Both the replication and transcription of the influenza virus are catalyzed by the viral polymerase complex. The polymerases of most avian influenza A viruses have poor performance in mammalian cells, which is considered to be one of the important species barriers. Pigs have been long considered as important intermediate hosts for interspecies transmission of the avian influenza virus, because of their susceptibility to infection with both avian and mammalian influenza viruses. However, the molecular basis of influenza polymerase adaptation in pigs remains largely unknown. ANP32A and ANP32B proteins have been identified as playing fundamental roles in influenza virus replication and host range determination. In this study, we found that swine ANP32A (swANP32A), unlike swine ANP32B or other mammalian ANP32A or B, shows stronger supporting activity to avian viral polymerase. Knockout of ANP32A in pig cells PK15 dramatically reduced avian influenza polymerase activity and viral infectivity, suggesting a unique feature of swANP32A in supporting avian influenza viral polymerase. This species-specific activity is mapped to two key sites, 106V and 156S, in swANP32A. Interestingly, the amino acid 106V is unique to pigs among all the vertebrate species studied, and when combined with 156S, exhibits positive epistasis in pigs. Mutation of 106V and 156S to the signature found in ANP32As from other mammalian species weakened the interaction between swANP32A and chicken viral polymerase, and reduced polymerase activity. Understanding the molecular basis of ANP32 proteins may help to discover new antiviral targets and design avian influenza resistant genome edited pigs. The pig is considered to be a “mixing vessel” for influenza viruses because it can be infected by both human and avian influenza viruses. This mixing of viruses could potentially produce novel recombinant strains that are more adaptable to humans or other mammals. The permissive mechanism that allows pig cells to be infected with avian influenza virus is largely unknown. Here we reveal that the pig cellular protein ANP32A has a unique 106V/156S signature, different from that of ANP32A proteins from other mammals, enabling the protein to bind strongly to avian influenza polymerase at the post-entry step, and promoting avian virus replication. This species-specific 106V/156S epistasis of swANP32A likely determines the susceptibility of pigs to avian influenza infection. Our findings provide novel insights into the molecular basis of interspecies transmission of avian IAV between chickens and pigs. Influenza A viruses (IAVs) are highly infectious respiratory pathogens that can infect many species, posing a great threat to both veterinary and human public health. Aquatic birds are considered to be the largest natural reservoir for IAVs. The four influenza pandemics that occurred in the past century  all originated in whole or in part from non-human reservoirs, and were associated with the genetic recombination of avian and other influenza viruses . Due to For interspecies transmission of avian influenza virus to mammals, the virus needs to overcome two main host barriers: HA receptor-binding specificity; and the ability of the viral polymerase to replicate in mammalian cells , 11. PigThe influenza viral polymerase complex is composed of the PB2, PB1, and PA proteins. Together with the viral genome, and the nucleoprotein (NP), the polymerase forms viral ribonucleoprotein complex (vRNP) to carry out viral replication and transcription in infected cells , 16. In Many host factors are involved in mediating vRNP trafficking and promoting vRNP functions in infected cells , 43, 44.In this study, we found that swine ANP32A (swANP32A) showed stronger supporting activity to avian viral polymerase than did either swine ANP32B (swANP32B) or ANP32A and B proteins from other mammals. Molecular mapping revealed two mutations at amino acid positions 106 and 156, which determine this activity. An amino acid Valine in position 106, which is unique to swANP32A, combined with a Serine at position 156, is critical in supporting avian influenza polymerase activity and therefore virus infectivity in pig cells by enhancing the interaction between swANP32A and avian polymerase. Our work reveals the role of ANP32A by which pigs can serve as intermediate hosts and can allow avian influenza virus replication, and gives further insights into the evolution of mammalian ANP32A, that determines the host range of the avian influenza A virus.ANP32 genes from different species, including chicken, human, pig, horse, and dog, and analyzed their ability to support polymerase activity of influenza A viruses from above mentioned species in DKO cells. The results showed that influenza A virus polymerases from all species tested were active in the presence of chicken ANP32A (chANP32A), but that none of the polymerases were active in the presence of chANP32B, which was consistent with previous studies [ZJ13) [ZJ12) [ZJ13 and H9N2ZJ12 (NC08) [GD12) [XJ07) [Results from our previous work, and that of other labs, suggest that the host factors ANP32A and ANP32B play an important role in influenza A virus polymerase activity and virus replication in different animals , 48, wit studies  Fig 1)..ANP32 ges [ZJ13)  and A/ch) [ZJ12) . SurprisH9N2ZJ12 . MammaliH9N2ZJ12 , swine i2 (NC08)  Fig 1D)ANP32 gen) [GD12)  Fig 1E)ANP32 gen) [XJ07)  Fig 1F)ANP32 genZJ13 polymerase while huANP32A had only a weak interaction with H7N9ZJ13 polymerase  cell line by using CRISPR-Cas9 system targeting the second exon of the 32A gene . The PK132A gene . There w32A gene . The pol present . AdditioswANP32A , showingswANP32A , indicatANP32A proteins have been found expressed in many species through the animal, plant and protist kingdoms, although not in yeast or other fungi . These pZJ13 polymerase into DKO cells. The results showed that the V106I and S156P mutants slightly impaired the ability of swANP32A in supporting avian H7N9ZJ13 polymerase, while mutations at position 228, P228L and P228V, both showed no effect on the function of swANP32A  are zoonotic agents, capable of crossing the species barrier. The viral polymerase is a major determinant of host range , 11, 62.ANP32 proteins, including ANP32A and ANP32B, are key functional co-factors affecting IAV polymerase adaption and activity, and are also important in the replication of other viruses such as HIV-1 . ANP32A Only a few studies have focused on the replication ability of avian IAV polymerase in pig cells. Moncorge and his coauthors observed that avian IAV polymerase could not work well in pig NPTr or human 293T cells, compared with its activity in avian DF-1 cells, which was a controversial result due to the fact that the pigs can be infected by some avian viruses. This may be due to an imperfect detection system (lacking the control luciferase) and varies transfection efficiency in different kinds of cells as mentioned by the authors . The ANPPhylogenetic analysis of ANP32A proteins suggested that avian and mammalian ANP32A formed different evolutionary linages . ANP32A To date, some key sites in ANP32 proteins have been identified. Avian ANP32A has an additional 33 amino acid insertion located in the LRRCT region, which is crucial for its ability to support AIV polymerase activity . DeletioIn summary, we provide evidence in this study that species differences in cellular ANP32A determine avian influenza A virus polymerase host restriction. We also identify a unique key site, 106V, in swine ANP32A but not in ANP32A from any other mammals or bird studied, which, together with 156S, enables an interaction between swine ANP32A and avian viral polymerase, promoting viral replication. Our work may help to understand the interplay between mammalian ANP32A proteins and avian viral polymerase complex and the molecular mechanism by which pigs are “mixing vessels” for avian and mammalian influenza viruses.The 9-day-old embryonated chicken eggs were obtained from Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. The protocols for influenza virus propagation on chicken embryos were reviewed and approved by the Animal Ethics Committee of Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences (SYXK (Hei) 2017–009).2 atmosphere. Plasmids carrying the polymerase from influenza A viruses from different species were used in this study, and included those from: H1N1 human influenza virus A/WSN/1933 ; H7N9 chicken influenza virus A/chicken/Zhejiang/DTID-ZJU01/2013; H1N1 swine influenza virus A/swine/North Carolina/3793/08 ; H3N2 canine influenza virus A/canine/Guangdong/1/2011 ; H9N2 avian influenza virus A/chicken/Zhejiang/B2013/2012 ; and A/equine/Xinjiang/1/2007 . pCAGGS-ANP32 plasmids were generated by gene synthesis  based on the sequences retrieved from GenBank, and included: chicken ANP32A ; human ANP32A ; swine ANP32A ; equine ANP32A ; canine ANP32A ; chicken ANP32B ; human ANP32B ; swine ANP32B ; and canine ANP32B . Mutants of these plasmids were generated by overlapping PCR and were identified by DNA sequencing.Human embryonic kidney ANP32A and ANP32B double-knockout 293T cell lines  and pig kidney epithelial  cells were cultured in the growth medium  with 10% fetal bovine serum  and 1% Penicillin-Streptomycin (Gibco)), and kept at 37°C in a 5% COswANP32A were designed online (http://crispr.mit.edu/) [PK15 swANP32A-knockout (named PK15-AKO) cell lines were generated by CRISPR/Cas9 system, as described previously . Brieflyit.edu/)  and inseit.edu/) . PK15 ceRenilla luciferase expression plasmids , using Lipofectamine 2000 transfection reagent following the manufacturers’ instructions. Cells were lysed with passive lysis buffer (Promega) at 24 h after transfection, and Firefly and Renilla luciferase activities were measured using a commercial kit  with Centro XS LB 960 luminometer (Berthold technologies). The expression levels of polymerase PB2 and NP in different groups were assessed by western blotting using specific antibodies.To test the effect of various ANP32 proteins on activity of polymerases from different viruses, DKO cells were seeded in 12-well plates and transfected with plasmids carrying PB1 (80 ng), PB2 (80 ng), PA (40 ng) and NP (160 ng), together with 80 ng minigenome reporter   and 10 nFirefly and Renilla luciferase activities were measured.To analyze the activity of the influenza viral polymerase in PK15 cells, a reporter plasmid pHH21-pgPolI-vLuc containing swine RNA polymerase I promoter was constructed, based on the plasmid pHH21-huPolI-vLuc. PK15 cells were used to extract swine genomic DNAs by QlAamp DNA Mini Kit . The swine RNA polymerase I promoter sequence was then amplified from genomic DNA by using primer pairs, based on a previous study . PCR fraFirefly luciferase RNAs driven by influenza polymerase was amplified by a Reverse Transcription Kit (Cat.RR047A). Specific primers were then used in the RT reaction, as follows: 5’- CATTTCGCAGCCTACCGTGGTGTT-3’ for the Firefly luciferase vRNA; 5’-AGTAGAAACAAGGGTG-3’ for the Firefly luciferase cRNA; oligo-dT20 for the Firefly luciferase mRNA. The cDNA samples were quantified using real-time PCR with specific primers F (5’-GATTACCAGGGATTTCAGTCG-3’) and R (5’-GACACCTTTAGGCAGACCAG-3’) using SYBR Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa Cat: RR820A). Fold change in levels of RNA was calculated using double-standard curve methods and with β-actin as an internal control.This assay was performed as described previously . Total RZJ12 and human influenza virus WSN were propagated in 9-day-old specific pathogen-free (SPF) embryonated chicken eggs as described previously [ZJ12 virus as described above. The growth data shown are the average results of three independent experiments.Avian influenza virus H9N2eviously  and usedeviously . To deteDKO cells were transfected with the indicated plasmids, and cells were lysed in lysis buffer  at 24h post-transfection, then centrifuged at 13,000× g and 4°C for 10 min. The lysates were incubated with Anti-FLAG M2 Magnetic Beads  at 4°C for 2h. Immunocomplexes were eluted using a 3X Flag peptide and boiled in the SDS-PAGE loading buffer, subjected to SDS-PAGE and analyzed by western blotting. Signals were detected using a LI-COR Odyssey Imaging System . The cell samples were also digested by RNase A/T1 Mix  to confirm whether the interactions between ANP32 proteins and viral polymerase were RNA dependent. This experiment was carried out using the following primary antibodies: Influenza A Virus PA antibody , Influenza A Virus PB1 antibody , Influenza A Virus PB2 antibody , Anti-Flag antibody .http://weblogo.berkeley.edu/logo.cgi) was used to construct WebLogo diagrams.We used the BLASTP algorithm to retrieve ANP32A protein sequences from 53 vertebrates  with human ANP32A protein as the query. ANP32A proteins were aligned using MAFFT v7.271 , and theData analysis was performed in GraphPad Prism, version 7 . Statistical significance was assessed using One-way ANOVA followed by a Dunnett’s post-test. Error bars represent the standard deviation (SD) or the standard error of the mean (SEM), as indicated in figure legends. NS, not significant (p>0.05), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. All the experiments were performed independently at least three times.S1 Fig(A) DKO cells were transfected with different ANP32A (0.6μg) and polymerase plasmids  from avian influenza viruses H7N9ZJ13. The cells were lysed at 24 h post-transfection. Cell lysates with or without treatment with 2μL RNase A/T1 Mix  were incubated with Flag beads overnight at 4°C. Immunocomplexes were eluted using a 3X Flag peptide and boiled in the SDS-PAGE loading buffer, subjected to SDS-PAGE and analyzed by western blotting. (B) The cellular RNA extracted from equal number of cells used in the IP assay was digested with or without the treatment with 2μL RNase A/T1 Mix overnight at 4°C. Then the samples were assessed by 1.5% agarose gel electrophoresis.(EPS)Click here for additional data file.S2 FigThe height of each stack corresponds to the level of nucleotide conservation at that position. When the nucleotide is invariant, only one letter is shown; when the nucleotide is variable, the most common substitutions are noted. The figure depicts the key sites involved in supporting the influenza viral polymerase activity. The universal site 129/130 is indicated with red diamonds, the unique sites 106 and 156 are indicated with red circles, and the 33-aa insertion, including SLS (SIM-like sequence) and 27-aa repeat, is labeled in line.(TIF)Click here for additional data file.


