Stromal cell-derived factor 1 (SDF1) has crucial role in the regulation of angiogenesis and ocular neovascularisation (NV). The purpose of this study was to evaluate the association between SDF1-3’G(801)A polymorphism and NV complications of retinal vein occlusion (RVO).SDF1-3’G(801)A polymorphism was detected by PCR-RFLP. Allelic prevalence was related to reference values obtained in the control group consisted of 125 randomly selected, age and gender matched, unrelated volunteers . Statistical analysis of the allele and genotype differences between groups  was determined by chi-squared test. P value of <0.05 was considered statistically significant.130 patients with RVO  were enrolled in this study. In the RVO group, 40 (30.8%) patients were diagnosed with NV complications of RVO and 90 (69.2%) patients without NVs. The median follow up period was 40.3 months (range: 18–57 months). The SDF1-3’G(801)A allele and genotype frequencies of RVO patients were similar to controls AA: 5.4% vs 4.8%, SDF1-3’(801)GG: 60.8% vs 63.2%). The frequency of SDF1-3’(801)AA and SDF1-3’(801)GA genotypes, as well as the SDF1-3’(801)A allele frequency were higher in RVO patients with NV versus in patients without NV complication (SDF1-3’(801)AA+AG genotypes: 57.5% vs 31.1%, p = 0.008; SDF1-3’(801)A allele: 35.0% vs 16.7%, p = 0.002) or versus controls (SDF1-3’(801)AA+AG genotypes 57.5% vs 36.8%, p = 0.021; SDF1-3’(801)A allele: 35.0% vs 20.8% p = 0.01). Carrying of SDF1-3’(801)A allele increased the risk of neovascularisation complications of RVO by 2.69 .Hardy-Weinberg criteria was fulfilled in all groups. The SDF1-3’(801)A allele plays a role in the development of neovascular complications in retinal vein occlusion.These findings suggest that carrying  Retinal vein occlusion (RVO) is the most common form of retinal vascular disease following diabetic retinopathy with a prevalence rate of 5.2/1000, and may result in permanent vision loss. The frequency of SDF1-3’(801)A allele: 16.7% vs 20.8%, p = 0.282; SDF1-3’(801)AA genotypes: 2.2% vs 4.8% p = 0.473; SDF1-3’(801)AA+AG genotypes 31.1% vs 36.8%, p = 0.386). However in RVO patients diagnosed with NV complications, both the frequency of the SDF1-3’(801)A allele and SDF1-3’(801)A carrying genotypes (SDF1-3’(801)A allele: 35.0% vs 20.8%, p = 0.01; SDF1-3’(801)AA+AG genotypes 57.5% vs 36.8%, p = 0.021) was higher than in control group.Neither the allele nor the genotype frequencies were different in RVO patients without NV when compared to controls (SDF1-3′G(801)A polymorphism in patients with retinal vein occlusion. We found significant association between the development of neovascular complications and the presence of the SDF1-3′G (801)A allele.According to our knowledge this is the first investigation focusing on the relevance of Neovascular complications usually occur in the first 7–8 months in CRVO and 6–12 months in BRVO –7 In ordSDF1-3’(801)A allele in RVO patients with NV complications was higher than in RVO patients without NV complications resulting in an almost tripled risk increase. The frequencies of homozygous SDF1-3’(801)AA and SDF1-3’(801)GA heterozygous genotypes were also higher in RVO patients with NV complications than in patients without NV and controls. These findings suggest that carrying of SDF1-3’(801)A allele predisposes for the development of NV complications in patients with RVO.In this present study the frequency of the SDF1 is essential for the migration, homing and differentiation of circulating HSCs and EPCs during the retinal neovascularisation process. [SDF1 was found to be expressed and upregulated in an ischemia model of the retina in rats. [SDF1 induced CXCR-4 (seven-transmembrane domain G-protein coupled receptor) activation leads to β-integrin expression and the binding of vascular cell adhesion molecule (VCAM)-1 to endothelial cells. [SDF1 expression resulted in a higher VCAM-1 production [SDF1 had an effect on retinal endothelial cells and on the occludin gap junction protein, which is responsible for the tight junctions between endothelial cells to prevent leakage of the vessel. The retinal endothelial expression of occludin is decreased with increasing SDF1 level. [process. , 20–21 Iin rats. –23 SDF1 l cells. –25 In reoduction , which poduction , 26 It h1 level. –12SDF1 in ocular diseases with neovascularisation complications. [SDF1 levels in patients with proliferative diabetic retinopathy than in non-diabetic controls. Furthermore in proliferative diabetic retinopathy patients the vitreous concentration of SDF1 correlated well with the VEGF level. [SDF1 promoted neovascularization further [SDF1 alpha level was also increased in eyes with vascularly active stage 4 retinopathy of prematurity (ROP) versus either inactive stage 4 ROP or control eyes. [Only a few previous studies have investigated the vitreous level of cations. , 27–29 C further –12. VitrSDF1 levels were measured in the presence of NV complications compared to RVO patients without NV complications or controls. [In RVO patients higher intravitreous ontrols. SDF1 is a prominent contributor in the progression of retinal neovascularisation with a special emphasis on the angiogenic changes during RVO. [All these data suggest that ing RVO.  Higher VSDF1 gene  is located on human chromosome 10q11.1. In the single nucleotide polymorphism of SDF1-3’G(801)A is mutated to A at position 801 in 30-untranslated region in the b transcriptional splice variant. [variant. –17SDF1-3’G(801)A was reported to vary between 0.16–0.21 in Caucasian population which is in line with the prevalence of SDF1-3’(801)AA and AG genotypes in our study population. [SDF1–3’(801)AA than those with GA or GG genotypes.The allelic frequency of ulation. –33 Gu XLulation.  recentlySDF1-3’(801)AA genotype is more frequent in patients with proliferative diabetic retinopathy and pointed to a possible role of this allelic variant in the development of proliferative diabetic retinopathy. [Djuric Z et al. found that the nopathy. The incidence of diabetes mellitus in our RVO patients was 19.2%, in previous studies it ranged from 14% to 34% , 34 AccoThe effect of diabetes mellitus on the risk of developing NV in RVO is controversial. Previous studies found higher rates of NV in diabetic patients with CRVO than in non-diabetic patients with CRVO. –36 OtherWe did not exclude diabetic patients from our study only those patients who had diabetic retinopathy that could mimic RVO and induce neovascular proliferation. We did not find significant difference in the presence of diabetes mellitus among the RVO patients with and without NV in our study. In order to include the persence of diabetes mellitus as possible confounding systemic risk factor in the statistical analysis, a larger sample size would be needed. This is a limitation of our study.None of our BRVO patients did develop neovacular glaucoma. It is in concordance with observation published by Hayreh et al. They found that in BRVO the retina and optic disc were the major sites of NV and none of their BRVO eyes  did develop neovascular glaucoma. None of our patients with CRVO did develop retinal NV. This finding is in concordance with the observation published by Hayreh et al. that in CRVO the chance for the development of anterior segment NV is much greater than for posterior segment NV.  In this There are certain limitations to our study. First, it should be noted that all of our study participants were Hungarian white Caucasians, and thus, the possibility of ethnicity as a confining factor could be excluded. Second, here we evaluated hypertension and diabetes mellitus as possible risk factors. Other systemic risk factors such as hyperlipidemia, cardiovascular diseases, blood hyperviscosity and diseases with hypercoagulation were not included. Further data collection is in progress to analyze these values as possible limiting factors. Third, due to our relatively small sample size, these are rather preliminary results which should be confirmed in larger cohorts and in subgroups of different types of RVO as well.SDF1-3’G(801)A polymorphism in patients with retinal vein occlusion. We found significantly higher frequencies of the SDF1-3’(801)A allele and SDF1-3’(801)AA and SDF1-3’(801)GA genotypes in RVO patients with NV compared to patients without NV and to controls. Since SDF1-3’G(801)A polymorphism has been shown to increase the production of SDF1 protein, which could promote neovascularisation by causing a disruption of the blood-retinal barrier, presence of SDF1-3’(801)A allele may be genetic risk factors for NV complications after RVO.In summary, this is the first study evaluating S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.


Human influenza A(H3N2) viruses that predominated during the moderately severe 2014-2015 influenza season differed antigenically from the vaccine component, resulting in reduced vaccine effectiveness (VE). To examine antibody responses to 2014-2015 inactivated influenza vaccine (IIV) and live-attenuated influenza vaccine (LAIV) among children and adolescents, we collected sera before and after vaccination from 150 children aged 3 to 17 years enrolled at health care facilities. Hemagglutination inhibition (HI) assays were used to assess the antibody responses to vaccine strains. We evaluated cross-reactive antibody responses against two representative A(H3N2) viruses that had antigenically drifted from the A(H3N2) vaccine component using microneutralization (MN) assays. Postvaccination antibody titers to drifted A(H3N2) viruses were higher following receipt of IIV  versus LAIV . In 9- to 17-year-olds, the highest MN titers were observed among IIV-vaccinated individuals who had received LAIV in the previous season. Among all IIV recipients aged 3 to 17 years, the strongest predictor of antibody responses to the drifted viruses was the prevaccination titers to the vaccine strain. The results of our study suggest that in an antigenically drifted influenza season, vaccination still induced cross-reactive antibody responses to drifted circulating A(H3N2) viruses, although higher antibody titers may be required for protection. Antibody responses to drifted A(H3N2) viruses following vaccination were influenced by multiple factors, including vaccine type and preexisting immunity from prior exposure. Neutralizing antibodies against hemagglutinin (HA) on the surfaces of influenza viruses have been considered the major immune mechanism that provides protection against influenza infection , 2. Howe3–Among all seasonal influenza virus subtypes, HA of influenza A(H3N2) has the fastest evolutionary rate with new antigenic clusters emerging on average every 3.3 years , 9. In a10–Even when seasonal influenza vaccines are antigenically mismatched to circulating influenza viruses, vaccination may still provide partial protection by inducing cross-reactive antibody responses to circulating strains