed site to site analysis to identify positive selection sites and cytotoxic T-lymphocyte (CTL) escape mutation positions. MSM was found to be the predominant risk factor for all four of the CRF07_BC epidemic pol segment clusters with the largest number of infections. Two of those clusters had higher growth in the effective number of infections, and two clusters had slower growth. Analysis of all four clusters showed no significant differences in the mean substitution rates and dN/dS selection pressure ratios. However, a site to site codon analysis found thirteen significant positive selection sites. Ten of these sites are CTL escape mutations. The two clusters with the higher growth in infections had seven and eight pol segment CTL escape mutation sites respectively, while the two with slower growth had only one or two. Our findings demonstrated differences in the CTL escape mutation and divergent evolution of several CRF07_BC clusters circulating among men who have sex with men in China.

Utilizing an integrative computational drug discovery approach, we predicted that nelfinavir is a potential inhibitor of SARS-CoV-2 main protease. Further docking nelfinavir to 30 potential target proteins of COVID-19, we found that nelfinavir is most probably a multi-target agent. The half-maximal effective concentration (EC50) of nelfinavir mesylate against SARS-CoV-2 was 2.89±0.65 μM while that of remdesivir was 1.00±0.34 μM, both drugs showed similar dose-response curves. Based on its high potency against SARS-CoV-2 at cellular level, its higher exposure in lung than in plasma, its good safe profile and its potential to reduce inflammation, nelfinavir deserves further exploration for the treatment of COVID-19.

Background With the outbreak of unknown pneumonia in Wuhan, China in December 2019, a new coronavirus (SARS-CoV-2) attracted worldwide attention. Although coronaviruses typically infect the upper or lower respiratory tract, discovery of the virus in plasma is common. Therefore, the risk of transmitting coronavirus through transfusion of blood products remains. As more asymptomatic infections are found in COVID-19 cases, blood safety is shown to be particularly important, especially in endemic areas. Study Design and MethodsBX-1, an ‘AIDS treatment instrument’ based on methylene blue (MB) photochemical technology, developed by Boxin (Beijing) Biotechnology Development LTD, has proven that inactivation of lipid-enveloped viruses such as HIV-1 in plasma has high efficiency, without damage to other components in the plasma, and proved safe and reliable in clinical trials of HIV treatment. In order to confirm the inactivation effect of BX-1 in SARS-CoV-2, we used the SARS-CoV-2 virus strain isolated from Zhejiang University for plasma virus inactivation studies. Results and ConclusionBX-1 can effectively eliminate SARS-CoV-2 within 2 mins, and the virus titer decline can reach 4.5 log10 TCID50/mL. Faced with the expanding epidemic, BX-1 is safe for blood transfusion and plasma transfusion therapy in recovery patients, and the inactivated vaccine preparation has great potential for treatment in the current outbreak.

The outbreak of COVID-19 become enormous threat to human beings, showing unclear virus mutation during dissemination. We found, in our 788 confirmed COVID-19 patients, the decreased rate of severe/critical type, increased liver/kidney damage and prolonged period of nuclear acid positivity, when compared with Wuhan. To investigate underlining mechanisms, we isolated one strain of SARS-CoV-2 (ZJ01) in mild COVID-19 patient and found the existence of 35 specific gene mutation by gene alignment. Further phylogenetic analysis and RSCU heat map results suggested that ZJ01 may be a potential evolutionary branch of SARS-CoV-2. We classified 54 strains of viruses worldwide (C/T type) based on the base (C or T) at positions 8824 and 28247. ZJ01 has both T at those sites, becoming the only TT type currently identified in the world. The prediction of Furin cleavage site (FCS) and the sequence alignment of virus family indicated that FCS may be an important site of coronavirus evolution. ZJ01 had mutations near FCS (F1-2), which caused changes in the structure and electrostatic distribution of S protein surface, further affecting the binding capacity of Furin. Single cell sequencing and ACE2-Furin co-expression results confirmed that Furin level was higher in the whole body, especially in glands, liver, kidney and colon while FCS may help SARS-CoV-2 infect these organs. The evolutionary pattern of SARS-CoV-2 towards FCS formation may result in its clinical symptom becoming closer to HKU-1 and OC43 (the source of FCS sequence-PRRA) caused influenza, further showing potential in differentiating into mild COVID-19 subtypes.

Continuous surveillance has shown that H6 subtype avian influenza viruses (AIVs) are prevalent in poultry and occasionally break the species barrier to infect humans. It is therefore necessary to establish a specific, rapid and sensitive method to screen H6 AIVs. In this study, a panel of monoclonal antibodies (mAbs) against the hemagglutinin (HA) of an H6 AIV isolate was produced. The purified mAbs have high affinity and specificity for H6 AIVs. An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) and immunochromatographic strip were developed based on two mAbs (1D7 and 1F12). The AC-ELISA results showed high sensitivity with a limit of detection (LOD) of 3.9 ng/ml for H6 HA protein and 0.5 HAU (HA units)/100 µl for live H6 subtype AIVs. The average recovery of the AC-ELISA with allantoic fluid, respiratory specimens, and cloacal swabs was 91.907 ± 1.559%, 82.977 ± 1.497% and 73.791 ± 2.588%, respectively. The intra- and inter-assay coefficient of variation was less than 10%. The LOD of immunochromatographic strip was 1 HAU when evaluated by the naked eye, and the detection time was less than 10 min without any equipment. Storage at room temperature or 4 °C for 30 days or 60 days had no effect on sensitivity and specificity of the strip. Thus, the AC-ELISA and immunochromatographic strips described here could be a secondary method to diagnose H6 AIV infections with high specificity, sensitivity, and stability.