Background: Influenza A (H3N2) clade 3C.3a was the predominant influenza virus in Israel throughout the 2018-2019 season, constituting a drift from the influenza A (H3N2) vaccine. We estimated the end-of season vaccine effectiveness (VE) by age, among community patients with influenza-like illness (ILI), considering the hemagglutinin (HA) gene mutations and amino acid substitutions of influenza A (H3N2) viruses detected. Methods: Nose-throat samples were analyzed for the presence of influenza virus, type/subtype, and HA gene sequence. HA gene sequences and amino acid substitutions were compared to the influenza A/Singapore/INFIMH-16-0019/2016 (H3N2)-like 2018-2019 vaccine virus, and a phylogenetic tree was generated. Influenza VE against influenza A (H3N2) was estimated using the test-negative design. VE was estimated by age group and by 15 year moving age intervals. Results: In total, 90% of the influenza A (H3N2) viruses belonged to the 3C.3a clade, constituting a unique situation in the northern hemisphere. Adjusted all-age influenza A (H3N2) VE was −3.5% (95% CI: −51.2 to 29.1). Although adjusted VEs were very low among infants, children, and young adults, a VE of 45% (95% CI: −19.2 to 74.6) was estimated among adults aged ≥45 years old. Conclusions: The higher VE point estimates among older adults may be related to previous exposure to similar influenza viruses.  In recent years, influenza A H3N2) viruses have presented a significant challenge for vaccine selection. In fact, the recommendation regarding the 2019–2020 northern hemisphere influenza A (H3N2) vaccine component was made one month later than for the influenza A (H1N1)pdm09 and the influenza B vaccine components  viruses . This raIsrael has a population of 9.1 million, and is located in the Middle East. Influenza activity in Israel usually lasts from December to March , in pattth 2019. ILI was defined as a temperature of ≥37.8 °C with at least one of the following symptoms: sore throat, coryza, cough, and muscle ache. Medical teams were granted discretion concerning the inclusion of other symptoms and signs that appeared relevant [Combined nose-throat samples were collected from ILI patients visiting the Israel Influenza Surveillance Network (IISN) 32 outpatient sentinel clinics. The study period lasted from September 30th 2018 to March 30https://www.gisaid.org). . . The 201VE was estimated for individuals ≥6 months of age by means of the test-negative design using the formula (1-OR) x 100 . Sample Influenza A (H3N2)-positive ILI patients were labeled “cases”, whereas influenza-negative ILI patients were labeled “controls”.VE was calculated for all ages and for specific age groups. In order to gain insight into the VE trend at various ages, adjusted VE was also calculated using moving 15 year intervals, beginning at the first year of life and progressing by 1 year increments . A cubicχ2 test was applied for comparison of demographic and medical characteristics between cases and controls. The Pearson’s A univariate logistic regression model was applied to calculate the odds ratio for crude VE estimates, including the 95% confidence intervals. A multivariate logistic regression model was applied to adjust for age, gender, number of days from disease onset to sampling, calendar month of sample collection, and underlying chronic conditions placing patients at risk for severe influenza or complications. Wherever the number of patients was too small in a particular calendar month, instead of adjusting according to calendar month, adjustment was performed for the high influenza activity period  and low influenza activity period . Statistical analyses were performed using SAS Enterprise Guide 7.1 (SAS Institute Inc.) and R version 3.5.2  software.Influenza surveillance in Israel, including the laboratory confirmation, is implemented under the Israel Public Health Ordinance, and does not entail informed consent.During the 2018–2019 study period, 1476 combined nose-throat samples were collected from ILI patients. A total of 594 (40%) were positive for influenza. Of these, 451 (75.9%) were positive for influenza A (H3N2), 134 (22.6%) for influenza A (H1N1)pdm09, 5 (0.8%) for influenza B, and 4 (0.7%) were positive for both influenza A (H1N1)pdm09 and influenza B .HA gene sequencing analysis was performed on the original samples of 60 influenza A (H3N2)-positive ILI patients presenting to sentinel clinics at various times throughout the 2018-2019 influenza season. Of these, a total of 54 (90%) viruses belonged to the 3C.3a clade, which differed from the 3C.2a1 subclade of the influenza A/Singapore/INFIMH-16-0019/2016 (H3N2)-like virus included in the 2018-2019 influenza vaccine. Only five sequenced influenza A (H3N2) viruses belonged to subclade 3C.2a1, and one belonged to clade 3C.2a.Analysis of amino acid (AA) substitutions of the 3C.3a clade influenza A (H3N2) viruses as compared to the cell-grown influenza A/Singapore/INFIMH-16-0019/2016 (H3N2)-like virus, in a representative subset of viruses, is shown in A total of 50 individuals were not eligible for VE analysis due to their age (<6 months old), 14 due to unknown vaccination status, 33 due to vaccination <14 days before symptom onset, 38 due to sampling >7 days after symptom onset, 6 due to missing critical data, and 21 children due to partial vaccination . After eAs a result of its predominance, VE was estimated against influenza A (H3N2). No VE analysis was carried out for influenza A (H1N1)pdm09 or influenza B due to the small number of cases.In order to gain insight into VE trends at various ages, adjusted VE estimates were calculated according to moving 15 year age intervals, beginning at the first year of life and progressing by 1 year increments . We thenThe adjusted moving influenza A (H3N2) VE estimates are shown in The influenza A (H3N2) was the dominant influenza virus circulating in Israel throughout the 2018–2019 season. Most of these viruses belonged to the 3C.3a clade, a clade different from that of the 2018–2019 influenza A (H3N2) vaccine virus component. The 3C.3a influenza A (H3N2) viruses also differed from influenza A (H3N2) viruses that circulated in most locations in the northern hemisphere, especially in the early and middle parts of the season , and hadPrior to the 2018–2019 season, influenza A (H3N2) viruses belonging to the 3C.3 clade circulated in Israel only during the 2013–2014 season . AlthougThe low all-ages influenza A (H3N2) VE estimate observed in Israel, was consistent with the genetic and antigenic analysis of these viruses. In addition, it was consistent with the all-ages influenza A (H3N2) clade 3C.3a VE estimates in the USA , Canada Despite the low all-ages influenza A (H3N2) VE estimate in Israel, VE point estimates were higher in adults as compared with younger individuals . SpecifiEstimation of VE for predetermined age groups is valuable; however, given the distinct character of such groups, it may not provide enough information regarding the trend of VE at various ages. Using the 15 year moving interval allowed us to evaluate VE at various ages despite the fact that our sample size may be smaller than that of larger countries ,22,23. TThe difference in VE estimates between the younger and older age groups in our study is interesting. The low VE among infants, children, and young adults can be explained by the vaccine mismatch. However, the higher VE point estimates among older adults may be explained by past exposure to similar influenza A (H3N2) viruses, which may have allowed the mismatched 2018-2019 vaccine to provide them with an immunological boosting effect. Furthermore, it has been proposed that influenza viruses that circulate during a person’s childhood can confer lasting protection against new influenza viruses that belong to a similar phylogenetic group . In thisAlthough both the 2016-2017  and 2018Recent studies by Skowronski et al.  and KissIn the present study, no statistical significance was noted for any of the age intervals showing negative VE point estimates. The negative VE point estimates observed here among the younger individuals , were similar to smaller declines in VE estimates of younger birth cohorts observed in the study of Skowronski et al. . Thus, tChildren are thought to have the highest rates of illness and complications resulting from influenza ,29. In aThis study has several advantages. The predominance of the 3C.3a clade in Israel throughout the 2018–2019 season resulted in a sample size that enabled the analysis of VE trends by age against this clade. Additionally, vaccination status and vaccination dates of ILI patients included in our study were based entirely on medical records’ data. It is also important to realize that Israel is the only country in the Middle East that performs yearly assessments of influenza vaccine effectiveness. Thus, these VE estimates may be relevant also to neighboring countries in the region. In this regard, influenza A (H3N2) circulated during the 2018–2019 season in Lebanon, Jordan, and the Palestinian authority, all of which share borders with Israel .This study was limited by the unavailability of data regarding influenza vaccination in prior seasons. Although studies examining the effect of prior influenza vaccination on current season VE have shown variable results ,39,40,41Confidence intervals are used to describe statistical significance of VE estimates. In general, caution should be exercised when VE estimates are not statistically significant, as in such cases, the possibility of ‘no protective effect’ (zero VE) cannot be ruled out . HoweverThe patterns of genetic evolution and divergence of influenza A (H3N2), as well as their impact on influenza VE, are likely to continue to challenge the selection of a suitable influenza A (H3N2) vaccine component. In the absence of a universal influenza vaccine, we support the concurrent inclusion of influenza A (H3N2) viruses from more than one clade in future influenza vaccines, as was recently suggested by others .The genetic changes of the influenza A (H3N2) virus will likely continue to present a challenge for the selection of an optimal influenza A (H3N2) vaccine component. New solutions should be considered to overcome this challenge.


