Hericium erinaceus was used in traditional Chinese medicine for physiologically beneficial medicines. Recently, it has become a candidate in causing positive brain health-related activities. We previously reported that Hericium erinaceus mycelium ameliorates Alzheimer’s disease (AD)-related pathologies. To reveal the role of the cyanthin diterpenoid and sesterterpene constituents on this effects, erinacine A and S were isolated and their effects on attenuating AD-related pathology in APPswe/PS1dE9 transgenic mice were investigated. A 30 day short-term administration of erinacine A and S were performed to explore the effect of each erinacine on AD-related pathology including amyloid β production and degradation, plaque formation, plaque growth, glial activation and neurogenesis deterioration. Our results indicated the benefit effects of both erinacine A and S in cerebrum of APPswe/PS1dE9 mice, including: (1) attenuating cerebral plaque loading by inhibiting plaque growth; (2) diminishing the activation of glial cells; (3) raising the level of insulin degrading enzyme; and (4) promoting hippocampal neurogenesis. Moreover, erinacine A reduced the level of insoluble amyloid β and C-terminal fragment of amyloid precursor protein which was not mediated by erinacine S. We further performed a long term administration of erinacine A and found that erinacine A recovered the impairment in the tasks including burrowing, nesting, and Morris water maze. Our data pointed out that although both erinacine A and S reduce AD pathology via reducing amyloid deposition and promoting neurogenesis, erinacine A can also inhibit amyloid β production and is worth to be further developed for AD therapeutic use. Alzheimer’s disease (AD) is characterized by progressive cognitive decline, neurofibrillary tangles, amyloid plaques, neuro-inflammation, and decline of adult neurogenesis ,2,3. AmyIncreasing evidence has indicated that adult hippocampal neurogenesis play the major role in cognitive function . The APPHericium erinaceus is an edible and medicinal mushroom with various pharmacological activities, including anti-neurodegenerative and neuroprotective activities [H. erinaceus fruiting body ameliorated Aβ-induced cognitive declined in mice and people with mild cognitive impairment [H. erinaceus mycelium ameliorated Aβ-induced cognitive decline in mice [H. erinaceus mycelium are erinacine A (HE-A), C (HE-C), and S (HE-S) which are belong to cyanthin diterpenoid (both HE-A and HE-C) and sesterterpene (HE-S). For verifying the effect of these different constituents of H. erinaceus mycelium on AD-related pathologies, HE-A and HE-S were isolated and their effects were compared in the present study.tivities ,12. Erintivities . Recent pairment ,15. Our  in mice . The majThe APPswe/PS1dE9 mouse model (APP/PS1), co-expressed Swedish, mutated human APP695 and human mutated presenilin 1 (PS1) in which exon 9 is deleted , exhibitIn the present study, the potentials of HE-A and HE-S on amyloid pathology in 5 months old APP/PS1 mice were investigated and compared. Our data suggests that although both HE-A and HE-S were active on reducing Aβ plaque growth, diminishing neuroinflammation and increasing the level of insulin-degrading enzyme (IDE), only HE-A reduced the level of insoluble amyloid β and APP C-terminal fragment (CTF) and decreased the initiation of Aβ formation.n = 8), HE-A or HE-S  at 5 months of age for 30 days. For control, female Wild type mice (WT) were administrated by gavage with vehicle (n = 5) at 5 months of age for 30 days. Male mice were also used to verify the difference between male and female, and no significant difference was observed. BrdU (5-bromo-2′-deoxyuridine) was injected intraperitoneally at 50 mg·kg−1·day−1 during the last 7 days. The detail Aβ-related pathological changes in mice brain were examined using immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and immunoblotting (n = 8) or HE-A  to APP/PS1 mice at 5 months of age for 100 days (n = 5) at 5 months of age for 100 days.APP/PS1 transgenic mice was used as AD animal model to examine the effects of HE-A and HE-S A on amelblotting B. For be100 days C. For coBurrowing task was performed during the time between the 70th and 79th day after drug administration; nesting task was performed during the time between the 80th and 89th day after drug administration; Morris water maze was performed during the time between the 90th and 100th day after drug administration. The animal were sacrificed at 100th day after drug administration and then the Aβ-related pathological changes were inspected by immunoblotting.−1·day−1 of HE-A and HE-S for 30 days were employed to explore the effects of HE-A and HE-S on Aβ plaque deposition ; and for HE-S group is y = 1.03x + 44.84, R2 = 0.80 (p < 0.001 for difference in slopes). This result indicates that both HE-A and HE-S eliminated the peripheral region of AB10-P, but not the compact core region . We have also performed immunohistochemical analysis on long term animals, and similar results were observed.To understand the three-dimentional structure of amyloid plaque in brain of APP/PS1 mice, both ThS and AB10 antibody were used to stain the amyloid plaque. In a previous study, ThS stained the compact core region with β-structure (ThS-P) and AB10 imunostained the peripheral region without β-sheet structure (AB10-P) . The pla1–40 and Aβ1–42 and SDS-soluble Aβ1–40 in the cerebral cortex. Nevertheless, the administration of HE-S had no significant effect on the level of both SDS-soluble and SDS-insoluble Aβ. To determine the effect of HE-A and HE-S on Aβ production, the levels of CTF-β, the precursor of Aβ, were detected by Western blotting. Again, the result showed that administration of HE-A, but not HE-S, significantly reduced the levels of CTF-β , were detected by immunoblot. We found that the level of cortex IDE in the vehicle-treated trangenic mice (Veh group) is significantly increased to 136.6% of WT group. The level was further increased to 303.5% and 269.8% of WT group after treated with HE-A and HE-S, respectively C. On theNext, we evaluated the effects of HE-A and HE-S on activation of microglia and astrocytes. To determine the both plaque-dependent and independent activations of microglia and astrocytes in the cerebrum of APP/PS1 transgenic mice, Aβ plaque, microglia and astrocytes were examined by immunostaining of AB10, Iba-1, and glial fibrillary acidic protein (GFAP), respectively. We found that plaque-associated microglia in the cerebral cortex immersed their processes into AB10-P and contacted with the ThS-P. Likewise, the plaque-associated astrocytes extended their processes to contact AB10-P. By contrast, the non-plaque-associated microglia showed more ramified morphology indicating their resting state, and the non-plaque-associated astrocytes hardly express GFAP.There is about 50 plaque-associated glial clusters in a section of hemi-cerebral sphere been observed, and both HE-A and HE-S treatments decreased the number of plaque-associated microglial and astroglial clusters A,B. MoreWe hypothesized that the erinacine-mediated decline of AB-10-P burden and microglial activation may subsequently promote hippocampal neurogenesis. To certify this hypothesis, we assessed newly born granular neurons and proliferating type 2 progenitors in the subgranular zone (SGZ) by immunohistochemical staining using anti-doublecortin (DCX) and anti-BrdU antibody, respectively. There is a decline of the number of DCX-positive newly born granular neurons and BrdU-positive proliferating type 2 progenitors in SGZ of APP/PS1 mice as compared with that of WT mice, and which were recovered by treatment of HE-A and HE-S A,C.Because the dendrite growth is important for neuronal integration in neurogenesis, we further analyzed the dendritic complexity of DCX positive cells by laminar quantification method . We foun−1·day) for 100 days. The tasks of burrowing and nesting were then initiated at the 70th and 80th day after orally administration, respectively . APP/PS1 mice showed deficit in spontaneous burrowing behavior which was significantly recovered by administrating HE-A at both 10 and 30 mg·kg−1. APP/PS1 mice also showed deficit on nesting behavior evaluated by nest score and unshredded nestlet. The impaired nesting behavior was significantly recovered by administration of HE-A at 30 mg·kg−1 dose, but not at 10 mg·kg−1 dose . In MWM task, APP/PS1 mice showed a longer escape latency to find the hidden platform than WT mice during the training phase, suggesting that APP/PS1 mice showed a spatial learning impairment at 8 months-old. This obvious impairment was significantly recovered by the treatment of HE-A at 30 mg·kg−1 dose, but not at 10 mg·kg−1 dose . Two way repeated measurement analysis of variant (ANOVA) analysis confirmed an interaction between groups and days of training on Escape latency to find the plateform . It is significantly different among the days of training , among groups , and in subject . In Bonferroni posttests, it is significantly different between WT and vehicle groups at the fifth  and sixth  day of training. It is significantly different between vehicle and A-10 groups at the fifth day of training . It is significantly