Additional file 2:Table S2. The optimal concentrations of N8 primers and probe. a. The most optimal concentrations of N8 primers and probe.

Additional file 1: Table S1. The optimal concentrations of H5 primers and probe. a. The most optimal concentrations of H5 primers and probe.

The sudden outbreak of the severe acute respiratory syndrome–coronavirus (SARS-CoV-2) has spread globally with more than 1,300,000 patients diagnosed and a death toll of 70,000. Current genomic survey data suggest that single nucleotide variants (SNVs) are abundant. However, no mutation has been directly linked with functional changes in viral pathogenicity. We report functional characterizations of 11 patient-derived viral isolates. We observed diverse mutations in these viral isolates, including 6 different mutations in the spike glycoprotein (S protein), and 2 of which are different SNVs that led to the same missense mutation. Importantly, these viral isolates show significant variation in cytopathic effects and viral load, up to 270-fold differences, when infecting Vero-E6 cells. Therefore, we provide direct evidence that the SARS-CoV-2 has acquired mutations capable of substantially changing its pathogenicity. Funding: This work was supported by funds from Major Project of Zhejiang Provincial Science and Technology Department #2020C03123, National Science and Technology Major Project for the Control and Prevention of Major Infectious Diseases in China (2018ZX10711001, 2018ZX10102001, 2018ZX10302206), and start-606 up funds from Life Sciences Institute at Zhejiang University. Conflict of Interest: None. Ethical Approval: The study was approved by the Clinical Research Ethics Committee of The First Affiliated Hospital, School of Medicine, Zhejiang University (Approval notice 2020-29) for emerging infectious diseases.

In recent years, H5 and H7 subtypes of highly pathogenic avian influenza viruses (HPAIVs) have been identified in poultry worldwide, resulting in large economic losses to poultry production. Furthermore, H9N2 low pathogenic AIVs are reported to provide internal genes for generating novel reassortant AIVs, leading to potential pandemic risks. To establish an accurate, sensitive and convenient diagnostic method for H5, H7 and H9 subtype AIVs in Eurasian lineage, four groups of specific primers and probes were designed based on the conserved fragments of M, H5, H7 and H9 genes, and a multiplex real-time RT-PCR (RRT-PCR) method was established. High sensitivity was achieved for the multiplex RRT-PCR approach, with a detection limit of 1-10 copies (plasmid DNA) per reaction. The specificity of the method was evaluated using diverse subtypes of AIVs and other avian respiratory viruses isolated in eastern China over the last 9 years. Compared with virus isolation, a higher consistency was achieved when assessing 135 field samples and 126 clinical samples. The results showed that the multiplex RRT-PCR method is a fast, convenient and practical method for AIV clinical detection and epidemiological analysis.

In recent years, avian-origin H10 influenza viruses have proved capable of infecting human beings, and they pose a potential public health threat. Seven H10 avian influenza viruses (AIVs), H10N3 (n = 2), H10N7 (n = 1), and H10N8 (n = 4), were isolated from chickens in Zhejiang Province, Eastern China, during surveillance of AIVs in live poultry markets in 2016 and 2017. Phylogenetic analysis indicated that Zhejiang H10 strains received gene segments from H10, H3, and H7 viruses from birds in East Asia. Animal inoculation tests showed that these isolates have low pathogenicity in mice and can replicate in this species. Our findings suggest these H10 AIVs have the ability to adapt to chicken or other poultry, and highlight the need of long-term surveillance.

Background/aims Monoclonal antibodies (mAbs) are presently the most promising treatment against Ebola virus disease (EVD), and cocktail of two or more antibodies likely confers protection through complementary mechanisms. Zaire Ebolavirus (EBOV) glycoprotein (GP) and viral protein 40 (VP40) are targets for designing neutralizing antibodies. Currently, the antiviral therapeutics of mAb-cocktails are still limited solely to anti-GP antibodies，there is no Abs cocktail against Zaire EBOV GP and VP40, which both have important interactions with host cellular membrane. Methods We used hybridoma technology to produce anti-Zaire EBOV GP mAb against GP receptor binding domain, and anti-Zaire EBOV VP40 mAbs against the N-terminal domain, the C-terminal domain, respectively; synthesized Zaire EBOV transcription and replication competent virus like particles (trVLPs), which model even all aspects of the EBOV life cycles in order to evaluate the anti-viral effect of mAbs. Then, we characterized the anti- Zaire EBOV trVLPs effect of anti-GP and VP40 mAbs in vitro by real time-PCR, immunofluorescence assay and western blot analysis. Results Our results demonstrate that anti-GP or anti-VP40 mAbs effectively inhibit trVLPs replication. The cocktails of anti-GP and anti-VP40 mAbs, or between anti-VP40 mAbs, had synergistic anti-trVLPs effect. Meanwhile, the detailed DNA and amino acid sequences of the mAbs were checked. Conclusion The study verifies neutralizing efficacy of anti-GP or anti-VP40 mAb, report promising cocktail of anti-GP and anti-VP40 mAb, or cocktail of two anti-VP40 mAbs. To our knowledge, this is the first account to report the important anti-viral effect of cocktails of anti-GP and anti-VP40 mAbs in vitro.