Siloxane-based materials have a wide range of applications. Cage-type oligosiloxanes have attracted significant attention as molecular building blocks to construct novel siloxane-based nanoporous materials with promising applications such as in catalysis and adsorption. This paper reviews recent progress in the preparation of siloxane-based nanoporous materials using alkoxy- and silanol-functionalized cage siloxanes. The arrangement of cage siloxanes units is controlled by various methods, including amphiphilic self-assembly, hydrogen bonding of silanol groups, and regioselective functionalization, toward the preparation of ordered nanoporous siloxane-based materials. Ordered nanoporous materials have applications in various fields such as catalysis, adsorption, optics, electronics, and medicine ,2,3. SilThe use of well-defined oligosiloxanes as molecular building blocks has received great interest in overcoming these issues ,7. The cNumerous efforts have been made on the synthesis of nanoporous materials by cross-linking cage siloxanes with siloxane bonds or organic linkers. This paper focuses on the construction of nanoporous siloxane frameworks by connecting cage siloxanes with siloxane bonds. In addition to the D4R siloxanes, other cage siloxanes have also been used. Among various functional groups that can be employed for the siloxane bond formation  ,21, alko1.5)n  can be obtained when the hydrolysis and polycondensation reactions of alkoxysilanes or chlorosilanes are performed under specific conditions. Anionic cage silicate with Si-O– groups are formed in basic aqueous silicate solutions containing quaternary ammonium cations. For example, in the presence of tetramethylammonium (TMA) cations, cubic silicate (Si8O208−) with a D4R structure is almost quantitatively formed (12O3012−) can be obtained as an inclusion complex with α-cyclodextrin [3Si–CH2–Si(OEt)3] in the presence of TMAOH H7[Si8O20]·5.33H2O crystals for silylation. The amount of H2O per D4R silicate is much smaller than that in the TMA-D4R silicate hydrate crystal (65H2O per D4R); therefore, a bifunctional silylating agent Me2SiCl2 was successfully used to form (Si8O12)(OSiMe2Cl)8. More importantly, Nozawa et al. succeeded in the isolation of silanol-modified D4R siloxane (Si8O12)(OH)8 by cation exchange of (Si8O20)(NMe4)8 using Meldrum’s acid in N,N-dimethylacetamide 8 and Si8O12[OSi(OEt)3]8) gave non-porous xerogels under identical conditions ,348O127, n = 16, 18, and 20) 7). Self-assembly and polycondensation of these hydrolyzed species occurred upon solvent evaporation, and alkyl chains were finally removed by calcination to form mesopores. The pore diameter was 2.8, 3.0, and 3.3 nm when the alkyl chain length was n = 16, 18, and 20, respectively. Similarly, mesoporous silica has been successfully produced using alkyl-substituted cage siloxanes with a D5R structure (C16H33Si10O15(OEt)9) . T. TnH2n+15(OEt)9) . This meOne effective method to avoid thermal deterioration of the cage siloxane units by calcination is the introduction of an alkyl ester group instead of simple alkyl groups b 40]. T. T40]. TThe above-mentioned microporous xerogels and mesoporous materials prepared using cage siloxanes do not have crystalline frameworks. It is necessary to establish a new methodology to achieve a regular arrangement of the cage siloxanes to construct crystalline nanoporous materials like zeolites. In the past decade, some progress has been made in the field of POSS-based inorganic–organic hybrid porous materials ,46,47,48One promising approach to crystalline materials is solid-state polycondensation of molecular crystals. Iyoki et al. synthesized an alkoxy-functionalized cage siloxane having an adamantoxy group and seven methoxy groups . Hierarc8O12(CH=CHC6H4OSiPh2OH)8); however, condensation of the silanol groups was not performed . Th. Th60]. ensation . These cThe recent development in the construction of siloxane-based porous materials using cage-type siloxanes as molecular building blocks has been presented. Significant progress has been made on the syntheses of molecularly ordered nanoporous materials by the controlled connection of cage siloxanes based on H-bonding assembly and regioselective modification with silanol groups. This building block approach allows for the creation of novel nanoporous materials with diverse structures and compositions that cannot be achieved by conventional synthetic methods, leading to various applications.


C. albicans and the human liver cancer cell line HepG2, respectively. The highly enriched fractions were also analyzed by high performance liquid chromatography coupled with high resolution mass spectrometry (HPLC-HRMS) and their effect on the HepG2 cells was assessed via transcriptomics and with a proliferation assay.Marine endophytic fungi (MEF) are good sources of structurally unique and biologically active secondary metabolites. Due to the increase in antimicrobial resistance, the secondary metabolites from MEF ought to be fully explored to identify candidates which could serve as lead compounds for novel drug development. These secondary metabolites might also be useful for development of new cancer drugs. In this study, ethyl acetate extracts from marine endophytic fungal cultures were tested for their antifungal activity and anticancer properties against We demonstrated that the fractions could reduce proliferation in HepG2 cells. The detailed transcriptome analysis revealed regulation of several cancer- and metabolism-related pathways and gene ontologies. The down-regulated pathways included, cell cycle, p53 signaling, DNA replication, sphingolipid metabolism and drug metabolism by cytochrome P450. The upregulated pathways included HIF-1 signaling, focal adhesion, necroptosis and transcriptional mis-regulation of cancer. Furthermore, a protein interaction network was constructed based on the 26 proteins distinguishing the three treatment conditions from the untreated cells. This network was composed of central functional components associated with metabolism and cancer such as TNF, MAPK, TRIM21 and one component contained APP.C. albicans and S. cerevisiae alone or both and reduced proliferation of the human liver cancer cell line HepG2 implicating regulation of several cancer- and metabolism-related pathways. The data from this study could be instrumental in identifying new pathways associated with liver cancer anti-proliferative processes which can be used for the development of novel antifungal and anti-cancer drugs.The purified fractions from MEF investigated in this study showed antifungal activity against  The majority of neglected tropical diseases are caused by fungal pathogens, as well as helminths and protozoans. Efforts towards drug discovery that utilizes fungal cells as the target organism has the potential of identifying compounds that exclusively target fungal pathogens and compounds that share cellular targets with other non-pathogenic fungal eukaryotes. Fungi residing in terrestrial and marine environments are being explored for the development of novel antifungal compounds due to their potential to produce biologically active metabolites . MetabolSaccharomyces cerevisiae is used as a model for investigating many cellular processes in humans. These processes include cell cycle progression, DNA replication and segregation, maintenance of genomic integrity and stress responses. In cancer, these processes are affected by genetic and epigenetic alterations. Hence, yeast can be used as a suitable model organism for identifying novel compounds with anti-cancer activity during screening of chemical libraries [Metabolites from fungal isolates can also be screened for anticancer activities using target-oriented approaches. This has led to the approval of many molecularly targeted anticancer drugs. Development of cancer chemotherapeutics has focused on identifying compounds which target many cancer pathways while causing minimal toxicity to non-cancer cells . Humans ibraries .In this project, extracts from marine endophytic fungi were partially purified to obtain fractions containing potential novel compounds. The effect of these fractions on proliferation and transcriptome of the human liver cancer cell line HepG2 were investigated. The evidence presented in this study will inform future effort to scale up the fermentation of this fungal isolate to purify the potential novel compounds for detailed characterization.Candida albicans  and selected fractions from the second round of the preparative TLC  were analyzed by high performance liquid chromatography coupled with high resolution mass spectrometry (HPLC-HRMS). The HPLC-HRMS analysis of the FD K1V1V1 fraction revealed 11 major peaks; 5 of these 11 major peaks were important for this study because of their interesting fragmentation patterns , FD K1V5V3 (V3) and FD K2V3V5 (V5) fractions on proliferation of HepG2 cells was also investigated. All the three fractions reduced proliferation of HepG2 cells. The cells were first observed using light microscopy, comparing control cells to the treated cells. It could be observed how the untreated cells grew in the typical cell morphology; triangular and growing in islands or clumps, whereas the treated cells were somehow dissociated, were rounder in shape and could be found in smaller clumps, as well as single cells. The extent of reduction in cell proliferation seemed to be dose-dependent for fraction V5 whereas there were deviations from the dose-dependent effect for fractions V1 and V3 Fig. . In the HepG2 cells were treated with 2% of the partially purified MEF 134 fractions (FD K1V1V1 (V1), FD K1V5V3 (V3) and FD K2V3V5 (V5)) in order to assess the effect of these fractions on cell viability. Following treatment, the mRNA expression profile of 4 key markers  of cell viability were analyzed and compared to the expression profile from the untreated cells  appears on top of several compounds’ analysis results were sorted by significance of the E-value. Transcription factors of the HOX e.g. HOXA13, HOXC13), KLF , SP , CDX  TF families appear as prominent results across the components  or down-regulated genes  from the intersection sets of the pairwise Venn diagrams. The analysis demonstrated that treatment of HepG2 cells with either V1, V3 or V5 had impact on many cancer- and metabolism-related pathways  but not in the untreated HepG2 cells were further analyzed for protein interaction networks based on the Biogrid database. Most of these 26 genes  is absent in most transformed cells [For V5-treated HepG2 cells, the up-regulation of genes from the ed cells . More geed cells . Thus, iThe protein interaction network derived from the set of 26 genes expressed in common in all treatments  but not in the untreated HepG2 cells revealed communities  were associated with metabolism and cancer. It is anticipated that the data from this study would propel synthesis of the prominent compounds detected from the partially purified fractions. The synthesis of these compounds would be instrumental in identifying new compound(s) exhibiting antifungal or anti-proliferative activities. Such compounds could be used as starting material for the development of novel antifungal and anti-cancer drugs.In this study, crude extract from a marine endophytic fungal culture was fractionated to obtain partially purified fractions. These fractions showed antifungal activity against Pure isolates of MEF 134 were inoculated into 20-l culture vessels containing 10 l of Yeast extract, Peptone, Malt, Dextrose  broth prepared with filtered seawater. The inoculated broth was incubated at 30 °C for 4 months . The secThe MEF 134 crude extract was fractionated using a modified Kupchan fractionation method followed by preparative Thin Layer Chromatography (TLC). The modified Kupchan fractionation was performed by partitioning the crude extracts among seven different solvent systems . These sSaccharomyces cerevisiae