through shared epitopes on HA or other viral proteins . The levHere, we investigated immune responses of children and adolescents enrolled in an observational study. We measured serum antibody responses to 2014-2015 live-attenuated and inactivated influenza vaccines, evaluated the levels of neutralizing antibodies to antigenically drifted influenza A(H3N2) strains, and explored factors that may influence cross-reactive antibody responses to drifted A(H3N2) viruses following vaccination.Healthy children aged 3 to 17 years were recruited from three health centers  from the University of Pittsburgh Medical Center (UPMC) Health System in 2014. The criteria used for enrollment in the study were as follows: (i) the child had not received and was planning to receive 2014-2015 influenza vaccine; (ii) no contraindications for LAIV; (iii) known vaccination status for the 2013-2014 influenza season ; and (iv) weight of ≥17 kg. Due to potential concerns about LAIV safety in those with uncontrolled asthma, children with a history of severe asthma episodes were excluded.Eligible children were recruited by two age groups, 3 to 8 years and 9 to 17 years. All children received 2014-2015 influenza vaccines either LAIV or IIV according to parent, child, or clinician preference. Children receiving 2014-2015 LAIV were enrolled regardless of prior season vaccination status. Children receiving 2014-2015 IIV were enrolled as the comparison group. All participants were enrolled and vaccinated between September and December 2014. Sera were collected prior to vaccine administration (day 0) and 19 to 33 days postvaccination  assays were performed with pre- and postvaccination serum specimens as previously described  using 0.Viruses that represent Northern Hemisphere 2014-2015 influenza season quadrivalent vaccine components were used in HI assays: A/California/7/2009 A(H1N1), A/Texas/50/2012 A(H3N2), B/Massachusetts/02/2012 (B/Yamagata lineage), and B/Brisbane/60/2008 (B/Victoria lineage). All viruses used in HI assays were propagated in 9- to 11-day-old embryonic chicken eggs. B virus antigens were ether treated prior to the HI assay.50]) were added to serum dilutions, incubated at 37°C with 5% CO2 for 1 h, and used to infect 1.5 × 105 MDCK-SIAT1 cells per ml. After overnight incubation, the presence of the viral proteins was detected by an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies specific to the influenza A virus nucleoprotein. MN titers were defined as the reciprocal of the highest dilution of serum that yielded at least 50% neutralization.Microneutralization (MN) assays were performed as previously described , except http://www.mbio.ncsu.edu/bioedit/bioedit.html).Pre- and postvaccination sera were tested against three A(H3N2) viruses: A/Texas/50/2012 , A/Switzerland/9715293/2013 (group 3C.3a), and A/Nebraska/04/2014 (group 3C.2a). All viruses used in MN assays were propagated in MDCK-SIAT1 cells. Sequence analyses of the viruses were performed using BioEdit alignment editor  vaccination status. Separate linear regression models restricted to IIV-vaccinated subjects examined predictors of IIV response, including prior season vaccine type. Predictors of seroconversion were examined by logistic regression. Statistical analyses were conducted using SAS for Windows .For statistical analyses, specimens with reciprocal HI or MN titers of <10 were assigned a titer of 5. Geometric mean titers (GMTs), fold rises or increases , and 95% confidence intervals (95% CIs) were calculated using repeated-measure linear mixed models as previously described . SeroconP > 0.05). At enrollment in the study, the majority of subjects had preexisting HI antibodies to vaccine viruses: HI GMT antibody titers among both IIV and LAIV recipients were 60 to influenza A(H1N1), 119 to A(H3N2), 72 to B/Yamagata, and 35 to B/Victoria vaccine components and were not statistically different between IIV and LAIV recipients  and 53 received quadrivalent IIV  in the 2014-2015 influenza season . There wcipients . Preexiscipients .P < 0.01 for all three comparisons; P < 0.001; As measured by HI antibody titers, children who received IIV had higher postvaccination GMTs, seroconversion rates, and fold rises to all four vaccine viruses than those vaccinated with LAIV , with reciprocal MN titers being slightly higher  than the corresponding HI titers: HI titers against A/Texas/50/2012 of 1:40 corresponded to MN titers of ∼1:54.Antibody titers to the influenza A(H3N2) vaccine component measured by HI assay with egg-propagated viruses and by MN assay with MDCK-SIAT1 cell-propagated viruses were highly correlated  than in those with high preexisting titers (≥1:40) for all four vaccine viruses (Compared to the A(H3N2) vaccine strain A/Texas/50/2012, A/Nebraska/04/2014 (genetic group 3C.2a) and A/Switzerland/9715293/2013 (group 3C.3a) viruses have mutations in several positions in HA proteins, including antigenic sites A and B, and near the receptor binding pocket ; both are antigenically distinct from the A(H3N2) vaccine strain characterized with ≥8-fold reduction in HI titers when tested with ferret antisera against the vaccine strain (data not shown).Vaccination with influenza A/Texas/50/2012 induced cross-reactive neutralizing antibodies to drifted A(H3N2) viruses from genetic groups 3C.2a and 3C.3a, with MN titers against drifted viruses 7- to 11-fold lower than against vaccine virus . Among IAmong IIV recipients, ≥70% and ≥40% had postvaccination MN titers of ≥40 and ≥110 against drifted influenza A(H3N2) viruses, respectively, although postvaccination GMTs reached only 63  for A/Nebraska/04/2014 (group 3C.2a) and 68  for A/Switzerland/9715293/2013 (group 3C.3a) compared to 565  for the vaccine virus. Among LAIV recipients, approximately 40% and 10% had postvaccination MN titers of ≥40 and ≥110 against the drifted H3N2 vaccine virus, respectively, with MN GMT around 22 postvaccination; pre- and postvaccination GMTs were not significantly different .P = 0.002 for A/Nebraska/04/2014; P = 0.07 for A/Switzerland/9715293/2013) in children vaccinated with LAIV in the 2013-2014 season than in those vaccinated with IIV in the prior season. Among all children vaccinated with IIV in the 2014-2015 season, prevaccination MN titers to the A(H3N2) vaccine strain were the strongest predictor of seroconversion to drifted 3C.2a and 3C.3a viruses (P < 0.05), with higher likelihood of seroconversion at lower prevaccination titers against the A(H3N2) vaccine virus .Receipt of LAIV in the 2014-2015 influenza season was not associated with increased MN titers against drifted A(H3N2) viruses, even after controlling for age group, prevaccination MN titer, and prior season vaccine type . HoweverDespite the poor antigenic match between the 2014-2015 influenza A(H3N2) vaccine component and circulating influenza A(H3N2) viruses, children and adolescents vaccinated with inactivated influenza vaccine exhibited significant antibody rises and seroconversion to A(H3N2) vaccine virus as well as to newly emerged, antigenically drifted influenza A(H3N2) viruses that circulated during the 2014-2015 influenza season. Overall, more than 70% of IIV-vaccinated children had MN titers of ≥40 after vaccination and 38 to 45% seroconverted to A(H3N2) vaccine and drifted viruses. However, postvaccination GMTs for cross-reactive neutralizing antibodies to drifted A(H3N2) viruses remained almost 10-fold lower than antibodies against the vaccine component, with geometric mean MN titers ranging from 63 to 68 to drifted A(H3N2) viruses. In contrast to IIV, receipt of LAIV did not significantly increase titers to vaccine or cross-reactive neutralizing antibodies to drifted viruses. Data from the U.S. Influenza Vaccine Effectiveness network reported low VE in children and adolescents for this season: only −5%  VE for LAIV and 13%  VE for IIV against influenza A(H3N2)-related illness . Thus, MImmune correlates of protection are not well established among children and may differ between inactivated and live attenuated vaccines. Traditionally, a postvaccination HI titer of 40 has been associated with 50% reduction in influenza infection in adults –27. The –in vitro, whereas the HI assay is considered only a surrogate assay. Our data and several previous studies demonstrated higher sensitivities for the MN assay than for the HI assay  viruses. Traditional HI assays are not possible for many A(H3N2) viruses belonging to genetic group 3C.2a, due to their altered receptor binding properties to red blood cells, resulting in insufficient hemagglutination activity for characterization by HI , 31. FewHI assay , 32. ChiHI assay . It is aHI assay , the incWe examined factors that may contribute to levels of cross-protective antibodies to drifted viruses postvaccination. An individual's antibody repertoire accumulated from past priming history to influenza viruses can shape the responses to current influenza vaccination , 34. Pre37–Consistent with low vaccine effectiveness observed for LAIV against influenza A(H3N2)-related illness during the 2014-2015 influenza season , receiptOur study has several limitations. Surveillance was not conducted for influenza illnesses among study participants, so we cannot correlate antibody levels with actual protection. Enrollees were not randomized by vaccine type, and there may be differences between LAIV and IIV recipients that affect immune responses, although when comparing characteristics such as age, sex, and preexisting titers to vaccine antigens, there was no statistical difference between LAIV- and IIV-vaccinated children. In addition, vaccination history was determined for only one prior season (2013-2014 season). We do not have vaccination information from earlier seasons and also lack information on possible infections. The absence of this information may contribute to the lack of association between 2013-2014 vaccination history and prevaccination titers. Prior infection and repeated vaccination may change the antibody profile and affect the cross-reactive antibody responses to the antigenically mismatched circulating strains. Also, we were able to enroll only a small number of children previously vaccinated with LAIV or unvaccinated children who chose to receive IIV in the 2014-2015 season. When stratified by both age categories and prior season vaccination, sample sizes in strata were small.In summary, IIV induced cross-reactive neutralizing antibodies to drifted A(H3N2) viruses that predominated during the 2014-2015 influenza season in children, but higher thresholds may be required for heterologous protection. Further research is needed to better understand the correlate of protection against antigenically drifted viruses, and ultimately to develop improved vaccines and vaccination strategies that can offer broader immune protection to overcome the challenges from constant antigenic drift of seasonal influenza viruses.