The H7N7 avian influenza viruses can infect humans and poses a great threat to human health. To identify the amino acid substitutions that are associated with adaptation of avian-origin H7N7 virus to mammals, adaptation of the H7N7 virus was carried out by serial lung-to-lung passage in mice. Genomic analysis of the mouse-adapted virus revealed amino acid changes in the PB2 (E525G, M645I, and D701N), NP (I475V), HA(D103N), and NA(K142E) proteins. The adapted H7N7 virus was more virulent in mice than the wild-type virus. Our results suggest that continued surveillance of poultry populations for these substitutions in the H7N7 virus is required.

Background Elite controller refers to a patient with human immunodeficiency virus infection with an undetected viral load in the absence of highly active antiretroviral therapy. Studies on gene expression and regulation in these individuals are limited but significant, and have helped researchers and clinicians to understand the interrelationships between HIV and its host. Methods We collected CD4 T-cell samples from two elite controllers (ECs), two HIV-positive infected patients (HPs), and two healthy controls (HCs) to perform second-generation transcriptome sequencing. Using the Cufflinks software, we calculated the Fragments Per Kilobase of transcript per Million fragments mapped (FPKM) and identified differentially expressed (DE) mRNAs and long non-coding RNAs (lncRNAs), with corrected P value < 0.05 (based on a false discovery rate (FDR) < 0.05). We then constructed a protein-protein interaction network using cytoHubba and a long non-coding RNA-mRNA co-expression network based on the Pearson correlation coefficient. Results In total, 1109 linear correlations of DE lncRNAs targeting DE mRNAs were found and several interesting interactions were identified as being associated with viral infections and immune responses within the networks based on these correlations. Among these lncRNA-mRNA relationships, hub mRNAs including HDAC6, MAPK8, MAPK9, ATM and their corresponding annotated co-expressed lncRNAs presented strong correlations with the MAPK-NF-kappa B pathway, which plays a role in the reactivation and replication of the virus. Conclusions Using RNA-sequencing, we systematically analyzed the expression profiles of lncRNAs and mRNAs from CD4+ T cells from ECs, HPs, and HCs, and constructed a co-expression network based on the relationships among DE transcripts and database annotations. This was the first study to examine gene transcription in elite controllers and to study their functional relationships. Our results provide a reference for subsequent functional verification at the molecular or cellular level.

High precision sulfur isotope data on eight eucrites and two diogenites are reported to explore the provenance of sulfur and processes that affected the sulfur isotope composition and sulfur abundance within the HED ( H owardite, E ucrite, D iogenite) parent body (Asteroid 4 Vesta) or parent bodies for eucrite-like meteorites. Sulfur isotope values for a subset of six of the eucrites (EET 90020, PCA 82502, BTN 00300, MIL 11290, QUE 97014, and Sioux County) and two diogenites (Johnstown and Shalka) converge on a singular sulfur isotope composition, yielding a mean δ34S of 0.26 ± 0.17‰ (2 SD), Δ33S of 0.010 ± 0.007‰ (2 SD), and Δ36S of −0.22 ± 0.21‰ (2 SD). The variances on these mean values can be entirely accounted for by analytical uncertainties. The homogenous positive Δ33S for these samples suggests that sulfur in Asteroid 4 Vesta has a Δ33S of 0.010 ± 0.002‰ (2 SE) which is consistent with the occurrence of a process that allowed for promoted-isotopic-exchange within the parent body to homogenize Δ33S. This process was previously recognized in the homogenous Δ17O composition that has been linked to large-scale silicate melting and mixing. The positive δ34S of this group of samples is attributed to fractionations associated with sulfur loss, and the homogeneity of sulfur isotopes suggests that this loss predates the mixing event. The sulfur isotope data alone do not differentiate whether this loss occurred prior to or post core formation, or even prior to accretion. Two eucrites (GRA 98098 and CMS 04049) yield isotopic compositions that are distinct from this mean for δ34S, and in the case of CMS 04049, for Δ33S as well. GRA 98098 yields δ34S of 0.81 ± 0.18‰ (2 σest, estimated external precision), Δ33S of 0.010 ± 0.008‰ (2 σest) and Δ36S of 0.01 ± 0.34‰ (2 σest). CMS 04049 yields δ34S of −0.22 ± 0.18‰ (2 σest), Δ33S of 0.024 ± 0.008‰ (2 σest), and Δ36S of −0.17 ± 0.34‰ (2 σest). The more positive δ34S of GRA98098 suggests fractionation of sulfur (34S/32S) by a combination of parent body processes that may be linked to evaporation. The more positive Δ33S and the negative δ34S of CMS 04049 suggests that this meteorite may be from another ‘Vesta-like’ parent body.

H10 subtype avian influenza viruses have caused several epidemics in poultry and mammals, and specific, rapid and sensitive methods for detection are urgently needed. Herein, TaqMan minor groove binder (MGB) probes and multiplex real-time RT-PCR primers were designed based on gene regions encoding conserved domains of the nucleoprotein and H10 hemagglutinin. The developed multiplex real-time RT-PCR assay displayed high specificity, repeatability, and a detection limit of 10 copies per reaction. This diagnostic method could prove valuable for the rapid detection of H10 subtype AIVs in China.