and Candida albicans, grown in 50 ml nutrient broth, were diluted to an optical density at 600 nm (OD600) of 0.7. The diluted cultures were uniformly inoculated on to YPDA  plates and the discs were placed at defined positions on the agar plate. Zones of inhibition were measured in mm, 12 h after incubation of inoculated plates at 30 °C.Antifungal activity of the crude extract and the dichloromethane fractions (FD) were tested by the agar plate disc-diffusion method. A volume of 30 μl (0.05 g) and 120 μl (0.2 g) from stock solution of crude extract and FD, respectively, were added on to sterile filter paper disc which were 5 mm in diameter. All other fractions were tested by this method at 0.01 g/disc. The discs were allowed to air-dry prior to the assay. Overnight cultures of 2 in a humidified atmosphere. Cells were split with trypsin (Gibco) when they reached confluency. Monolayers were treated with the FD K1V1V1, FD K1V5V3 and FD K2V3V5 fractions at the indicated concentrations . Cell proliferation was determined by the resazurin metabolic assay [Hepato-carcinoma (HepG2) cells (ATCC®HB-8065TM) were cultured in DMEM low glucose, 10% FCS, 1% Penicillin/Streptomycin (P/S)  at 37 °C and 5% COic assay . After tHepG2 cells were lysed in 300–500 μl Trizol and total RNA was isolated with the Direct-zol™ RNA Isolation Kit (Zymo Research) according to the user’s manual including the optional on-column DNase digestion. The TaqMan Reverse Transcription (RT) Kit (Applied Biosystems) was used for cDNA synthesis. Real time PCR was performed in technical triplicates of biological duplicates using Power SYBR Green Master Mix (life technologies) on a VIIA7 (life technologies). Mean Ct values were calculated and normalized to RPL37A as a housekeeping gene. Fold change was calculated relative to the untreated control. The data were shown as mean values (log2).p value was less than 0.05. The detection p value was calculated as described in the supplementary methods in Graffmann et al. [p-value in compound-treatment and control less than 0.05, limma-p-value for differential expression less than 0.05 and ratio greater than 1.33. Down-regulated genes were detected via the criteria detection-p-value in compound-treatment and control less than 0.05, limma-p-value for differential expression less than 0.05 and ratio less than 0.75.Untreated HepG2 cells and cells treated with FD K1V1V1 (V1), FD K1V5V3 (V3) and FD K2V3V5 (V5) were hybridized in duplicates on the Affymetrix Human Clariom S Array  at the BMFZ (Biomedizinisches Forschungszentrum) core facility of the Heinrich-Heine Universität, Düsseldorf. Data analysis of the Affymetrix raw data was performed in the R/Bioconductor  environmn et al. . Up-reguP-values were adjusted by the q-value method by Storey et al. [Based on the transcriptome analysis, over-represented gene ontology terms and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways  were dety et al. . Visualiy et al. .We identified transcription factors (TFs) via motif enrichment analysis in the differentially expressed genes employing the CentriMo (version 5.1.0) tool from the MEME suite . BrieflyHomo sapiens) were filtered from the Biogrid database version 3.4.161 [n = 30 interacting proteins with the most interactions to proteins coded by genes from the original set. These interactions were plotted employing the R package network [Based on the Venn diagram analysis, a protein interaction network was constructed using the set of 26 genes that were expressed in common in all treatments  but not in the untreated HepG2 cells. Interactions annotated with the taxonomy id 9606  Table S2. Venn diagram HepG2 V3 vs. HepG2 control sets Table S3:. Venn diagram HepG2 V5 vs. HepG2 control sets Table S4. Venn diagram HepG2 V1, HepG2 V3, HepG2 V5 and HepG2 control sets Table S5. Overrepresented KEGG pathways in the down-regulated genes between HepG2 V1 vs. HepG2 control sets Table S6. Overrepresented KEGG pathways in the up-regulated genes between HepG2 V1 vs. HepG2 control set Table S7. Overrepresented KEGG pathways in the down-regulated genes between HepG2 V3 vs. HepG2 control sets Table S8. Overrepresented KEGG pathways in the up-regulated genes between HepG2 V3 vs. HepG2 control sets Table S9. Overrepresented KEGG pathways in the down-regulated genes between HepG2 V5 vs. HepG2 control sets Table S10. Overrepresented KEGG pathways in the up-regulated genes between HepG2 V5 vs. HepG2 control sets Table S11. Overrepresented gene ontologies in the subset of 26 genes expressed in HepG2 V1, V3 and V5 but not in HepG2 control from the venn diagram in Fig. Table S12. Detailed listing and functional annotation via the DAVID functional annotation of the subset of 26 genes expressed in HepG2 V1, V3 and V5 but not in HepG2 control from the venn diagram in Fig. Table S13. Results of CentriMo (MEME suite) motif enrichment analysis of the 300 bp upstream of the transcription start site for the genes down-regulated in HEPG2 treated with V1 vs. untreated HEPG2. Table S14. Results of CentriMo (MEME suite) motif enrichment analysis of the 300 bp upstream of the transcription start site for the genes down-regulated in HEPG2 treated with V3 vs. untreated HEPG2. Table S15. Results of CentriMo (MEME suite) motif enrichment analysis of the 300 bp upstream of the transcription start site for the genes down-regulated in HEPG2 treated with V5 vs. untreated HEPG2. Table S16. Results of CentriMo (MEME suite) motif enrichment analysis of the 300 bp upstream of the transcription start site for the genes up-regulated in HEPG2 treated with V1 vs. untreated HEPG2. Table S17. Results of CentriMo (MEME suite) motif enrichment analysis of the 300 bp upstream of the transcription start site for the genes up-regulated in HEPG2 treated with V3 vs. untreated HEPG2. Table S18. Results of CentriMo (MEME suite) motif enrichment analysis of the 300 bp upstream of the transcription start site for the genes up-regulated in HEPG2 treated with V5 vs. untreated HEPG2. Table S19. Summary of the 20 most significant results of all CentriMo (MEME suite) motif enrichment analyses.Additional file 3: Figure S1. KEGG pathway chart of pathway Steroid Biosynthesis in genes down-regulated in HepG2 cells treated with V1 vs. control.Additional file 4: Figure S2. KEGG pathway chart of pathway Drug metabolism – cytochrome P450 in genes down-regulated in HepG2 cells treated with V1 vs. control.Additional file 5: Figure S3. KEGG pathway chart of pathway p53 signaling pathway in genes down-regulated in HepG2 cells treated with V1 vs. control.Additional file 6: Figure S4. KEGG pathway chart of pathway cell cycle in genes down-regulated in HepG2 cells treated with V3 vs. control.Additional file 7: Figure S5. KEGG pathway chart of pathway viral carcinogenesis in genes up-regulated in HepG2 cells treated with V3 vs. control.Additional file 8: Figure S6. Fig: KEGG pathway chart of pathway cell cycle in genes up-regulated in HepG2 cells treated with V5 vs. control.Additional file 9: Figure S7. Dendrogram of community clustering of protein interaction networks of HepG2 cells treated with V1, V3 and V5.Additional file 10: Figure S8. Pairwise scatter plots of logarithmic (base 2) expression values of all samples of HepG2 cells treated with V1, V3, V5 and untreated versus each other.


Rabies virus (RABV) matrix protein (M) plays crucial roles in viral transcription, replication, assembly, and budding; however, its function during the early stage of virus replication remains unknown. Here, we mapped the protein interactome between RABV M and human host factors using a proteomic approach, finding a link to the V-type proton ATPase catalytic subunit A (ATP6V1A), which is located in the endosomes where RABV first enters. By downregulating or upregulating ATP6V1A expression in HEK293T cells, we found that ATP6V1A facilitated RABV replication. We further found that ATP6V1A was involved in the dissociation of incoming viral M proteins during viral uncoating. Coimmunoprecipitation demonstrated that M interacted with the full length or middle domain of ATP6V1A, which was dependent on the lysine residue at position 256 and the glutamic acid residue at position 279. RABV growth and uncoating in ATP6V1A-depleted cells was restored by trans-complementation with the full length or interaction domain of ATP6V1A. Moreover, stably overexpressed ATP6V1A enhanced RABV growth in Vero cells, which are used for the production of rabies vaccine. Our findings identify a new partner for RABV M proteins and establish a new role of ATP6V1A by promoting virion uncoating during RABV replication. Lyssavirus in the Rhabdoviridae family and has a non-segmented negative-strand RNA genome of approximately 12 kb in length. The viral genome consists of five genes, encoding five viral proteins: the nucleoprotein (N), the phosphoprotein (P), the matrix protein (M), the glycoprotein (G), and the large polymerase protein (L) , is an important and devastating infectious disease with an almost 100% mortality rate . RABV betein (L) , 4. The tein (L) .The RABV M protein is the smallest of the virion proteins  and form+ from the cytoplasm to the lumen  , 16, 17.he lumen , 20. Thehe lumen  and playhe lumen . Systemahe lumen . ATP6V1Hhe lumen .+ transport  as described previously (B). We identified a total of 2672 host proteins that coimmunoprecipitated with the M protein; 492 proteins coimmunoprecipitated with both M-NF and M-CF, 1761 proteins coimmunoprecipitated with M-NF only, and 419 proteins coimmunoprecipitated with M-CF only. The MS data have been deposited in the IMEx consortium through IntAct with the accession code IM-27674 (http://www.imexconsortium.org) (https://david.ncifcrf.gov/). The analysis showed that total 38 ATP-powered pump subunits interacted with the M protein  and identified host proteins that interacted with the M proteins by using mass spectrometry (MS) A as desceviously . Proteineviously B. We ide protein , which sC, together with five matched peptides detected in the MS analysis (highlighted in red). ATP6V1A was detected in eluted lysates of cells transfected with plasmids expressing FLAG-tagged M proteins but not in those of cells transfected with control vectors, as confirmed by using Western blotting with an ATP6V1A-specific antibody (D).We focused on the catalytic subunit of V-ATPase-ATP6V1A for further study based on previous findings that ATP6V1A influences the pH of endosomes and that the acidic endosomal environment is vital for the early stage of viral infection , 21. TheFigure 1antibody D.A). There was no significant difference in cell viability between ATP6V1A and scrambled siRNA-treated cells based on the quantification of ATP or NADH by using CellTiter-Glo  reagent or Cell Counting Kit-8 (CCK8) , respectively (B). Viral titration with cell supernatants showed that ATP6V1A knockdown decreased viral titers by 5.1-fold at 6 h post infection and decreased RABV replication in HEK293T cells at all later times post infection (C). The RABV P protein in cell supernatants and lysates was detected by Western blotting at different times post infection. Viral P protein in cell supernatants was first detected at 18 h post infection, with a considerable reduction in ATP6V1A-knocked-down cells compared with that in control cells (D). ATP6V1A knockdown resulted in less accumulation of viral proteins in cells (D). We also infected ATP6V1A-knocked-down and scrambled siRNA-transfected HEK293T cells with ERA virus at an MOI of 0.01. The results showed that knockdown of ATP6V1A decreased RABV replication in the supernatant of infected cells by 6-fold at 36 h post infection (E). Strikingly, the P protein was almost undetectable at 36 h post infection in the culture supernatants of ATP6V1A-knocked-down cells but was clearly detectable in the culture supernatants of the control cells (F), indicating that the knockdown of ATP6V1A suppressed viral replication.To investigate whether ATP6V1A is involved in RABV replication, HEK293T cells were transfected with ATP6V1A siRNA and infected with RABV Evelyn-Rokitnicki-Abelseth (ERA) strain at a multiplicity of infection (MOI) of 1. As a control, additional cells were transfected with a scrambled siRNA and infected with virus A. There ectively B. Viral nfection C. The RAol cells D. ATP6V1in cells D. We alsnfection E. Strikiol cells F, indicaG) by 29%, 61%, and 49% at 36, 48, and 60 h post infection, respectively .To further confirm the role of ATP6V1A in RABV replication, we infected ATP6V1A-knocked-down and scrambled siRNA-transfected HEK293T cells with ERA-eGFP, a recombinant RABV ERA strain that expresses enhanced green florescence protein, at an MOI of 0.01. Cells expressing eGFP were seen as indicators of viral infection by using a high-content imaging-based screen as described previously . The resFig. 