The goal of this study was to characterize the ligandome displayed by HLA-E following infection with Mycobacterium tuberculosis (Mtb) using an in-depth mass spectrometry approach. Here we identified 28 Mtb ligands derived from 13 different source proteins, including the Esx family of proteins. When tested for activity with CD8+ T cells isolated from sixteen donors, nine of the ligands elicited an IFN-γ response from at least one donor, with fourteen of 16 donors responding to the Rv0634A19-29 peptide. Further evaluation of this immunodominant peptide response confirmed HLA-E restriction and the presence of Rv0634A19-29-reactive CD8+ T cells in the peripheral blood of human donors. The identification of an Mtb HLA-E ligand that is commonly recognized may provide a target for a non-traditional vaccine strategy.HLA-E is a non-conventional MHC Class I molecule that has been recently demonstrated to present pathogen-derived ligands, resulting in the TCR-dependent activation of αβ CD8 Mycobacterium tuberculosis (Mtb), remains a leading cause of infectious mortality worldwide, accounting for 9.6 million new cases and 1.5 million deaths in 2014 . Although the incidence rates worldwide are slowly declining, treatment of active cases alone is not likely to lead to the eradication of TB [Tuberculosis (TB), caused by + T cells and proinflammatory cytokines such as IFN-γ and TNF-α are essential in the control of Mtb [+ T cells, through their direct recognition of the infected cell, could play a unique role in a protective immune response. Classically restricted CD8+ T cells are characterized by their activation via peptides presented in the context of the highly polymorphic HLA-Ia molecules. In contrast, non-classically restricted CD8+ T cells are characterized by their dependence on molecules that are not restricted to a specific donor. We and others have shown that non-classical CD8+ T cells restricted by HLA-E, MR1, and CD1 molecules can recognize antigens presented by Mtb [+ T cells can recognize Mtb-infected cells via HLA-E [+ T cells elicited by cytomegalovirus vector vaccination of rhesus macaques were restricted by HLA-E, which presented a diverse range of SIV peptides [While CD4l of Mtb , 6, vaccd by Mtb –11. Thesd by Mtb , 12, CD1d by Mtb . Althougia HLA-E  and is nia HLA-E  and thuspeptides . Additiopeptides –18, furt+ T cells [+ T cells from donors with Mtb infection, latent Mtb infection (LTBI), as well as healthy donors. The response was blocked with an anti-Class I antibody and could be elicited using antigen presenting cells that did not express matched HLA-A, B, or C alleles. Furthermore, CD8+ T cells expanded with the peptide could be stained with an HLA-E tetramer. As HLA-E is highly conserved and not down-regulated in HIV, these Mtb-derived HLA-E epitopes could be used in novel vaccination strategies targeting donor-unrestricted T cells.As with other non-classical Class I molecules, HLA-E presents both self and pathogen-derived antigens to CD8 T cells –21. The  T cells . Althoug T cells . Here, wThis study was conducted according to the principles expressed in the Declaration of Helsinki. Samples were collected under protocols approved by the Institutional Review Board (IRB) at Oregon Health Science University . All study participants were adults. Written informed consent was obtained for all participants and samples were assigned a random three-digit code by the study coordinator. Peripheral blood mononuclear cells (PBMC) were obtained by apheresis from healthy, LTBI, and Mtb-infected adult donors recruited from OHSU as previously described , or via M. tuberculosis expressing GFP was a kind gift from David Sherman . Bacteria were grown in Middlebrook 7H9 broth supplemented with Middlebrook ADC (Fisher), 0.05% Tween-80, and 0.5% glycerol. Before infection, bacteria were passaged through a tuberculin syringe to obtain a single cell suspension. For infection of the bioreactor, 10 ml of a culture containing 5e9 bacteria was added to 1L of RPMI 1640 with 10% heat inactivated FBS and fed to the bioreactor over the course of 24 hours.The H37Rv strain of + T cells were isolated from PBMC by positive selection using CD8 antibody-coated magnetic beads per the manufacturer’s instructions (StemCell Technologies). The D160 1–23 HLA-E restricted CD8+ T cell clone was expanded and maintained as previously described [A549 cells were obtained from ATCC CCL-185) and cultured in F12K with 10% heat inactivated FBS. U373 cells were a kind gift from the lab of Dr. Lewis Lanier  and were cultured in DMEM with 10% heat inactivated FBS. Monocyte-derived dendritic cells (DC) were prepared from PBMC as described . Briefly5 and culTo select a cell line for soluble HLA-E production, we tested fifteen human cell lines to determine how well each cell type was able to present Mtb-derived antigens to a human HLA-E restricted T cell clone compared to primary human dendritic cells (DC), as well as how efficiently each cell type produced recombinant HLA-E molecules. The HLA-E restricted T cell clone D160 1–23 was able to make IFN-γ in response to Mtb-infected U373 cells at levels similar to DC , and U37After validating our model system, U373 cells were transfected with a previously described soluble form of HLA-E*01:03 . Cells wClass I HLA ligands are complex and have similar lengths and biochemistry, so a two-dimensional HPLC separation was utilized for the best sensitivity, as recently described . Brieflyde novo identification of HLA-E ligands, data-dependent acquisition (DDA) spectra from both infected and uninfected cells were analyzed using PEAKS 7.0 and Mascot. For both algorithms, three databases were used: the M. tuberculosis NCBInr for Mtb ligands, H. sapiens NCBInr for non-HLA human ligands, and a custom HLA sequence database for leader peptide sequences. For PEAKS and Mascot, n-terminal acetylation, deamidation of N and Q, oxidation of M,W,H, pyro-glu from Q, and sodium adducts of D,E and c-terminus were used as variable modifications. All positive identifications were made using a 1% FDR at a mass error tolerance of 50 ppm with most positive identifications within 25 ppm of theoretical mass. For all Mtb sequences, a synthetic peptide (Genemed Synthesis) with the corresponding sequence was analyzed under the same LCMS conditions. Only peptides that matched the fragment pattern, were within 50 ppm, and eluted within 5 min of the synthetic were further characterized.For the H. sapiens Protein Pilot 4.5 search of the DDA data with no score threshold set. SWATH data for all first-dimension fractions from both infected and uninfected cells was searched using the spectral library described above with Peakview 2.0 with the SWATH 2.0 microapp. Intensity values were generated using the sum of the top 5 fragment ions within 50 ppm and a RT window of 5 minutes. Intensities from modifications and different charge states were summed for each distinct peptide. To eliminate false-positives the intensity data for every peptide in every first dimension fraction was only analyzed further if the intensity value was above 10000 counts. For the infected and uninfected samples, the thresholded intensity values for each first-dimension fraction were summed. Next, to account for total peptide quantity, the intensity data was normalized to the TIC. The log10 fold increase or decrease was then calculated for each peptide.For the MS quantitation experiment, a spectral library was generated using the ligands from both infected and uninfected cells. The spectral library consisted of only ligands identified at a 1%FDR by PEAKS and Mascot. Fragment ion information for each peptide was extracted from a + T cells isolated as described above were used a source of responder T cells and tested in duplicate at 250,000 cells/well. In some cases, autologous DCs  were used as antigen presenting cells (APC), In other assays, U373 cells or HLA-mismatched A549 cells were used. Peptides were synthesized by Genemed Synthesis and resuspended in DMSO at a concentration of 20mg/ml. DC or A549s were pulsed with peptide  and added to the assay. U373 cells were infected with H37Rv-GFP at MOI:3,10, or 30. T cells (250K/well) were incubated with