different between vehicle and A-30 groups at the fourth , fifth  and sixth  day of training.Burrowing and nesting behaviors, which engage a broad network of brain regions, have previously been applied on evaluating the activities of daily living (ADL) skills of AD transgenic mice. In the present study, five month old APP/PS1 mice were orally administered with HE-A  mice, APPswe/PSEN1(A246E) mice, and Tg2576 mice in parallel with Aβ accumulation ,36. TherWhile it is clear that astrocytes and microglia cluster around amyloid plaques in AD, whether they are primarily attracted to amyloid deposits or are just reacted to plaque-associated damages neurites remains elusive . MicroglIn the present study, we found that HE-A and HE-S promote neurogenesis. APP could influence neurogenesis via sAPPβ and AICD . Adminis−1·day−1 for 100 days and the behavior assays of burrowing, nesting and MWM were performed started at 7.3, 7.6, and 8.0 months old, respectively.A previous study found that APP/PS1 transgenic mice increased Aβ production and plaque formation at around 3–5 months of age ; developTo verify the effects of HE-A on behavior impairment in APP/PS1 mice, we focus on species-specific burrowing and the nest building activity because those are spontaneous multiple brain regions-dependent behaviors ,47, and BrdU, formic acid, and Thioflavin S were purchased from Sigma-Aldrich . General chemicals were purchased from Sigma-Aldrich  or Merck .H. erinaceus mycelia. Mycelia (2 kg) were refluxed with 95% ethanol. The ethanol solution was concentrated in vacuum to give a brown extract which was partitioned with H2O/ethyl acetate (EtOAc) (1:1) to afford H2O layer and EtOAc layer. The EtOAc layer was chromatographed on a silica gel column , and eluted with a gradient system of n-hexane/EtOAc  to give seven fractions (Fr. I−VII). Fraction III, the elute of n-hexane-EtOAc (3:2), was rechromatographed on Sephadex LH-20 and silica gel columns to afford HE-S (525 mg). Fraction VI, the eluate of n-hexane/EtOAc (1:2), was separated on a Sephadex LH-20 column eluting with MeOH to yield two sub-fractions (Fr. VI-1 and VI-2). Sub-fraction VI-2 was rechromatographed on Sephadex LH-20 (70% MeOH) and RP-18 (60% MeOH) columns to afford HE-A (8.4 g). The efficiency of extraction of HE-S and HE-A are 525 mg and 8.4 g from 2 kg H. erinaceus mycelia, respectively. The identification of HE-A and HE-S in mycelia using NMR and LC-MS-MS have been previously described [Erinacines were prepared from the ethanol extract of escribed ,23,52. Tn = 8), HE-A or HE-S  at 5 months of age for 30 days. For behavior effect assays, the experiment is designed to the long-term administration by oral gavage of vehicle, HE-A  to APP/PS1 mice at 5 months of age for 100 days. For control, female Wild type mice (WT) were administrated by gavage with vehicle at 5 months of age for 30 and 100 days (n = 5 for each group). BrdU was injected intraperitoneally at 50 mg·kg−1·day−1 during the last 7 days.The Institutional Animal Care and Use Committee at the National Research Institution of Chinese Medicine approved the animal protocol (IACUC No: 103-417-1 (02.01.2014), 104-417-1 (02.01.2015) and 105-417-1 (02.01.2016)). All experimental procedures involving animal and their care were carried out in accordance with Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH). APP/PS1 was purchased from Jackson Laboratory (No. 005864). Breeding gender ratio was a male with two females in one cage. Experiments were conducted using wild type siblings and AD transgenic female C57BL/6J mice. The animals were housed under controlled room temperature (24 ± 1 °C) and humidity (55–65%) with 12:12 h (07:00–19:00) light-dark cycle. All animal experimental procedures were performed based on Guide for the Care and Use of Laboratory Animals (NIH). APP/PS1 was purchased from Jackson Laboratory (No. 005864). Breeding were conducted using female transgenic mice and the male wild type siblings. The animals were housed under temperature (24 ± 1 °C) and humidity (55%–65%). Light-dark cycle was 12:12 h (07:00–19:00). All mice were provided with commercially available rodent normal chow diet and water ad libitum. For studying the therapeutic effect in short-term administration, female APP/PS1 mice were administrated by oral gavage with vehicle . Mice after anesthetized were sacrificed by transcardial saline perfusion. Mice brain was removed and half brain was homogenized in homogenization buffer containing 20 mM Tris-HCl (pH 7.4), 320 mM sucrose, 2 mM EDTA, 1 mM PMSF, 5 μg·mLThioflavin S  is used to detect the deposition of amyloid plaque as described previously . Briefly1–16 , and glial fibrillary acidic protein ; and goat polyclonal antibody to anti-ionized calcium-binding adaptor molecule-1 (Iba-1) antibody  overnight at 4 °C. Sections were then incubated in antibody dilution buffer containing Hoechst33258 , Fluorescein isothiocyanate- or rhodamine red X (RRX)-conjugated donkey anti-mouse IgG, RRX-conjugated donkey anti-rabbit IgG or Alexa Fluor 647-conjugated donkey anti-goat IgG  at room temperature for 2 h. After washed with PBS containing 0.01% Triton X-100, sections were mounted with Aqua Poly/Mount  for microscopic analysis using a Zeiss LSM 780 confocal microscopy . Representative confocal images were a 10-μm depth with maximal projection. Quantification of amyloid plaque was performed using ImageJ software (https://imagej.nih.gov/ij/). Amyloid plaque burden was calculated by the ratio of AB10-reactive or ThS-positive area to the total area.Immunohistochemistry was performed as described previously . BrieflyNeurogenesis were detected by incubating tissue sections in sodium citrate buffer  for 30 min and 2 M HCl at 37 °C for 30 min, and then in blocking buffer and antibody buffer with primary antibodies and corresponding secondary antibodies, as mentioned above. Primary antibody include mouse anti-BrdU antibody  and rabbit anti-doublecortin (DCX) antibody . BrdU- or DCX-positive cell number in the subgranular zone (SGZ) was quantified and the linear density of BrdU- or DCX-positive cells per millimeter of SGZ was calculated. The dendritic complexity of newborn neurons in DG was analyzed by laminar quantification of disjointed dendrites [g for 1 h at 4 °C. The pellet (defined as SDS-insoluble fraction) was re-suspended in 70% formic acid, and then centrifuged at 100,000× g for 1 h at 4 °C. The supernatant (defined as SDS-soluble fraction) was collected and neutralized with Tris buffer . Both fractions were stored at −80 °C until ELISA analysis. Aβ level was measured by a human Aβ1−40 and Aβ1–42 ELISA kit . The detailed experiments were performed according to the manufacturer’s protocol.The level of Aβ was determined as described previously . BrieflyImmunoblots was performed as described previously . BrieflyAfter oral gavage administration for 70 days, mice were assessed for burrowing test as described previously , with miOne day after burrowing test, nesting test was performed as described previously . In brieAfter 90-day treatment, spatial memory performance was evaluated using a Morris water maze (MWM) test as described previously ,56, withSpatial memory test was conducted to study the spatial memory performance of the mice. All mice were trained in the MWM for 6 days. The platform was always placed in the center of the southwest quadrant. Each mouse was trained to find the platform with four trials a day with an inter-trial interval of 20 min. On each trial, the mouse was lowered gently into the water facing the pool wall at one of the three fixed locations according to a semi-random schedule. In case the mouse did not succeed within 60 s, it was aided onto the platform. At the conclusion of each trial, the mice were allowed to remain on the platform for 30 s whether it had found the platform or not. The escape latency to find the platform were measured in each trial and averaged over four trials.vs. periphery zones in the probe trial version of the MWM examined. The periphery zone is defined as the area between wall and the circle apart by 10-cm from the wall [A spatial probe test was performed wherein the extent of memory was assessed . The timthe wall .t test or one-way analysis of variance (ANOVA) with post-hoc Bonferroni’s multiple comparisons tests. The nonparametric data, including crossing times of the platform in MWM test, amount of food pellets in burrowing test and nest score in nesting test, were analyzed using the Kruskal-Wallis ANOVA followed by post hoc Dunnett's multiple comparisons test.The results are expressed as the mean ± standard error of the mean (SEM) and processed for statistical analysis using GraphPad Prism 5 software . The parametric data were analyzed by unpaired two-tailed Student’s Taken together, these results suggest that HE-A and HE-S reduce cortical and hippocampal amyloid plaque growth and promoting hippocampal neurogenesis, putatively by inhibiting glial cells and increasing IDE expression in APP/PS1 mice. However, only HE-A was able to decrease Aβ production and the initiation of plaque formation. We also show that HE-A recovers behavioral deficits in APP/PS1 mice. These findings raise the possibility that HE-A may have therapeutic potential for treating AD.