MARID (Mica-Amphibole-Rutile-Ilmenite-Diopside) and PIC (Phlogopite-Ilmenite-Clinopyroxene) xenoliths are thought to be formed by intense “primary” mantle metasomatism. These rocks also display secondary features, such as cross-cutting veins and geochemical zonation of matrix minerals, which probably reflect later metasomatic events. To investigate the nature and origin(s) of these secondary features, 28 MARID and PIC xenoliths from southern African kimberlites and orangeites have been studied. MARID-hosted veins contain both carbonate and Ti-rich phases (e.g., titanite, phlogopite), suggesting that they formed by the infiltration of a carbonated silicate melt. Elevated TiO2 contents in MARID matrix mineral rims are spatially associated with carbonate-dominated veins, suggesting a genetic relationship between vein formation and geochemical zonation. Spongy rims around primary MARID and PIC clinopyroxene are depleted in Na2O and Al2O3 relative to their cores, possibly reflecting mineral dissolution in the xenoliths during ascent and emplacement of the entraining kimberlite. The preservation of compositional differences between primary and secondary phases in MARID and PIC xenoliths indicates that metasomatism occurred shortly before, or broadly coeval with, kimberlite/orangeite magmatism; otherwise, at typical mantle temperatures, such features would have quickly re-equilibrated. Increased Na2O in some mineral rims (e.g., K-richterite) may therefore reflect equilibration with a more Na-enriched primitive kimberlite melt composition than is commonly suggested. Vein-hosted clinopyroxene 87Sr/86Sri (0.70539 ± 0.00079) in one MARID sample is intermediate between primary clinopyroxene in the sample (0.70814 ± 0.00002) and the host Bultfontein kimberlite (0.70432 ± 0.00005), suggesting that vein minerals are derived from interactions between primary MARID phases and kimberlite-related melts/fluids. Sulfur isotope compositions of barite (δ34SVCDT = +4.69 ‰) and sulfides (δ34SVCDT = −0.69 ‰) in carbonate veins reflect equilibration at temperatures of 850–900 °C, consistent with sulfur-rich melt/fluid infiltration in the lithospheric mantle. In contrast, vein carbonate C-O isotope systematics (δ13CVPDB = −9.18 ‰; δ18OVSMOW = +17.22 ‰) are not typical of kimberlites or other mantle carbonates (δ13CVPDB = −3 to −8 ‰; δ18OVSMOW = 6 to 9 ‰), and may represent post-emplacement hydrothermal interactions of the cooling kimberlite with crustal fluids. These constraints suggest protracted metasomatism of MARID rocks shortly before and during entrainment by the host kimberlite.

Background/Aims: Since the first case of novel H7N9 infection was reported, China has experienced five epidemics of H7N9. During the fifth wave, a highly pathogenic H7N9 strain emerged. In order to assess whether the H7N9 vaccine based on A/Zhejiang/DTID-ZJU01/2013(H7N9) was effective in protecting against highly pathogenic H7N9, we conducted this study. Methods: Groups of mice were immunized twice by intraperitoneal injection with 500 µl of either split vaccine alone or MF59-adjuvanted vaccine. Serum was collected 2 weeks after the second vaccine booster. The hemagglutinin inhibition test was conducted on vaccine seed and highly pathogenic H7N9 to evaluate the neutralization of highly pathogenic H7N9. We also immunized mice and challenged them with highly pathogenic H7N9. Mice were observed for illness, weight loss, and death at 1 week and 2 weeks post-infection. Then, the mice were sacrificed and lungs were removed. Antibody responses were assessed and pathological changes in the lung tissue were evaluated. Results: The ability of serum to neutralize highly pathogenic H7N9 was reduced. In mice, highly pathogenic H7N9 was more virulent than A/Zhejiang/DTID-ZJU01/2013(H7N9). After challenge with highly pathogenic H7N9, all mice died while mice challenged with A/Zhejiang/DTID-ZJU01/2013(H7N9) all recovered. The A/ZJU01/PR8/2013 split H7N9 avian influenza vaccine was able to protect against infection with highly pathogenic H7N9 in mice, with or without MF59. Moreover, H7N9 vaccine adjuvanted with MF59 produced high antibody levels, which lead to better protection. Conclusions: The A/ZJU01/PR8/2013 split H7N9 avian influenza vaccine based on A/Zhejiang/DTID-ZJU01/2013(H7N9) is effective in protecting against highly pathogenic H7N9. H7N9 vaccine adjuvanted with MF59 offers better protection against infection with highly pathogenic H7N9. In order to make the H7N9 vaccine applicable to humans, further clinical trials are required to evaluate MF59 adjuvanted vaccine. Meanwhile, the vaccine strain should be updated based on the highly pathogenic H7N9 gene sequence.

H9N2 avian influenza viruses (AIVs) can cause respiratory symptoms and decrease the egg production. Additionally, H9N2 AIVs can provide internal genes for reassortment with other subtypes. During the monitoring of live poultry markets in 2016, a total of 32 (32/179, 17.88%) H9N2 AIVs were isolated from poultry in Eastern China, and seven representative strains were selected based on the isolation time, isolation location and sequence homology for further characterization. Phylogenetic analysis of hemagglutinin and neuraminidase showed that these H9N2 AIVs clustered into the Y280 sublineage. And the phylogenetic trees of six internal genes showed that the source of these gene fragments was more abundant, suggesting that extensive reassortment has occurred in these H9N2 viruses. Molecular analysis showed that multiple specific amino acid mutations occurred that increased H9N2 AIVs' infectivity, transmissibility, and affinity to mammals, including Q226L and Q227M in hemagglutinin, E627K in polymerase basic protein 2 (PB2), L13P in polymerase basic protein 1 (PB1), and A70V and S409N in polymerase acidic protein (PA). Pathogenicity tests in mice showed these H9N2 AIVs could replicate in lungs and exhibited slight to moderate virulence. The continuous circulation of these H9N2 viruses suggests the necessity for persistent surveillance of the H9N2 AIVs in poultry.