DC or A549 cells, or for U373 cells, the HLA-E restricted human T cell clone, D160 1–23, in the presence peptide or infection with Mtb. Negative and positive controls were included in each assay and consisted of wells containing T cells and A549/DCs without antigen, or with inclusion of phytohemagglutanin . For all assays, responding T cells were incubated with APC overnight. A positive ELISPOT response was defined as one in which the peptide specific response was at least two standard deviations above the background control. The peptide specific T cell response was then determined by subtracting the mean of the background from the positive response.The IFN-γ ELISPOT assay was performed as described previously . CD8+ T 19-29 peptide at 2ug/ml final concentration for 24 hours at 37 degrees C. Autologous CD8+ T cells were isolated from D481 PBMC as described above and added to the peptide pulsed DC at 1e6/ml with 5ng/ml IL-2. After 6 days, CD8+ T cells were harvested from wells containing peptide pulsed DC and labeled with CFSE. 1e6 CFSE-labeled CD8+ T cells were then transferred to an ultra-low adherence plate containing 1e5 Rv0634a19-29 peptide-pulsed autologous macrophages and incubated with 5ng/ml IL-2 for 6 days. After 6 days, the CD8+ T cells were sorted on CFSE staining. CFSE-dim cells were collected and tested for response to the Rv0634a19-29 peptide by IFN-γ ELISPOT assay. The CD8+ T cell line expanded from D481 as described above was also incubated with either a control HLA-B15 tetramer (recognizing a peptide from the Mtb protein CFP1075-83) or the Rv0634a19-29-HLA-E tetramer  at a 1:50 dilution for 1 hour at 37 degrees C. The cells were then stained with LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen) and subsequently stained with antibodies to CD8 (APC Cy7), CD3 (BV650), and CD4 (BV421) for 30 min at 4 degrees C.Dendritic cells (DC) were prepared from D481 PBMC as described above and plated in ultra-low adherence plates at 1e5/ml. DC were incubated with Rv0634aAt present, HLA-E ligands presented by the Mtb-infected cell remain largely unknown. Because of the size and complexity of the Mtb genome, bioinformatic identification of HLA class I (HLA I) epitopes is challenging and often does not cover the entire genome. We have previously pioneered the use of stably transfected cells expressing soluble HLA molecules to define both exogenous and endogenous ligands from virally infected cells –29. ThisLike other HLA molecules, cell surface expression of HLA-E depends on the presence of a suitable peptide , 30. HLAIn addition to the leader peptides we observed a combined total of 1263 host cell-derived non-leader ligands from 324 source proteins from both infected and uninfected cells. Ligands ranged from 8 to 33 amino acids in length with nonamers being the most common length . Among t). The Mtb-derived ligands, discussed below, were also a minor ligand category, accounting for 0.4% of the total ligand intensity. Thus, after Mtb infection the HLA-C5 leader peptide displaces the host-cell minor ligand pool, with Mtb-derived ligands accounting for a small fraction of the total ligand pool.In prior work, we found that viral infection can alter the repertoire of host-derived HLA-I ligands . To asseFig 3B19-29) source protein, with the corresponding synthetic peptide are shown in We identified 28 Mtb-derived sequences originating from 13 proteins  in the H+ T cells. CD8+ T cells isolated from PBMC from Mtb-infected (n = 4), LTBI (n = 8), or healthy (n = 4) donors were tested ex vivo with peptide-pulsed autologous DC used as APC for their ability to produce IFN-γ. Of the 28 peptides tested, 12 were recognized by at least one of the 16 donors tested , and 14 of the 16 donors screened responded to at least one of the peptides  IFN-γ spot forming units (SFU) per 2.5e5 CD8+ T cells; CD8+ T cells from LTBI donors responded to 7/28 peptides with an average of 42.6(+/-32.1) IFN-γ /SFU per 2.5e5 CD8+ T cells; and CD8+ T cells from Mtb-infected donors responded to 5/28 peptides with an average of 25.7(+/-20.8) IFN-γ /SFU per 2.5e5 CD8+ T cells.To determine whether or not Mtb-derived HLA-E ligands were immunogenic in the setting of infection with Mtb, each peptide was tested for its ability to elicit a response by human CD8s tested , and 14 peptides . The res19-29 peptide was the most commonly recognized peptide, with CD8+ T cells from 13 of 16 donors responding. For 10 of those 13 donors, the response to Rv0634A19-29 was also the strongest, with an average of 46.5(+/-39.9) IFN-γ /SFU per 2.5e5 CD8+ T cells across all donors. Because this peptide had both the most common and strong response, we chose to analyze it further to validate that T cell responses to the peptide were HLA-E restricted. Given the HLA-A2 motif of many of the peptides, classically restricted CD8+ T cell responses to peptide-pulsed autologous DC could not be ruled out for the two HLA-A2 donors (D454 and D603). As such, we analyzed the ex vivo responses of CD8+ T cells from representative donors that could be tested with an HLA-mismatched APC. CD8+ T cells isolated from donor PBMC were added to Rv0634A19-29 peptide-pulsed A549 cells, a line that is mismatched with the selected donors at the HLA-A, B, and C alleles. Responses by CD8+ T cells from representative HLA-mismatched TB (D481), LTBI (D673), and healthy (D519) donors are shown in + T cells from each of the donors, further supporting the HLA-E restriction of these responses to the Rv0634A19-29 peptide, although not ruling out HLA-F or–G restricted responses.The Rv0634A19-29 was HLA-E restricted, and to demonstrate that Rv0634A19-29 responsive, HLA-E restricted CD8+ T cells were present in donor PBMC, we generated tetrameric HLA-E with the Rv0634A19-29 peptide. The Rv0634A19-29 peptide was used to generate a T cell line from the CD8+ T cells of one of the Mtb-infected donors, D481. CFSE-diluted CD8+ T cells generated after culture with peptide-pulsed autologous macrophages and DC were sorted and stained with the Rv0634A19-29 HLA-E tetramer  and Joosten et al. (2010) , 16. Int and JoosM. bovis BCG may limit the presentation of mycobacterial ligands on HLA-E, thus reducing the expansion of HLA-E restricted T cells in the context of BCG vaccination.Although we identified 28 Mtb peptides bound to HLA-E in the context of infection, these peptides were a very small proportion (0.4%) of the ligand pool. The small contribution of Mtb ligands to the total ligand pool may be a reflection of the intracellular localization of Mtb. Following infection, viral proteins are translated in high quantities by host cell machinery in the cytosol, where they are readily available to be processed and presented through the same peptide loading complex as host cell peptides . Mtb, in+ T cells. One simple explanation is that we have not screened enough donors to find responses to all of the identified peptides. Another possibility is that T cells recognizing the remaining peptides have different effector functions. Interestingly, recent studies that followed up on the Mtb HLA-E binding peptides predicted through bioinformatics analysis [+ T cell clones have unconventional phenotypes that more closely resemble Th2 type functions [Here, we used IFN-γ as a readout for T cell activity in response to peptide stimulation. Using this readout, 12 of the 28 Mtb-derived HLA-E binding peptides elicited an IFN-γ response, suggesting that T cells recognizing these peptides have Th1 like functions. This leaves the question as to why the other 16 Mtb HLA-E binding peptides did not elicit any IFNγ release by CD8unctions , 18. TheHLA-E is an attractive candidate for novel and innovative vaccine strategies for TB. As previously mentioned, it is highly monomorphic, is not down-regulated after HIV infection, and has recently been shown to play an important role in generating SIV-reactive T cells following vaccination. We have identified several Mtb epitopes that are presented by HLA-E in the context of intracellular Mtb infection. Further analysis of the T cells elicited through recognition of these peptides will increase our understanding of the HLA-E-restricted cellular response to infection with Mtb.