Here, to further investigate whether these two transporters are functionally coupled, we compared the respective knockout (KO) of either LAT1 or ASCT2 in colon (LS174T) and lung (A549) adenocarcinoma cell lines. Although ASCT2KO significantly reduced glutamine import (>60% reduction), no impact on leucine uptake was observed in both cell lines. Although an in vitro growth-reduction phenotype was observed in A549-ASCT2KO cells only, we found that genetic disruption of ASCT2 strongly decreased tumor growth in both cell lines. However, in sharp contrast to LAT1KO cells, ASCT2KO cells displayed no amino acid (AA) stress response (GCN2/EIF2a/ATF4) or altered mTORC1 activity (S6K1/S6). We therefore conclude that ASCT2KO reduces tumor growth by limiting AA import, but that this effect is independent of LAT1 activity. These data were further supported by in vitro cell proliferation experiments performed in the absence of glutamine. Together these results confirm and extend ASCT2's pro-tumoral role and indicate that the proposed functional coupling model of ASCT2 and LAT1 is not universal across different cancer types.The transporters for glutamine and essential amino acids, ASCT2  and LAT1 , respectively, are overexpressed in aggressive cancers and have been identified as cancer-promoting targets. Moreover, previous work has suggested that glutamine influx via ASCT2 triggers essential amino acids entry  Commonly known as the building blocks of proteins, amino acids (AAs)3–9–+-dependent transporter that exchanges small neutral AAs    and the alanine-serine-cysteine transporter 2 (ASCT2/SLC1A5) have emerged as major pro-tumoral transporters with increased expression levels correlating with poor patient prognosis in a large number of cancer types –16. ASCT–and Asn) . ASCT2 hand Asn) . Indeed,and Asn) , 20, 21.et, Tyr) . LAT1 foet, Tyr) , 23. Woret, Tyr) , 25. Impet, Tyr) , 26. Intet, Tyr) , 27. Theet, Tyr) .In 2009, Nicklin and colleagues  proposed32–in vitro. Interestingly, ASCT2 ablation reduced tumor growth in vivo but this effect does not appear to be linked to a reduced LAT1 activity because no AA stress response and only a minor reduction of mTORC1 activity were detected in tumor tissue analyses. Together these findings demonstrate that the proposed functional coupling of ASCT2 and LAT1 is not obligatory nor a generalized phenomenon across cancer types. However, ASCT2 is required for optimal tumor growth in vivo and its ablation sensitized A549 cells to the LAT1 inhibitor JPH203 (Here we report that knockout of ASCT2 in two independent cancer cell lines (LS174T and A549) does not alter LAT1 transport activity. Moreover, whereas LAT1 is essential for cell proliferation by promoting AA homeostasis and mTORC1 activity, ASCT2 is dispensable r JPH203 , 35, indASCT2 knockout (KO) was achieved in colon (LS174T) and lung (A549) adenocarcinoma cell lines using the CRISPR-Cas9 technique. To minimize clonal heterogeneity, experiments were performed on two independent clones. LAT1KO cells were obtained from a previous study (A), genomic DNA analysis, and sequencing of the CRISPR-targeted site demonstrated disruptive mutations in the ASCT2 gene (Table S1). In both cell lines, removal of either ASCT2 or LAT1 does not appear to consistently influence the expression of their proposed functional partner (A). The impact of ASCT2 disruption on glutamine transport activity was then assessed (B). ASCT2KO in both cell lines (white bars) reduced glutamine transport activity to ∼35% of WT cells (dashed bars), demonstrating that ASCT2 is the major glutamine transporter expressed in LS174T and A549 cells (B). Two different pharmacological inhibitors were then used to identify the transporters responsible for the residual glutamine transport activity in ASCT2KO cells. These included JPH203, a specific LAT1 inhibitor (N-methylaminoisobutyric acid (MeAIB), an inhibitor of the system “A” AA transporters   demonstrating that LAT1 and SNATs contribute to the residual transport activity detected in ASCT2KO cells (B).us study . Lack ofus study A, genomi partner A. The imassessed B. ASCT2K49 cells B. Two dinhibitor , 35, andSLC38A5) . SNAT trine 143B . UtilizaKO cells B.+-independent rate of leucine transport (C). Surprisingly, despite the fact that glutamine uptake is strongly reduced, ASCT2KO cells display the same leucine transport rates as WT cells (C). Combined, these results suggest that, even if its abolition strongly reduces glutamine uptake, ASCT2 is not required for LAT1 expression and activity in both LS174T and A549 cells.To investigate the functional coupling of these transporters, the impact of the genetic disruption of ASCT2 on LAT1 activity was analyzed by measuring the Naransport C. SurpriWT cells C. CombinLAT1KO cells demonstrated that genetic disruption of LAT1 induces an AA stress response (GCN2/EIF2a/ATF4) and decreases mTORC1 activity . In these two conditions, a strong induction of the GCN2 pathway and decreased mTORC1 activity was detected in LAT1KO cells . As expected, LAT1KO cells display a slower activation of mTORC1 whereas no significant differences were detected in ASCT2KO cells, further confirming the data mentioned above. Furthermore, experiments performed with re-addition of glutamine alone following 24 h of glutamine starvation (see extended description below) revealed the same timing of mTORC1 activation between LS174T-WT and ASCT2KO cells. Overall, these results demonstrate that the genetic disruption of ASCT2 does not mimic the effect of LAT1 knockout in terms of AA homeostasis and mTORC1 activity.Previous analysis of activity . Becauseactivity , 29, we KO cells . In nutrKO cells . In addis tested . Next, cKO cells . Again, nchanged . Finallywhite bars), ASCT2KO (light and dark gray bars), and LAT1KO (black bars) cells (A). LS174T-ASCT2KO cells displayed the same proliferation rate as WT cells in the three different media tested whereas LAT1KO cell proliferation is strongly reduced (below 20% of WT rate in 0.3× and 0.1× media) (A). Interestingly, A549-ASCT2KO cells exhibit the same reduced proliferation rate as LAT1KO cells in standard DMEM (∼50%) (A). However, in AA reduced media (0.3× and 0.1×) A549-ASCT2KO cells exhibited higher rates of proliferation matching the rate of WT cells whereas A549-LAT1KO cell proliferation was below 20% the WT rate in 0.3× and 0.1× media) (A). Cell proliferation rates were further confirmed using 15-day clonogenecity assays (B). Together these findings demonstrate that genetic invalidation of ASCT2 does not phenocopy the LAT1KO cells in vitro, suggesting that ASCT2 is dispensable for LAT1-dependent AA homeostasis, mTORC1 activity, and proliferation.We then compared the proliferation phenotype (-fold increase at 72 h) of WT (s) cells A. LS174T× media) A. IntereM (∼50%) A. Howeve× media) A. Cell py assays B. TogethC). LS174T-ASCT2KO and WT cell proliferation rates were again the same in the absence of external glutamine whereas A549-ASCT2KO cells maintained a lower proliferation rate in comparison to WT cells. The effect of glutamine starvation on mTORC1 activity and AA stress response was also monitored. Removal of external glutamine removed phosphorylation of S6K1 after 6 h and induced phosphorylation of GCN2 by 24 h in both WT and ASCT2KO cells , indicating that cellular glutamine synthesis is not sufficient to maintain mTORC1 activity and is not induced as an adaptive mechanism in ASCT2KO cells. Inhibition of glutamine synthetase with methionine sulfoximine (MSO) in LS174 cells decreased mTORC1 activity after 1 h and continued for 6 h . ASCT2KO cells demonstrated the same mTORC1 activity patterns as WT cells in response to MSO treatment. Therefore, we conclude that the intracellular glutamine pool and synthesis rate cannot explain the inability of ASCT2KO cells to phenocopy the AA stress response of LAT1KO cells.To investigate the contribution of internal glutamine synthesis we monitored proliferation in the absence of external glutamine in standard DMEM. Glutamine removal reduced WT cell proliferation by ∼50% in both LS174 and A549 C. LS174TASCT2KOin vivo we utilized a mouse xenograft model. LS174T-WT, A549-WT and ASCT2KO cells were injected subcutaneously into nude mice to monitor tumor growth (A). Surprisingly, although they display a normal proliferation rate in vitro, LS174T- and A549-ASCT2KO tumors exhibited a reduced growth rate by ∼50 and 85%, respectively, compared with WT (A). To assess if this decrease in tumor size is linked to a reduced LAT1 activity, protein analysis was performed in three independent tumors of each cell type (B). LS174T-ASCT2KO tumors exhibit a slightly reduced mTORC1 activity (observed via S6 phosphorylation) compared with the control tumors whereas the GCN2 pathway is not altered (observed via ATF4 levels). Remarkably, even with a near abolishment of growth (A), A549-ASCT2KO tumors did not display any alterations in the AA sensing pathways mTORC1 and GCN2 (B). Previously we demonstrated that LAT1KO tumor growth is strongly reduced (>90%) because of markedly decreased mTORC1 activity and activation of the GCN2 pathway . To confirm this increased sensitivity in the context of AA stress we investigated the dose-response effect of JPH203 on the two AA sensing pathways investigated previously (GCN2 and mTORC1) in A549-WT and ASCT2KO cells (C). Induction of the AA stress response pathway (GCN2) begins at 10 μm and is further increased at 30 μm in A549-ASCT2KO cells whereas only a slight induction is detected in A549-WT cells at the highest dose. In terms of mTORC1 activity, 30 μm of JPH203 strongly decreased the phosphorylation of S6K1 and S6 in A549-ASCT2KO cells whereas it has no effect on A549-WT. This increased sensitivity to LAT1 inhibition in ASCT2KO cells appears to be cell line–specific because the sensitivity of LS174T-ASCT2KO to JPH203 was not consistently altered . Together these results demonstrate that ASCT2 disruption may sensitize certain tumor cell types to LAT1 inhibition suggesting that even if ASCT2 expression is not an obligatory requirement for LAT1-dependent AA homeostasis and proliferation, a certain level of functional coupling could exist. However, the difference between A549- and LS174T-ASCT2KO cells in terms of JPH203 sensitivity suggests that other AA transporters or mechanisms may be involved in this functional AA transport collaboration.As pharmacological inhibitors for LAT1 are progressing toward the clinic  we wantean 30 μm , A and BKO cells C. InductTumor cells that are “oncogenically rewired” for rapid growth continually sense overall nutrient availability. To survive, anabolic and catabolic processes are tightly controlled to efficiently coordinate growth with nutritional and energetic status. The key molecule within this coordination, often up-regulated in cancers, is mTORC1, a conserved serine-threonine kinase among eukaryotes , 38. mTOASCT2KO was correlated with normal LAT1 leucine transport activity (C) and mTORC1 activity (S1) in both cell lines. Furthermore, whereas LS174 LAT1KO cells exhibit a severe reduction in growth, ASCT2KO cells are unaffected in vitro (A and B). This absence of growth phenotype in vitro with ASCT2KO was correlated with normal LAT1 activity (C) and mTORC1 activity (S1). A549-ASCT2KOin vitro cell proliferation is indeed reduced in normal DMEM (A and B) whereas mTORC1 activity was unaffected (C and S2) show the same reduced growth rates for ASCT2KO cells, indicating that the reduced growth phenotype is independent of glutamine transport as discussed further below. We also observed that in these glutamine-free growth conditions, de novo synthesis of glutamine enables low levels of cell proliferation; however there is no obvious compensation for up-regulated synthesis in ASCT2KO cells as an adaptive pathway. We further confirmed that glutamine synthesis contributes to mTORC1 activity via use of the glutamine synthetase inhibitor MSO with no discernable differences between WT and ASCT2KO cells .Previously, our lab has demonstrated the essential role of LAT1 in promoting tumor growth via the maintenance of AA homeostasis and mTORC1 activity . Becauseactivity C and mTOactivity  and S1 iin vitro , A and Bactivity C and mTOactivity  and S1. mal DMEM , A and Baffected . Howeveraffected C and S2 ASCT2KO has also shown that