2A). The expression of P protein was primarily detected at 6 h post infection in the cell lysates and at 18 h post infection in the cell supernatants, and the expression level increased substantially over time in the cells overexpressing ATP6V1A (B). Overexpression of ATP6V1A also enhanced RABV growth in cells infected with ERA virus at an MOI of 0.01. Viral titers in the supernatants of cells overexpressing ATP6V1A were roughly 1-log higher than those of control cells at 36 and 48 h post infection (C). Viral P protein expression in cell supernatants or lysates was also enhanced by 7-fold in cells overexpressing ATP6V1A at 36 h post infection, which was consistent with the observed enhanced virus growth (D). These results suggest that ATP6V1A is important for the replication of RABV in cells.We then evaluated whether overexpression of ATP6V1A affects RABV replication. HEK293T cells transfected with pA-Myc to overexpress ATP6V1A were infected with ERA virus at an MOI of 1 at 24 h post transfection. Virus and P protein in the cell supernatants and lysates were titrated or detected at different times. The results showed that overexpression of ATP6V1A promoted the growth of ERA virus and increased the viral titers by 5-fold at 18 h post infection A. The ex ATP6V1A B. Overexnfection C. Viral s growth D. These +-ATPase pump, BAF A1 blocks endosomal acidification and eliminates the trigger of the conformational rearrangements in the RABV G protein that are necessary for membrane fusion (A). ATP6V1A distributed in the cytoplasm, which was consistent with previous report (B), indicating that more virus particles remained intact in the ATP6V1A-knocked-down cells. To quantify the dissociation of RABV M proteins in virus-infected cells, we chose 130 cells from each group to count high-intensity punctate staining spots by using Imaris software  and found that more than 70% of the cells treated with BAF A1, or transfected with ATP6V1A siRNA, contained more than 10 high-intensity punctate staining spots in each cell (C) and maintained RABV particles without uncoating. No cells transfected with scrambled siRNA contained more than 10 high-intensity punctate staining spots (C). Taken together, these data demonstrate that ATP6V1A knockdown inhibits the dissociation of RABV M proteins during the early stage of viral life cycle.RABV enters cells through receptor-mediated CME. Virus-endosome membrane fusion is stimulated by RABV G proteins, which are conformationally changed after being exposed to acidic conditions in the endosome . After me fusion , 32 . Under acidic conditions allowing membrane fusion, depletion of ATP6V1A still inhibited the dissociation of viral M proteins in particles, indicating that ATP6V1A plays a role in RABV uncoating after membrane fusion ; the observation also found that, although a small amount of aggregated M proteins remained in cell plasma membrane, most of the aggregated M proteins trafficked to the cytoplasm . The dissociation of RABV M proteins was quantified by using Imaris software as described above, and the results showed that 82% of the cells treated with BAF A1 in neutral conditions contained more than 10 spots in each cell , whereas no cells treated with BAF A1 in acidic conditions had more than 10 spots , which indicated that virion under acidic conditions bypassed endocytic requirements and completed membrane fusion directly. However, 75% of the cells transfected with ATP6V1A siRNA and incubated in acidic conditions still contained more than 10 spots in each cell , whereas no cells transfected with scrambled siRNA contained more than 10 spots . Collectively, these findings suggest that ATP6V1A is required for RABV uncoating.Viral M proteins dissociate and release from vRNPs in the step of RABV uncoating . RABV, sFig. 5A). In addition, FLAG-tagged M protein was coimmunoprecipitated with Myc-tagged ATP6V1A in a reverse Co-IP experiment (B). These results indicate that ATP6V1A interacts with RABV M protein in cells.To further examine the interaction between ATP6V1A and the M protein, we performed coimmunoprecipitation (Co-IP) and Western blotting analysis. HEK293T cells were transfected with plasmids expressing FLAG-tagged RABV M (pM-NF) and Myc-tagged ATP6V1A (pA-Myc) together, or individually. Myc-tagged ATP6V1A was eluted and coimmunoprecipitated with FLAG-tagged M protein but not FLAG peptide when they were coexpressed A. In addperiment B. These C). These results suggest that RABV M protein interacts with endogenous ATP6V1A during RABV replication.Next, we asked whether endogenous ATP6V1A interacts with RABV M protein during RABV infection. HEK293T cells were infected with ERA virus at an MOI of 5 or uninfected. At 24 h post infection, we performed the Co-IP assay with lysates of infected cells and beads conjugated with IgG or antibodies to M protein. Endogenous ATP6V1A was immunoprecipitated with RABV M protein in virus-infected cell lysates C. These S-transferase (GST)-fused M protein and C-terminal His-tagged ATP6V1A. We then used GST pull-down and immunoprecipitation analysis to reveal that GST-fused M protein directly binds to His-tagged ATP6V1A (D). This direct interaction was confirmed with His-tagged pull-down that GST-fused M protein immunoprecipitated with His-tagged ATP6V1A (E). Our findings prove that ATP6V1A directly interacts with M protein.To determine whether the interaction between M and ATP6V1A was physically direct, we expressed and purified C-terminal glutathione- ATP6V1A D. This d ATP6V1A E. Our fiA). The four constructs were, respectively, cotransfected with pM-NF into HEK293T cells. At 24 h post transfection, cell lysates were prepared for Co-IP assays with beads conjugated with mAbs to FLAG. All four truncated constructs of ATP6V1A were well expressed (B–E), and only the fragment of amino acids 160 to 309 of ATP6V1A (A160-309) (C) interacted with the M protein. We also performed the reverse Co-IP assay using beads conjugated with mAbs to Myc and confirmed that also only A160-309 coimmunoprecipitated with the M protein (F). These results demonstrate that the middle domain of ATP6V1A, namely, amino acids 160 to 309, is responsible for the interaction of ATP6V1A with the M protein.To define the key domain in ATP6V1A that determines its interaction with M protein, we generated four truncated ATP6V1A constructs, pA1-159, pA160-309, pA310-464 and pA465-617, that, respectively, expressed amino acids 1 to 159, 160 to 309, 310 to 464, and 465 to 617, and fused each of them with a Myc-tag on the C terminus A. The foxpressed , B–E, an160-309) C interac protein F. These G), suggesting that the residues at positions 256 and 279 in A160-309 are critical for the interaction with the M protein.Previous studies have shown that the K256Q mutation in ATP6V1A results in an unstable V-ATPase complex and that the E279Q mutation affects primary ATP hydrolysis . InteresFig. 7H). Both truncates were fused to the C-terminal eGFP that was tagged with Myc. We, respectively, transfected pM1-76 and pM77-202 with pA160-309-HA into HEK293T cells and lysed them at 24 h post transfection for the Co-IP assay. The results showed that the N-terminal domain of the M protein interacts with A160-309 (I) rather than the C-terminal domain (J).To define the domain of the M protein that interacts with A160-309, we generated two constructs, pM1-76 and pM77-202, that, respectively, expressed two truncated M protein fragments, namely, amino acids 1 to 75 (M1-76) and amino acids 77 to 202 (M77–202) H. Both tA160-309 I rather l domain J.A). Transfection with pA-Myc or pA160-309 obviously enhanced virus replication in ATP6V1A RNAi cells compared with pMu160-309 or control vector pMyc at an MOI of 1 or 0.01 (B–E). There was no significant difference in ERA virus titers between the trans-complementation of pA-Myc and pA160-309 or pMu160-309 and control vector pMyc.To verify the effect of ATP6V1A on the replication of RABV, HEK293T cells were transfected with siRNA to suppress the expression of endogenous ATP6V1A. At 48 h post transfection of siRNA, cells were transfected with pA-Myc, pA160-309, pMu160-309, or pMyc. At 24 h post transfection, cells were infected with ERA virus at an MOI of 1 or 0.01. Western blotting showed that transfection with pA-Myc recovered ATP6V1A expression in siRNA-transfected cells A. Transf or 0.01 , B–E. ThD and E), which is comparable with that in mock knockdown cells. The results demonstrate that trans-complementation with ATP6V1A or A160-309 restores the effect of ATP6V1A on RABV replication in ATP6V1A-knocked-down cells.Transfection of pA-Myc and pA160-309 also resulted in more than 8-fold higher expression of the P protein compared with transfection of pMu160-309 or the control vector pMyc in ATP6V1A RNAi cells that were infected with ERA virus at an MOI of 0.01 at 60 h post infection , D and Etrans-complementation assay as described above. The cells were infected with ERA at an MOI of 100 and treated with acidic PBS for acid bypass assay as previously described , whereas in the cells trans-complemented with ATP6V1A or A160-309, the high-intensity punctate staining of viral M protein disappeared, indicating that the virion was uncoated (A). In acidic condition allowing membrane fusion of RABV, trans-complementation of ATP6V1A mutant failing to interact with M protein was unable to recover the dissociation of viral M protein during uncoating after membrane fusion (A). To quantify the dissociation of RABV M proteins, we used Imaris software to count high-intensity punctate staining spots in 130 virus-infected cells from each group. The results showed that, in acidic conditions, more than 65% of the cells trans-complemented Mu160-309 or without trans-complementation still contained more than 10 high-intensity punctate staining spots (B), whereas no cell has more than 10 spots when it trans-complemented with ATP6V1A or A160-309 (B). These findings further indicated that ATP6V1A participates in the replication and uncoating of RABV, which is associated with its interaction with M protein.To test the effect of ATP6V1A on RABV uncoating, we performed escribed . We founith pMyc A, whereauncoated A. In acie fusion A. To quang spots B, whereaA160-309 B. These A) and there is no apparent difference of M or G protein level between the VLPs produced in HEK293T cells depleted ATP6V1A or not . To detect the integrity of rabies VLPs, purified pellets were detected by Western blotting in non-denaturing and non-reducing conditions. Rabies VLPs were detected in the band larger than 250 kDa size, and depletion of ATP6V1A in cells has no obvious effect on the production of rabies VLPs . Then, we observed the morphology and specification of rabies VLPs by immunoelectron microscopy. Consistently with previous research . Knockdown of ATP6V1A in cells did not influence the morphology of rabies VLPs . These results showed that ATP6V1A has no effect on the production and morphology of rabies VLPs.To evaluate the influence of ATP6V1A on RABV VLP production, we produced and detected rabies VLPs by transfecting plasmids expressing viral M and G protein, respectively . WesternFig. 