Objectives. This study aimed to explore the expression of GLP-1 receptor in hypothalamus and gastrointestinal tissues after Roux-en-Y gastric bypass (RYGB) surgery in diabetic rats. Methods. Male 12-week-old Wistar rats (control) and Goto-Kakizaki rats (diabetic) were randomly divided into two groups, respectively: control sham surgery group (C), control RYGB group (C + R), diabetic sham surgery group (D), and diabetic RYGB group (D + R). Body weight and blood glucose were monitored before and after surgery every week. Eight weeks after surgery, all rats were sacrificed and the serum fasting GLP-1 concentrations were measured by ELISA. GLP-1R and DPP-4 expression in hypothalamus and ileum were measured by RT-PCR. Results. The body weight and fasting/random blood glucose in the D + R group decreased significantly compared with the D group (P < 0.05). Serum GLP-1 levels in diabetic rats treated with RYGB were higher than the corresponding sham surgery rats. The expression of GLP-1R of hypothalamus in RYGB-treated diabetic rats was significantly higher than those of the sham surgery diabetic rats and both control group rats (P < 0.05). We found a negative correlation between hypothalamus GLP-1R mRNA and blood glucose level. No significant difference was seen in ileum GLP-1R and DPP-4 expression among all groups. Conclusions. RYGB efficiently promoted serum GLP-1 levels and the expression of GLP-1 receptor in the hypothalamus in diabetic rats. These data suggest that the hypothalamus GLP-1R may play an important role in the GLP-1 system for improving glucose homeostasis after reconstruction of the gastrointestinal tract. Roux-en-Y gastric bypass (RYGB) has a rapid and drastic effect in type 2 diabetic patients, many of whom show complete remission within days of the procedure , 2. A nuIn our study, we explored the impact of RYGB on GLP-1R expression in hypothalamus and ileum tissues in both diabetic and nondiabetic rats, aimed to identify the effect of GLP-1 receptor on glucose metabolism, and uncover the mechanisms behind the RYGB surgery after RYGB.n = 12) were randomly assigned to a RYGB surgery of control rats  and sham surgery of control rats . Goto-Kakizaki rats (n = 20) were also randomly assigned to RYGB surgery of diabetic rats  and sham surgery of diabetic rats . Six rats died within three days after RYGB surgery. All the animal procedures were performed in accordance with the ethical principles in animal research adopted by the Department of Laboratory Animal Science, Jiaotong University School of Medicine, Shanghai, China.Male 12-week-old Wistar and Goto-Kakizaki (GK) rats were purchased from SLACK Laboratories, SIBS, Shanghai, China. Animals were housed at an ambient temperature of 22 ± 2°C and maintained under a normal 12 hr light/dark cycle with free access to food and water. Rats were allowed 2 weeks for acclimation before the start of experiments. Wistar rats . A dose of 200 mg/kg penicillin sodium  was given intravenously as prophylactic antibiotic. Midline laparotomy was performed under sterile conditions. Gastric bypass surgery was performed as previously described  and in tBlood glucose was detected weekly by the electronic glucometer . At 2, 5, and 8 weeks after surgery, blood glucose was detected at the overnight fasting condition. One week before surgery and at 3, 4, 6, and 7 weeks, random blood glucose was detected. Body weight was measured using an electronic balance weekly.μL dipeptidyl dipeptidase IV inhibitor, measured at room temperature by using ELISA kits (Shanghai Westang).Eight weeks after surgery, tail blood was obtained to assess the total GLP-1 levels; the tubes for blood samples of GLP-1 contained 20 μL reactions from 200 ng of total RNA following the manufacturer's instructions. The integrity of the cDNA was confirmed by amplifying β-actin. The real-time PCR was conducted by a LightCycler 96  employing SYBR Green I as the dsDNA specific binding dye for continuous fluorescence monitoring. The PCR protocol comprised 5 min at 95°C and 45 cycles of 15 s at 95°C, 15 s at 58°C, and 15 s at 72°C. The sequences of the GLP-1R primers are as follows: forward 5′-AGT AGT GTG CTC CAA GGG CAT-3′, reverse 5′-AAG AAA GTG CGT ACC CCA CCG-3′; and β-actin forward 5′-GCC CCT CTG AAC CCT AAG-3′, reverse 5′-CAT CAC AAT GCC AGT GGT A-3′; DPP-4 forward 5′-CAA ATC ACC GCT CCT GCA T-3′, reverse 5′-TCA TAG TCG CAG ATC GCC A-3′. The mRNA levels of GLP-1R were compared by calculating the crossing point (Cp) value and normalized by the reference genes (β-actin).Hypothalamus and ileum samples were separated after rats were sacrificed. Hypothalamus was separated according to previous study ; perpendP < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS 19.0 statistical software.Measurement data were expressed as the mean ± SD. The data were analyzed with a repeated-measures one-way ANOVA. P < 0.01) and then increased gradually. The fasting blood glucose and random blood glucose in the D + R group decreased significantly compared with the D group after surgery (P < 0.05). However, no significant difference in fasting/random blood glucose was seen between C and C + R groups  than control rats, whereas GLP-1 concentrations were greater in RYGB-treated diabetic rats (P < 0.01) than group D rats. The GLP-1 concentrations were not significantly different between the C + R and C group . We found a negative correlation between hypothalamus GLP-1R mRNA and blood glucose level. There was no statistical difference in GLP-1R mRNA between D group, C group, or C + R groups (P > 0.05)  surgery is one of the most effective therapies to achieve sustained improvement in T2DM patients , 9, 10. P < 0.05, compared to that of the control group rats). These findings demonstrated that RYGB surgery could increase the levels of GLP-1 and GLP-1R in the diabetic rats, implying that RYGB may improve glucose homeostasis by upregulating GLP-1/GLP-1R expressions. We also found a negative correlation between hypothalamus GLP-1R expression and blood glucose level. This is an interesting finding, which, for the first time, showed evidence that hypothalamus GLP-1R was involved in the glucose regulation after RYGB, directly or indirectly. These results demonstrated the potential role of GLP-1/GLP-1R in high glucose environment.GLP-1R is widely expressed in various tissues. Given the fact that the plasma half-life of the intact GLP-1 peptide is short in vivo , the GLPHowever, no significant difference in ileum expression of GLP-1R was seen after RYGB from real-time PCR analysis. The reason that ileum GLP-1R did not change is unknown yet. One possibility is that the GLP-1R protein level, which was not measured in this study, may have changed after RYGB. Another possibility is that the expression of GLP-1R in other tissues may play a role in glucose regulation. A recent research found that GLP-1R expression is reduced in gastric glands from T2DM patients . The expDipeptidyl peptidase-4 (DPP-4), which has been proposed as a target for pharmacological intervention in patients with type 2 diabetes , was priIn conclusion, our data illustrated a negative correlation between hypothalamus GLP-1R mRNA and blood glucose level after RYGB surgery. The upregulated GLP-1R expression in hypothalamus may contribute to the regulation of glucose homeostasis. This research can provide ideas and information for research to further study the GLP-1 receptor-associated signaling pathways regulation and GLP-1 receptor expression in CNS. Further research is needed to evaluate the potential beneficial effects of RYGB surgery, the mechanism behind the tissue receptor regulation, and further role in the therapeutic range.