Sulfur has multiple stable isotopes (32S, 33S, 34S, and 36S), but little research has been conducted to investigate the information that low abundance sulfur isotope ratios (i.e., 33S/32S and 36S/32S) may carry on ore-forming processes in post-Archean deposits. In this study, the Cretaceous Mina Justa iron oxide–copper–gold deposit, was selected to examine the capability of multiple sulfur isotopes to trace ore-forming processes in post-Archean mineral deposits. In situ secondary ion mass spectrometry was applied to the pyrite of the magnetite–pyrite stage in the deposit to characterize the spatial variation of multiple sulfur isotopes at the microscale. This revealed a clear co-variation trend between δ34S (= 1000 × [(34S/32S)sample/(34S/32S)V-CDT − 1]) and Δ33S (= δ33S − 1000 × [(1 + δ34S/1000)0.515 – 1]). Simple modeling suggests that the triple sulfur isotope data recorded is best explained by mixing magmatic and externally derived (e.g., basinal brine) fluids. The contribution of external fluids is also supported by the trace element distribution patterns in pyrite, as revealed by laser ablation-inductively coupled plasma-mass spectrometry. This study is the first attempt to use multiple sulfur isotopes to trace ore-forming processes in post-Archean deposits and demonstrates that multiple sulfur isotopes are a faithful recorder of ore-forming processes at the microscale, especially when multiple fluids mix.

In 2015, an H5N1 influenza virus was isolated from a pig in Zhejiang Province, Eastern China. This strain was characterized by whole-genome sequencing with subsequent phylogenetic analysis. Phylogenetic analysis showed that all segments from this strain belonged to clade 2.3.2 and that it had received its genes from poultry influenza viruses in China. A Glu627Lys mutation associated with pathogenicity was observed in the PB2 protein. This strain was moderately pathogenic in mice and was able to replicate without prior adaptation. These results suggest that active surveillance of swine influenza should be used as an early warning system for influenza outbreaks in mammals.

// Huilin Ou 1,* , Wei Yao 2,* , Dongshan Yu 1,* , Tianhao Weng 1 , Frederick X.C. Wang 3 , Xiaoxin Wu 1 , Haibo Wu 1 , Linfang Cheng 1 , Xiangyun Lu 1 , Nanping Wu 1 , Honglin Chen 4 , Lanjuan Li 1 and Hangping Yao 1 1 State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China 2 Zhejiang Tianyuan Bio-Pharmaceutical Co., Ltd., Hangzhou, China 3 Department of Bioengineering, Erik Jonsson School of Engineering and Computer Science, The University of Texas at Dallas, Dallas, Texas, USA 4 State Key Laboratory for Emerging Infectious Diseases, Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong, China * These authors have contributed equally to this article Correspondence to: Hangping Yao, email: // Lanjuan Li, email: // Keywords : H7N9, adjuvant vaccine, MF59, immunogenicity, protective immune responses Received : May 26, 2017 Accepted : July 29, 2017 Published : August 08, 2017 Abstract The influenza virus is a serious threat to public health worldwide. A novel avian influenza A (H7N9) virus with a mortality rate of approximately 30% has been identified as an unusually dangerous virus for humans by the World Health Organization. Pathogenic H7N9 continue to represent a public health concern, and several candidate vaccines are currently in development. We generated candidate H7N9 vaccine strains using reverse genetics, consisting of hemagglutinin and neuraminidase genes derived from a human H7N9 virus and the remaining genes from the PR8 (A/PuertoRico/8/34 (H1N1)) virus. This H7N9 vaccine exhibited superior efficacy when combined with MF59 compared to other adjuvants. Immunized BALB/c mice were followed to determine the duration of the protective immune response. Antibody levels decreased to between one-half and one-eighth of the peak values four months after the final dose of the vaccine. Previously vaccinated mice received an A/Zhejiang/DTID-ZJU01/2013 H7N9 challenge six months post-vaccination, and all remained protected. We also verified that MF59 enhanced the HI, MN, and IgG antibody titers to influenza antigens. The humoral immune response and Th2 cytokine production following influenza challenge was potently induced in the animals that received the split vaccine. Therefore, the split H7N9 influenza vaccine with the MF59 adjuvant could effectively induce antibody production and protect mice from H7N9 virus challenge even after six months.

From 2013 to 2015, 32 H1-subtype avian influenza viruses (AIVs), H1N2 (n = 12), H1N3 (n = 14), H1N4 (n = 4) and H1N9 (n = 2), were isolated from poultry in Zhejiang Province in eastern China. These strains were characterized by whole-genome sequencing with subsequent phylogenetic analysis and genetic comparison. Phylogenetic analysis of all eight viral genes showed that these strains clustered in the AIV Eurasian lineage. These strains were found to be minimally pathogenic in mice and were able to replicate in mice without prior adaptation. Continued surveillance is needed, considering the important role of poultry in AIV reassortment.