In vitro differences include acid sensitivity, optimal growth temperature and tissue tropism, which reflect a preferential in vivo replication in the respiratory or gastrointestinal tract and are thus key determinants of EV virulence. To investigate the underlying cause of these differences, we generated chimeras at the capsid-level between EV-D68 (a respiratory EV) and EV-D94 (an enteric EV). Although some chimeras were nonfunctional, EV-D94 with both the capsid and 2A protease or the capsid only of EV-D68 were both viable. Using this latter construct, we performed several functional assays, which indicated that capsid proteins determine acid sensitivity and tropism in cell lines and in respiratory, intestinal and neural tissues. Additionally, capsid genes were shown to also participate in determining the optimal growth temperature, since EV-D94 temperature adaptation relied on single mutations in VP1, while constructs with EV-D68 capsid could not adapt to higher temperatures. Finally, we demonstrate that EV-D68 maintains residual binding-capacity after acid-treatment despite a loss of infectivity. In contrast, non-structural rather than capsid proteins modulate the innate immune response in tissues. These unique biophysical insights expose another layer in the phenotypic diversity of one of world’s most prevalent pathogens and could aid target selection for vaccine or antiviral development.Despite their genetic similarities, enteric and respiratory enteroviruses (EVs) have highly heterogeneous biophysical properties and cause a vast diversity of human pathologies.  in vitro tropism and optimal growth temperature. We also show that while capsid proteins are central to the biophysical viability of EV, they elicit only minor effects on the innate immune response. This work identifies key players in the pathogenesis of these highly prevalent viruses, which not only improves our understanding of their biology, but may also better guide the selection of new targets for vaccine and antiviral innovations.Enteroviruses (EV) are one of the most frequent human pathogens worldwide, causing a broad spectrum of diseases, ranging from the common cold to fatal flaccid paralysis. Surprisingly, this vast phenotypic diversity is not reflected at the genetic level, where despite intensive research, the factors contributing to these variations remain poorly understood. By generating novel chimeric viruses derived from a respiratory (EV-D68) and an enteric (EV-D94) EV, we highlight the crucial role of capsid proteins in acid sensitivity,  Enteroviruses (EVs) represent one of the leading causes of human disease worldwide and are associated with a broad spectrum of clinically distinct syndromes. While most infections are benign or asymptomatic, a small proportion may lead to life-threatening disease. Apart from the successful vaccines against poliovirus, there are currently no efficient antiviral treatments or prophylaxes against these common and potentially fatal pathogens.Picornaviridae family, the Enterovirus genus is composed of over 300 different genotypes distributed among 13 species (EV-A to EV-J and RV-A to RV-C), seven of which include human pathogens [cis-acting elements necessary for replication and translation. An internal ribosomal entry site (IRES) is required for cap-independent translation, which gives rise to a precursor polyprotein that is subsequently cleaved into 11 mature proteins by viral proteases. This precursor polyprotein encompasses 3 major regions (P1-P3). The P1 region encodes capsid proteins (1A to 1D or VP4 to VP1), while the P2 and P3 regions encode non-structural proteins (2A to 2C and 3A to 3D). The 3’UTR is marked by a poly-A tail and contains elements involved in the regulation of replication [Classified in the in vitro: for example, unlike enteric EVs, respiratory EVs exhibit particle instability at low pH, a lower optimal replication temperature, and a restricted cell tropism [Although closely related at a genetic level, viruses within the EV genus have distinct transmission routes and replication sites. They also display remarkably different phenotypic characteristics and associated clinical syndromes , 4. Base tropism .in vivo; however, the exact genomic regions providing resistance to higher body temperatures have not yet been defined [Despite intensive research, the genetic roots of this phenotypic variability are still poorly understood. Acid treatment of RVs was shown to result in a loss of infectivity due to a conformational change in the capsid proteins, leading to the expulsion of VP4 . Studies defined , 10. Sim defined , the preIn vitro examinations revealed a stability of the virion under acidic conditions and a remarkably wide tissue and cell tropism [The aim of this work is to identify the genetic regions responsible for differences in key determinants of viral viability  through the creation of unique viral chimeras. We thus exchanged the viral capsid proteins between respiratory and enteric EVs. As only intraspecies chimeric combinations are viable at the polyprotein level , we sele tropism . EV-D94  tropism , 19. ICA tropism .in vitro using cell lines and ex vivo with reconstituted human tissues), acid sensitivity, temperature adaptation and finally interplay with host innate immunity of the chimeras. Taken together, this work clarifies the diverse biophysical and immunogenic roles of capsid proteins in EV pathogenesis and may guide the search of therapeutic targets.We thus exchanged the capsid regions of EV-D68 and EV-D94 and assessed the viability, tropism  EV-D68/D94P1 which integrated the P1 region of EV-D94 into the framework of EV-D68, C) EV-D94/D68P1-2A which integrated both the P1 and 2A regions of EV-D68 into the framework of EV-D94 and finally D) EV-D94/D68VP1 with VP1 of EV-D68 integrated into the framework of EV-D94. After in vitro transcription and cell transfection, 2 blind passages were performed and virus viability was assessed by cytopathic effect (CPE) examination and confirmed by immunofluorescence (IF). EV-D94/D68P1 and EV-D94/D68P1-2A gave rise to viable replicating viruses, causing CPE in HeLa cells; however, although EV-D94/D68P1 growth was similar to EV-D94 and EV-D68, only few cells were infected with EV-D94/D68P1-2A and viral loads measured by quantitative PCR after the first passage was more than 100’000 times higher for EV-D94/D68P1 than for D94/D68P1-2A. The full genomes of EV-D94, EV-D68 and EV-D94/D68P1 obtained in HeLa cells after 3 passages were sequenced and several adaptation mutations were observed, particularly for EV-D94  observed in the P1 region of the parental EV-D94 virus , both the capsid and protease regions together (P1 and 2A) or VP1. The following 4 permutations were studied : A) EV-Dr EV-D94 . Since E94 virus  was alsoP1 was used in functional assays, and together with its parental viruses (EV-D68 and EV-D94), underwent acid sensitivity testing. A standard protocol was used to test particle stability after acid (pH 3) exposition. While EV-D94 infectivity was unchanged, acid pre-treatment of EV-D68 resulted in a total loss of infectivity. Interestingly, the chimera carrying the EV-D68 P1 region (EV-D94/D68P1) was similarly affected by acid pre-treatment. To identify the step of the viral cycle impaired by acid treatment, we performed several assays to test the binding, entry and replication steps based on time post infection  of each viral stock and, after extensive washing at 4 hpi, apical viral release was measured daily as previously described [To better replicate viral tropism in humans, the same infection assays were performed on complex tissue models escribed , 22. Resescribed .7 and 108 viral particles were used to infect primary neurons or engineered neural tissues [Between 0.5x10 tissues ; but as P1 was significantly higher than EV-D94 from day 1 to day 4 (st panel). This higher viral replication was paralleled by an inhibition of mucociliary clearance (MCC) (nd panel). Similarly, we observed a decrease in cilia beating frequency from day 3 in EV-D68 and EV-D94/D68P1 infected tissues while neither MCC nor cilia beating were affected by infection with EV-D94. These findings can be linked to the massive loss of ciliated cells observed by IF at 4 dpi caused by EV-D68 and EV-D94/D68P1 as opposed to EV-D94 (rd panel).In respiratory tissues, viral production peaked between day 1 and 3 for all viruses. However, viral replication of both EV-D68 and EV-D94/D68P1 exhibited the same characteristics of its EV-D68 P1 donor parent strain in the intestinal tissue model. The same replication profile was observed in in vitro-reconstituted neural tissues, where no increase in cell-associated virus was measured 4 dpi with EV-D68 and the chimera EV-D94/D68P1 in either neuron monolayers  RNAs for RIG-I) was systematically more efficient at 33°C than at 37°C . Titration of viral stocks prepared at 33°C revealed a systematic growth advantage over those prepared at 37°C  were observed in EV-D94 adapted to 33°C when compared to the original plasmid construct  and 1 mutation in 2A (2A A144V) were identified , we demonstrate that capsid-encoding genes determine viral tropism and acid sensitivity. We also show that EV-D68 capsid presents a major obstacle to adaptation to higher growth temperature while EV-D94 capsid does not. We finally highlight a significant and differential modulation of innate immune response by non-capsid proteins, and thus clarify mechanisms of several layers of EV pathogenesis from biophysical properties to host-pathogen interaction.EV genotypes are co-circulating in humans and are responsible for a wide variety of diseases . The phein vitro as well as in nature [in vitro studies on poliovirus [ATPase. This enzyme is believed to interact directly with the VP3 capsid protein to incorporate newly synthesised genomes and a highly specific protein-protein interaction has been shown to be an important constraint of recombinant compatibility [P1 viral stocks revealed