The A/H3N2 influenza viruses circulated in humans have been shown to undergo antigenic drift, a process in which amino acid mutations result from nucleotide substitutions. There are few reports regarding the charged amino acid mutations. The purpose of this paper is to explore the relations between charged amino acids, N-glycosylation and epitopes in hemagglutinin (HA) and neuraminidase (NA).A total of 700 HA genes (691 NA genes) of A/H3N2 viruses were chronologically analyzed for the mutational variants in amino acid features, N-glycosylation sites and epitopes since its emergence in 1968.1 increased respectively 1.54-fold (+7.0 /+17.8) and 1.08-fold (+8.0/+16.6) and the number of NGS in nearly doubled (7/12). As great diversities occurred in 1990s, involving Epitope A, B and D mutations, the charged amino acids in Epitopes A, B, C and D in HA1 mutated at a high frequency in global circulating strains last decade. The charged amino acid mutations in Epitopes A (T135K) has shown high mutability in strains near years, resulting in a decrease of NGT135-135. Both K158N and K160T not only involved mutations charged in epitope B, but also caused a gain of NYT158-160. Epitope B and its adjacent N-glycosylation site NYT158-160 mutated more frequently, which might be under greater immune pressure than the rest.It was found that both the number of HA N-glycosylation sites and the electric charge of HA increased gradually up to 2016. The charges of HA and HAThe charged amino acid mutations in A/H3N2 Influenza play a significant role in virus evolution, which might cause an important public health issue. Variability related to both the epitopes (A and B) and N-glycosylation is beneficial for understanding the evolutionary mechanisms, disease pathogenesis and vaccine research. Influenza is an acute respiratory infectious disease caused by influenza virus, which affects millions of people annually and results in moderate mortality. Among the different types and subtypes of influenza virus, the A/H3N2 subtype has dominated a lot of human influenza outbreaks worldwide since its emergence in 1968 . Both ev1 and HA2 while former is an important functional region. Viral NA is responsible for cleaving the terminal sialic acid residues, which helps to release viruses from the host cell ; the; the18];Viral RNA molecules from H3N2 strains from Guangdong collected during 2012–2016 were extracted using a QIAamp Viral RNA mini kit . Reverse transcription-polymerase chain reaction (RT-PCR) assays were conducted using Qiagen Sensiscript Reverse Transcriptase and Takara PyroBest Taq. After purification using a Qiagen Gel Extraction Kit, amplicons were sequenced using an ABI PRISM 3100 Genetic Analyzer. Amplifications were performed on a 96-Well Thermal Cycler PCR system  under the following conditions: 30 min at 55°C; 2 min at 94°C; 40 cycles of 15 s at 94°C, 30 s at 55°C, and 60 s at 68°C; and a final extension of 5 min at 72°C.Viral nucleotide sequences were analyzed by means of the Maximum Composite Likelihood and phylogenic trees were generated based on the Neighbor-Joining (NJ) in the MEGA 7.0.21 package . The relwww.datamonkey.org). Four different methods include the Single-likelihood ancestor counting (SLAC), Fixed effects likelihood (FEL), Internal Fixed effects likelihood (IFEL) and Random effects likelihood (REL), The SLAC, FEL, and REL are used to detect sites under selection at external branches of the phylogenetic tree, while the IFEL investigates sites along the internal branches. The SLAC is intensive for large alignments but appears to underrate the substitution rate and the REL is more suited to intermediate-sized datasets than SLAC, but it may be unsuitable for small alignments. The FEL method gives a lower rate than REL of false positives for data sets of few sequences [Evolutionary selection pressure was conducted using the Datamonkey web-server (equences . The SLAThe amino acids based on their charges were classified into three groups including acidic amino acids [Aspartic acid (D) and Glutamic acid (E)], basic amino acids  and neutral AA. At a specific physiological pH, each acidic/basic AA is ionized alternatively with a single negative/positive net charge, respectively . For HA http://zhanglab.ccmb.med.umich.edu/I-TASSER/) [www.cgl.ucsf.edu/chimera/) [The molecular models of H3 HA and N2 NA was performed with the HA of A/Hong Kong/1/ 1968 (HA PDB: 4WE4 and NA PDB: 2hty) (TASSER/) . The thrhimera/) .http://www.cbs.dtu.dk/services/Net-NGlyc/). The scores of two pluses (++) or three pluses (+++) in the N-Glyc results were classified as strong potential NGSs . Based on the server-based analysis, ~76% of positively scored loci are modified by N-Glycans that contain Thr[The potential NGSs  were analyzed using the NetNGlyc 1.0 Server  and two additional conserved neutralizing epitopes (L and R) ,14. The P<0.05), used SPSS 20.0 . Calculated electric charge, the net charge Snc = Σni, where n is the respective number of acidic AAs (D and E) and basic AAs  in each monomer or epitope, and i is each charge of the five charged AA.Correlation and regression were statistically performed and statistical significance was depended on its significance probability , Epitope B , Epitope C (N278K) and Epitope D (K173Q and N225D). Based the variants/vaccine strains, the spatial positions of charged amino acids on HA epitopes were shown , respectively. Only sites 121, 137, 138, 145, 159 and 262 in HA genes were considered as the positive selection with above methods. For NA genes, only sites 370 and 435 were done as the positive selection by three methods.Used the Datamonkey web-sever, the numbers of 10, 15 and 18 residues in HA genes were identified as the positive selection sites by the methods of SLAC, FEL and IFEL  to +12.0 (1986), then kept around +10 (to 1998). But they decreased rapidly from +9.4 (1998) to +5.9 (2000), then it rose slightly.122-124 and NGT133-135 and remained up to date. As to the HA gene of the vaccine strain A/Hong Kong/4801 /2014, a total of 11 NGSs were identified, such as NST008-010, et al , but its uncertain potentiality accounting for 22.0% (99/452) got a lower N-Glyc score (<++), especially during 2003–2007. Remarkably, due to the mutation K160T, the NYT158-160 emerged in 2014 and got prevalent quickly worldwide . The NST008-010, NGT022-024, NVT165-167, NCT063–065 and NGS285-287 in HA genes were relatively conversed and remained stable potentiality during the whole period , which indicated that the amino acid mutations involved charge changing were highly correlated with NGS domains here.A total of 15 potential NGSs were identified in HA genes spanning 48 years. The HA NGSs showed a gradual increase, ranging from 7 to more than 12 (14 domains). The spatial position of NGSs on HA proteins in variants/vaccine strains were shown in 0, et al . It was e period , whereas093-095 was due to point mutation K93N during 1997–2000 and a loss was due to point mutation N93D/G since 2008. There were 8 NGSs emerging in NA of A/Hong Kong/4801/2014, such as NIT061-063, et al . Both NIT061-063 and NGT234-236 were the strongest and conservative glycosylated potential since 1968 .The NGSs of NA genes changed more slightly than those of HA, including 8–11 NGSs . An acqu3, et al . The NDT142G and S144N), epitope B (Y159F/S and K160T), epitope C (D053N and K278N) and epitope E (Y094H). Most notably, mutations Q197K (epitope B) and N171K (epitope L) emerged and became prevalent in 2016. Among the 17 mutations , S144N , Y159F  and K160T  occurred during 2012–2016.Referred to the reference of HA gene of A/Hong Kong/4801/2014, mutations of HA antigenic epitopes were identified during 2007–2016 . The preutations , 88% , A and E , and D and E . It indicated that the positive correlation between the epitope A and B charges, the negative correlation between the epitope A and E charges to some degree, and the slight positive correlation between epitope D and E charges.As to average and standard deviation (SD) values, the charges of epitope A  substitutions. Charged AA (either acidic or basic one) mutations could alter the physical characteristics of a specific domain or the whole protein, furthermore some mutations resulted in a gain or loss of NGSs. Compared to strain A/Hong Kong/1/ 1968, the HA charges in this study increased and peaked at 1.54-fold and HA to 2010 .1 is positively charged in this study and the HA/HA1 charge values were highly correlated with NGSs number , which suggested that HA/HA1 amino acid mutations related to their charge