10research , envelopA), and this overexpression did not affect cell viability determined by the quantification of ATP or NADH using CellTiter-Glo  reagent or Cell Counting Kit-8 (CCK8) , respectively . We infected Vero-ATP6V1A and Vero cells with ERA virus at an MOI of 5 and found that viral titers in the culture supernatants of the Vero-ATP6V1A cells were 2.8- to 4.0-fold higher than those of Vero cells at different time points post infection . The level of P protein in the Vero-ATP6V1A cell supernatants was 1.9-fold higher than that in Vero cell supernatants at 24 h post infection . P protein in the lysates of Vero-ATP6V1A cells and Vero cells was detectable at 6 h post infection, and the P protein level was higher in the Vero-ATP6V1A cell lysates than in the Vero cell lysates at 24 h post infection . Steady overexpression of ATP6V1A in Vero-ATP6V1A cells infected with ERA virus at an MOI of 0.05 yielded about 4-fold increase in viral growth titers  and the P protein expression . These data suggest that overexpression of ATP6V1A in Vero cells promotes the propagation of RABV, which could be a potential strategy to improve the production of RABV vaccine.The Vero (African green monkey kidney) cell line is the most widely accepted continuous cell line for vaccine production. It was introduced to produce the inactivated rabies vaccine in 1982 and has been in use for over 30 years . To examFig. 11The functions of RABV M protein in regulating the balance between viral transcription and translation  and viriDissociation of viral M proteins is an indication of enveloped virus uncoating, and the cellular distribution of dissociated M protein related with its function on viral early stage of life cycle . M proteThe uncoating of RABV occurs after viral particle entry into the host cell endosomes via endocytosis and membrane fusion , 46. A pTrans-complementation with the interacting domain of ATP6V1A restores RABV infection as ATP6V1A did in knocked-down cells. Mutation of the lysine residue at position 256 and the glutamic acid residue at position 279 in the interacting domain of ATP6V1A destroyed the interaction activity with the M protein, which then blocked the ATP6V1A-induced promotion of RABV replication. It is interesting to note that the N-terminal domain of the M protein, which is responsible for its interaction with ATP6V1A, also contains the late budding domain PPEY motif that is responsible for efficient virion release and the pathogenicity of RABV , baby hamster kidney cells  stably expressing T7 polymerase (BSR-T7/5), Vero (African green monkey kidney) cells , and a Vero cell line stably overexpressing ATP6V1A protein (Vero-ATP6V1A) were cultured in Dulbecco's modified Eagle's medium  with 10% fetal bovine serum .RABV Evelyn-Rokitnicki-Abelseth (ERA) strain was maintained in our laboratory. Recombinant ERA expressing eGFP (ERA-eGFP) was generated as described previously .Escherichia coli to express and purify the M protein for pull-down assays. Two pCAGGS constructs were generated to express two truncates of the M protein, amino acids 1 to 76 and 77 to 202, respectively, which were fused to the C terminus of eGFP tagged with Myc and named pM1-76 and pM77-202, respectively. pcDNA plasmids  was cloned into pCAGGS in-frame with a Myc-tag sequence at the C terminus and named pA-Myc. Four truncated constructs based on pCAGGS were generated to express four truncates of ATP6V1A, specifically amino acids 1 to 159, 160 to 309, 310 to 464, and 465 to 617, each of which was tagged with Myc at the C terminus, and named pA1-159, pA160-309, pA310-464, and pA465-617, respectively. The construct based on pA160-309 with the two mutations of K256Q and E279Q was named pMu160-309. The pCAGGS construct expressing amino acids 160 to 309 tagged with HA at the C terminus was named as pA160-309-HA. The pGEX-4T-1 , mouse anti-Myc , rabbit anti-ATP6V1A , mouse anti-β-actin , mouse anti-HA epitope tag , mouse anti-GST , mouse anti-His epitope tag , rabbit IgG  and the secondary antibodies  ) and gold-labeled goat anti-mouse IgG antibody  were purchased from the indicated vendors. Murine sera against M, P, or G protein were produced in our laboratory.HEK293T cells were transfected with pM-NF or pM-CF, or the control plasmids pNF or pCF by using the TransIT-293  transfection reagent according to the manufacturer's instructions. The cells were incubated with lysis buffer  for 1 h at 4 °C at 24 h post transfection. Supernatants, clarified by centrifugation, were incubated with an anti-FLAG affinity gel  for 18 h at 4 °C. Proteins bound to the affinity gel were eluted with elution buffer  for 1 h at 4 °C. Then, a 10% (vol/vol) volume of the eluted proteins was subjected to SDS-PAGE and silver staining ; the remaining 90% (vol/vol) volume of eluted proteins was subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis to identify coimmunoprecipitated host proteins. Samples were mixed with trypsin and digested at 37 °C overnight. LC-MS/MS was implemented by using an EASY-nLCTM 1200 UHPLC system  coupled with an Orbitrap Q Exactive HF-X mass spectrometer  at Shanghai Applied Protein Technology Co Ltd in China. The MS/MS signals were then processed against the Uniprot human protein database  using the Mascot algorithm  with the following parameters: dynamical modifications: oxidation (M); fixed modifications: carbamidomethyl: (C); maximum missed cleavages, 2; filter by score ≥20.g/min, 10 min) at 4 °C, and clarified lysates were incubated with beads conjugated with anti-FLAG  or anti-Myc  affinity gel on a flip shaker for 18 h at 4 °C. Incubated beads were washed five times with lysis buffer, followed by SDS-PAGE and Western blotting.For the ERA-M and ATP6V1A coimmunoprecipitation (Co-IP) assay, HEK293T cells were cotransfected with plasmids expressing ERA-M protein or the M truncates, and plasmids expressing ATP6V1A protein or the ATP6V1A truncates by using TransIT-293 transfection reagent. At 24 h post transfection, the cells were lysed with ice-cold lysis buffer on a flip shaker for 1 h at 4 °C. Cell lysates were centrifuged . ATP6V1A fused with a His tag at the C terminus was expressed in E. coli and purified by using a Ni-NTA column . For the GST pull-down assay, purified M-GST protein (10 μg) or GST protein at equimolar ratios was incubated with glutathione 4B-Sepharose beads on a flip shaker for 4 h at 4 °C. After three washes with lysis buffer, the beads were incubated with ATP6V1A-His protein (10 μg) on a flip shaker for 8 h at 4 °C. After three more washes with lysis buffer, the beads were assessed by Western blotting. For the His-tag pull-down assay, purified ATP6V1A-His protein (10 μg) was incubated with beads conjugated with His antibody on a flip shaker for 4 h at 4 °C. The beads were washed three times and incubated with M–GST proteins (10 μg) on a flip shaker for 8 h at 4 °C. The beads were then washed again and assessed by Western blotting.For the ERA-M and ATP6V1A pull-down assays, ERA-M protein fused with C-terminal GST was expressed in g/min, 10 min) at 4 °C at the indicated time points post infection. Viral titers in the centrifuged culture medium were detected as focus forming unit per milliliter (FFU/ml) in BSR-T7/5 cells. The levels of viral P protein in the centrifuged culture medium, and P protein, ATP6V1A, or β-actin in the lysate of cells were detected by Western blotting.HEK293T cells were transfected with small interfering RNA (siRNA) pool oligonucleotides  that targeted four different sequence domains of ATP6V1A (ATP6V1A siRNA) or were non-targeting to any gene in the human genome (scrambled siRNA) at a concentration of 30 nM by using the Lipofectamine RNAiMAX transfection reagent  according to the manufacturer's instructions. At 48 h post transfection, the cells were infected with ERA at the indicated MOI, and culture medium or lysates of cells were collected and centrifuged . At 48 h post transfection, the cells were incubated with 5 μM protein synthesis inhibitor cycloheximide  for 1 h at 37 °C and then incubated with ERA virus at an MOI of 100 for 1 h at 37 °C in the presence of CHX. Unabsorbed virus was removed by replacing the medium three times with fresh medium, and the cells were incubated for an additional 2 h at 37 °C. Note that CHX and/or BAF A1 were maintained on the cells throughout the incubation. Cells were fixed and permeabilized and then incubated with mouse anti-M protein serum and rabbit anti-ATP6V1A antibody . After staining with FITC-labeled anti-mouse secondary antibody  and Alexa Fluor 647-labeled goat anti-rabbit IgG (H + L)  for 1 h, the cells were examined by means of confocal microscopy with an LSM880 system .trans-complementation assay, at 48 h post transfection of siRNA, the cells were transfected with plasmids of pA-Myc or pA160-309 or pMu160-309 or pMyc, respectively. At 24 h post transfection, the cells were infected with ERA virus at an MOI of 100 and treated as previously described  transfection reagent according to the manufacturer's instructions and cultured for 48 h. Culture supernatants were harvested and ultracentrifuged as previously described  to generate packaged retroviruses in HEK293 cells following the manufacturer's instructions and by using TransIT-Lenti Transfection Reagent . Vero cells were transduced with packaged retroviruses and cultured in medium supplemented with puromycin for selection. The surviving cell clone was isolated, propagated, and examined for stable expression of ATP6V1A by Western blotting.For siRNA interfered cells' viability measurement, HEK293T cells were seeded in opaque-walled 96-well plates and transfected with siRNAs. At 48 h post transfection, the viability of cells in culture were determined based on the quantification of ATP by using CellTiter-Glo  reagent. A volume of 100 μl of CellTiter-Glo reagent was added directly into each well by following the manufacturer's instruction, and the contents were mixed for 10 min on a shaker at room temperature to induce cell lysis. After stabilizing luminescence signal at room temperature for 10 min, luminescence was measured with a GloMax 96 microplate luminometer . For the viability measurement of Vero cells stably overexpressing ATP6V1A (Vero-ATP6V1A), Vero-ATP6V1A cells and Vero cells were seeded in opaque-walled 96-well plates. Twenty-four hours later, cells were treated with CellTiter-Glo reagent and luminescence was measured according to the manufacture's instruction.Cell viability was also determined based on the quantification of NADH by using Cell Counting Kit-8 (CCK8)  according to the manufacturer's instruction. Cells were seeded in 96-well plates and transfected with siRNAs. At 48 h post transfection, culture medium was removed, 10 μl CCK8 solution mixed with 90 μl fresh medium was added and incubated for 2 h in 37 °C. The absorbance was measured at 450 nm. For the viability measurement of Vero cells stably overexpressing ATP6V1A (Vero-ATP6V1A), Vero-ATP6V1A cells and Vero cells were seeded onto 96-well plates. Twenty-four hours later, cells were treated with 10 μl CCK8 solution mixed with 90 μl fresh medium and absorbance was measured at 450 nm.t test. A p value of 0.05 was considered statistically significant.All experiments were performed two or three times, statistical analysis was performed for each experiment every time, and the data from one independent experiment were shown. Results were analyzed for statistical significance by using Student's http://www.imexconsortium.org). All other data are contained within the manuscript and also deposited to Zenodo with accession number 10.5281/zenodo.4279152.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner The authors declare that they have no conflicts of interest with the contents of this article.The corresponding author had full access to all the data and accepts the final responsibility for the decision to submit this manuscript for publication.