ASCT2 is not required for mTORC1 activity . These results suggest that A549-ASCT2KO cells may rely on SNATs for glutamine uptake in the absence of ASCT2 as shown previously for 143B cells (B). Our results combined with the literature suggest that the apparent absence of obligatory functional coupling between ASCT2 and LAT1 will require consideration of multiple glutamine transporters but also non-glutamine transporting mechanisms in future studies to obtain more precise information on mTORC1 activation. Our lab is currently exploring alternative glutamine transporter(s) or anabolic sources of AA capable to maintain import of leucine via LAT1. Consequently, it appears that variation between cancer types with respect to glutamine transport and potential cooperation with LAT1 may provide a formidable barrier to development of an effective, broad-reaching cancer-specific drug in this area. Nevertheless, ASCT2 inhibition could still prove to be useful as an additive therapy as A549-ASCT2KO cells exhibited a notable increased sensitivity to LAT1 inhibition (A–C). However, this increased sensitivity is not reproducible in LS174T-ASCT2KO cells , reinforcing the notion that other factor(s) can play a role in LS174 acquired resistance. Therefore effective LAT1 inhibition alone appears to be an area better suited for further research as it avoids the variable nature of transport cooperativity between cancer cell types and directly stops growth by inhibiting mTORC1 and the import of essential AAs . This growth reduction does not correspond however to what was previously described for LAT1KO tumors (ASCT2KO tumors (B), correlating with our in vitro results demonstrating that ASCT2 is dispensable for LAT1-dependent AA homeostasis and mTORC1 activity. These results highlight the metabolic differences between in vitro and in vivo conditions for cancer cells and suggest that in vivo, ASCT2 AA transport activity is required for tumor growth independently of LAT1. Historically, ASCT2 literature has focused on its role in glutamine uptake, leading to the assumption that ASCT2 disruption would alter glutamine metabolism and thus tumor growth . Indeed, ASCT2 carries a number of other substrates and notably the two semi-essential AAs: serine and cysteine  500 μm (0.3×) and 166 μm (0.1×). For the final concentration of AA in the different media used in this study and comparison with plasma concentrations see Table S2.Colon adenocarcinoma LS174T cells , and A549 cells  were used in all experiments. These two cell lines have been authenticated by DNA profiling of eight highly polymorphic short tandem repeat loci . Cells were grown in DMEM (Gibco) supplemented with 7.5% dialyzed FBS, penicillin (10 units/ml), and streptomycin (10 μg/ml) and MEM NEAA (Gibco). 0.3× media was obtained by mixing 2 volumes of DMEM lacking all amino acids with 1 volume of DMEM without glutamine. The 0.1× media was obtained by mixing 9 volumes of DMEM lacking all amino acids with 1 volume of DMEM without glutamine. Glutamine was added fresh just before cell culture experiments respectively at 4 mTable S1). LAT1 knockout cells were obtained from our previous study (LS174 and A549 WT cells were transfected with PX458 plasmids containing CRISPR-Cas9 targeting regions of the fifth exon of the ASCT2 (SLC1A5) gene using JetPRIME (Polyplus). The pSpCas9(BB)-2A-GFP (PX458) plasmid was a gift from Dr. Feng Zhang (Addgene plasmid no. 48138) . The sgRus study .ASCT2KO cells was followed for 72 h as described below. Glutamine starvation (in otherwise complete DMEM supplemented with MEM NEAA) was also performed for analysis of the AA stress response and mTORC1 activation. Finally, the glutamine synthetase inhibitor MSO was used (3 mm) in complete DMEM supplemented with MEM NEAA for observation of mTORC1 activity during 0–6 h.Cells were exposed to glutamine deprivation experiments in the following manner. For cell proliferation studies, the effect of glutamine-free DMEM on LS174 and A549 WT+m Tris-HCl pH7.4, 150 mm NaCl) and incubated with the following anti-human antibodies: rabbit LAT1 , rabbit ASCT2 ), mouse GCN2 , mouse phospho-GCN2 , rabbit EIF2α , mouse phospho-EIF2α , rabbit ATF4 , rabbit p70-S6K1 , rabbit phospho-p70-S6K1 , rabbit S6 , and rabbit phospho-S6 . Detection of tubulin  or ARD1 . Protein extracts were separated by electrophoresis on 10% SDS-polyacrylamide gel and transferred onto polyvinylidene difluoride membranes (EMD Millipore). Membranes were blocked in 5% nonfat milk in PBS buffer (50 m or ARD1  was used5) were seeded onto 35-mm dishes, in triplicate per cell line and used for uptake experiments 24 h after seeding. Culture media were removed and cells were carefully washed with prewarmed Hanks' Balanced Salt Solution (HBSS) , pre-incubated in 1.0 ml of prewarmed HBSS at 37 °C for 5 min before adding substrates for the uptake experiment. Cells were then incubated at 37 °C for 5 min in 1 ml of HBSS containing 10 μm L-[14C]-amino acid (0.03 μCie/ml) (PerkinElmer Life Sciences). Subsequently, cells were washed three times with ice-cold Na+-free HBSS containing 1.0 mm nonradiolabeled amino acid. Cells were then lysed with 500 μl of 0.1 N NaOH and mixed with 3 ml of Ultima Gold (PerkinElmer). Radioactivity was measured using a β-scintillation counter. Specifically, L-[14C]-leucine uptake was performed with a Na+-free HBSS media . Inhibition experiments were performed in the presence of 10 mm MeAIB and/or 30 μm JPH203.Cells (5 × 104 cells for A549 and 4 × 104 for LS174) were seeded onto 6-well plates in triplicate per cell line and per condition. We measured proliferation by trypsinizing the cells and counting them daily with a Coulter Z1 (Beckman) during 3 days. The cell proliferation index was calculated as -fold increase by standardizing each measurement to the cell number obtained 24 h after seeding (Day 0).The different cell lines (2.5 × 104 cells for A549 and 4 × 104 for LS174) were seeded onto 6-well plates in triplicate for each cell line and JPH203 concentrations indicated. We measured proliferation as described above.The different cell lines  supplemented with 7.5% dialyzed serum and containing JPH203 for LAT1 inhibition experiments. Media were changed every 2 days. Dishes were stained with 5% Giemsa (Fluka) for 30–45 min to visualize colonies.LS174 and A549-derived mutants (1000 cells) were plated in 60-mm dishes and incubated at 37 °C, 5% CO6 cells) suspended in 500 μl of serum-free DMEM supplemented with insulin-transferrin-selenium (Life Technologies) were injected subcutaneously into the backs of 8-week-old female athymic mice (Janvier). Tumor dimensions were measured at least twice a week using calipers and the tumor volume was determined by using the formula: (4π/3) × L/2 × W/2 × H/2, . When the tumor volume reached 1000 mm3, mice were euthanized and the tumors were excised. For protein analysis, tumors were lysed directly after harvesting. Tumors were incubated in cell extraction buffer  supplemented with Halt protease inhibitor mixture  and lysed using a Precellys homogenizer. Animal housing was done in compliance with the European Union directive 2010/63/EU. Briefly, each cage contained five mice with an enriched environment, food and water were given ad libitum, and the litter was changed on a weekly basis. Animal care met the European Union directive 2010/63/EU ethical criteria. The animal experimentation protocol was approved by the local animal care committee. The different stable cancer cell lines  using the Bonferroni and Dunnett's post hoc tests where appropriate. Differences between groups were considered statistically significant when Y. C., P. A. M., M. V., S. K. P., and J. P. conceptualization; Y. C., P. A. M., M. V., and S. K. P. data curation; Y. C., P. A. M., M. V., S. G., S. K. P., and J. P. formal analysis; Y. C., P. A. M., M. V., S. G., E. T., J. D., V. V., and S. K. P. investigation; Y. C., M. V., S. K. P., and J. P. methodology; Y. C., S. K. P., and J. P. writing-original draft; Y. C., M. V., S. K. P., and J. P. writing-review and editing; H. E. and M. F. W. resources; S. K. P. and J. P. supervision; J. P. funding acquisition; J. P. project administration.


CLN2 gene encoding a soluble lysosomal enzyme, tripeptidyl peptidase 1 (TPP1), cause late infantile NCL/CLN2 disease. The genotype of the original mouse model of CLN2 disease, Cln2-/-, excludes mutation guided therapies like antisense oligonucleotides and nonsense suppression. Therefore, the purpose of this study was to develop a model of CLN2 disease that allows for the assessment of all therapeutic approaches. Nonsense mutations in CLN2 disease are frequent, the most common being CLN2R208X. Thus, we created a mouse model that carries a mutation equivalent to the human p.R208X mutation. Molecular assessment of Cln2R207X/R207X tissues determined significant reduction in Cln2 transcript abundance and TPP1 enzyme activity. This reduction leads to the development of neurological impairment (e.g. tremors) and neuropathology (e.g. astrocytosis). Collectively, these assessments indicate that the Cln2R207X/R207X mouse is a valid CLN2 disease model which can be used for the preclinical evaluation of all therapeutic approaches including mutation guided therapies.The Neuronal Ceroid Lipofuscinoses (NCLs), also known as Batten disease, result from mutations in over a dozen genes. Although, adults are susceptible, the NCLs are frequently classified as pediatric neurodegenerative diseases due to their greater pediatric prevalence. Initial clinical presentation usually consists of either seizures or retinopathy but develops to encompass both in conjunction with declining motor and cognitive function. The NCLs result in premature death due to the absence of curative therapies. Nevertheless, preclinical and clinical trials exist for various therapies. However, the genotypes of NCL animal models determine which therapeutic approaches can be assessed. Mutations of the  Children across the globe are affected by any one of more than 40 different lysosomal storage disorders (LSDs); many of them resulting from deficiencies in either a lysosomal membrane protein or lysosomal enzyme , 2. ThesCLN1, CLN2, and CLN3 causing classic Infantile Neuronal Ceroid Lipofuscinosis (cINCL), classic Late Infantile Neuronal Ceroid Lipofuscinosis (cLINCL), and classic Juvenile Neuronal Ceroid Lipofuscinosis (cJNCL) respectively , and then homogenized using a Bertin Technologies Precellys 24 homogenizer. Samples were then transferred to clean 1.5-ml microcentrifuge tubes and pulse sonicated using a Branson Sonifier 250. Following sonication, samples were incubated on ice for 30 min and then centrifuged at 12,000g for 10min. Sample supernatants were then transferred to clean pre-chilled 1.5mL microcentrifuge tubes and stored at -80°C. Total protein concentrations were quantified using the Pierce 660 nm Protein Assay (Thermo Fisher Scientific).Under the NIH and Sanford Research IACUC guidelines, 1-month-old Cln2R207X/R207X and Cln2+/+ total protein samples were evaluated for TPP1 enzyme activity  for 1 h at room temperature. Blocking buffer was removed and sections were incubated with the primary antibody diluted in blocking buffer  at room temperature overnight. Primary antibody was removed and slices were washed three times in PBS. The secondary antibody [anti-rabbit IgG coupled to Alexa Fluor 568 (Thermo Fisher Scientific: z25306)] diluted 1:1500 and 4’,6-Diamidino-2-Phenylindole (DAPI) (Thermo Fisher Scientific: D1306) diluted 1:1000 all in PBS was added to each sample. The plates were covered in aluminum foil and incubated at room temperature for 1 h. The secondary antibody and DAPI were removed and slices were washed three times in PBS. Floating sections were then transferred to labeled glass slides, mounted using DAKO mounting medium, and covered with cover glass. Slides were allowed to dry at room temperature and then stored at 4°C.Serial sections of Double staining for co-localization was conducted using the same protocol. Anti-mitochondrial ATP synthase subunit c 1:100 (Abcam: ab181243) and anti-lysosomal associated membrane protein 1 (LAMP-1) 1:1000 (Santa Cruz: sc-20011) diluted in blocking buffer were used as the primary antibodies. The secondary antibodies were: anti-rabbit IgG coupled to Alexa Flour 568 (Thermo Fisher