changing mainly focused on N-glycan modification.Electrostatic interactions are important for the viral RBD binds to the host cell sialic acid receptors. However, viral N-glycans could shield the RBD, and the highly negatively charged sialic acids and sulfuric acids on the viral N-glycans might cause electrostatic repulsion of the host cell, which has an adverse influence on the HA binding function. Since t126-128 glycosylation site is interesting in the context of biological evolution, but the one plus (+) potential reduced its significance. The NGSs of NA were more conserved than those of HA, which suggested that NA undergo less immune pressure than HA. It was the current opinion that an increase of HA NGSs in A/H3N2 might lead to reduction of viral virulence and decline in the severity of illness [Some novel NGSs (such as Asn-91) might be useful candidates for functional analyses to identify innovative genetic modifications for beneficial phenotypes acquired in human lineages . For H3 158K, Y159F/S, K160T, K189N and Q197K; 2) four of the five mutations contained charged modification; 3) mutations in position 158, 159 and 160 resulted in the gain of NGS NYT158-160. It’s noteworthy that both K158N and K160T were not only charged AA mutations in epitope B, but also caused a gain of NYT158-160. Previous study have shown that epitope B played a vital role in both antigenic phenotype and receptor specificity [135K occurred in epitope A, resulting in deleting a NGT135-135, which might be a significant mutation as well. The 1990s was also the special time for all epitopes’ net charges, which coincided with our phylogenetic diversities of HA genes. Epitopes A and B, including residues of 135, 142, 144, 159 and 160, also showed high mutability in strains isolated from Guangdong, Southern China during 2012–2016.A B-cell epitope is defined as a region of an antigen recognized by either a particular B-cell receptor or that can subsequently elicit antibody in a humoral response. Epitopes A, B, C, E and L in HA genes of A/H3N2  have mutcificity . Mutatio053N and K278N (epitope C), might cause electrical repulsion of antibody binding, while the synthetic effect on net charge was neutral. Besides mutations in epitope B, the R142G (A), D053N (C), K278N (C), Y094H (E) and N171K (L) were prevalent or emerged charged mutations in recent five years as well. The accumulation of antigenic epitope mutations could, to a great extent, spark a local or provincial epidemic and/or outbreak [Antigenic mutations are based on amino acid substitutions, where the net charge value and the order in AAs for each epitope are crucial. Just like the relation between key and lock, the charged AAs located on epitope must electrically and spatially correspond to that located on antibody’s complementarity determining region, even if the general net charge is appropriate. Some opposite charged mutations emerging in one epitope simultaneously, such as K160T and Q197K (epitope B) and Doutbreak . Overall135K), Epitope B (K158N and K160T), Epitope C (Q311H) and Epitope D (K173Q and N225D) during last decade. We should continue to survey that A/H3N2 variations in AA features, NGSs and antigenic epitopes of could, to some degree, be sufficient to evade immune protection in humans. Molecular monitoring of NGSs and antigenic epitopes of the influenza A/H3N2 virus is beneficial for understanding the evolutionary mechanisms that govern influenza viruses. Moreover, monitoring of viral charge might enlighten vaccine development, including the selection of vaccine delivery vector and immunologic adjuvant.The A/H3N2 virus circulated and dominated worldwide for more than four decades, meantime it still evolves rapidly at the genetic level. Due to its polymerase and segment reassortment, HA segments originated from equine H3 influenza might become associated with cross-species transmission even contribute to appearance of new strains , 32. TheS1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.


MMP-2/MMP-9 gene polymorphism with ulinastatin (UTI) efficacy in treating severe acute pancreatitis (SAP). A total of 276 SAP patients were assigned into the control (n=135) and observation (n=141) groups. PCR-restriction fragment length polymorphism (PCR-RFLP) was used for genotype and allele frequency distribution. Relevance of MMP-2/MMP-9 genotypes with UTI efficacy was analyzed. The observation group showed lowered duration in symptoms  than the control group. Laboratory analysis  were decreased, while serum albumin levels increased after 7th day of therapy. The total effective rate of UTI for patients with MMP-2 C-1306T C/C genotype was higher than those with C/T and T/T genotypes after the 7th day of therapy, which was lower in patients with MMP-9 C-1562T C/C and C/T genotypes than those with T/T genotype. The duration for symptoms in patients with MMP-9 C-1562T T/T genotype was shorter than those with C/C and C/T genotypes, which was less in patients with MMP-2 C-1306T C/C genotype than those with C/T and T/T genotypes. The improvement values of APACHE-II and Balthazar CTIS scores for patients with MMP-2 C-1306T C/C genotype were higher than those with C/T and T/T genotypes, which for patients with MMP-9 C-1562T C/C and C/T genotypes were lower than those with T/T genotype. These results demonstrated that MMP-2/MMP-9 gene polymorphism was associated with UTI efficacy for SAP.We aim to explore the associations between matrix metalloproteinase (MMP)  Acute pancreatitis (AP) is severe disease involving pancreatic inflammation that leads to the activation and release of inflammatory cytokines resulting in systemic inflammation . AP is cSAP is a clinically common acute abdominal disease characterized by its rapid progression and complications. Despite the current treatment methods, patients suffering from SAP still have a 10–20% mortality due to severe pancreatic and extrapancreatic necrosis ,4. The pMMP-1, MMP-2, and MMP-9) levels in the beginning phase of inflammation induce pancreatitis, and MMPs may have a predictive value in assessment of histological severity [MMP-9 level can be considered as a valuable marker for the assessment of severity of SAP [MMP-2 and MMP-9 are classified as the gelatinases and play important roles in the degradation and regeneration of extracellular matrix mainly through secretion into extracellular matrix and participating in matrix degradation upon activation [UTI is a glycoprotein that functions as a trypsin inhibitor. It can be extracted from human urine or synthetically used . Not onlseverity . It alsoy of SAP . MMP-2 ativation .MMP-2 and MMP-9 genes have been verified and MMP-2 promoter polymorphisms −1306C/T and −735C/T, and MMP-9 promoter polymorphism −1562C/T have been proved to be functional [MMP-2 are correlated with a greater risk of cancer, such as non-Hodgkin’s lymphoma, gastric cancer, and colorectal cancer, via promotion of angiogenesis by degradation of the ECM and promoting cell migration and motility [C−1562T gene in MMP-9 has also been confirmed to be able to regulate gene expression [MMP-2 and MMP-9 gene polymorphism on clinical outcomes of UTI in treating SAP are seldom reported. Thereby, the study aims to explore the associations of MMP-2 and MMP-9 gene polymorphism with the efficacy of UTI in treating SAP with the expectation to lay a theoretical foundation for the treatment of SAP.In recent years, several single nucleotide polymorphisms (SNPs) have played a key role in detecting genetic variations in many individuals and how they may provide prognostic markers to diseases in terms of their susceptibilities and prognosis ,16. An Snctional . It was motility –22. The pression ,24. Due The present study was performed with the approval of Clinical Ethical Committee in Yishui Central Hospital of Linyi. All procedures were conducted strictly in accordance with the Declaration of Helsinki. The present study was approved by the Ethics Committee of Yishui Central Hospital of Linyi. All participating patients in this investigation agreed to and signed informed consents.9/l, lactic acid intoxication higher than 600 U/l, blood glucose higher than 180 mg/dl, albumin <32 g/l, urea nitrogen higher than 16.065 mmol/l; PaO2 60 mmHg, serum calcium lower than 2 mg/l, and APACHE-II score more than 8 points [A total of 276 SAP patients  hospitalized in Yishui Central Hospital of Linyi were recruited between January 2011 and December 2016. Their ages ranged from 20 to 66 years and the mean age was 36.84 ± 7.63 years. All patients conformed to the diagnostic