a non-conservative mutation in VP3, mapping to the interior of the viral capsid , noliovirus , 27. Furliovirus . The factibility . Sequencl capsid  and posseric RVs , althougeric RVs  and may eric RVs , 32. FinP1 exhibited identical acid sensitivity to that of its P1 donor virus, EV-D68, proving the essential involvement of capsid proteins in this biophysical characteristic. While acid treatment completely inhibited viral entry, we showed that sialic-acid dependent binding was still effective. Our observation that binding of acid-treated EV-D68 virions could be further reduced by sialidase cell-treatment contradicts previous studies in RV-A2, concluding that acidity induces a conformational change in the capsid  that makes it incapable of cell binding [The chimeric virus EV-D94/D68 binding . However binding , 34. In P1 chimera, we were able to conclude that tropism is probably determined by virus-receptor interactions of the capsid proteins. Both EV-D68 and EV-D94 are known to bind sialic acid on cells [As the cellular tropism of EV-D68 was completely transferred to the EV-D94/D68on cells ; while son cells –20. Our in vitro-reconstituted human tissue models essential to EV research [in vitro studies have limitations, as can be seen by our observation that the in vivo EV cellular tropism of both EV-D68 and EV-D94 was not reflected in their respective immortalized cell lines. Although ex vivo models do not fully reproduce the complexity of human physiology, the first steps of infection can be still accurately studied. As expected, EV-D68 proved to be an efficient respiratory virus, reaching 1000-fold higher titres than EV-D94 in respiratory tissues. The fact that these high replication levels were transferred to the EV-D94/D68P1 chimera further underlines the essential role of the P1 capsid region in the EV-D68 phenotype. This similarity between EV-D68 and the EV-D94/D68P1 chimera was also seen in ciliated cell toxicity and impaired mucociliary clearance, which is in line with our recently published work on these tissues [P1 was comparable to EV-D94, replication levels rather than collateral damage by the host immune response likely explain the pathogenesis observed.As until now no animal model is able to accurately replicate the EV pathogenesis in humans, this makes research . Indeed, tissues . InteresP1 chimera were able to efficiently infect the neural tissue models. Of note, absolute replication levels vary considerably among tissue types. Though these values are not directly comparable due to different experimental settings linked to differences in tissue composition , EV-D94 was shown to reach the highest viral loads in intestinal tissues. This correlates with preferred replication of enteric EVs in the gastrointestinal tract. Taken together, these experiments show that, in contrast to immortalised cell lines, ex vivo model systems are reliable surrogates to investigate the early stages of EV pathogenesis and, more importantly, reveal the central involvement of capsid proteins in determining tissue tropism.In line with the expected tropism of EV-D94, this virus was able to infect both neural and gastrointestinal tissues, supporting its probable role in AFP cases in humans. While a potential causal role of EV-D68 in AFP has been suggested by epidemiological studies, recent murine models have suggested that only recently emerged strains (and not the Fermon prototypic strain used in this study), have this neuroinvasion ability  which waP1, studies investigating the innate immune response were standardised in this model. As EV-D68 was the only virus to induce both type I and III IFN, whereas EV-D94 and EV-D94/D68P1 did not lead to strong IFN induction either at the protein or the mRNA level, we could conclude that this feature is not determined by the P1 genomic region. Chimeras with exchanges of non-structural proteins could help to identify the elements involved in the innate immune response. Of note, innate immunity was not induced at all in cell lines, showing again the limitations of this model.Since respiratory tissues sustain important and comparable replication of both EV-D68 and EV-D94/D68P1 could be transfected or adapted to 37°C, strongly implicates the P1 capsid region in EV thermal stability. Concerning EV-D94, our selected strain (E210) was only passaged once in L20B cells and was thus not adapted to grow in HeLa [Similarly to the other observations, the fact that neither EV-D68 nor EV-D94/D68 in HeLa . Acquisi in HeLa , 37 and  in HeLa , 39 and  in HeLa  and the  in HeLa . In agrein vitro between EV-D68, a respiratory EV and EV-D94, an enteric EV, we proved that capsid proteins are both necessary and sufficient to determine the acid sensitivity and restricted tropism of the respiratory EV, as well as its optimal growth temperature. In contrast, we showed that innate immunity induction is rather modulated by non-structural proteins. The same experimental approach could be effective to explore the role of other EV proteins and exchanges between other pairs of EVs could help to generalize our findings to all EVs. Such approaches open the door to an improved understanding of these highly prevalent but poorly understood pathogens.To conclude, using recombinants produced (i) Infectious clones. Full genomes of EV-D94  and EV-D68  introduced in a pcDNA3.1 and pBMH plasmid, respectively, were purchased from Biomatik . Silent mutations were added in order to introduce restriction sites in between each gene. A T7 RNA polymerase promoter lacking the last G and a PstI restriction site were added before the viral 5’ end and after the polyA sequence respectively for in vitro transcription. (ii) EV-D94/D68P1chimeric construction. The EV-D94/D68P1 chimeric construction was obtained by exchanging the P1 region of pcDNA3.1-EV-D94 with that of pBMH-EV-D68 using SfoI and BamHI restriction sites. (iii) EV-D68/D94P1chimeric construction. This construct was obtained by 3 different methods: first, the P1 region of pBMH-EV-D68 was exchanged with that of pcDNA3.1-EV-D94 in the pBMH-EV-D68 vector using SfoI and BamHI restriction sites. Second, PCR amplifications were performed of the 5’UTR of EV-D68 ATCC Fermon strain after reverse transcription of viral RNA extracted from a viral stock (PCR1: primers RV156-RV144), P1 of pcDNA3.1-EV-D94 infectious clone (PCR2: RV158-RV159) and P2/P3/3’UTR of EV-D68 (PCR3: RV160-RV140). The resulting PCR products were gel-purified and PCR1+2 were fused together using nested primers (RV139-146). The fusion PCR product was then fused further with PCR3 (RV157-RV161). The forward primer of the final fusion (RV157) contains a floating tail with the T7 RNA polymerase promoter sequence, whereas the reverse (RV161) contains a floating-24T sequence. The final PCR product was used as template for in vitro transcription. Finally, an infectious clone composed of the genome of EV-D68 Fermon strain (Genbank AY426531) but the P1 region of EV-D94  in a pMA-7-Ar vector was purchased from Invitrogen GeneArt Gene Synthesis. (iv) EV-D94/D68VP1chimeric construction. 5’UTR-VP3 and P2-P3-3’UTR regions of EV-D94 were PCR amplified from the pcDNA3.1-EV-D94 infectious clone , whereas VP1 region of EV-D68 was PCR-amplified from the EV-D68 ATCC Fermon strain (PCR 2: RV171-RV167). The resulting PCR products were gel-purified. PCR1 and 2 were fused together using primers (RV150-RV153) and the fusion product was fused again with PCR3 (RV157-RV169). (v) EV-D94/D68P1-2Achimeric construction. P1-2A region from the EV-D68 ATCC Fermon strain was PCR-amplified with modified forward and reverse primers containing SfoI and AvrII restriction sites, respectively (Ent1.87 and Ent1.88). The resulting PCR product was subcloned in a pCR 2.1-TOPO vector  before subcloning in a pcDNA3.1-EV-D94 vector opened with SfoI and AvrII.All primers used in this study are listed in the D94G, VP1P165R on the 33°CP1-adapted EV-D94 mutant with primers RV192-193 and RV194-195. All PCR reactions were performed using the taq Phusion  and absence of mutation was checked by sequencing .RNA of EV-D94 adapted to 33°C and 37°C (6 passages) was extracted and reverse-transcribed. PCR amplification of either the P1 or 2A region was performed and the resulting PCR products were cloned in a pCR2.1-TOPO vector . Cloning in the pcDNA3.1-EV-D94 vector was undertaken with either SfoI/BamHI or BamHI/AvrII for P1 or 2A inserts, respectively. Site-directed mutagenesis, as described in  was usedRNA was synthesised using the RiboMAX Large Scale RNA Production System-T7 kit  either on Pst1 linearized plasmids or on PCR products according to manufacturer’s instruction. 2ug of transcribed RNA were transfected in 6-well plates, each well containing 80% confluent HeLa Ohio cells, using the TransMessenger transfection reagent kit , at either 33°C or 37°C.