Scientific: z25306) 1:1500 and anti-mouse IgG coupled to Alexa Fluor 488 (Thermo Fisher Scientific: z25002) 1:1500.All slides were viewed and images collected using a Nikon Eclipse Ni-E microscope equipped with Photometrics CoolSNAP MYO CCD camera. Cerebral and cerebellar sections were viewed and imaged using a 20X-objective. Cortical regions of cerebral sections were visually divided into superficial and deep cortical layers. Four images were collected per superficial and deep cortical layers per cerebral section. This approach was used for all cerebral immunostainings. Sections double immunostained for co-localization were viewed and imaged using an oil immersion 100X objective.Images were analyzed and processed with the Cellomics Scan (Thermo Fisher Scientific) software using the Spot Detector BioAssay (Version 4). Subunit c positive staining was measured in a region of interest created based on the DAPI signal and expanded by 10 pixels using a threshold based analysis. Individual cells were then identified and counted based on the nuclei (DAPI), subunit c signal intensity was then measured in the region of interest and positive puncta were counted and measured for size. Averages of 6000 cells were counted per group. In GFAP analysis, cells stained positive for GFAP were used to measure the total staining intensity, the total area of GFAP staining, and the average area of a GFAP positive cell. In Iba1 analysis, cells stained positive for Iba1 were used to measure the total staining intensity, the average area of a Iba1 positive cell, and the average staining intensity per cell.All data was graphed and statistically analyzed using GraphPad PRISM Version 5.04 software.CLN2 mutations [CLN2 p.Arg208X directed us to generate the murine equivalent, Cln2R207X.To establish mutation type frequencies  in CLN2 disease patients, the University College London’s online NCL mutation database was assessed for recorded utations . Missensutations , 31. TheCln2R207X/R207X mouse model generation is represented in Cln2R207XNeo/+ mouse founders. To create these founders, a Cln2 targeting vector carrying a C to T transition in exon 6 and a Neomycin selection cassette flanked with FRT sites between exon 6 and exon 7, all within Cln2, was placed into mouse embryonic stem cells. Through homologous recombination the targeting vector inserted into the embryonic stem cell genome. Cln2R207XNeo/+ mouse founders were then bred to create Cln2R207XNeo/R207XNeo. The Neomycin selection cassette was removed via FRT-Flippase mediated excision by breeding Cln2R207XNeo/R207XNeo mice with FLPo-10 mice obtained from Jackson Laboratory. The Cln2R207X/+ mice obtained following Neomycin cassette removal were then used to generate our Cln2+/+ and Cln2R207X/R207X colonies. Sequencing results confirmed preservation of the point mutation . Cln2R207X/R207X mice demonstrated a 60–90% reduction in Cln2 transcript levels when compared to Cln2+/+ mice (Transcripts containing nonsense mutations/premature termination codons (PTCs) are targeted for degradation via the nonsense mediated decay (NMD) pathway; thus minimalizing the abundance of PTC-containing transcripts and reducing the production of truncated and potentially harmful proteins. To assess NMD in +/+ mice .Cln2 transcript reduction, the activity of TPP1, the enzyme encoded by Cln2, should be significantly reduced in Cln2R207X/R207X mice. Using a previously established TPP1 fluorogenic enzyme activity assay, TPP1 activity was evaluated in Cln2R207X/R207X mice  were used to assess the activity of palmitoyl-protein thioesterase 1 (PPT1), the lysosomal enzyme associated with cINCL, and sphingomyelinase, the lysosomal enzyme associated with Niemann-Pick disease. Overall, Cln2R207X/R207X mice were found to have similar PPT1 and sphingomyelinase activity when compared to Cln2+/+ mice . This device tracks the movement of mice by tracing the changes in the force applied to the plate. Several parameters including distance traveled, area covered, number of left and right turns, focused stereotypes, low mobility bouts, and tremor index are measured. The average force, closely related to the weight of the mice, did not differ between Cln2+/+ and Cln2R207X/R207X mice at 1 and 3 months . Nevertheless, a principal component of cLINCL storage material is mitochondrial ATP synthase subunit c, a substrate of TPP1 [Cln2+/+ and Cln2R207X/R207X brain sections were evaluated for mitochondrial ATP synthase subunit c accumulation. The cerebral cortex of Cln2R207X/R207X mice had a pronounced accumulation of mitochondrial ATP synthase subunit c throughout superficial and deep cortical layers; whereas, Cln2+/+ brain sections had little immunoreactivity , increased frequency per cell , and a greater size   . Likewis to none . To confh LAMP-1 .Cln2+/+ and Cln2R207X/R207X cerebral sections were immunostained for GFAP. Superficial and deep cortical layers in sections from Cln2R207X/R207X mice demonstrated significantly increased GFAP immunostaining as well as hypertrophy of astrocytes when compared to sections from Cln2+/+ mice (Cln2R207X/R207X mice were found to have a 2.52 fold increase in GFAP intensity (p = 0.0008) and a 2.04 fold increase in mean astrocyte area (p = 0.0002), whereas deep cortical layers of Cln2R207X/R207X mice showed a 3.12 fold increase in GFAP intensity (p = 0.0004) and a 2.19 fold increase in mean astrocyte area (p < 0.0001)  . An addilia size . AlthougCln2+/+ and Cln2R207X/R207X mice were sectioned, hematoxylin/eosin stained, and sent to a veterinary pathologist for examination. The veterinary pathologist was blinded from all identifying information. According to the pathology report, the brain was the only tissue with identifiable abnormalities which consisted of hypereosinophilic granules and sporadic neurodegeneration  from 4-month-old neration . ConsequCln2-/- mouse model which carries an uncommon missense mutation [Cln2-/- mice are not suitable to test therapies such as read-through compounds, nonsense mediated decay inhibitors, and clinically relevant mutation targeting antisense oligonucleotides. Therefore, the primary purpose of this study was to generate an alternative model of CLN2 disease that allows for the evaluation of all therapies including those previously excluded.Current preclinical therapeutic studies for CLN2 disease utilize the mutation . Cln2-/-Cln2R207X/R207X mouse was rigorously characterized to confirm the presence of CLN2 disease-associated anomalies. Molecular and biochemical characterization confirmed that the presence of the p.R207X mutation targeted Cln2 transcripts for the governing nonsense mediated decay pathway; this reduction in combination with TPP1 truncation resulted in a significant decrease in ubiquitous TPP1 activity. These alterations in TPP1 activity are similar to what is seen in CLN2 disease patients [Cln2R207X/R207X cerebral and cerebellar sections did not show autofluorescent storage material at the same time point as symptomatic onset (3 months). Nevertheless, similar sections strongly demonstrated the accumulation of mitochondrial ATPase synthase subunit c. Co-localization of mitochondrial ATPase synthase subunit c and LAMP-1 indicates lysosomal accumulation of non-autofluorescent material prior to the accumulation of autofluorescent components. Secondary pathological findings such as astrocytosis in 3-month-old Cln2R207X/R207X cerebral sections confirm previous findings of NCL. Overall, both biochemical and histological assessments of Cln2R207X/R207X mice demonstrate similar findings to the Cln2-/- mouse model and CLN2 disease patients.The new model was chosen to have the most common nonsense mutation that occurs in CLN2 disease patients, p.R207X. To be considered a model of CLN2 disease, the patients , 58, 59.patients , 60–62. Cln2R207X/R207X mice identified motor and strength deficits starting at 3 months of age. Unfortunately most Cln2R207X/R207X mice did not survive to be tested for motor coordination at either 4 or 5 months, those that could be tested failed immediately. In addition to motor and strength deficits, Cln2R207X/R207X mice at 3 months of age developed a tremor that progressively worsened and became significantly debilitating. This behavioral anomaly is being characterized as a tremor based on presentation; although it cannot be ruled out as seizure activity without further specialized testing. Overall, these behavioral abnormalities contribute to a significant shortening of the Cln2R207X/R207X mouse lifespan. We tried to test visual acuity  in 1 and 3 month old wildtype and mutant mice. However, the hyperactivity of the mutant mice prohibited adequate assessment of visual acuity. Cln2R207X/R207X mice will be given to an expert in NCL vision loss and retinal pathology to investigate visual changes in these new Cln2 mutant mice.Severe deficiency in TPP1 activity and neuropathological abnormalities lead CLN2 disease patients to develop incapacitating neurological deficits and ultimately death , 16. BehCln2R207X/R207X mice clearly demonstrates the successful generation of a new CLN2 disease model. Unlike previous mouse models, this model carries a common disease causing CLN2 mutation. In addition, this model allows for the assessment of all therapies. Preclinical studies investigating the use of read through compounds and nonsense mediated decay inhibitors have shown positive outcomes in other disease models including lysosomal storage disorders [Cln2R207X/R207X mouse model, preclinical studies into these therapies could not be evaluated for CLN2 disease. The generation of this new CLN2 disease model advances preclinical studies into CLN2 disease therapies.Collectively, the biochemical, histological, and behavioral characterization of isorders , 63–69. S1 FigCln2+/+ (n = 3) and Cln2R207X/R207X (n = 3) mice. In the sphingomyelinase activity assay, three technical replicates were performed using the three biological samples obtained from Cln2+/+ and Cln2R207X/R207X mice; whereas, the PPT1 activity assays used four technical replicates from the three biological samples. Cln2R207X/R207X sphingomyelinase and PPT1 activity was normalized to Cln2+/+ activity levels. Columns and bars represent mean ± SEM. Statistical significance was determined using an unpaired t-test (*p < 0.05).Fluorogenic enzyme activity assays for sphingomyelinase (A) and PPT1 (B) were used to measure endogenous activity in five different tissues from 1-month-old (TIF)Click here for additional data file.S2 FigCln2+/+ (n = 14) and Cln2R207X/R207X (n = 20) mice. Both cohorts spent similar amounts of time in the dark. Columns and bars represent mean ± SEM. Statistical significance was determined using an unpaired t-test.The light/dark box test was used to assess for anxiety in 3-month-old (TIF)Click here for additional data file.S3 FigCln2R207X/R207X mice when compared to Cln2+/+ controls. Pronounced accumulation is present in the Purkinje cell layer (at the border of ML and GL) in Cln2R207X/R207X mice.Images of the molecular (ML) and granular (GL) cerebellar layers demonstrate accumulation of mitochondrial ATP synthase subunit c in (TIF)Click here for additional data file.S4 FigCln2R207X/R207X cerebral sections immunostained with anti-subunit c (red) and anti-LAMP-1 (green) reveal co-localization.(TIF)Click here for additional data file.S5 FigCln2+/+ and Cln2R207X/R207X mice show minimal differences in immunostaining for the microglial marker, Iba1. (B) Images from 3-month-old Cln2+/+ (n = 4) and Cln2R207X/R207X (n = 5) mice were blindly collected and analyzed for Iba1 immunostaining total intensity, average intensity per Iba1 positive microglia, and average microglia size. Columns and bars represent mean ± SEM. Statistical significance was assessed using an unpaired t-test.(A) Superficial and deep cortical layers from 3-month-old (TIF)Click here for additional data file.S6 FigCln2+/+ (n = 3) and Cln2R207X/R207X (n = 3) mice were sectioned, hematoxylin/eosin stained, and evaluated by a veterinary pathologist under blinded conditions. The pathology report solely identified cerebral cellular hypereosinophilic inclusions (arrows) and signs of neurodegeneration (arrow heads) in Cln2R207X/R207X mice (A-C) when compared to Cln2+/+ controls (D).Tissues from (TIF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.