Epidemiologic analyses have shed light on an association between type 2 diabetes (T2D) and pancreatic ductal adenocarcinoma (PDAC). Recent data also suggest a potential relationship between T2D and insulinoma. Under rare circumstances, type 1 diabetes (T1D) can also be implicated in tumorigenesis. The biological mechanisms underlying such relationships are extremely complex. Some genetic factors contributing to the development of T2D are shared with pancreatic exocrine and endocrine tumors. Obesity and overweight can also contribute to the initiation and severity of T2D, while aging may influence both endocrine and exocrine tumors. Finally, pharmacological treatments of T2D may have an impact on PDAC. On the other hand, some treatments for insulinoma can trigger diabetes. In the present minireview, we discuss the cellular and molecular mechanisms that could explain these interactions. This analysis may help to define new potential therapeutic strategies. Diabetes is a metabolic disorder characterized by chronic hyperglycemia. Type 1 diabetes (T1D) is less frequent 5.6%) than type 2 diabetes (T2D) and is caused by autoimmune destruction of pancreatic beta-cells. T2D represents 91.2% of diabetes cases and is generally associated with insulin resistance and compensatory hyperinsulinemia, an early indicator of metabolic dysfunction. In the longer term, T2D leads to progressive functional defects of beta-cells. The remaining cases are primarily gestational diabetes, which are represented by hyperglycemia that generally disappears after delivery. Pancreatic adenocarcinoma (PDAC), the most frequent (95%) exocrine pancreatic cancer, is also the most lethal, with a 5-year overall survival of less than 8% . This li.6% than T2D and PDAC have common determinants, including aging, obesity, and genetic factors . T2D andAging is the time-dependent deterioration of physiological functions affecting both diabetes and PDAC. The incidence of diabetes increases with age: with 1.8 million patients aged 20–39, 11.7 millions at 40–59, to 19.3 millions at 60–79 in the European Union in 2017 . Islet aG12D model of PDAC, knock-out of the senescence-inducing factor SIN3B reduced the initiation and progression of pancreatic lesions, while decreasing secretion of the SASP factor IL-1α  of 30 kg/mdiabetes . Indeed,diabetes , 35. Thediabetes . Moreovediabetes .Lipocalin2 decreases PDAC incidence in KRAS-G12D transgenic mice . Some variants of PDX1 and HNF1α are also associated with increased risks of T2D  have been used to identify relationships between diabetes and pancreatic cancers. Several pancreatic developmental genes, for PDAC . Moreoves of T2D , 60, obes of T2D , or hypes of T2D . The ants of T2D , is overs of T2D , suggestigenesis . This efMAFA that prevents GSK3-mediated MAFA phosphorylation  in 30% of tumors . To delineate the molecular mechanisms involved in this protective effect of metformin, animal models were used. Metformin significantly decreased the incidence of PDAC promoted by diet-induced obesity in the conditional KRAS-G12D knock-in mouse  (Grant number 26866).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Trypanosoma cruzi (T. cruzi) is the etiologic agent of Chagas disease. Previously, we have identified T. cruzi antigens TcG2 and TcG4 as potential vaccine candidates, cloned in eukaryotic expression vector pCDNA3.1 (referred as p2/4) and tested their ability to elicit protection from T. cruzi infection. In the present study, we subcloned the two antigens in a nanoplasmid that is optimized for delivery, antigen expression, and regulatory compliance standards, and evaluated the nanovaccine (referred as nano2/4) for prophylactic protection against repeat T. cruzi infections. For this, C57BL/6 mice were immunized with two doses of p2/4 or nano2/4 at 21 days interval, challenged with T. cruzi 21 days after 2nd immunization, and euthanized at 10- and 21-days post-infection (pi) corresponding to parasite dissemination and replication phase, respectively. Some mice were re-challenged 21 days pi and monitored at 7 days after re-infection. Without the help of a vaccine, T. cruzi elicited delayed and sub-par T cell activation and low levels of effector molecules that failed to control tissue dissemination and replication of the parasite and provided no protection against repeat challenge infection. The nano2/4 was most effective in eliciting an early activation and production of IFN-γ by CD4+T effector/effector memory (TEM) cells and cytolytic perforin (PFN) and granzyme B (GZB) molecules by CD4+ and CD8+ TEM subsets at 10 days pi that was followed by robust expansion of CD4+ and CD8+ TEM and TCM cells with further increase in IFN-γ production at 21 days pi. Consequently, nano2/4-immunized mice exhibited potent control of parasite dissemination at 10 days pi, and tissue parasite burden and tissue inflammatory infiltrate and necrosis were barely detectable at 21 days pi. Furthermore, nano2/4-immunized mice responded to re-challenge infection with high levels of effector molecules production by CD4+ and CD8+ TEM subpopulations that offered even better control of tissue parasite burden than was observed after 1st infection. In comparison, non-vaccinated/infected mice exhibited clinical features of sickness and 59% mortality within 7 days after re-infection. In conclusion, we show that delivery of TcG2 and TcG4 in nanoplasmid offers excellent, protective T cell immunity against repeat T. cruzi infections.A parasitic protozoan  Trypanosoma cruzi (T. cruzi or Tc) is the etiological agent for Chagas disease (CD). The prolonged burden of CD primarily occurs in Latin American countries, Mexico, and USA  to offer antibiotic-free approach for selection and amplification of the recombinant DNA , and optT. cruzi TcG2 and TcG4 antigens into a NTC9385R-MCS nanoplasmid and used the recombinant plasmids as a nano2/4 vaccine. As re-infection might provoke tissue inflammation due to insults from higher parasite burden and intensify tissue necrosis in the host  were included as controls so that we can study the effects (positive or negative) of the changes in the vector backbone on the immunogenic potential of the selected candidates against repeated T. cruzi infections. We examined whether and how nano2/4 modulated the systemic CD4+ and CD8+ T cell immunity against T. cruzi repeat infections, and whether the nano2/4 induced immunity was effective in arresting systemic parasitemia, prevent tissue invasion and replication, and parasite-induced tissue inflammation and injury.In this study, we have cloned the genes encoding the host , 17, we We followed the National Institutes of Health guidelines for housing and care of laboratory animals. All animal experiments were conducted in accordance with the protocols that were approved by the Institutional Animal Care and Use Committee (protocol number 08-05-029) at the University of Texas Medical Branch at Galveston. All experiments were conducted in ABSL2/BSL2-approved laboratory and all personnel have received appropriate ABSL2/BSL2 training.TcG2 and TcG4 are deposited in the GenBank . The full-length cDNAs for the two antigens were previously cloned into pCDNA3.1 eukaryotic expression plasmid and the recombinant plasmids were purified by anion exchange chromatography by using a Qiagen Endo-free maxi prep kit   . A CMV prth, CA) . The rec process .T. cruzi SylvioX10/4 strain were amplified in vitro in C2C12 cells (Mouse myoblast C2C12 cells (ATCC CRL-1772) were propagated in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.1 mM sodium pyruvate. Trypomastigotes of 12 cells .T. cruzi infection; (3) two doses of p2/4 followed by T. cruzi infection; and (4) two doses of nano2/4 followed by T. cruzi infection. The size of the backbone of NTC9385R nanoplasmid is approximately half of the size of backbone of eukaryotic expression plasmid pcDNA3.1. To compensate for the differences in backbone size and deliver similar amounts of antigen-encoding DNA, the vaccine referred as p2/4 was constituted of 25 µg each of pCDNA3.1-TcG2 and pCDNA3.1-TcG4 and the vaccine referred as nano2/4 was constituted of 12 µg each of NTC9385R-TcG2 and NTC9385R-TcG4. Both vaccines were constituted in 50 µl PBS and delivered by intramuscular injection in the hind thighs in two doses at 21 days interval. Mice in groups 2, 3, and 4 were challenged with T. cruzi trypomastigotes  via intraperitoneal route at 21 days after the 2nd immunization and euthanized on day 10 and day 21 post-infection (pi). The rational for choosing 10 days and 21 days pi was to check the efficacy of the vaccine against parasite dissemination and replication, respectively, based on our previous published studies (C57BL/6 mice (6-week-old) were purchased from Jackson laboratory , and randomly distributed in four groups: (1) mock treatment/mock infection; (2) mock treatment/ studies .To examine the vaccine efficacy against re-exposure, some mice in groups 2, 3, and 4 were re-challenged on day 21 after first infection and euthanized 7 days after the second parasite exposure. Mice were daily monitored for mortality during the study period. Sera and splenocytes were obtained for evaluating the cytokines and T cell responses in vaccinated and non-vaccinated mice after infection and reinfection. Heart, skeletal muscle, and spleen tissues were collected to monitor the parasite burden and histopathology. The schematic timeline of immunization, infection and re-challenge is presented in Tc18SrDNA  or murine Gapdh , and a real-time quantitative PCR was performed for 40 cycles on an iCycler thermal cycler. The threshold cycle (TC) values for Tc18SrDNA were calculated and normalized to Gapdh reference cDNA. The relative burden of T. cruzi in each sample was calculated by the following formula: To evaluate the parasite burden at various stages of infection and reinfection, total DNA was purified from spleen, heart and skeletal muscles tissues by standard procedures. Total DNA (50 ng) was used as template and added to the reaction mix consisting SYBR Green Supermix (Bio-Rad) and oligonucleotides specific for ed by us .Heart and skeletal muscle tissue sections were fixed by incubation in 10% buffered formalin for 24 h and dehydrated in absolute alcohol. Tissue samples were then cleared in xylene and embedded in paraffin. Slides containing 5-micron paraffin-embedded tissue sections were stained with hematoxylin (stains nuclei blue) and eosin (stains extracellular matrix and cytoplasm pink). The H&E stained tissue slides were imaged at 20 × magnification by using an Olympus BX-15 microscope  equipped with digital camera and Simple PCI software . The inflammatory infiltrate and tissue damage were scored as described previously . Briefly4/50 ml) were incubated in dark with the fluorochrome-conjugated monoclonal antibodies to surface molecules (concentration determined by titration) for 30 min at 4°C, washed twice in cold staining buffer, fixed and permeabilized by incubation with fixation and permeabilization solution  for 20 min, and washed with perm wash buffer (BD Biosciences)  and incubated for 10 min with Fc Block . Splenocytes (5 × 10ciences) . For anaciences) . All ant5 of CD3+ T live cells per sample by FlowJo Downsample platform. The resultant cumulative dataset was analyzed by FlowSOM FlowJo plugin, with the grid dimensions set to 7 × 7 that created a minimum spanning tree with 49 nodes  and T central memory (TCM) sub-populations were further examined for median fluorescent intensity for intracellular IFN-g cytokine and PFN and GZB cytotoxic activity markers.T effector/effector memory .t test (comparison of 2 groups) and one-way analysis of variance (ANOVA) with Tukey’s post-hoc test or Kruskal–Wallis H/Dunn’s post-hoc test (comparison of multiple groups). Unless specified otherwise, significance is annotated in figures as *T. cruzi vs. control, ^T. cruzi vs. p2/4.Tc, #T. cruzi vs. nano2/4.Tc, and &p2/4.Tc vs. nano2/4.Tc, and one, two, and three symbol characters were used to represent the p-values of ≤0.05, ≤0.01, and ≤0.001, respectively.All datasets were acquired from a minimum of five mice per group (minimum of duplicate observations per mouse per experiment) and managed in GraphPad Prism5 software. Data are presented as mean value ± SEM. Significance was analyzed by Student’s Schematics of immunization and challenge protocol is presented in T. cruzi exhibited very high levels of parasite burden in heart and skeletal tissue and lower levels of parasite burden in spleen at day 10 and day 21 post-infection . The nano2/4 exhibited better efficacy in controlling tissue dissemination and replication of the parasite than was noted with p2/4 vaccine (&p < 0.05).We first determined the nanovaccine efficacy by qPCR evaluation of tissue parasite burden. Mice infected with ction ^,#p <0.01. ^,#p < 0.001) and skeletal muscle  (&p < 0.01) and preserving the tissue integrity in infected mice. The uninfected control mice exhibited no detectable parasite DNA and inflammatory infiltrate in their tissues  and skeletal muscle (histology score: 3.33) of infected (vs. non-infected) mice at 21 days pi ^,#p < 0.001We enumerated the T cell profile in mice at 10 days pi to examine if vaccine triggers the T cell activity immediately after infection and at 21 days pi to examine if nanovaccine enhances the potency of T cell activity against replicating parasite. For this, splenocytes of all groups of mice were analyzed by flow cytometry as detailed in Materials and Methods.