To determine the safety and immunogenicity of a novel recombinant adenovirus type 5 vector based Ebola virus disease vaccine (Ad5-EBOV) in Africans in China.A phase 1, dose-escalation, open-label trial was conducted. 61 healthy Africans were sequentially enrolled, with 31 participants receiving one shot intramuscular injection and 30 participants receiving a double-shot regimen. Primary and secondary end points related to safety and immunogenicity were assessed within 28 d after vaccination. This study was registered with ClinicalTrials.gov (NCT02401373).Ad5-EBOV is well tolerated and no adverse reaction of grade 3 or above was observed. 53 (86.89%) participants reported at least one adverse reaction within 28 d of vaccination. The most common reaction was fever and the mild pain at injection site, and there were no significant difference between these 2 groups. Ebola glycoprotein-specific antibodies appeared in all 61 participants and antibodies titers peaked after 28 d of vaccination. The geometric mean titres (GMTs) were similar between these 2 groups (1919.01 vs 1684.70 P = 0.5562). The glycoprotein-specific T-cell responses rapidly peaked after 14 d of vaccination and then decreased, however, the percentage of subjects with responses were much higher in the high-dose group (60.00% vs 9.68%, P = 0.0014). Pre-existing Ad5 neutralizing antibodies could significantly dampen the specific humoral immune response and cellular response to the vaccine.The application of Ad5-EBOV demonstrated safe in Africans in China and a specific GP antibody and T-cell response could occur 14 d after the first immunization. This acceptable safety profile provides a reliable basis to proceed with trials in Africa.

The prevalence of HIV-1 is increasing rapidly among the population of men who have sex with men (MSM) in China. Here, we report a novel HIV type 1 recombinant form consisting of CRF01_AE and CRF07_BC detected in a male patient through homosexual behavior. A phylogenic analysis revealed that this unique recombinant form (URF) exhibits a complex genomic structure with three CRF07_BC regions inserted into the CRF01_AE backbone. Recently, several second-generation recombinant forms (e.g., CRF01_AE/CRF07_BC) have been identified among MSM in China. The emergence of such URFs highlights the complexity of the HIV-1 epidemic among this population. Therefore, further molecular epidemiological investigation is required to track the genetic evolution of HIV-1 strains.

// Dong-Shan Yu 1, 2, * , Tian-Hao Weng 1, 2, * , Xiao-Xin Wu 1, 2 , Frederick X.C. Wang 3 , Xiang-Yun Lu 1, 2 , Hai-Bo Wu 1, 2 , Nan-Ping Wu 1, 2 , Lan-Juan Li 1, 2 and Hang-Ping Yao 1, 2 1 State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China 2 Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China 3 Department of Bioengineering, Erik Jonsson School of Engineering and Computer Science, The University of Texas at Dallas, Dallas, TX, USA * These authors contributed equally to this work Correspondence to: Hang-Ping Yao, email: yaohangping@zju.edu.cn Lan-Juan Li, email: ljli@zju.edu.cn Keywords: Ebola virus, EBOV proteins, EBOV lifecycle Received: April 21, 2017 Accepted: May 29, 2017 Published: June 15, 2017 ABSTRACT Ebola haemorrhagic fever causes deadly disease in humans and non-human primates resulting from infection with the Ebola virus (EBOV) genus of the family Filoviridae . However, the mechanisms of EBOV lifecycle in host cells, including viral entry, membrane fusion, RNP formation, GP-tetherin interaction, and VP40-inner leaflet association remain poorly understood. This review describes the biological functions of EBOV proteins and their roles in the lifecycle, summarizes the factors related to EBOV proteins or RNA expression throughout the different phases, and reviews advances with regards to the molecular events and mechanisms of the EBOV lifecycle. Furthermore, the review outlines the aspects remain unclear that urgently need to be solved in future research.

MicroRNAs are potent gene expression regulators involved in regulating various biological processes, including host-pathogen interactions. In this study, we used high-throughput sequencing to investigate cellular miRNA signatures related to HIV-1 replication and latent infection in CD4+ T cell lines, which included HIV-1-replicating H9/HTLV-IIIB, HIV-1-latently-infected CEM-Bru cells, and their parental uninfected H9 and CEM-SS cells. Relatively few miRNAs were found to be modulated by HIV-1 replication or latent infection, while the cell-lineage-specific miRNA difference was more pronounced, irrespective of HIV-1 infection. In silico analysis showed that some of our HIV-1 infection-regulated miRNA profiles echoed previous studies, while others were novel. In addition, some of the miRNAs that were differentially expressed between the productively and latently infected cells seemed to participate in shaping the differential infection state. Thus, the newly identified miRNA profiles related to HIV-1 replication and latency provide information about the interplay between HIV-1 and its host.

Objectives MicroRNA-155 (miR-155) regulates T-cell differentiation and activation. It has also been associated with HIV infection. However, it remains unclear whether miR-155 is related to the T-cell response in HIV-infected individuals (e.g. T-cell activation and exhaustion). Methods We performed a cross-sectional study involving 121 HIV-1-infected patients on highly active antiretroviral therapy (HAART) and 43 HAART-naive patients. MiR-155 levels in the peripheral blood were determined by quantitative reverse transcription–polymerase chain reaction (PCR). T-cell immune activation, exhaustion, and homeostasis were measured by determining the expression of CD38, programmed death 1 (PD-1) and CD127 via flow cytometry. Results The levels of miR-155 in total peripheral blood mononuclear cells, CD4 T cells and CD8 T cells from HIV-1-infected patients were increased (P < 0.01). Nonresponders and HAART-naive patients also exhibited a higher percentage of CD8+CD38+ T cells and a lower percentage of CD4+CD127+ and CD8+CD127+ T cells (P < 0.05). We also found higher levels of PD-1 expression on the CD4+ and CD8+ T cells of HIV-1-infected patients (P < 0.05). Conclusions Our findings suggest that miR-155 levels in the peripheral blood of HIV-1-infected patients are increased and associated with T-cell activation. Therefore, miR-155 is a potential biomarker of the immune response following HIV-1 infection.