Toxoplasma gondii, as a zoonotic protozoan parasite, develops sophisticated strategies to manipulate hosts for efficient intracellular survival. After successful invasion, T. gondii injects many effector proteins into host cells for various purposes. TgROP16 (T. gondii rhoptry protein 16), which is secreted from rhoptries into host cells, can activate the host STAT  signaling pathway through phosphorylation of STAT3 and STAT6. However, whether there are other host proteins modulated by TgROP16 is currently unknown. In this study, yeast two-hybrid (Y2H) screen was used to look for additional host proteins interacting with TgROP16. Yeast cells expressing a mouse cDNA library cloned into the prey vector were used to mate with yeasts expressing ROP16 without signal peptide. Two mouse proteins, Dnaja1 (DnaJ heat shock protein family member A1) and Gabra4  were identified to interact with ROP16 from this screen. Further analysis suggested that the Predomain of ROP16 played key roles in mediating interactions with these host proteins, whereas the contribution from the Kinase domain was minor. The interactions between Dnaja1 and different parts of ROP16 were also estimated in vivo by co-immunoprecipitation. The results showed that the Predomain of ROP16 was the major region to interact with Dnaja1, which is consistent with the Y2H results. Based on the gene ontology analysis, Dnaja1 is predicted to participate in stress response while Gabra4 is involved in the system development process. The discovery of new host proteins that interact with ROP16 of T. gondii will help us to further investigate the functions of this effector proteins during T. gondii infection. Toxoplasma gondii, as a model organism of Apicomplexa parasites, can infect all warm-blooded animals and result in toxoplasmosis worldwide  cells. Tachyzoites were harvested and purified by filtration through 3 μm membranes  before total RNA extraction. Seven-week-old female Kunming mice, were purchased from the Center for Disease Control (CDC) of Hubei Province in China for mice brain tissue RNA extraction. HEK293T cells were cultured in RPMI 1640 medium  supplemented with 10% FBS  and 1% penicillin/streptomycin .ROP16-F (5′-GAATTCCGATACATGTCGTTTGAGGAAGC) and mROP16-R (5 ′-GTCGACGCTACATCCGATGTGAAGAAAG) for full length mature ROP16; ROP16-Pre-F (5′-GAATTCCGATACATGTCGTTTGAG) and ROP16-Pre-R (5′-GTCGACAGCGATCGGCACT) for the Predomain; ROP16-Kinase-F (5′-GAATTCCTATACAATCGTGGGCAC) and ROP16-Kinase-R (5′-GTCGACCTACATCCGATGTGAAGA) for the Kinase domain. These PCR products were then digested with EcoRI and SalI, and then ligated into similarly digested pGBKT7.Total RNA was extracted from purified RH tachyzoites using TRIzol Reagent  according to manufacturer’s instructions. Then one microgram of total RNA was reverse transcribed to cDNA using the ReverTra Ace reverse transcription kit . The bait plasmids expressing different parts of TgROP16 were constructed by cloning one of the following three fragments of ROP16 into the pGBKT7 vector: the 2055 bp fragment encoding the full length mature ROP16 , the 1056 bp fragment encoding the Predomain  and the 999 bp fragment encoding the Kinase domain . Using the cDNA above as templates, these three fragments were PCR amplified using specific primers mROP16-F (5′–GGAATTCATGGAGGAGCAGAAGCTGATCTCAGAGGAGGACCTGCGATACATGTCGTTTGAGGA) and pCDNA3.1-ROP16-R (5′-CCGCTCGAGCTACATCCGATGTGAAGAAAGTT) for Myc tagged full length mature ROP16; pCDNA3.1-ROP16-F and pCDNA3.1-ROP16-Pre-R (5′-CCGCTCGAGCTAAGCGATCGGCACTCCGTTGG) for Myc tagged Predomain of ROP16; pCDNA3.1-ROP16-Kinase-F (5′-GGAATTCATGGAGGAGCAGAAGCTGATCTCAGAGGAGGACCTGCTATACAATCGTGGGCACCT) and pCDNA3.1-ROP16-R for Myc tagged Kinase domain of ROP16. The corresponding PCR products were digested with EcoRI and XhoI sites, and cloned into pCDNA3.1 vector.For co-immunoprecipitation (Co-IP) assays, the three fragments of ROP16 mentioned above were tagged with Myc epitope and cloned into the pcDNA3.1 vector. The primers used to amplify these fragments for cloning were listed as follow: pCDNA3.1-Dnaja1 gene from mouse, total RNA was extracted from mouse brain and reverse-transcribed into cDNA as above. Subsequently, the Dnaja1 gene was amplified from cDNA using Dnaja1-F (5′-GCTAGCGCTACCGGACTC AGATGGGCAAGGACTACTATCA) and Dnaja1-R (5′-TCCTCGCCCTTGCT CACCATTATGGGAAGAACCTGCTCCA) as primers, and was cloned into pEGFP-N1 using ClonExpress homologous recombinase . The linearized vector for such cloning was generated by amplification of pEGFP-N1 using primers pEGFP-N1-F (ATGGTGAGCAAGGGCGAGGA) and pEGFP-N1-R (CTGAGTCCGGTAGCGCTAGC).To clone the Saccharomyces cerevisiae strain Y2H Gold according to the Yeastmaker Yeast Transformation System 2 User Manual . Transformants were then plated onto agar plates containing appropriate selection media, including SD/-Trp, SD/-Trp/X  or SD/-Trp/X/A . The plates were incubated at 30°C for 3 days and the growth (color and size) of transformants were recorded. The empty vector pGBKT7 was included as a control.To test autoactivation and toxicity of bait protein TgROP16 in yeasts, bait plasmids containing the above mentioned three fragments of ROP16 were individually transformed into 600 ≈ 0.6. Then total proteins were extracted using the Urea/SDS method  were inoculated into SD/-Trp broth and cultured until ODS method  and subsSaccharomyces cerevisiae Y2H Gold and Y187 strains, as well as the medium were purchased from Clontech Co.  for Y2H screen. The Mate & Plate Universal Mouse  cDNA library  was cloned into the pGADT7-RecAB vector and expressed in Y187 yeast cells. To screen host proteins interacting with ROP16, Y2H Gold cells expressing ROP16 and Y187 cell expressing mouse proteins were mixed to mate for 20–24 h at 30°C. Subsequently the mated cultures were plated onto SD/-Leu/-Trp plates supplemented with 40 μg/ml X-α-Gal and 125 ng/ml Aureobasidin A (DDO/X/A) and incubated at 30°C for 3 days, part of the mated cultures were also plated on SD/-Trp, SD/-Leu, SD/-Trp/-Leu (DDO) plates to calculate the number of clones and the mating efficiency according to manufacturer’s instructions. Afterwards, blue colonies were picked up and tested on higher stringency SD/-Ade/-His/-Leu/-Trp/X-α-Gal/AbA (QDO/X/A) plates.1 to further estimate the function of the mouse proteins.To identify the genes in positive hits, prey plasmids in positive hits were extracted using TIANprep yeast plasmid DNA kit , then sequenced by T7 primer and 3′ AD primer. Sequencing results were blasted with NCBI databases to obtain gene information. Meanwhile, according to GO Classifications, molecular function and biological process of the prey genes were performed by searching the Mouse Genome Informatics (MGI) databaseTo further confirm the potential positive hits, bait plasmids containing ROP16 and prey plasmids were transformed into Y2H Gold and Y187 strains, respectively, according to manufacturer’s instructions for small-scale transformation. Transformants were cultured on SD/-Trp and SD/-Leu plates for 3 days at 30°C and single clones picked from each plate were used for mating. Mated heterozygotes were spread on SD/-Leu/-Trp/X-α-Gal (DDO/X) and QDO/X/A plates and grown for 3–5 days at 30°C. The empty bait vector pGBKT7 without ROP16 was used as a negative control.To study the interaction between the prey protein and different domains of TgROP16, different fragments of ROP16 cloned in pGBKT7 and prey plasmids were transformed into Y2H Gold and Y187 strain, respectively. Singles clones were mated and tested as above.in vivo, plasmids pcDNA3.1 containing different parts of ROP16 and prey plasmid pEGFP-N1 containing mouse gene Dnaja1 were co-transfected into HEK293T cell for Co-IP assay. Bait and prey plasmids were mixed at the ratio of 1:1 and transfected into HEK293T cells using Lipofectamine 2000  according to the manufacturer’s instructions. Empty vectors were included as controls. The tranformants were grown for 28–36 h, harvested and lysed by ultrasonication in RIPA buffer  containing 1 mM phenylmethylsulfonyl fluoride  and protease inhibitor cocktail . Supernatants of cell lysates were incubated with mouse anti-Myc tag antibody  overnight, followed by Protein G agarose beads  incubation at 4°C for 6 h. Subsequently, the agarose beads were washed with RIPA buffer and proteins bound to agarose beads were solubilized in 1× SDS-sample buffer containing dithiothreitol and boiled for 10 min. Using Western blotting method, ROP16 was detected by mouse anti-Myc tag antibody and the bound Dnaja1 were detected by mouse anti-GFP . GAPDH detected by a mouse monoclonal antibody  was included as a loading control.To verify the identified protein–protein interactions T. gondii ROP16, three different fragments of ROP16, including the mature full length ROP16 without signal peptide (mROP16), the Predomain (ROP16-Predomain) and the Kinase domain (ROP16-Kinase)  were amplified from cDNA of RH tachyzoites and cloned into the pGBKT7 vector. These ROP16 expressing constructs were individually introduced into yeast cells and used as baits for two-hybrid screens. Before the screen, the autoactivation and toxicity of ROP16 in yeasts were estimated. To do that, bait plasmids expressing mROP16, ROP16-Predomain, ROP16-Kinase domain were transformed into Y2H Gold yeast cells, respectively. Transformants were grown on SD/-Trp, SD/-Trp/X, or SD/-Trp/X/A plates . Toxicity was indicated by poor growth on SD/-Trp plates, whereas autoactivation was determined by growth of colonies on SD/-Trp/X/A plates and blue colonies on SD/-Trp/X plates. Compared with the empty vector pGBKT7 control, all bait plasmids transformed yeast cells were growing well on SD/-Trp plates. Colonies on SD/-Trp/X plates were white or orange colored instead of blue. No colony growth on SD/-Trp/X/A plates was observed . Taken together, these data indicated that no obvious autoactivation and toxicity were detected for ROP16 baits.To screen for mouse proteins interacting with Figure 1C). The molecular weight of the mROP16 bait protein was approximately 111 kDa composed of mROP16 protein (90 kDa) fused to the DNA-BD protein (21 kDa), which was consistent with what was detected on the Western blot.To further check the expression of ROP16 bait in yeasts, Y2H Gold cells transformed with mROP16 or the empty vector were subject to Western blot analysis. Total proteins were extracted from transformed cells by the Urea/SDS method. Western blotting analysis was performed using a Myc-tag antibody. The results indicated that mROP16 and control protein DNA-BD were expressed successfully in Y2H Gold cells . From the colonies grown on DDO/X/A plates, thirty blue clones were selected and tested on higher stringency QDO/X/A plates. Two clones (Clones #9 and #19) out of thirty still maintained blue  while the other 28 failed to give blue colonies, indicating that two potential positive hits were obtained.To screen the host proteins that interact with TgROP16, Y187 yeast cells expressing a mouse cDNA library were used to mate with Y2H Gold cells expressing mROP16, as described in Section “Materials and Methods”. According to the manufacturer’s instruction, mating efficiency was calculated to be 13.3%, while 4.6 × 10Figure 2D). Further sequencing of the inserts and Blast analysis showed that clone #9 contained the Dnaja1 (AB028273) gene and clone #19 contained the Gabra4 (NM_010251) gene. According to GO classification, Dnaja1 likely participates in biological processes  and performs molecular function like carbohydrate derivative binding, enzyme regulation, and receptor binding. Gabra4, on the other hand, may be involved in system development as a receptor or a transporter.Plasmids in the two positive clones were extracted and PCR amplified using the T7 primer and 3′ AD primer to estimate the sizes of inserts. Gels electrophoresis results indicated that clone #9 contained a 1.5 Kb insert, whereas clone #19 had an insert of 2.1 Kb . Dnaja1 also interacted with the Predomain of ROP16. But the interaction was probably not as strong as with full length ROP16, since less blue colonies were observed during ROP16-Predomain mating . Gabra4, on the other hand, interacted with both full length and the Predomain of ROP16 .To examine the interactions between Dnaja1/Gabra4 and each domain of ROP16, ROP16-Predomain and ROP16-Kinase domain expressing plasmids were transformed into Y2H Gold cells, respectively, and mated with Dnaja1 or Gabra4 expressing Y187 cells. Mated cultures were plated on the above-mentioned indication plates. The results showed that Dnaja1 mainly interacted with full length ROP16, as blue colonies were only observed during mROP16 mating  and detected by anti-GFP monoclonal antibodies . To assess protein interactions by Co-IP, each of the ROP16 fragment expressing plasmids was mixed with pEGFP- Dnaja1 at the ratio of 1:1, and co-transfected into HEK293T cells. Cell lysates prepared from transfectants 24 h post transfection were immunoprecipitated with an anti-Myc antibody. The GFP-Dnaja1 protein bound to precipitated ROP16 was detected by an anti-GFP antibody. Consistent with the Y2H results, GFP-Dnaja1 was co-precipitated with full length mature ROP16, as well as the Predomain of ROP16 . Dnaja1 did not show obvious co-precipitation with the Kinase domain of ROP16, consistent with the lack of interaction of the two . The interactions detected by Co-IP were likely to be specific, since negative controls using GFP alone or Myc tag alone did not give detectable Co-IP products . Together these results indicated that Dnaja1 was able to be interact with full length, as well as Predomain of ROP16.In order to verify the interactions between ROP16 and host proteins T. gondii rhoptry protein ROP16 was involved in the activation of host STAT signaling pathway when it reached host cell nuclei  in most vertebrates . Many prll lines . Previoull lines  as well ll lines . Thus, Dll lines . Moreovell lines , while Hll lines . With thGabra4, which encodes the α4 subunit of gamma-aminobutyric acid A receptor, belong to alcohol-regulated genes (ARGs) that show remarkable plasticity in response to alcohol . Previous studies demonstrated that type I and type III ROP16 could modify STAT3 by phosphorylation, but type II ROP16 could not. A single polymorphic amino acid (L503S) in the Kinase domain of ROP16 determined STAT3 activation (Toxoplasma infection.As a protein kinase, ROP16 is composed of a Predomain and Kinase domian with a signal peptide at the N-terminus (tivation . On the In summary, two mouse proteins, Dnaja1 and Gabra4 were identified to interact with TgROP16 by Y2H screen. The interaction between ROP16 and Dnaja1 was further verified by Y2H, as well as Co-IP methods. These results provided important clues to further investigate the functions of TgROP16.JZ conceived and designed the study. JZ and YZ revised the manuscript critically. MP performed the studies and wrote the manuscript. YW assisted in the yeast two-hybrid screen. LL, YS, and LH contributed to yeast mating and Co-IP assay. All authors read and approved the final manuscript.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.