-1, 100μg of gentamicin ml-1, 20μg of vancomycin ml-1, and 10% fetal calf serum (FCS). Parental or chimeric viruses were propagated in these cells in McCoy’s 5A medium  supplemented with 2% or 5% FBS and incubated at 33°C or at 37°C, respectively, in a 5% CO2 atmosphere.RD , Vero , Caco-2 , and SH-SY5Y  cells were cultured as previously described . A549  and cultured at the air-liquid interface as previously described , 22. Inthttp://www.epithelix.com/products/mucilair) and EpiIntestinal tissues  were respectively ordered from Epithelix and MatTek, two biotechnology companies. There, the tissues are developed from anonymized samples and after Ethical approval. Neural tissues were also developed from anonymized samples and following ethical approval. The study was conducted according to the Declaration of Helsinki on biomedical research , and the research protocol was approved by our local ethics committee.Respiratory  was observed or at a maximum 7 dpi, supernatants and cells were collected and subjected to 3 freeze-thaw cycles before a 2h method . For tem8 RNA copies of each virus. The viral inoculum was washed out 3 times at 4 hpi. After these 3 washes and then every day, 200μl of medium was applied apically for 20 minutes at 33°C  for sample collection. Basal medium was also collected daily and replaced with 500μl of fresh medium. RNA was extracted and viral replication quantified by RT-qPCR.MucilAir and EpiIntestinal tissues were infected apically with 100μl of medium containing 107 to 108 RNA copies) diluted in 100μl or 40μl of culture medium respectively. Due to tissue fragility, the viral inoculum was not washed out. Tissues (without culture medium) were lysed directly after infection  and 4 dpi to measure cell associated virus. Total RNA was extracted and viral replication was quantified by RT-qPCR.Primary neurons and 3D neural tissues were infected with equivalent amount of each virus (0.5x108 to 2.5*105 copies/ml) of the in vitro transcribed full-length pBMH-EV-D68 were used as a quantitative reference standard for each run.RNA was extracted using the NucliSens easyMAG magnetic beads system  according to the manufacturer’s instructions. Retro-transcription was performed with Superscript II (Invitrogen) and either random hexamer primers (Roche) or oligo(dT) primers (Invitrogen), as described . RT-qPCR—1% bovine serum albumin was realized for 45 minutes at 37°C. Intensive PBS washings were performed prior to second incubation with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG antibody . Then cells were washed 3 times with PBS, stained with 4,6-diamidino-2-phenylindole at room temperature for 5 minutes and washed with PBS a final time. Coverslips were mounted in Fluoroprep mounting medium  and analyzed using standard microscopy.Infected and control cells were washed twice with phosphate-buffered saline (PBS), fixed and permeabilized for 20 minutes in a methanol-acetone mixture (1:1) at -20°C and then air dried at room temperature during a few minutes. Incubation with the primary mouse J2 monoclonal antibody , specific for double-stranded RNA longer than 40 bp, and diluted 1:500 in PBSAt 5 dpi, tissues were washed 3 times with PBS and fixed with 4% paraformaldehyde at room temperature (RT) for 15 minutes. Tissues were then washed 3 times and permeabilized with Perm/wash buffer , then (after 3 more wash steps) the first antibodies were added and incubated for 1 hour at 37°C. Viral staining was performed using the mAbJ2 diluted 1/500 whereas ciliated cells were stained with the rabbit anti-beta IV tubulin Ab . The Alexa 594-goat anti-mouse Ab  and the Alexa 488-goat anti-rabbit Ab  were used as secondary antibodies and incubated for 1 hour at 37°C. After rinsing with PBS, tissues were stained with DAPI, washed with PBS and mounted onto glass slides in Fluoroprep . Images were acquired with a Zeiss LSM 700 Meta confocal microscope with a 63.6/1.4 objective, processed by Imaris and are presented in 3D projections.Polystyrene microbeads (Sigma 84135) displacement velocity at the apical surface was measured with contrast phase microscopy and image ProsPlus software, as already described . Cilia bVirus binding assay. HeLa cells were seeded at 5x104 cells/well in 96-well plates. The following day, medium was removed, cells were washed with cold Hanks' Balanced Salt Solution (HBSS) and 200ul of binding buffer (HBSS containing 0.1% sodium azide and 1% BSA) were added on cells, which were then chilled on ice for 10 minutes. Buffer was removed and 100ul of virus mixture were added. Wells were washed 3 times with 200ul of binding buffer directly after inoculation or after 1 hour incubation on ice to assess residual or specific binding respectively, and after 3 washes with PBS, cells were lysed in 200ul of easyMAG lysis buffer to quantify bound RNA by RT-qPCR. Residual RNA was subtracted to bound RNA and percent of bound RNA was calculated relative to non-treated control for each virus.Based on a standard protocol , 20 μl oα2–3,6,8,9 Neuraminidase A  was diluted 1:100 in a EDB-0.5%BSA buffer, as described , added t-ΔΔCt method [IFN lambda  was measured in the apical medium of respiratory tissues by enzyme-linked immunosorbent assay ELISA according to manufacturer’s instructions . mRNA of IFN-β and IFN-λ in tissue lysate were quantified using TaqMan Gene Expression Assay  for IFN-β and with primers and probes described previously for IFN-λ . The IFNt method .Values are expressed as mean (± SEM). Experiments were done at least in biological duplicates and in each experiment, conditions were run in duplicate except for intestinal and 3D neural tissues where biological replicates were done without intra-experimental replicate. Ordinary one-way or two-way ANOVA tests (without matching or pairing) and multiple comparisons were performed with GraphPad Prism 7.02 software. For S1 Fig50 in HeLa cells (dilutions starting from 106 TCID50 were performed) and an endpoint dilution assay was performed in various cell lines. Endpoint dilution (expressed in log) was assessed at 5 dpi.Viral stocks were normalized based on TCID(TIF)Click here for additional data file.S2 Fig-ΔΔCt method. In A549/pr(IFN-β) GFP reporter cells [RD, Hela and A549 cells were infected with the indicated virus. For HeLa and RD cells, RNA was extracted from cell lysate 24 hpi and IFNβ and λ mRNAs were quantified by quantitative real-time PCR. The IFN Ct values were normalized to those of the RNAse P housekeeping gene  and relative quantification was calculated using the 2er cells , IFN ind(TIF)Click here for additional data file.S3 Fig50 method at 5 dpi. All viruses preferentially replicate at 33°C compared to 37°C. Experiments were run as biological duplicates.Viral stocks obtained after 3 passages in HeLa cells grown at 33°C were titrated at either 33°C or 37°C. Viral titers (expressed in log) were determined using the TCID(TIF)Click here for additional data file.S4 Fig(A) and EV-D94 adapted to 37°C (B). Titration of the two stocks in HeLa, RD, Vero and A549 cells was performed at 33°C and 37°C to define their optimal growth temperature in each cell line.EV-D94 was transfected and passaged 6 times at 33°C or 37°C in HeLa to give rise to EV-D94 adapted to 33°C (TIF)Click here for additional data file.S5 FigG94D,P165R(A) and EV-D94 VP1H284Y,A241T,G94D, P165R(B) in HeLa and RD cells.Replication of EV-D94 VP1(TIF)Click here for additional data file.S6 FigP1 (A) or EV-D94 after amplification in cell culture at different temperatures (B). A. Residue VP3H35 (highlighted in yellow) is imaged in the context of VP3 (in red) and VP4 (in grey). The interior and exterior of the capsid are indicated. B. Mapping of residues mutated in EV-D94 based on homology with EV-D68. Amino acid G94D, P165R, A241T and H284Y in EV-D94 VP1 protein correspond to amino acid S82, L153, E227 and R270 in EV-D68. For A and B, mapping were performed on the available 3D structure of the EV-D68 capsid (PBD accession 4WM8 and 5BNP) thanks to Accelrys Discovery Studio Visualizer 3.5 .Mapping of the capsid residues mutated in EV-D94/D68(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


ZnT-1) and apoptosis . Our results strongly suggest that the zinc-Schiff base-Novicidin complex has great potential as a novel anticancer drug.Prostate cancer cells control energy metabolism by chelating intracellular zinc. Thus, zinc delivery has been a popular therapeutic approach for prostate cancer. Here, we propose the use of the membrane-penetrating peptide Novicidin connected to zinc-Schiff base as a carrier vehicle for the delivery of zinc to prostate cells. Mass spectrometry, electrochemistry and spectrophotometry confirmed the formation/stability of this complex and provided insight regarding the availability of zinc for complex interactions. This delivery system showed minor toxicity in normal PNT1A cells and high potency towards PC3 tumor cells. The complex preferentially penetrated PC3 tumor cells in contrast to confinement to the membranes of PNT1A. Furthermore, zinc uptake was confirmed in both cell lines. Molecular analysis was used to confirm the activation of zinc stress (e.g.,  Prostate cancer (PCa) is the most common malignant tumor in males in the developed world, and its prevalence is steadily increasing . PCa thaGlandular epithelial cells of the human prostate gland can accumulate a high level of zinc, two- to five-fold greater than the zinc level found in other tissues . Zinc isThe challenge associated with zinc deficiency in malignant cells can be addressed through the use of various delivery systems. In the search for new delivery systems, cell-penetrating peptides (CPP) have attracted attention due to their ability to deliver intracellularly a wide range of molecules . CPP areTo carry the metal ion cargo, Schiff bases are organic compounds with versatile biological properties. The type of metal and the complexity of the Schiff base play a major role in the potency of these compounds as chemotherapeutic agents. Schiff bases bound to metal ions interact directly with the cell membrane and cause oxidative stress damage. The binding of the Schiff base-metal ion complex with other compounds can neutralize this oxidative capability and permit delivery within biological systems ,16. AccoZnT-1) and apoptosis  genes. For clarity, protein names were shown in normal fonts and gene names were shown in italics.Consequently, we were interested in synthesizing a zinc delivery system to increase zinc availability in PCa cells. Here, we investigated the molecular changes that occur after delivery by studying the genes involved in zinc stress. This delivery system showed minor toxicity in normal PNT1A cells and a high potency towards PC3 tumor cells, as shown by a preference to penetrate PC3 cells in contrast to localized confinement to membranes of PNT1A. Furthermore, zinc uptake was confirmed in both cell lines. Molecular analyses were used to confirm the activation of zinc stress  corresponds to the ionized Zn-S complex, whereas the peak m/z = 439 Da is assigned to the perchlorate adduct of the molecular ion indicating the formation of a hydrogen bond between perchlorate ions and nitrogen