Substantial increases in human immunodeficiency virus (HIV) have been reported worldwide in recent years, particularly among men who have sex with men (MSM). We conducted a matched case-control study to examine the factors associated with HIV diagnosis among MSM in one sample of eastern China. Between February 2012 and December 2014, we used surveillance records to identify MSM diagnosed with HIV (case participants); we also recruited MSM who did not have HIV (controls) and then matched them (2:1) with control cases in terms of age (±3 years). Multivariate logistic regression models were used to assess the factors associated with HIV diagnosis. According to a multivariate analysis using logistic regression model involving 101 cases and 202 matched controls, a lack of comprehensive knowledge of HIV (adjusted odds ratio [OR] = 0.40; 95% confidence interval [CI] = 0.18, 0.89), a monthly income of ≥4,000 RMB (adjusted OR = 2.99; 95% CI = 1.45, 6.16), having at least two male sexual partners in the past 6 months (adjusted OR = 2.85; 95% CI = 1.28, 6.31), participating in at least four anal sex experiences with a man in the past month (adjusted OR = 3.56; 95% CI = 1.64, 7.73), and having a current syphilis infection (adjusted OR = 3.30; 95% CI = 1.06, 10.25) were associated with an increased risk for HIV diagnosis. MSM with a comprehensive knowledge of HIV were at reduced risk of HIV diagnosis, whereas those with more male sexual partners, more male anal sexual experiences (including receptive or/and insertive anal intercourse, rimming, and fisting), and a current syphilis infection were at increased risk of HIV diagnosis. Focus on protection and safer sex behaviors during male sexual activity (i.e., consistent condom use, pre-exposure prophylaxis, closed sexual networks among MSM) would likely be effective for reducing the HIV transmission rate.

Arginine (Arg) deprivation is a promising therapeutic approach for tumors with low argininosuccinate synthetase 1 (ASS1) expression. However, its efficacy as a single agent therapy needs to be improved as resistance is frequently observed. Methods: A tissue microarray was performed to assess ASS1 expression in surgical specimens of pancreatic ductal adenocarcinoma (PDAC) and its correlation with disease prognosis. An RNA-Seq analysis examined the role of ASS1 in regulating the global gene transcriptome. A high throughput screen of FDA-approved oncology drugs identified synthetic lethality between histone deacetylase (HDAC) inhibitors and Arg deprivation in PDAC cells with low ASS1 expression. We examined HDAC inhibitor panobinostat (PAN) and Arg deprivation in a panel of human PDAC cell lines, in ASS1-high and -knockdown/knockout isogenic models, in both anchorage-dependent and -independent cultures, and in multicellular complex cultures that model the PDAC tumor microenvironment. We examined the effects of combined Arg deprivation and PAN on DNA damage and the protein levels of key DNA repair enzymes. We also evaluated the efficacy of PAN and ADI-PEG20 (an Arg-degrading agent currently in Phase 2 clinical trials) in xenograft models with ASS1-low and -high PDAC tumors. Results: Low ASS1 protein level is a negative prognostic indicator in PDAC. Arg deprivation in ASS1-deficient PDAC cells upregulated asparagine synthetase (ASNS) which redirected aspartate (Asp) from being used for de novo nucleotide biosynthesis, thus causing nucleotide insufficiency and impairing cell cycle S-phase progression. Comprehensively validated, HDAC inhibitors and Arg deprivation showed synthetic lethality in ASS1-low PDAC cells. Mechanistically, combined Arg deprivation and HDAC inhibition triggered degradation of a key DNA repair enzyme C-terminal-binding protein interacting protein (CtIP), resulting in DNA damage and apoptosis. In addition, S-phase-retained ASS1-low PDAC cells (due to Arg deprivation) were also sensitized to DNA damage, thus yielding effective cell death. Compared to single agents, the combination of PAN and ADI-PEG20 showed better efficacy in suppressing ASS1-low PDAC tumor growth in mouse xenograft models. Conclusion: The combination of PAN and ADI-PEG20 is a rational translational therapeutic strategy for treating ASS1-low PDAC tumors through synergistic induction of DNA damage.

H7N9 low pathogenic avian influenza viruses (AIVs) emerged in China in 2013 and mutated into highly pathogenic strains in 2017, causing disease in infected birds and humans. Thus, the development of rapid, specific, and sensitive detection methods is urgently required. Herein, two specific monoclonal antibodies against H7N9 AIV were produced to develop a colloidal gold-based immunochromatographic test strip to detect H7N9 AIV. High specificity, repeatability, and sensitivity were achieved, with a detection limit of two hemagglutinin units or 102.55 50% tissue culture infective dose. This assay may represent a powerful tool to rapidly detect H7N9 influenza viruses in the future.

Persistence of human immunodeficiency virus 1 (HIV-1) latency and residual immune activation remain major barriers to treatment in patients receiving highly active antiretroviral therapy (HAART). In the present study, we investigated the molecular mechanisms of persistent HIV infection and residual immune activation in HAART-treated patients. We showed that the expression level of B-cell CLL/lymphoma 11B (BCL11B) was significantly increased in CD4+T cells from HIV-infected patients undergoing HAART, and this was accompanied by increased expression of BCL11B-associated chromatin modifiers and inflammatory factors in comparison to healthy controls and untreated patients with HIV. In vitro assays showed that BCL11B significantly inhibited HIV-1 long terminal repeat (LTR)-mediated transcription. Knockdown of BCL11B resulted in the activation of HIV latent cells, and dissociation of BCL11B and its related chromatin remodeling factors from the HIV LTR. Our findings suggested that increased expression of BCL11B and its related chromatin modifiers contribute to HIV-1 transcriptional silencing, and alteration of BCL11B levels might lead to abnormal transcription and inflammation.