Similar to its parent strain, P10 remained no transmissible between guinea pigs. Using genome sequencing and alignment, multiple amino acid substitutions, including PB2 E627K, PB2 T23I, PA T97I, and HA R239H, were found in the adaptation of H5N6 to mice. In summary, we identified amino acid changes that are associated with H5N6 adaptation to mice.At least 15 cases of human beings infected with H5N6 have been reported since 2014, of which at least nine were fatal. The highly pathogenic avian H5N6 influenza virus may pose a serious threat to both public health and the poultry industry. However, the molecular features promoting the adaptation of avian H5N6 influenza viruses to mammalian hosts is not well understood. Here, we sequentially passaged an avian H5N6 influenza A virus (A/Northern Shoveler/Ningxia/488-53/2015) 10 times in mice to identify the adaptive amino acid substitutions that confer enhanced virulence to H5N6 in mammals. The 1st and 10th passages of the mouse-adapted H5N6 viruses were named P1 and P10, respectively. P1 and P10 displayed higher pathogenicity in mice than their parent strain. P10 showed significantly higher replication capability  Due to the antigenic differences of HA and NA, influenza A virus is classified into 18 HA subtypes and 11 NA subtypes. Except for H17N10 and H18N11 isolated from bats , all infAvian H5N6 influenza virus was first isolated from mallards in North America in 1975 . In ChinMice are the most widely used animal model for studying the pathogenesis and mammalian adaptation of avian influenza A viruses , for whiIn 2015, we isolated a novel H5N6 virus, named A/Northern Shoveler/Ningxia/488-53/2015, from wild birds fences in Ningxia, China. To identify the amino acid changes related to the adaptation of this H5N6 virus to mammal hosts, we sequentially passaged the virus 10 times in mice and identified the amino acid substitutions associated with the adaptation of H5N6 to mice. We also evaluated the pathogenicity and transmissibility of mouse-adapted H5N6 viruses.All animal studies were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals of the Military Veterinary Research Institute. All animal experiments were approved by the Animal Care and Use Committee of the Military Veterinary Research Institute.50) using the Reed–Muench method. All experiments involving viruses were performed in Animal Biosafety Level-3 (ABSL-3) facilities.The avian H5N6 influenza virus (A/Northern Shoveler/Ningxia/488-53/2015) was isolated from wild birds fences in Ningxia, China and stored in our laboratory. All viruses were grown in specific-pathogen-free (SPF) embryonated eggs and stored at -80°C. Viruses were titrated in 9-day-old embryonated eggs to determine the median egg infectious dose  with 50 μL of 10Viral RNAs were extracted from the homogenized lung tissue supernatants using an RNeasy Mini-kit  according to manufacturer’s protocol. Viral RNAs were reverse transcribed into cDNAs using influenza virus-specific U12 primers and an RT reagent kit  and viral genes were amplified using a PCR kit  according to the manufacturer’s protocol. The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 min, then followed by 35 cycles of 94°C for 40 s, 55°C for 40 s, 72°C for 2 min, prior to a final extension step at 72°C for 10 min. The primers used for PCR are listed in Supplementary Table 6 EID50/mL. PBS by itself was used as the control. Body weight changes and mouse survival rate were monitored daily for 14 days, and mice that lost more than 25% of their body weight were euthanized.To evaluate the pathogenicity of the mouse-adapted H5N6 influenza viruses, groups of six 6-week-old female BALB/c mice were anesthetized with ether and inoculated i.n. with 50 μL of two mouse-adapted viruses passage 1 (P1) and passage 10 (P10) and WT H5N6 virus at 10in vivo, groups of 12 six-week-old female BALB/c mice were inoculated i.n. with 50 μL of the P1, P10, and WT H5N6 virus at 106 EID50/mL, while three naïve mice were inoculated with PBS as control. On days 3 and 5 post-infection (dpi), three mice were sacrificed and tissue samples, including lung, heart, kidney, spleen, liver, and brain tissues, were harvested in 1 mL of PBS. These tissue samples were then homogenized and centrifuged, viral titers in the homogenized tissue supernatants were determined by the Reed–Muench method using 9-day-old embryonated eggs.To detect the tissue distribution of mouse-adapted influenza virus variants To assess pathological lesions from mice infected with the mouse-adapted influenza virus variants, their lungs were harvested at 3 dpi and fixed with 4% formalin. The sectioned lungs were stained with hematoxylin and eosin and then subjected to pathological examination with light microscopy.6 EID50/mL and then housed in a cage inside an isolator. Three naïve guinea pigs were co-housed with the three infected guinea pigs 24 h post-inoculation, and nasal washes were collected from each guinea pig every other day, beginning at 2 dpi. Viruses in the nasal washes were titrated in 9-day-old embryonated eggs and the experiment conditions for these studies were set at 19–23°C with 30–50% relative humidity.To measure the contact transmissibility of a mouse-adapted influenza virus variant, three guinea pigs were inoculated i.n. with 200 μL of P10 virus (100 μL in each nostril) at 106 EID50/mL. Each inoculated guinea pig was housed in a separate cage placed in an independent isolator. The next day, three naïve guinea pigs were paired with the three inoculated guinea pigs. Each naïve guinea pig was housed in a new cage adjacent to the cage housing its respective infected partner, and the distance between the two adjacent cages was 5 cm apart. Nasal washes were collected from each guinea pig every 2 days, beginning at 2 dpi. Viruses were titrated in embryonated eggs.To evaluate the aerosol transmissibility of a mouse-adapted influenza virus variant, three guinea pigs were inoculated i.n. with 200 μL of P10 virus in PBS (100 μL in each nostril) at 10P-values less than 0.05 indicated significant differences.Quantitative data were analyzed with GraphPad Prism 5.0 software using the one-way or two-way ANOVA method, and statistical comparisons between two groups were tested using the Student–Newman–Keuls (SNK) method. Survival analysis data were analyzed with GraphPad Prism 5.0 software using log-rank tests. 6 EID50/mL WT H5N6. Three days later, the infected mice were sacrificed, and their homogenized lung tissue supernatants were subsequently used to inoculate i.n. the next three mice (passage 2). These procedures were repeated until passage 10, and 10 passages of mouse-adapted H5N6 viruses were isolated from the homogenized lung supernatants.To study the adaptation of H5N6 virus isolated from wild birds to mammals, we serially passaged WT H5N6 influenza A virus in mice. Three female BALB/c mice (passage 1) were first inoculated i.n. with 50 μL of 106 EID50/mL. Body weight loss and mortality were successively monitored for 2 weeks, and the PBS-inoculated group served as the control. All mice infected with WT virus survived, and their average body weight decreased approximately 10% at 1 dpi before gradually recovering to normal . In contrast, all mice inoculated with P10 virus rapidly lost weight and succumbed to death by 7 dpi, while mice infected with P1 virus showed moderate pathogenicity . The body weights of mice infected with P1 virus were rapidly reduced approximately 25% during the first 4 dpi before finally recovering to the normal state . Two of six mice infected with P1 virus died .We compared the pathogenicity of WT H5N6 with mouse-adapted P1 and P10 viruses. Groups of six 6-week-old female BALB/c mice were infected with 50 μL of P10, P1, or WT viruses at 10Figure 2B). A slight infiltration of inflammatory cells and alveolar wall thickening was observed in P1-infected mice  while acidophilic protein-like exudation, epithelial cell shedding and moderate pneumonia with neutrophilic granulocyte infiltration were observed in P10-infected mice . No obvious histopathological changes were observed in the negative control (mock infected) mice . Histological analysis demonstrated that the mouse-adapted viruses exhibited more severe histopathological changes than the WT-infected mice.Additionally, the WT-infected mice showed no obvious pathological changes, as determined by histopathological analysis . In contrast, P10 replicated very well in mouse lungs, with titers approximately 5.1 and 5.5 log10 EID50 at 3 and 5 dpi , which was approximately 1000-fold higher than that of WT virus. P1 replicated in the lungs to a titer of 3.6 and 3.8 log10 EID50 at 3 and 5 dpi , which was much higher than that of WT virus. Therefore, the mouse-adapted H5N6 viruses, especially P10, displayed much higher replication capability in mouse lungs. Systemically, in addition to being detected in the lungs of mice, WT virus was detected in their hearts, livers, and kidneys at only 5 dpi, while it was undetectable in their spleens and brains . In sharp contrast, except for the brain at 3 dpi, P10 was found in all tested tissues . Specifically, P10 was detected in the brains of mice at 5 dpi, with a higher titer of 4.4 log10 EID50 being observed , whereas no virus was detected in the brains of mice infected with P1 in the 7 days of animal experiments. Thus, the mouse-adapted H5N6 viruses, especially the P10 virus, displayed higher replication abilities than their parental virus strain in mice.To determine whether the replication capacities of mouse-adapted H5N6 viruses were significantly enhanced, we compared the replication capabilities and system viral spreads of two mouse-adapted H5N6 viruses with WT virus. Groups of six 6-week-old female BALB/c mice were infected with P10, P1, and WT viruses. At 3 and 5 dpi, three mice were sacrificed and their lungs, hearts, livers, kidneys, spleens, and brains were harvested. Viral titers in the collected tissues were determined by the Reed–Muench method. WT virus could replicate in mouse lungs, with titers ranging from 1.5 to 2.5 log6 EID50/mL P10. The next day, three naïve guinea pigs were co-housed with the three infected animals. To assess the virus transmission in guinea pigs, nasal washes from all guinea pigs were collected at 2-day intervals, and the viruses were titrated in 9-day-old embryonated eggs. Viruses were detected in all nasal washes from the inoculated guinea pigs from 1 to 5 days post-contact, but no virus was detected in the three naïve guinea pigs of the contact group .We evaluated the transmissibility of P10 in guinea pigs by contact. Three guinea pigs were inoculated i.n. with 200 μL of 106 EID50/mL P10 virus. Twenty-four hours later, three naïve guinea pigs were paired with the three inoculated guinea pigs and each naïve guinea pig was housed in a new cage adjacent to the P10-infected guinea pigs at a distance of 5 cm. Viral titers in the nasal washes collected from all guinea pigs at different time points post-exposure were determined by Reed–Muench method. No virus was found in nasal washes of the naïve guinea pigs of the aerosol group . Therefore, similar to its parental strain WT H5N6 strain, P10 did not acquire the ability to be transmitted between guinea pigs by contact or aerosol.We also measured the aerosol transmissibility of P10 in guinea pigs. Three guinea pigs were inoculated i.n. with 200 μL of 10Table 1). Most (five of eight) amino acid changes, including HA I67V, PA S388C, PB2 E627K, NP H52Y, and NP A430T, first occurred in the P1 virus, indicating that the WT H5N6 virus rapidly adapted in the presence of selective pressure in mice. PB2 T23I was identified in P2, HA R239H was found in P6 and T97I was first observed in P7.To identify the amino acid substitutions associated with the enhanced pathogenicity and