donors that have been previously reported for complex ions+, where the presence of sodium ion can be due to its presence in the matrix, whereas the peaks at m/z = 2585 Da and 2484 Da are likely to be perchlorate adducts of [Zn-NVC+Na]+. Although we cannot confirm the type of bonding that occurred between the metal and the peptide by MALDI-TOF, complexation between NVC and Zn-S occurred.As described below, we exploited the interaction of the Schiff base–zinc complex (Zn-S) with the NVC peptide to produce a stable complex for zinc delivery to cells. To confirm this interaction, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied. The observed signals for Zn-S are shown in plex ions,20. Fig -1, i.e., near the most intense vibration of free peptide  . This slight increase in gallic acid equivalents supports the notion that oxidative stress is not the main cause of cell death, which indicates that the Schiff base is not the main toxic component of the delivery system.SP1 plays a dual role in regulating stress to heavy metals and inducing apoptosis, possibly as a competitor antagonist toward other transcription factors involved in ion stress and apoptosis. Previous studies have reported that overexpression of the transcription factor Sp1 leads to cell cycle inhibition at the G1 phase before the onset of apoptosis -N-[2-[(E)-2-pyridylmethylene-amino]ethyl]ethanamine)] was prepared by mixing 2-pyridinecarboxaldehyde (1902 μL) and diethylenetriamine (1080 μL) with stirring and heating under reflux in methanol (35 mL) for 6 h. The color turned to orange; after cooling, methanol was added to 50 mL.Zinc perchlorate hexahydrate (0.372 g) was dissolved in 50 mL of water, and Schiff base S (5 mL) was added with stirring to obtain Zn-S Schiff base complex. The light orange solution was heated at 80°C for 2 h, the solution was then filtered, and water was added to 100 mL.To synthesize NVC peptide, Liberty Blue peptide synthesizer was used . The sequences and monoisotopic molecular weight of the synthesized peptide were as follows, respectively: KNLRRIIRKGIHIIKKYF and—2296 Da.A stock solution of NVC peptide (1 mM) was mixed with Zn-S (1 mM) at a 1:1 ratio. The final concentration of the complex in PBS buffer was 1 mM. The sample was incubated for 60 min at 25°C. After incubation, unbound Zn-S was removed using the fluorescein fast protein liquid chromatography (FPLC) system, Biologic DuoFlow .Mass spectrometry experiments were performed using a MALDI-TOF MS Bruker Ultraflextreme  equipped with a laser operating at a wavelength of 355 nm with an accelerating voltage of 25 kV, cooled with nitrogen, with a maximum energy of 43.2 μJ and a repetition rate of 2000 Hz in linear and positive mode. Data were acquired and processed using mass spectra flexControl version 3.4 and flexAnalysis version 2.2 software. The 2,5-dihydroxybenzoic acid matrix (DHB) was used for MALDI-TOF .–1) were recorded on a Bruker Tensor 27 spectrometer  equipped with a platinum-ATR accessory with a diamond crystal.The interactions between Zn-S and NVC were determined using the multifunctional microplate reader, Tecan Infinite 200 PRO . The absorbance spectra were measured within the range from 230–850 nm. For the fluorescence spectra, 230 nm was used as the excitation wavelength, and the fluorescence scan was measured within the range from 260–650 nm. The complex stability was determined by spectra recorded within the range from 190–850 nm using quartz cuvettes  on a SPECORD 210 spectrophotometer  at 20°C. Spectra were recorded after 0, 1, 3 and 7 days of incubation. The FTIR spectra .Electrochemical measurements were performed with the AUTOLAB Analyzer  connected to VA-Stand 663 , using a standard cell with three electrodes. Stability experiments for the Zn-S-NVC complex were performed using cyclic voltammetry in acetate buffer . To prepare a viable cell standard, a 100-μL cell suspension was mixed with 10 mL of Casy Tone. All subsequent measurements were performed with a 100-μL cell suspension diluted 100×. The background was subtracted prior to each measurement.Two human prostatic cell lines were used in this study: the PNT1A human cell line and the PC3 human cell line were purchased from Health Protection Agency Culture Collections . PNT1A cells were cultured in RPMI-1640 with 10% FBS. PC3 cells were cultured in Ham's F12 medium with 7% FBS. All media were supplemented with penicillin (100 U/mL) and streptomycin (0.1 mg/mL), and the cells were maintained at 37°C in a humidified incubator  with 5% COdimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT [5 mg·mL-1 in PBS]) and incubated for 4 h at 37°C. The MTT-containing medium was replaced with 200 μL of 99.9% dimethyl sulfoxide to dissolve MTT-formazan crystals. Then, 25 μL of glycine buffer was added to all wells, and the absorbance was determined at 570 nm . Six replicates were used to assess the Zn-S-NVC complex, and four replicates were used for Zn-S.The suspension of approximately 5000 cells was added to each well of microtiter plates (E-plates 16). Cultures were incubated for 2 days at 37°C to ensure cell growth. The medium was replaced with medium containing Zn-S-NVC complex (2–500 nM), and medium without agents served as a control. The plates were incubated for 24 h, and the medium was replaced with fresh medium three times daily. Additionally, the medium was replaced with 200 μL of fresh medium containing 50 μL of 3-/A100%-Ac)]×100, where At is the absorbance of the supernatant from samples incubated with Zn-S-NVC; Ac is the absorbance of the supernatant from the negative control ; A100% is the absorbance of the supernatant from the positive control (0.1% Triton X-100), which causes complete RBC lysis.Human blood was obtained from the Department of Physiology, Faculty of Medicine, Masaryk University, Czech Republic. We analysed the samples of healthy volunteers (n = 2), whereas the written consent of blood donors was granted. The research has been approved by the Independent ethics committee at University Hospital, Brno, Czech Republic. Red blood cells (RBCs) were obtained from whole blood according to Evans et al. . RBC susN-hydroxysuccinimide ester] and purified by FPLC prior to microscopy. An inverted microscope system, Olympus UIS2 series , was used to image complex internalization into the cells. The CPlanFLN 10× objective was used for 100× magnification, and the LUCPlanFLN 40× objective was used for 400× magnification. The images were captured using a Camera Olympus DP73 and processed with Stream Basic 1.7 Software.Zinc-Schiff base-Novicidin complex was conjugated to fluorescent dye [5(6)-carboxyfluorescein The total content of zinc in the samples was determined using a 280Z Agilent Technologies atomic absorption spectrometer  with electro-thermal atomization. An arsenic ultrasensitive hollow cathode lamp (Agilent) was used as the radiation source (lamp current 10 mA). The spectrometer was operated at a resonance of 193.7 nm with a spectral bandwidth of 0.5 nm.The medium was discarded, and the cells were washed with 2 mL of PBS. Approximately 2 mL of either PBS or PBS/Zn-S-NVC was added for both types of cell lines to a final concentration of 150 μM Zn-S-NVC complex. The four cultures were incubated for 40 min with occasional shaking. RNA was isolated using the RNeasy Mini Kit .18S ribosomal RNA (GenBank Accession X03205.1). The relative levels of transcription were calculated using the 2−ΔΔCT method.Gene expression was studied by quantitative RT-PCR using the SYBR Green Quantitative RT-PCR Kit  and the Mastercycler pro S instrument . The primer sets used to amplify the coding regions of different genes are shown in Table A in v/v) methanol) were used. The transfer was performed for 1 h at a voltage of 0.9 mA per 1 cm2 of the membrane. Anti-β-Actin (sc-130657) and anti-p53 (sc-126) primary antibodies were purchased from Santa Cruz Biotechnology  and diluted 1:200. Secondary antibodies were diluted 1:1000. The solution used for calorimetric detection for 1 membrane consisted of 5 mL of 0.01 M acetate buffer pH 5.4, 5 μL of hydrogen peroxide and 50 μL of 3-Amino-9-ethyl-carbazole in DMF.Proteins were lysed directly in culture plates with 2 mL of lysis buffer ) for 30 min. Proteins were electrophoresed in 12.5% polyacrylamide gel at 200 V for 33 min using a Mini Protean Tetra apparatus . For Western blotting, PVDF membranes and blotting buffer  and 100 ng of random hexamers. The obtained cDNA was biotinylated at its 3’ end using the Biotin 3’ End DNA labeling kit . Microarray was conducted according to Roth et al. . For hybS1 FileFig A. ElectraSense Array Image. Graphic showing the number of genes regulated in the PC3 and PNT1A cell lines in response to different treatments. PNT1A (PNT1A cell line before treatment with Zn-S-NVC complex), PNT1A-Zn-S-NVC (PNT1A cell line after treatment with Zn-S-NVC complex), PC3 (PC3 cell line before treatment with Zn-S-NVC complex) and PC3-Zn-S-NVC (PC3 cell line after treatment with Zn-S-5-NVC complex). Fig B. Stability testing of Zinc-Schiff base-Novicidin complex over one week under different pH conditions. A) pH = 3.8. B) pH = 6. C) pH = 7.2. D) pH = 9. Table A. Primers used for quantitative RT-PCR. Table B. Lists of up- and/or down-regulated genes in all possible combinations between treatments: A) PC3 vs. PNT1A. B) PNT1A-Zn-S-NVC vs. PNT1A. C) PC3-Zn-S-NVC vs. PC3. D) PC3-Zn-S-NVC vs. PNT1A-Zn-S-NVC. Table C. Lists of up- and/or down-regulated genes in biological processes in PC3 and PNT1A cell lines after treatment with Zn-S-NVC complex by gene ontology (GO) annotations. A) PC3 vs. PNT1A. B) PNT1A-Zn-S-NVC vs. PNT1A. C) PC3-Zn-S-NVC vs. PC3. D) PC3-Zn-S-NVC vs. PNT1A-Zn-S-NVC. Table D. Lists of up- and/or down-regulated genes in various pathways in PC3 and PNT1A cell lines after treatment with Zn-S-NVC complex by KEGG 10 software. A) PC3 vs. PNT1A. B) PNT1A-Zn-S-NVC vs. PNT1A. C) PC3-Zn-S-NVC vs. PC3. D) PC3-Zn-S-NVC vs. PNT1A-Zn-S-NVC.(DOCX)Click here for additional data file.