Biosynthesis of the pyrimidine nucleotide uridine monophosphate (UMP) is essential for cell proliferation and is achieved by the activity of convergent de novo and salvage metabolic pathways. Here we report the development and application of a cell-based metabolic modifier screening platform that leverages the redundancy in pyrimidine metabolism for the discovery of selective UMP biosynthesis modulators. In evaluating a library of protein kinase inhibitors, we identified multiple compounds that possess nucleotide metabolism-modifying activity. The JNK inhibitor JNK-IN-8 was found to potently inhibit nucleoside transport and engage ENT1. The PDK1 inhibitor OSU-03012 (also known as AR-12) and the RAF inhibitor TAK-632 were shown to inhibit the therapeutically-relevant enzyme dihydroorotate dehydrogenase (DHODH) and their affinities were unambiguously confirmed through in vitro assays and co-crystallization with human DHODH.

Human immunodeficiency virus (HIV) transcription is closely associated with chromatin remodeling. Retinoblastoma binding protein 4 (RBBP4) is a histone chaperone implicated in chromatin remodeling. However, the role of RBBP4 in HIV-1 infection and the underlying mechanism remain elusive. In the present study, we showed that RBBP4 plays a negative regulatory role during HIV-1 infection. RBBP4 expression was significantly increased in HIV-1-infected T cells. RBBP4 binds to the HIV-1 long terminal repeat (LTR)， represses HIV-1 LTR-mediated transcription through recruiting nuclear receptor subfamily 2 group F member 1(NR2F1) and histone deacetylase 1 and 2 (HDAC1/2) to HIV-1 LTR, and further controls local histone 3 (H3) deacetylation and chromatin compaction. Furthermore, the occupancy of RBBP4, HDAC1/2, and NR2F1 on LTR in HIV-latent J-lat cells was significantly higher than that in HIV-1-activated cells. In conclusion, our results establish RBBP4 as a new potent antiretroviral factor, which may provide theoretical basis for the treatment of HIV in the future.

Functional lysosomes mediate autophagy and macropinocytosis for nutrient acquisition. Pancreatic ductal adenocarcinoma (PDAC) tumors exhibit high basal lysosomal activity, and inhibition of lysosome function suppresses PDAC cell proliferation and tumor growth. However, the codependencies induced by lysosomal inhibition in PDAC have not been systematically explored. We performed a comprehensive pharmacological inhibition screen of the protein kinome and found that replication stress response (RSR) inhibitors were synthetically lethal with chloroquine (CQ) in PDAC cells. CQ treatment reduced de novo nucleotide biosynthesis and induced replication stress. We found that CQ treatment caused mitochondrial dysfunction and depletion of aspartate, an essential precursor for de novo nucleotide synthesis, as an underlying mechanism. Supplementation with aspartate partially rescued the phenotypes induced by CQ. The synergy of CQ and the RSR inhibitor VE-822 was comprehensively validated in both 2D and 3D cultures of PDAC cell lines, a heterotypic spheroid culture with cancer-associated fibroblasts, and in vivo xenograft and syngeneic PDAC mouse models. These results indicate a codependency on functional lysosomes and RSR in PDAC and support the translational potential of the combination of CQ and RSR inhibitors.

// Andrew H. Nguyen 1 , Irmina A. Elliott 1 , Nanping Wu 1 , Cynthia Matsumura 1 , Maria Vogelauer 2 , Narsis Attar 2 , Amanda Dann 1 , Razmik Ghukasyan 1 , Paul A. Toste 1 , Sanjeet G. Patel 1 , Jennifer L. Williams 3 , Luyi Li 1 , David W. Dawson 4 , Caius Radu 5 , Siavash K. Kurdistani 2 , Timothy R. Donahue 1, 5 1 Department of Surgery, David Geffen School of Medicine at UCLA, Los Angeles, California, USA 2 Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, California, USA 3 Department of Surgery, Harbor-UCLA Medical Center, Torrance, California, USA 4 Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA 5 Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, California, USA Correspondence to: Timothy R. Donahue, email: tdonahue@mednet.ucla.edu Keywords: histone deacetylase inhibitor, SAHA, AP-1, pancreatic cancer, cancer-associated fibroblasts Received: October 04, 2016 Accepted: November 07, 2016 Published: November 24, 2016 ABSTRACT Although histone deacetylase inhibitors (HDACi) are a promising class of anti-cancer drugs, thus far, they have been unsuccessful in early phase clinical trials for pancreatic ductal adenocarcinoma (PDAC). One potential reason for their poor efficacy is the tumor stroma, where cancer-associated fibroblasts (CAFs) are a prominent cell type and a source of resistance to cancer therapies. Here, we demonstrate that stromal fibroblasts contribute to the poor efficacy of HDACi’s in PDAC. HDACi-treated fibroblasts show increased biological aggressiveness and are characterized by increased secretion of pro-inflammatory tumor-supportive cytokines and chemokines. We find that HDAC2 binds to the enhancer and promoter regions of pro-inflammatory genes specifically in CAFs and in silico analysis identified AP-1 to be the most frequently associated transcription factor bound in these regions. Pharmacologic inhibition of pathways upstream of AP-1 suppresses the HDACi-induced inflammatory gene expression and tumor-supportive responses in fibroblasts. Our findings demonstrate that the combination of HDACi’s with chemical inhibitors of the AP-1 signaling pathway attenuate the inflammatory phenotype of fibroblasts and may improve the efficacy of HDACi in PDAC and, potentially, in other solid tumors rich in stroma.