replication, the genomes of viruses from all 10 passages were amplified by PCR and sequenced. Eight amino acid substitutions were identified, two in the HA subunit , two in the polymerase basic protein 2 (PB2) subunit , two in the polymerase acidic (PA) subunit  and two in the nucleoprotein (NP) subunit  . The other four mutations occurred in P1 was first identified in our work and their functions have yet to be explored. Thus the adaptation of different influenza A virus isolates to mice varied greatly.The PB2 E627K substitution found in P1 has been identified in many other mouse-adapted influenza viruses, including H5N1, H7N9, and H9N2 . This amThe PA T97I substitution observed in P7 has also been identified in many other mouse-adapted influenza A viruses, such as H5N2, H6N1, H7N9, and H10N7 . This muThe PB2 subunit of influenza virus polymerase bound to mitochondrial antiviral signaling protein (MAVS) through its N-terminal domain, specifically to its first 37 amino acids, to inhibit IFN-β expression . The PB2R239H (227 in H3 numbering) found in P6 was located in the 220 loop of influenza virus receptor binding sites. A previous study demonstrated that the HA Q227P substitution increased the ability of guinea pig-adapted H9N2 to bind an avian α2,3 receptor and did not affect its binding ability to a human α2,6 receptor, and demonstrated the critical effects of this mutation for influenza virus transmission in guinea pigs by direct contact . The sidConsidering the potential pandemic threat of avian H5N6 viruses to public health, more attention should be paid to the mammalian adaptive processes of H5N6 viruses. In the present study, we first reported the adaptation of an avian H5N6 influenza virus isolated from wild waterfowl in mice. Multiple amino acid substitutions associated with the adaptation of H5N6 influenza virus to mice were identified, and they conferred enhanced pathogenicity and higher replication capability to H5N6 in mammals.Most amino acid changes first occurred in P1, the early WT H5N6 passage step in mice indicating that H5N6 isolated from wild birds adapted rapidly in mammals. Wild birds are the natural reservoirs of influenza A viruses and many H5N6 viruses have also been isolated from wild birds . With thThe RNA-dependent influenza A virus RNA polymerase mediates the transcription and replication of the viral genome. Because this enzyme lacks proof-reading capacity, viral replication is characterized by a high mutation rate which allows the evasion of host immune defense and adaptation to new hosts upon interspecies transmission. The adaptation of viral polymerase plays important roles in the virulence and interspecies transmission of influenza viruses, as was shown for H5N1 . In our Receptor binding preference is considered to be a very important factor for influenza A virus transmission in different animal models . The humIn summary, we serially passaged an avian H5N6 influenza A virus strain 10 times in mice. P10 showed enhanced pathogenicity, but acquired no transmissibility in mammals. The identified PB2 E627K and PA T97I amino acid substitutions were shown to play important roles in the adaptation of H5N6 to mammals. The HA R239H mutation may be important for the attachment of adapted influenza viruses to their hosts airways; however, the specific effects of these amino acid substitutions on mammalian pathogenicity requires further study. Our study highlights the threat of H5N6, and constant surveillance is needed to monitor the evolution of H5N6 in wild birds and domestic poultry. Specifically, amino acid changes within the HA subunit and the RNA-dependent RNA polymerase complex should be monitored to gain advanced warnings for a better preventing and controlling H5N6 outbreaks.CZ, ZZ, and ZG designed and drafted the work. CZ, ZZ, ZG, JZ, JL, YY, SL, ZW, MZ, YF, XY, LL (Lina Liu), and YZ performed the experiments, analyzed the data, and interpreted the results. JQ, HC, LL (Linna Liu), and YH designed the work and revised it critically.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.


Mycobacterium chimaera is a nontuberculous mycobacterium species commonly found in the environment. Here, we report the first complete genome sequence of a strain from the investigation of invasive infections following open-heart surgeries that used contaminated LivaNova Sorin Stockert 3T heater-cooler devices. Mycobacterium chimaera is a nontuberculous mycobacterium (NTM) species within the Mycobacterium avium complex (MAC). M. chimaera is an emerging cause of infection, in particular due to increasing awareness of infections linked with exposure to contaminated LivaNova Sorin Stockert 3T heater-cooler unit (HCU) devices  at 36°C for approximately 1 week. The genome was sequenced using the Pacific Biosciences RSII  and Illumina MiSeq  platforms. A 10-kb library was generated with the SMRTbell template prep kit 1. The library was then bound to polymerase using the DNA/polymerase binding kit P6v2. The bound library was loaded on two SMRTcells and sequenced with C4v2 chemistry for 360-min movies on the RSII instrument. Sequence reads were filtered and assembled ersion 3 . The reson 2.3.1 . GenomicM. chimaera CDC2015-22-71 consists of four scaffolds, equaling 6,247,640 bp  and a G+C content of 67.6%. A total of 5,795 coding sequences were predicted, including 5,627 protein-coding genes and 168 pseudogenes. Our genome assembly contains 47 tRNAs, 3 noncoding RNAs, and 1 rRNA cistron consisting of the 16S, 23S, and 5S rRNA genes.The genome of M. chimaera CDC2015-22-71, Hawaiian respiratory strain AH16 , and IrCP012885 ) reveale1 genome ). The coundaries .M. chimaera genome assembly is the first complete NTM genome associated with an outbreak and will serve as a reference for epidemiological investigations of North American-based HCU contamination and postsurgical M. chimaera infections.This M. chimaera CDC2015-22-71 has been deposited in NCBI GenBank under accession numbers CP019221 through CP019224. PacBio and Illumina reads have been deposited in NCBI under BioProject number PRJNA344472 and BioSample number SAMN05824346.The genome sequence of


The Ag/TiO2 assemblies, which show large surface areas (119–128 m2·g−1) and narrow-sized mesopores (ca. 7.1–7.4 nm), perform as highly active catalysts for the reduction of nitroarenes, giving the corresponding aryl amines and N-aryl hydroxylamines with NaBH4 and ammonia-borane (NH3BH3), respectively, in moderate to high yields, even in large scale reactions (up to 5 mmol). Kinetic studies indicate that nitroarenes substituted with electron-withdrawing groups reduced faster than those with electron-donating groups. The measured positive ρ values from the formal Hammett-type kinetic analysis of X-substituted nitroarenes are consistent with the proposed mechanism that include the formation of possible [Ag]-H hybrid species, which are responsible for the reduction process. Because of the high observed chemo selectivities and the clean reaction processes, the present catalytic systems, i.e., Ag/MTA-NaBH4 and Ag/MTA-NH3BH3, show promise for the efficient synthesis of aryl amines and N-aryl hydroxylamines at industrial levels.In this study, we report the fabrication of mesoporous assemblies of silver and TiO The latter seems to be the most preferred method to sustain the stability and catalytic activity of nanoparticles -H active species. Such hybrid species are responsible for the rapid reduction of nitroarenes into the corresponding 2CH2O)20C16H33, Mn~1124), titanium tetrachloride (99.9%), AgNO3 (>99%), AgOTf, absolute ethanol (99.8%), NaBH4, NH3BH3, TMDS and DMPS were purchased from Sigma-Aldrich . Titanium(IV) isopropoxide (>98%) was purchased from Merck . TiO2 nanoparticles (P25) were purchased from Degussa AG .The aromatic nitro compounds used as substrates were of high purity and commercially available from Aldrich .Brij 58 surfactant (HO(CH2 nanoparticle assemblies (MTA) were prepared according to the method reported previous [2 (Ag/MTA) catalysts with different loading of AgNPs were obtained by photocatalytic reduction method. Typically, 0.2 g of MTA were dispersed into 10 mL of a CH3CN/H2O/ethanol (10:1:1 v/v) solution containing appropriate amounts of AgNO3. The suspension was then illuminated with a 5 mW ultraviolet lamp (λ = 365 nm) for 2 h under continuous stirring. The product was then collected by filtration, washed with ethanol, and dried at 60 °C for 12 h. A series of mesoporous Ag/MTA catalysts with different Ag loadings, i.e., x = 2, 3, 4 and 7 wt %, was prepared using 6.4, 9.6, 13.2 and 23.8 mg of AgNO3, respectively.The mesoporous TiOprevious . Ag-load6 filament) operating at 200 kV. The sample was dispersed in ethanol by sonication, and the dispersion was then dropped onto a Cu grid covered with carbon film. Nitrogen adsorption-desorption isotherms were measured at liquid N2 temperature (77 K) on a NOVA 3200e volumetric analyzer . Before analysis, samples were degassed overnight at 150 °C under vacuum (<10−5 Torr) to remove moisture. The specific surface areas were calculated using the Brumauer-Emmett-Teller (BET) method [P/Po) range. The total pore volumes were derived from the adsorbed volume at P/Po = 0.99 and the pore size distributions were obtained by the nonlocal density functional theory (NLDFT) method [The X-ray diffraction (XRD) patterns were collected using a Panalytical X’Pert Pro MPD X-ray diffractometer  with a Cu Kα radiation (λ = 1.5406 Å). Elemental microprobe analysis was performed on a JEOL Model JSM-6390LV scanning electron microscopy  system equipped with an Oxford INCA PentaFET-x3 energy-dispersive X-ray spectroscopy (EDS) detector . Data acquisition was performed several times using an accelerating voltage of 20 kV and 60 s accumulation time. Transmission electron microscopy (TEM) experiments were carried out with a JEOL model JEM-2100 electron microscope (LaB) method  based on4 (0.6 mmol) or NH3BH3 (0.25 mmol), and the reaction mixture was stirred at room temperature for a selected time. The reaction was monitored by thin layer chromatography (TLC), and after completion, the slurry was filtered under pressure through a short pad of silica to withhold the catalyst with the aid of ethanol or methanol (~5 mL). After solvent evaporation the corresponding products were formed in pure forms. Product analysis was conducted by 1H-NMR and 13C-NMR spectroscopy . Identification of the products was realized by comparing the NMR spectra with those of the commercially available pure substances. LC-MS 2010 EV Instrument  under Electrospray Ionization (ESI) conditions was used for the determination of the mass spectra.Supported silver catalyst Ag/MTA (10 mg) was placed in a 5 mL glass reactor, followed by the addition of ethanol (1 mL), nitro compound (0.1 mmol) and NaBH1, 1.2 mmol of NaBH4 and 20 mg of catalyst (4 mol % Ag)) was placed into a vial. Each catalytic reaction was stopped after 6 h and the catalyst was collected by filtration, washed with ethanol and dried in an oven at 100 °C for 12 h. Then, the recovered catalyst was used for the next catalytic run without any additional treatment.Reusability testing of the catalyst was conducted in the case of the 4% Ag/MTA sample through the reduction of 4-nitrotoluene (1). A 2 mL mixture of the feeding solution  or as the major products (with NH3BH3). Based on the observed high chemoselectivities and the fast and clean reaction processes, both catalytic systems, Ag/MTA-NaBH4 and Ag/MTA-NH3BH3, can be applicable to various hydrogenation reactions, including fine synthesis of amines and N-aryl hydroxylamines, respectively.In conclusion, we have shown that mesoporous titania supported silver nanoparticles (Ag/MTA) can effectively catalyze the chemoselective reduction of nitroarenes into the corresponding aryl amines and