criteria issued by Chinese Medical Association Pancreatic Surgery Group . Confirm8 points . The excMMP-2 and MMP-9 genes with FAST SNP. After gene locus screening, C-1306T and C-735T of MMP-2 gene and C-1562T of MMP-9 gene were selected for this investigation.The study was conducted based on the genomic data of Chinese Han population in HapMap. The gene locus screening was determined according to the three methods including literature retrieval, Tag-SNP selection, and observation of functional variation locus of 2, 2 μl dye, 1.0 U Taq-DNA polymerase, 200 μmol/l dNTPs, and 250 nmol/l forward and reverse primers. The reaction conditions for all the three methods were carried out differently. Conditions for C-1306T included: predenaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 58°C for 45 s and elongation at 72°C for 45 s (30 cycles), and a final elongation at 72°C for 10 min. Finally, a 193-bp sequence was obtained as the amplified product of PCR. A certain amount of product was taken out and incubated with restriction endonuclease XspI at 37°C overnight. A 3% agarose gel electrophoresis was carried out to isolate the product from enzymatic digestion and kept for later use. The reaction conditions for the C-735T gene were as follows: predenaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 61°C for 30 s, elongation at 72°C for 30 s (30 cycles), and a final elongation at 72°C for 10 min. The PCR product consisting of 391 bp was incubated overnight with restriction endonuclease HinfI at 37°C and was analyzed after 3% agarose gel electrophoresis. The reaction conditions for the last genotype, C-1562T were as follows: predenaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 62°C for 30 s and elongation at 72°C for 30 s (35 cycles), followed by a final elongation at 72°C for 10 min. The final amplified product was 435 bp, which was incubated in water at a constant temperature overnight with restriction endonuclease SphI. It was analyzed after 1.8% agarose gel electrophoresis.Venous blood (5 ml) was collected from each subject and transferred into a tube containing sodium citrate for anticoagulation and stored at 4°C. DNA extraction from WBC of peripheral blood was performed within 7 days after blood collection with a DNA extraction kit . PCR-restriction fragment length polymorphism (PCR-RFLP) technique was performed on the polymorphisms of C-1306T, C-735T, and C-1562T. PCR primers were designed by Premier 5.0 software and the sequences were synthesized by Sangon Biotech (Shanghai) Co. Ltd. The primer sequences and lengths are shown in n=135) and the observation group (n=141). Patients in the control group were treated with conventional therapy including: appropriate central venous fluid exchange, pain control, proton pump inhibitors, nasogastric tube, antibiotics, hemodynamic monitoring for parenteral or enteral nutrition support, and providing support for breathing and kidney. Patients diagnosed with severe biliary pancreatitis received endoscopic therapy  [9–27.5 × 109), serum amylase , urine amylase , serum calcium , and albumin , as well as the APACHE-II and Balthazar CTIS scores before and after therapy [Genotyped SAP patients were assigned into the control group ( stones) . Patient therapy . The APA therapy . On the The main symptoms, signs , and laboratory examinations  of patients before therapy and at the 3rd, 5th, 7th, and 10th day after therapy were recorded. The evaluation criteria were as following: the disappearance of any kind of abdominal pain and laboratory indexes returned to normal level within 3–5 days after therapy; the marked effectiveness evaluated by the decreased duration of abdominal pain, abdominal distension, tenderness, and rebound tenderness as well as an improvement in the laboratory indexes within the 7th day of therapy; the effectiveness referred to as the time of abdominal pain, abdominal distension, tenderness, and rebound tenderness was reduced and laboratory indexes were improved within the 10-day therapy; the ineffectiveness was referred to as the main symptoms and signs of patients were not improved, laboratory examinations did not return to normal after 10th day of therapy and some patients even became worse or died . The tot2 test. Count data were presented as a percentage or ratio and the statistical analysis was performed by a chi-square test. Measurement data were expressed as mean ± S.D. Comparisons between groups were performed with variance analysis. Comparisons of two groups were performed using ttest. P<0.05 was accepted as indicative of significant differences.SPSS 21.0 statistical software was used to analyze all the data. Hardy–Weinberg equilibrium was used to assess the group representativeness of genotype distribution. Comparison and analysis of allele and genotype frequencies was performed by χP>0.05).Baseline characteristics of patients from the control and the observation groups are shown in Results of enzymatic digestion demonstrated that the C allele in the C-1306T genotype had one XspI restriction site whereas the T allele had two restriction sites. C/C genotype products were 188 and 5 bp; T/T genotype products were 162, 26, and 5 bp; and C/T genotype products were 188, 162, 26, and 5 bp A. The C-Amongst the 276 cases of SAP patients, 206 cases belonged to the C-1306T C/C genotype, 45 cases belonged to the C/T genotype, and 25 cases were of the T/T genotype with a frequency of 74.64, 16.30, and 9.06% respectively. The frequency of C-allele occurrence was 82.79% and T allele was 17.21%. There were 171 cases (61.96%) of C-735 C/C genotype, 89 cases (32.25%) of C/T genotype, and 16 cases (5.80%) of T/T genotype. The frequency of C-allele occurrence in the patients was 78.08% and T allele was 21.92%. Finally, the C-1562T C/C genotype had 214 cases (77.54%), C/T genotype had 35 cases (12.68%), and T/T genotype had 27 cases (9.78%). The frequency of C-allele occurrence was 83.88% and T allele was 16.12%. Hardy–Weinberg showed that all genotypes and allele frequency had group representativeness in genetic equilibrium.P<0.05). WBC count, serum amylase, urine amylase, and serum calcium were all significantly decreased  in the observation groups as well as the scores of APACHE-II and Balthazar CTIS compared with the control group (both P<0.05)  .P<0.05). The effective rate of patients with C/C and C/T genotypes of MMP-9 C-1562T were 68.22 and 71.42% respectively, whereas 100.00% effective rate was calculated in patients with the T/T genotype . There were no significant differences in the efficacies amongst SAP patients with different genotypes of C-735T (P>0.05)  .MMP-2 gene C-1306T C/C genotype was obviously less than those in patients with C/T and T/T genotypes (both P<0.05). The duration of the symptoms in patients with MMP-9 gene C-1562T T/T genotype was 1.56 ± 0.85, 2.44 ± 1.74, and 1.82 ± 1.19, respectively, which was significantly lower than that in patients with C/C and C/T genotypes . The duration of symptoms for patients with different genotypes of C-735T had no significant differences   .MMP-2 C-1306T C/C genotype was obviously higher than those in patients with C/T genotype and T/T genotype (P<0.05). The proportion for SAP patients with C/C, C/T, and T/T genotypes of MMP-2 C-735T had no statistical differences (P>0.05). The proportion of blood amylase, urine amylase, albumin, WBC, and serum calcium returning to normal levels in patients with MMP-9 C-1562T T/T genotype was 100.00, 100.00, 92.31, 100.00, and 100.00% respectively, which was obviously higher than those in patients with C/C genotype and C/T genotype   .MMP-2 C-1306T C/C genotype  were significantly higher than those in patients with C/T genotype  and T/T genotype  . The improvement values of APACHE-II for patients with C/C, C/T, and T/T genotypes of MMP-2 C-735T were 5.67 ± 5.82, 5.00 ± 4.49, and 4.00 ± 2.61, respectively and of Balthazar CTIS were 1.70 ± 1.86, 1.24 ± 1.81, and 1.00 ± 1.67, respectively. The values of APACHE-II and Balthazar CTIS scores amongst the three genotypes showed no statistical differences . The values of APACHE-II and Balthazar CTIS scores for SAP patients with MMP-9 C-1562T C/C  genotype and C/T  were significantly lower than those in patients with T/T genotype    .MMP-2 and MMP-9 genes with UTI efficacy in treating SAP. Our results showed that MMP-2 and MMP-9 gene polymorphisms were directly related to the efficacy of UTI in treating SAP.In this investigation, we analyzed the genotype and allele frequency distribution amongst the included 276 SAP patients to