Bordetella pertussis, the causative agent of whooping cough. CyaA intoxicates target cells by a direct translocation of its catalytic domain (AC) across the plasma membrane and produces supraphysiological levels of cAMP, leading to cell death. The molecular process of AC translocation remains largely unknown, however. We have previously shown that deletion of residues 375–485 of CyaA selectively abrogates AC translocation into eukaryotic cells. We further identified within this “translocation region” (TR), P454 (residues 454–484), a peptide that exhibits membrane-active properties, i.e., is able to bind and permeabilize lipid vesicles. Here, we analyze various sequences from CyaA predicted to be amphipatic and show that although several of these peptides can bind membranes and adopt a helical conformation, only the P454 peptide is able to permeabilize membranes. We further characterize the contributions of the two arginine residues of P454 to membrane partitioning and permeabilization by analyzing the peptide variants in which these residues are substituted by different amino acids . Our data shows that both arginine residues significantly contribute, although diversely, to the membrane-active properties of P454, i.e., interactions with both neutral and anionic lipids, helix formation in membranes, and disruption of lipid bilayer integrity. These results are discussed in the context of the translocation process of the full-length CyaA toxin.The adenylate cyclase toxin CyaA is involved in the early stages of infection by  Bordetella pertussis, the causative agent of whooping cough  , 1,2-dioleoyl-sn-glycero-3-phosphocholine , 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] , 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine , and cholesterol  were from Avanti Polar Lipids . All experiments were performed in 20 mM HEPES, 150 mM NaCl, pH 7.4 (buffer A). ANTS , and DPX  were purchased from Molecular Probes .1-palmitoyl-2-oleoyl--W), and a tryptophan was added at its C-terminal extremity (underlined).The P19 peptide corresponds to the B-helix in AC, i.e., residues 19 to 35 of CyaA (IPAAVLDGIKAVAKEKN-W), with a tryptophan added at its C-terminal extremity (underlined).The P89 peptide corresponds to the D-helix in AC, i.e., residues 89 to 107 , and contains the native tryptophan W242.-W SLGEVSD- MAAVEAAELEMTRQVLHA), in which Phe-415 was substituted by a tryptophan residue (underlined).The P414 peptide corresponds to residues 414 to 440 of CyaA (S-The P454 peptide corresponds to residues 454 to 484 of CyaA (ASAHWGQRALQGAQAVAAAQRLVHAIALMTQ) and contains the single native tryptophan W458. Several P454-derived peptides were also synthetized, in which the two arginine residues R461 (hereafter referred to as R1) or R474 (referred to as R2), or both of them (referred to R12) were substituted by either lysines (R12K), glutamines , alanines (R12A), or glutamates (R12E).-W), and a tryptophan was added at its C-terminal extremity (underlined).The P495 peptide corresponds to residues 495 to 524 of CyaA (QEAASLSAAVFGLGEASSAVAETVSGFFRG-W), and a tryptophan was added at its C-terminal extremity (underlined).The P509 peptide is a shorter peptide from P495, corresponding to residues 509 to 524 of CyaA (EASSAVAETVSGFFRG-W), and a tryptophan was added at its C-terminal extremity (underlined).The P626 peptide corresponds to residues 626 to 647 of CyaA (LVQQSHYADQLDKLAQESSAYG-W), and a tryptophan was added at its C-terminal extremity (underlined).The P717 peptide corresponds to residues 717 to 733 of CyaA (IIEKLANDYARKIDELG-The P737 peptide corresponds to residues 737 to 771 of CyaA (AYFEKNLQARHEQLANSDGLRKMLADLQAGWNASS) and contains the single native tryptophan W767.-W) and a tryptophan was added at its C-terminal extremity (underlined).Finally, the P777 peptide corresponds to residues 777 to 793 of CyaA  and their purity, as their molecular weight, were controlled by HPLC and MALDI. All peptides were capped on the N-terminus with an acetyl group and on the C-terminus with an amide group. We studied 11 CyaA-derived peptides: P19, P89, P233, P414, P454, P495, P509, P626, P717, P737, and P777 peptides, as well as 6 P454-derived peptides . The peptides were selected according to their propensity to form helical structures, their local hydrophobicity, and their hydrophobic moment .-The P1The physical chemistry of these peptides is described in Small unilamellar vesicles (SUVs) were prepared at a lipid concentration of 10 mM from POPC and POPG at a molar ratio of 10:0 or 8:2 or from DOPC, DOPE, DOPG, Chol at molar ratio 10:0:0:0, 8:0:2:0, 7:3:0:0, 7:0:0:3, 6:3:0:1, and 4:3:2:1 in buffer A, as previously described . Brieflymax) and fluorescence intensity ratio at 330 nm over 370 nm (P454-derived peptides) or at 340 nm over 380 nm (CyaA-derived peptides) were used to report peptide-membrane interaction and to measure the partition coefficient KX.Fluorescence titrations were performed with a FP-8200 Jasco spectrofluorimeter, equipped with a Peltier-thermostated ETC-272T (25 °C). A bandwidth of 5 nm was used for both excitation and emission beams. Fluorescence experiments were carried out in a 109.004F-QS cuvette  with constant stirring. Fluorescence emission was recorded between 300 and 400 nm at a scan rate of 200 nm/min, with an excitation wavelength of 280 nm. Fluorescence emission spectra were corrected for SUV light scattering. Maximum emission wavelength , and T the temperature in Kelvin.The partition coefficient is related to the apparent dissociation constant as follows: Synchrotron radiation circular dichroism (SRCD) experiments were carried out on the DISCO beamline of Synchrotron SOLEIL . SRCD spectra were recorded at 25 °C with an integration time of 1.2 s and a bandwidth of 1 nm with a resolution of 1 nm. Each far-UV spectrum represents the average of three individual scans. QS cells  with a path length of 100 μm were used to record SRCD signal in the far-UV (from 190 to 250 nm). Peptides were tested at 100 μM in buffer A and in the presence of 20% of trifluoroethanol (TFE). Peptides were incubated in the presence of SUVs  at final concentration of 2 mM lipids. Buffer A, in the absence or presence of SUVs, was used as blank and subtracted from far-UV CD spectra of peptides. Secondary structure content was estimated using Bestsel . obs. The percentage of fluorescence intensity was calculated using the maximum intensity measured after complete disruption of the vesicles with addition of 0.12% (2 mM) of Triton. The concentration of membrane-bound peptide required to induce half-maximal ANTS fluorescence recovery (CP1/2) is extracted from the plot of permeabilization intensity as a function of peptide concentration (ANTS (fluorophore probe) and DPX (quencher) were encapsulated into large unilamellar vesicles (LUVs) to monitor membrane permeabilization induced by the peptides. The LUVs were prepared at a concentration of 10 mM lipid at the following molar ratio DOPC/DOPE/DOPG/Chol 4:3:2:1, POPC/POPG 8:2, or POPC containing 20 mM ANTS and 60 mM DPX. The multilamellar vesicle suspension was passed through 0.4- and 0.2-μm polycarbonate filters to produce LUVs of 200 ± 30 nm in diameter, as measured by DLS. The unencapsulated probes were removed by gel filtration through a Sephadex G-25 column 5 mL . For permeabilization assays, LUVs were incubated in buffer at 0.45 mM lipids at 25 °C in a 101-QS cuvette  and under constant stirring. The excitation wavelength was set to 390 nm and the emission of ANTS was continuously measured at 515 nm. A single exponential equation was fitted to kinetic traces to extract fluorescence intensity and observed rate constants, kntration A. −4 bar−1. Van der Waals interaction was cut off at 12 Å with a shift function applied beyond 9 Å. Electrostatics were cut-off at 14 Å with long range electrostatics treated by the particle mesh Ewald method [3. Each system was equilibrated over 20 ns in conditions identical to the lipid-water mixture, then simulated over 1 μs. Visualization of the trajectories was done with VMD 1.9.1 [The coarse-grained simulations were carried out with Gromacs 4.5.5  using thd method ,77 usingMD 1.9.1 . Their aMD 1.9.1 , R 2.14.MD 1.9.1 , and theMD 1.9.1 .


Despite abundant expression of miR-449a in developing thymus, miR-449a-mutant mice exhibited normal thymic development. This might be partially due to in miR-449a-mutant thymus the up-regulation of miR-34a which shared similar seed sequence with miR-449a. However, thymic expression of miR-449/34 sponge which was able to neutralize the function of miR-449/34 family members significantly reduced the number of mature Ly51-MHCIIhi mTECs. Taken together, our data suggested that miR-449a modulated mTEC differentiation, and members of miR-34 cluster functioned redundantly to rescue miR-449a deficiency in thymus development.Medullary thymic epithelial cells (mTECs) ectopically express a diversity of peripheral tissue-restricted antigens (PTAs) and provide unique cues for the expansion, maturation and selection of a repertoire of functionally diverse T lymphocytes. Genetic deletion of all mature microRNAs in thymic epithelial cells (TECs) results in premature thymic involution, progressive disorganisation of the thymic epithelium, and alteration in thymic T cell lineage commitment, consequently eliciting autoimmune disorders. In the present study, we identified that microRNA-449a (miR-449a), a member of miR-449 cluster, regulated mTEC differentiation. Expression of miR-449a was induced by RANK ligand in mouse fetal thymus. In  TECs consist of cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells (mTECs) which form discrete intrathymic microenvironments, thymic cortex and thymic medulla respectively. Each is specialized for mediating a particular aspect of thymocytes development3. The bone marrow-derived progenitors go through a consecutive process including release from bone marrow niches into the blood5 and exit from the circulation to settle in the thymus at the cortico-medullary junction (CMJ)7. On entering the thymus, these early thymic progenitors (ETP) undergo an ordered process of development from CD4/CD8 double negative stage (CD4−/CD8−) to double positive stage (CD4+/CD8+) during their migration from CMJ through the cortex to the outer subcapsular zone (SCZ) and then back to the cortex10. During this process, progenitors become committed to the αβ or γδT cell lineage12 at DN3 stage and undergo β-selection13; the resulting immature single positive (ISP) intermediate cells then differentiate into double positive cells and undergo positive selection by recognization of self-peptide/MHC complexes expressed on cTECs15. Positively selected thymocytes then enter the medulla, where T cells express T cell receptors with high affinity for self-antigens are clonally depleted by apoptosis18.The thymus containing thymic epithelial cells (TECs) that form a complex three-dimensional meshwork