−CD8− double negative (DN) T cells of naïve (P9: 0.76%–3.46%), TEM (P4: 0.25%–0.92%), or TCM (P6: 1.18%–3.12%) phenotypes constituted a small proportion of the total T cells in all the treatment groups. The non-vaccinated/infected (vs. vaccinated/infected) mice exhibited higher levels of premature CD4+To subset (P1) and a decline in the CD4+ T naïve (TN) subset (P2), while immunized mice exhibited significant increase in CD4+TEM subset . Regarding CD8+T cells, we observed two metaclusters of naive cells, including CD62LhiCD44lo (P8) subset that exhibited true naive phenotype and CD62LintermediateCD44low (P3) subset that was categorized as activated naïve subset + T cell subsets in any of the infected (vs. non-infected) groups of mice at 10 days pi. At a functional level, the IFN-γ expression was increased in CD4+TEM (P0) subset in vaccinated/infected (nano2/4.Tc > p2/4.Tc) mice  transition of the CD4+T cells into effector phenotype, b) IFN-γ production by CD4+TEM subset; and c) production of cytolytic molecules (PFN/GZB) by T cell subsets of TEM and TCM phenotypes in nano2/4.Tc (vs. p2/4.Tc or Tc only) mice.The group-specific tSNE plots of the ten metaclusters at the beginning of acute phase  are presented in + and CD8+ naïve T cells (P2 and P8) in all of the infected groups (vs. non-infected/control group) (+TEM subset (P0) was highest in p2/4.Tc mice while the +TEM subsets (P3 & P5) were highest in nano2/4.Tc mice (EM and TCM subsets in vaccinated/infected mice (nano2/4.Tc > p2/4.Tc) while only TEM subsets produced IFN-γ in non-vaccinated/infected mice (EM and TCM subsets of vaccinated/infected mice (nano2/4.Tc > p2/4.Tc), while fewer T cell subsets produced low levels of PFN in non-vaccinated/infected mice (+TEM subset only in vaccinated/infected mice (T. cruzi by delayed and sub-par T cell activation and low levels of production of effector molecules. Prophylactic immunization with nano2/4 (more effective than p2/4) enhanced the differentiation of naïve T cells toward CD4+ and CD8+ TEM and TCM phenotypes and increased the production of IFN-γ that is critical for induction of innate/adaptive immunity and of cytolytic molecules  that are required for controlling the intracellular pathogen.To capture the T cell profile in vaccinated and non-vaccinated mice during parasite replication phase, we defined and visualized the abundance of ten T cell meta-clusters in each group at 21 days pi  was decreased in non-vaccinated/infected mice, CD4+TN subset (P2) was decreased in vaccinated/infected mice (nano2/4.Tc > p2/4.Tc), and CD4+ TEM population (P0) was increased by 44.9% in p2/4.Tc and decreased by 14.5% in nano2/4.Tc mice at 21 days (vs. 10 days) pi. With respect to CD8+T cells, the CD8+TN subset was decreased and the +TEM (994%–2390%) subsets were increased in all infected groups at 21 days (vs. 10 days) pi, and maximal changes were noted in nano2/4-immunized mice. The CD8+TCM subset was increased in non-vaccinated/infected mice only at 21 days (vs. 10 days) pi (EM and TCM subsets in infected mice at 21 days (vs. 10 days) pi, and CD8+TEM and TCM subsets exhibited maximal increase in IFN-γ production in nano2/4.Tc mice (EM and TCM subsets in vaccinated/infected mice while fewer effector T cell subsets exhibited increase in PFN production in non-vaccinated/infected mice at 21 days (vs. 10 days) pi (+TEM subset exhibited increaser in GZB levels in vaccinated/infected groups at 21 days (vs. 10 days) pi , and 59% (10 out of 17) of non-vaccinated/infected mice succumbed within 7 days after re-infection  and skeletal muscle  tissue as compared to what was observed in non-vaccinated/infected mice  TcG2- and TcG4-based vaccines offer significantly better control of tissue parasite burden after re-infection than was observed after 1st infection; and c) vaccine-induced parasite control was associated with pronounced decline in tissue inflammatory pathology and tissue injuries.Extensive and large foci of tissue inflammatory infiltrate were observed in heart (histology score: 2.92) and skeletal muscle (histology score: 3.51) tissues of mice at 7 days post re-infection ^p < 0.001andst infection is presented in As above, we first defined and visualized the abundance of 10 meta-clusters of T cells in vaccinated and non-vaccinated mice at 7 days post reinfection by t-SNE plots  contracted in all infected (vs. control) groups, and CD4+To were primarily enhanced in non-vaccinated/infected mice (and+TEM subset (P0) was increased in all infected (vs. control) groups  was observed in nano2/4.Tc mice (+TEM subset (P5), though in contraction phase when compared to that observed at 21 days after 1st infection  mice but not in non-vaccinated/infected mice (EM and TCM subsets were very high after re-infection in vaccinated mice (nano2/4.Tc > p2/4.Tc) while non-vaccinated/infected mice continued to produce none-to-low levels of PFN/GZB after reinfection  in vaccinated groups  were contracting, CD4+TEM and CD8+TCM (P0 & P7) subsets exhibited increased GZB expression in vaccinated/infected mice (+ and CD8+ TEM sub-populations that produced high levels of effector molecules (nano2/4>p2/4). In comparison, non-vaccinated/infected mice responded to re-challenge with no increase in effector T cell populations and an overall decline in their production of effector/cytotoxic molecules.Upon re-challenge, mice reverted to T cell effector phenotypes as was observed at early stage of 1Tc ≥ p2/4.Tc > Tc only) and correlated with the control of parasite burden at all stages of infection and re-infection. The IL-6 levels were higher in p2/4.Tc group as compared to that noted in nano2/4.Tc group at all stages of infection and reinfection. In non-vaccinated/infected mice, IL-6 levels were high at 10 days pi and then it remained at low levels at 21 days pi and 7 days after reinfection. No detectable increase in IL-1β was observed in plasma of any of the infected groups of mice (data not shown). These results, along with the parasite burden data presented in T. cruzi infection induces systemic cytokine response, it is not maintained and fails to control parasite dissemination and replication as well as fails to respond to and provide protection from re-infection. Immunization with p2/4 and nano2/4 elicited sustained TNF-α– and IFN-g–dominated systemic immunity that correlated with control of parasite dissemination and replication as well as protection from repeat infections.Finally, we measured plasma levels of TNF-α, IFN-γ, IL-6, and IL-1β cytokines by an ELISA to determine if systemic secretion of cytokines offers an indication of anti-parasite protective immunity after infection and re-infection. We noted a potent increase in plasma levels of inflammatory cytokines in all infected groups of mice. The p2/4- and nano2/4-immunized mice elicited high plasma levels of TNF-α and IFN-γ at 10 days pi that were maintained in high range at 21 days pi and at 7 days after re-infection  and adults who might be acutely infected with T. cruzi , 29. SomT. cruzi , 31. YetT. cruzi , and theT. cruzi  and ChagT. cruzi –36. Furtse drugs , 38. It  disease , 40. His disease –44. Howe studies . In thisT. cruzi antigens and tested their potential as vaccine candidates in mice. Of the eleven candidates identified, TcG2 and TcG4 were most promising based on several selection features  at reinfection.We also assessed the efficacy of vaccine at another level by evaluating mortality as an outcome after re-infection at the peak acute phase of primary infection. We observed no death in vaccinated mice, whereas non-vaccinated mice exhibited >50% mortality associated with degeneration of organs due to high parasitemic burden after re-infection. The expansion of CD4+ and CD8+ effector T cell responses capable of providing protection against T. cruzi infection. We showed that nano2/4 enhanced the production of cytotoxic intracellular molecules by the existing T cells immediately after infection that was followed by rapid proliferation and further functional activation of CD4+ and CD8+ effector T cells, and it had the ability to keep the parasite dissemination and replication at a minimal level. Furthermore, nano2/4 vaccination shortened the time-course of development and early activation of most efficient CD8+ TEM cells for elimination of intracellular parasites. Most importantly, nano2/4-dependent quick, recall response prevented the severe increase in tissue parasite burden and injurious pathology that otherwise was observed in non-vaccinated mice after re-exposure to pathogen.In summary, we have demonstrated improved efficacy of nano2/4 in signaling polyfunctional DN, CD4T. cruzi infection is presented with development of Chagas cardiomyopathy. To address these limitations, we will aim to evaluate in future studies the vaccine efficacy against re-infection with diverse parasite isolates as well as against chronic phase of parasite persistence and Chagas cardiomyopathy and utilize advanced panel of antibodies for deeper analysis of T cell phenotypes.A limitation of this study is that vaccine protection was primarily examined against primary and re-challenge infection with the same parasite isolate, while insect vector carrying heterogenous parasites might circulate in the endemic areas, and, therefore, a host may encounter multiple parasite isolates during repeat infections. Further, this study focused on evaluating the vaccine efficacy against acute parasitemia, while clinical phase of The original contributions presented in the study are included in the article/All animal experiments were conducted following the National Institutes of Health guidelines for housing and care of laboratory animals and in accordance with protocols approved by the Institutional Animal Care and Use Committee (protocol number 08-05-029) at The University of Texas Medical Branch at Galveston. All experiments were conducted in ABSL2/BSL2-approved laboratory and all personnel have received appropriate ABSL2/BSL2 training.NG provided financial support and conceived the study. NL and IC designed and performed the experiments, analyzed the data, and confirmed the accuracy of their data presentation in the manuscript. NG and IC wrote and edited the manuscript. All authors contributed to the article and approved the submitted version.This work was supported in part by a grant from the National Institutes of Health/National Institute of Allergy and Infectious Diseases (R01AI136031) to NG.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.