explore the associations of different genotypes of MMP-2 and MMP-9 have been confirmed to be in a close association with acute inflammatory reaction [SAP is distinguished from mild AP not only in the hemorrhage and necrosis of pancreatic tissues, but also its life-threatening complications of shock, electrolyte imbalance, multiple organ failure, and inflammation . MMP-2 areaction .UTI is a drug that can act as an acid-resistant trypsin inhibitor, thus making it an ideal drug in the treatment of SAP. It works by stabilizing the lysosomal membranes, inhibiting excessive inflammatory response, and reducing tissue and organ damage, which makes it useful in treating the complications caused by SAP . By compMMP-2 and MMP-9 gene polymorphism was correlated with the efficacy of UTI in treating SAP. In a state of inflammation, hydrolytic enzymes would enter into the blood vessels, thereby causing the dysfunction in various organs and tissues [MMP-2 is a type IV collagenase that mainly degrades type IV collagen, the major structural component of the basement membrane and extracellular matrix [MMP-9, like MMP-2 is also a type IV collagenase. During the inflammation, MMP-9 is found to cause vascular endothelial injury and accelerate the migration and invasion of inflammatory cells, worsening the inflammatory response [MMP-9 and MMP-2 to function to have a protective role in tissues and organs [Moreover, the study also concluded that  tissues . MMP-2 ir matrix . MMP-9, response ,38. Throresponse . A studyresponse  has showd organs .MMP-2 and MMP-9 gene promoter factors existed –1306C/T and –1562C/T polymorphism, respectively, which may affect gene transcription and lead to changes in gene function. The transition C–T at locus −1306 or −735 in MMP-2 gene could disrupt a Sp-1-binding site, thus, resulting in decreased MMP-2 promoter activity. The transition of C–T at locus −1562 in the MMP-9 gene has been shown to lead to increased promoter activity [MMP-2 expression of patients with CC genotype was significantly higher than that of patients carrying CT and TT genotypes and individuals with the MMP-9 gene TT genotype had a higher risk for than those with the CC and CT genotypes [MMP-2 1306C/T and MMP-9 1562C/T polymorphism were correlated with cancer susceptibility. The study showed that the effective rates for patients with MMP-2 C-1306T C/C genotype and MMP-9 C-1562T T/T genotype were higher than that in patients carrying other genotypes, which indicated that MMP-2 and MMP-9 gene polymorphism had relevance with the efficacy of UTI in the treatment of SAP. Thus, this supports our study by showing that genotypic changes and MMP-2 and MMP-9 gene polymorphism may influence the expression of MMP-2 and MMP-9 in SAP patients and thereby affected the efficacy of UTI.activity ,41. A stenotypes . Furtherenotypes  found thMMP-2 and MMP-9 gene polymorphism was probably linked to the efficacy of UTI in treating SAP. However, some limitations existed in the present study. First, the sample size in our study still remains insufficient to obtain more accurate data, which requires further studies with larger sample sizes. In addition, confounding factors including smoking status, alcohol consumption, and dietary laws were not considered to a great extent and thus need to be considered in further investigations. Nonetheless, our findings provide evidence for clinical practice of UTI therapy in SAP patients.In summary, our study demonstrated that


Co-immunoprecipitation study using “hot-spot” p53 mutants suggested reduced G-actin association with cancer-associated p53 conformational mutants (R175H and R249S). Considering these findings, we hypothesized that point mutation in p53 structure, which diminishes p53:G-actin complexation results in mutant p53 altered subcellular localization. Our model suggests p53Arg249 form polar-contact with Arg357 of G-actin, which upon mutation, destabilizes p53:G-actin interaction and results in cytoplasmic retention of p53R249S.p53 preserves genomic integrity by restricting anomaly at the gene level. Till date, limited information is available for cytosol to nuclear shuttling of p53; except microtubule-based trafficking route, which utilizes minus-end directed motor dynein. The present study suggests that monomeric actin (G-actin) guides p53 traffic towards the nucleus. Histidine-tag pull-down assay using purified p53(1–393)-His and G-actin confirms direct physical association between p53 and monomeric G-actin. Co-immunoprecipitation data supports the same. Confocal imaging explores intense perinuclear colocalization between p53 and G-actin. To address atomistic details of the complex, constraint-based docked model of p53:G-actin complex was generated based on crystal structures. MD simulation reveals that p53 DNA-binding domain arrests very well the G-actin protein. Docking benchmark studies have been carried out for a known crystal structure,  Actin exists in 2 forms- monomeric G-actin and polymeric F-actin. Recently, an actin-dependent mechanism for long-range transport of vesicles has been reportedAlteration in the protein structure in course of mutation has been recognized as an important factor that affects p53 localization. Mutation in its DNA-binding domain (DBD) is a frequent event in cancer which contributes to the proliferation and differentiation of the neoplastic population. According to IARC TP53 mutation database, single point mutations in p53DBD accounts for more than 95% of the reported malignant mutationsOur study reveals monomeric G-actin directs p53 traffic towards the nucleus through protein-protein interaction. A characteristic perinuclear colocalization between p53 and G-actin is evident in response to genotoxic stress. Considering p53 ‘hot-spot’ mutants, our study suggests that structural mutants show reduced degree of association with G-actin, resulting in altered localization of mutant p53. Based on the experimental information, a hypothetical model of p53:G-actin complexation has been derived using molecular docking and MD simulation studies. Our study explores p53Arg249 to be situated at the p53:G-actin interface, which upon mutation into Serine (p53R249S) abrogates the protein-protein complex, and results in its cytosolic sequestration.upper panel). There was a significant negative correlation [r = (−)0.817] between p53 nuclear import and the level of total F-actin in Dox-treated cells (bottom panel).Tumor suppressor p53 is a short-lived protein, where genotoxic stress is responsible for increasing its half-lifeSince p53 nuclear transport was increased with the concomitant decrease in F-actin level, we next intended to test whether actin disruption has any direct effect on p53 transport. Different drugs have been chosen that alter the cellular actin dynamics; e.g., Cytochalasin-D (Cyt-D) - inhibits actin polymerization; Latrunculin-B (Lat-B) - impairs addition of actin subunits to the filament ends, and Jasplakinolide (Jas) - increases actin nucleation. The altered actin structure and respective 3D-surface plots in response to Cyt-D, Lat-B and Jas have been shown in Supple. Fig. 1b. In cases of Cyt-D and Lat-B treatment, fibrous actin became depolymerized as shown by the punctate phalloidin staining . When Cyet al., which suggested perinuclear staining pattern of G-actin upon treatment with etoposideNext, we sought to study the F/G-actin ratio in different actin perturbing drugs-treated conditions; to correlate the data with the degree of p53 nuclear import. For this purpose, A549 cells were treated with only Dox or in combination with Cyt-D/ Lat-B/ Jas. It was observed that in response to Dox, there was an increase in the level of G-actin, which showed an intense distribution in the perinuclear region . Plot prEarlier report suggests that actin stabilization retards nuclear transport of p53bottom panel). On the contrary, p53 did not show significant positive Pearson’s colocalization correlation with F-actin (upper panel). Pearson’s r obtained through quantitative image analysis was found to be significant (p < 0.005) for p53 and G-actin (0.80), compared to the values found in the case of p53 and F-actin (0.1), indicated in Co-transfection of A549 cells with wild-type p53 and EGFP-tagged wild-type actin revealed interaction between these two molecules . To findlower left). Colocalized pixels showed a distribution along the linear regression line of the scatter plot.Since our preliminary findings suggest that p53 colocalized with monomeric G-actin, next our intention was to study whether it