structure provides the microenvironment to drive the differentiation of bone marrow-derived hematopoietic precursors to mature T lymphocytes21. Recent studies have elucidated a battery of regulators underlying the differentiation and function of mTEC, among which the identification of autoimmune regulator (Aire) was a breakthrough in the study of mTEC biology19. Members of the tumor necrosis factor receptor (TNFR) family and their downstream canonical/alternative NF-κB pathways are involved in the differentiation and function of mTECs. Mice deficient with RANK23, CD4023, lymphotoxin β receptor25, NF-κB inducing kinase (NIK)26, IκB kinase (IKK) α27, RelB28, and Traf629 exhibited variable defects in mTEC.The crucial role of mTEC in establishing T cell central tolerance is attributed to the expression and presentation of a diversity of peripheral tissue-restricted antigens32. However, the molecular mechanisms of miRNAs underlying thymus development remain less understood. Adrian Liston’s group reported the function of thymic epithelial miRNA network in infection-associated thymic involution by Foxn1 mediated conditional knockout of Dicer in mouse model33. The deletion of Dicer and therefore all mature miRNAs in thymic epithelial cells result in premature thymic involution, progressive disorganisation of the thymic epithelium, alteration in thymic T cell lineage commitment, and consequently elicit autoimmune disorders34. In accordance with these discoveries, Foxn1 mediated conditional knockout of DGCR8, a gene whose product is responsible for canonical cleavage of miRNAs, results in severe loss of Aire+ mTECs and breaks down the negative selection in thymus35. A transcriptome analysis of murine thymic epithelial cells reveals miRNA expression profiling that is closely correlated with age-related thymic atrophy, indicating the function of miRNAs in control of thymic aging process36. Thus, miRNAs are reasonably playing crucial roles in thymic epithelial cell differentiation and function.MicroRNAs (miRNAs) are a class of small (19~22nt), noncoding RNAs that mediate sequence-dependent post-transcriptional gene repression by translational inhibition and/or mRNA destabilization. Approximately over 10,000 miRNAs have been identified to exert their effects in normal organism development and pathogenesisin vitro. Neutralization of miR-449a and other miR-449/34 family members reduced the number of mature MHCIIhi mTECs in thymus. Our results revealed a new function of miR-449a in regulation of mTEC differentiation.We identified miR-449a in 2-DG FTOC  treated Fetal Thymus Organ Culture) treated with recombinant human RANK ligand. Expression of miR-449a was induced by RANK ligand in fetal thymus and overexpression of miR-449a could induce TEPC differentiation Ins/Ins and miR-449aDel/Del) were generated by Shanghai Bioray Biotech Co., Ltd. All animals were housed and maintained in specific-pathogen-free conditions. All animal experiments were performed in compliance with the guide for the care and use of laboratory animals and were approved by the institutional biomedical research ethics committee of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.The miR-449a mutant mice  for 2 or 4 days.TSC  cells were established in our lab previouslyTo stably overexpress miR-449a in TSC cells, TSC cells were infected with empty control lentivirus or lentivirus expressing miR-449a and selected with 1ug/ml puromycin for 4 days. The stable cell lines were thereafter named as TSC Control or TSC miR-449a. Overexpression of miR-449a (>300 fold) was then confirmed by q-PCR.23. Fetal thymic lobes (2-DG FTOC) were cultured in RPMI1640-10%FBS medium and infected with lentivirus carrying miR-449a or miR-449/34 sponge expressing vectors. For in vitro differentiation, fetal thymic lobes (2-DG FTOC) were cultured with 100ng/ml recombinant human RANKL  for 4 days.Thymic lobes were isolated from E14.5 embryos and cultured for 4 days on nucleopore filters  placed in RPMI1640 (Invitrogen), supplemented with 10% fetal bovine serum (Invitrogen), 2 mM L-glutamine (Invitrogen), 50 µM 2-mercaptoethanol (Sigma-Aldrich) and 1.35 mM 2′-deoxyguanosine , as previously describedMiR-449a was amplified from mouse genome using forward primer: 5'-TGA ATT CAC TTA GCC TCA GCC ACT C-3' and reverse primer: 5'-TGT CTA GAT AAT GTC AAG CTA GGA C-3' and cloned into plvx-IRES-EGFP (Clontech).To clone miR-449/34 sponge, forward sequence: 5'-gatccACCAGCTAACTATCACTGCC ACGATACCAGCTAACTATCACTGCCAACGCGACCAGCTAACTATCACTGCCACGATACCAGCTAACTATCACTGCCAACG CGACCAGCTAACTATCACTGCCACGATACCAGCTAACTATCACTGCCAttttttg-3' and reverse sequence: 5'-aattcAAAAAATGGCAGTGATAGTTAGCTGGTATCGTGGCAGTGATAGTTAGCTGGTCGCGTTGGCA GTGATAGTTAGCTGGTATCGTGGCAGTGATAGTTAGCTGGTCGCGTTGGCAGTGATAGTTAGCTGGTATCGTGGCAGTGA TAGTTAGCTGGT g-3' were synthesized, annealed and cloned into plvx-shRNA2 (Clontech).The cells were harvested and washed with cold phosphate buffer solution (PBS) once. Add 100 µl lysis/loading buffer , boiling for 10 minutes.For each sample, 10-20 μl of protein lysate was separated by SDS-PAGE, transferred electrophoretically to a PVDF membrane  and immunoblotted with primary and peroxidase-conjugated secondary antibodies in 5% non-fatty milk. Detection of the bound antibody was performed by SuperSignal west pico Chemiluminescent Substrate (Pierce). Antibodies to Aire (N-20), GAPDH (G-9), SATB2 (SATBA4B10), RelB (A-9) and NF-κB p52 (K-27) were purchased from Santa Cruz Biotech Inc. Monoclonal antibody to DNMT3a was generated in Dr. Guoliang Xu’s lab .Virus expressing Control-GFP or miR-449/34 sponge-GFP were packaged according to Lenti-X™ shRNA Expression Systems User Manual (Clontech). Control-GFP virus and miR-449/34 sponge-GFP virus were concentrated by ultracentrifugation and stored at −80 °C. The GFP protein acted as a reporter gene of miR-449/34 sponge expression.3-week old C57BL/6 mice were narcotized, injected with about 30 μl of virus per lobe of thymus at the interstice between the second and third rib. Injection was conducted once every other day for 3 times. 2 days after the last injection, the thymus were harvested for analysis.−EpCAM+Ly51−MHCII+.The thymi were harvested, washed with cold PBS twice, minced and digested with Liberase TH (Roche) and DNase I (Roche). Thymic cells were stained with antibodies: EpCAM , Ly51 , MHCII , CD45  for 30 min on ice. mTECs were gated as CD45For Aire staining, cells were stained with Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and stained with Anti-Aire antibody  for 30 min on ice.Frozen thymuses embedded in OCT compound were sliced into 8 µm-thick sections. Frozen sections were fixed with cold acetone for less than 5 minutes and stained with the following antibodies: rabbit polyclonal antibodies to Alexa-488 K5 (Covance), Alexa-647 K8 , FITC-EpCAM , K14 (Covance) and Aire  followed by Alexa Fluor 568-conjugated anti-rabbit IgG antibody (Molecular Probes). Images were analyzed with TSC SP2 confocal laser-scanning microscope.RNA was isolated from cell lines or thymus samples using TRIzol Reagent (Invitrogen) and reverse transcripted using Transcript First Strand Synthesis Supermix (TransGen Biotech) according to the manufacturer’s instructions. Reverse transcription polymerase chain reactions (RT-PCR) were conducted using 2X Taq PCR MasterMix (TianGen Biotech).−ΔΔCt. Error bars represent standard deviation (SD), and statistical significance was calculated using a one-tailed, unpaired t-test. Relative mRNA or miRNA expression was summarized using mean ± SEM. All these results were calculated using student t-tests. p < 0.05 was considered to be significant. All the primers are listed in Table All quantitative PCR (q-PCR) were performed using a 7500 Fast Real-Time PCR System (Applied Biosystems) in SYBR Premix Ex Taq reaction system (TaKaRa). Each sample was analyzed in triple replication. Relative quantification (RQ) was derived from the difference in cycle threshold (Ct) between the target gene and tubulin (ΔCt) as compared to control cell lines using the equation RQ = 223. To see if downstream miRNAs of RANK signaling regulate thymus development, we treated fetal thymi with RANK ligand (RANKL) for 4 days in 2-DG FTOC system. By RANKL stimulation, miR-449a was dramatically induced in 2-DG FTOC  as an ble Fig. . MiR-449ble Fig. . In addible Fig. , indicatort Fig. 28. TheseWe further analyzed the temporal expression profiling of these miRNAs in thymus at development stages from E14.5 to 5-week old mice. Expression of miR-449a was dramatically increased at E18.5 and peaked at postnatal 10-day Fig. . However41. To see if miR-449a could induce mTEC maturation and Aire expression, we further stably overexpressed miR-449a in TSC cells (labeled as TSC miR-449a cells). Immunofluonrescence staining  and flow cytometry  were significantly decreased in TSC cells after miR-449a overexpression  bore a 20 bp insertion in miR-449a seed sequence following TGGCAGTGTATTGT while the deletion mutant strain (miR-449aDel/Del) bore a 30-bp deletion just after TGGCAGTGTATTGTTA . However, the expression of miR-449b was slightly increased and augmented GFP+ cortex (K8+GFP+) in GFP-expression area . However, control-GFP expression could be detected in both medulla and cortex . Statistic analysis of the K14+ and K8+ zone in GFP-expressing area revealed that K14+ GFP+ mTEC was significantly reduced, while K8+ GFP+ cTEC was increased after expression of miR-449/34 sponge . MiR-34a and miR-449a possessed similar seed sequence and had overlapped candidate targets as predicted. More importantly, expression of miR-34a was significantly up-regulated in miR-449a-mutant thymus, which in another way may indicate its compensation role.MiR-449a was highly expressed during thymus development and positively correlated with Aire expression. Furthermore, we demonstrated that miR-449a induced thymic epithelial progenitor cells differentiation into mature thymic epithelial cells in situ injection of lentivirus in 3-week old mice. Injection of miR-449/34 sponge virus resulted in reduced GFP+ medulla and augmented GFP+ cortex, reflected on the extensive miR-449/34 sponge-GFP expression in cortex. Flow cytometry analysis of TEC subsets in infected thymus also showed reduction of mature MHCIIhi mTECs. Taken together, these results indicated that interference of miR-449a and miR-449/34 cluster blocked normal differentiation of mTEC and may also have impact on cTEC development. Clues from the expression profiling of miR-449/34 cluster during thymus development, miR-34 may function at early stage before E15.5 while miR-449 may regulate late differentiation of mTECs. The generation of miR-449/34 sponge transgenic mice or miR-449/34 knock out mice may help to reveal the molecular mechanisms of miR-449/34 in regulation of thymus development.To further confirm the impact of miR-449a on mTEC development, we introduced miR-449/34 sponge through thymic Supplementary Information