involves protein-protein interaction. For this purpose, we selectively expressed non-polymerizable actin mutant (G13R-actin) to exogenously produce GFP-chimeric G-actin, defective in forming fibrous actinupper panel). Dox treatment further augmented such interaction (lower panel). With this observation, we were curious to identify whether this interaction is a direct protein-protein interaction or is mediated via any other protein. In order to establish this in vitro, histidine pull-down assay was performed using purified His6-p53 and G-actinupper panel). The purified His6-p53 protein was incubated with purified G-actin and the bound proteins were analyzed by SDS-PAGE followed by coomassie staining, which distinctly revealed bands for both His6-p53 and G-actin (lower right panel).Physical interaction between p53 and G-actin was validated by co-immunoprecipitation study when GFP-tagged G13R-actin was pulled down by p53 antibody (DO-1). Co-transfection of varying concentrations of wild-type p53 and G13R-actin-EGFP revealed that the degree of physical interaction between these two proteins increased with the higher amount of transfected cDNA (lower right panel), though wild-type p53 shuttled inside the nucleus. Colocalization correlation value for G13R actin-p53 wild-type pair is significantly higher compared to Lifeact-p53 wild-type pair (p < 0.005)  .PDB code: 4MZR for p53, fragment: 94–356 and 3HBT for G-actin) (PDB code: 1YCS) was carried out. 1YCS is the crystal structure of the complex between p53DBD and BP2 protein1YCS as well as to obtain the p53:G-actin complex. Importantly, ZDOCK has been reported to successfully reproduce protein-protein docking benchmark of 176 test cases (http://zdock.umassmed.edu/help.html). Furthermore, literature suggests that knowledge of interface residues constrain the initial search space of docking software, which considerably improves the accuracy of protein-protein docking1YCS. With this information, the crystal structure was reproduced as the top models  among the ZDOCK predictions . Schrödinger’s protein-protein docking module PIPER was also found to be successful in reproducing 1YCS for blind/constraint-driven docking calculation. Hence both the docking modules successfully reproduced 1YCS, irrespective of no constraint, or single point constraint. According to the energy-scoring function, these reproduced structures attained top ranking positions and showed minimum RMSD values as compared to 1YCS (third panel). By the ranking of the model and the fact whether it appears multiple times among the top 5 models with varied docking algorithms, we selected Model-1 (ZDOCK) and carried out all atoms Molecular Dynamics (MD) Simulation (AMBER14) in an implicit solvent for 50 ns. Details of docking benchmark studies, docking calculation, and MD simulation have been included in the Materials and Methods section. Since the parameters were proved to be successful for reproducing a known crystal structure 1YCS, with the help of different docking algorithms, our assumption is that the theoretically-derived p53:G-actin complex is most likely the probable structure of the real association. Post-simulation p53:G-actin model and the surface electrostatics of the complex are represented in To get the atomistic insight of p53:G-actin interaction, we undertook protein-protein docking approach using crystal structures (G-actin) . Here wedictions . Paralle to 1YCS . These o to 1YCS , Arg249  to 1YCS . Similar to 1YCS . ImportaCrucial amino acids contacts in p53:G-actin theoretical model (within 1.8 Å–3.9 Å) are shown in PDB code: 2A5X)left panel) was found to form contacts with p53 in the p53:G-actin dynamic model. Comparing these residues involved in making contacts, it is clear that the same interface (at protomer-3) of monomeric G-actin participates in both actin:actin and p53:G-actin interaction  and colocalized with G13R-actin close to the perinuclear region [Pearson’s r = (+)0.60 and (+)0.744 respectively]. Co-immunoprecipitation assay confirmed physical interaction of p53R273H with G13R-actin 2+ ionthird panel). Another structural mutant p53R249S shows a tendency to get accumulated in the cytoplasmbottom panel).Confocal imaging studies revealed that both p53WT and p53R273H shuttled into the nucleus . Hence mutation from p53Arg273 into His273 is not supposed to interfere with the p53:G-actin complexation. p53R73H forms a stable complex with G-actin and translocates into the nucleus. On the contrary, mutation at p53Arg249 into Serine, results in reduced p53:G-actin interaction. The simulation models for both p53wt:G-actin and p53R249S:G-actin were superimposed so as to find out the probable outcome of R249S mutation at the atomic level. Superimposition of post-simulated coordinates shows that in wild-type p53:G-actin complex, Arg249 (p53wt) forms a polar contact with Arg357 (G-actin). When mutated to Ser249, this crucial interaction is perturbed, whereas Arg357 forms another contact with p53Arg174. Hence the loss of Arg249-Arg357 interaction is compensated with the Arg174-Arg357 interaction. This change at the atomic level of p53:G-actin interaction can be hypothesized to be the reason for reduced degree of p53R249S:G-actin association. The movie of MD simulation trajectories has been included in the According to the p53:G-actin hypothetical model, p53Arg273 resides away from the most nearby residue (Glu152) of protein-protein interface (The tumor suppressor p53 is a 393 amino acid long transcription factor that mediates the cellular outcome in response to DNA damage3Here we report that monomeric G-actin regulates p53 nucleo-cytoplasmic shuttling in response to genotoxic stress. G-actin interacts with wild-type p53 and directs p53 traffic in reaching the nucleus. Co-expression of wild-type p53 and G-actin (G13R mutant) plasmids suggested that p53 interacts with the monomeric form of actin. A distinctive perinuclear pattern of colocalization was evident between p53 and G-actin, which inferred that the route towards perinuclear translocation of p53 is mediated by virtue of its interaction with G-actin. Histidine-pull down experiment confirmed that the binary interaction between p53 and G-actin is a direct protein-protein interaction. Superimposition of actin:actin and p53:G-actin model reveals that a common interface of protomer-3 of G-actin is involved in both the complex formation. This might account for reduced affinity of p53 towards F-actin. Interaction of p53 with G-actin is, therefore, a pre-requisite for p53 in order to get transported into the nucleus. Prior stabilization of actin with Jasplakinolide treatment, results in reduced conversion of F-actin to G-actin, which is assumed to be the reason for reduced nuclear translocation of p53. On the contrary, actin depolymerizing/G-actin sequestering drugs exert no such hindrance. The experimental results altogether, suggests a mechanism where G-actin directly interacts with p53 and escorts it towards perinuclear region. It is again to recall that previous studies suggested the role of actin in p53 nuclear transport, but our study distinctively revealed the contribution of G-actin in the p53 transport event.Wild-type p53 interacts with G-actin, shows perinuclear colocalization in response to genotoxic stress, and translocates to the nucleus.p53R273H interacts with G-actin and translocates to the nucleus. Since according to the hypothetical model Arg273 resides away from the interface, mutation of p53Arg273 does not seem to interfere with p53:G-actin complexation.p53R249S shows reduced interaction with G-actin. It fails to get transported into the nucleus and predominantly shows cytoplasmic localization. The model proposes that Arg249 of p53 forms polar interaction with Arg357 of G-actin. Since Arg-Arg pairings have been found to be very crucial in making protein structures thermodynamically stable2+ ion-binding domain, resulting in distortion of the native p53 structurep53R175H shows reduced interaction with G-actin, forms perinuclear aggregates and fails to get imported into the nucleus. It involves a mutation in the ZnRegarding p53 mutants, reports suggest that globally denatured p53 mutants are found as cytoplasmic aggregates in the perinuclear regionThe above mechanism accounts for the G-actin-mediated regulation of p53 localization that unifies poorly connected information regarding wild-type/mutant p53 localization and provides a new angle for the understanding of the same. In a nutshell, to prevent the aberrant proliferation of the tumor cells, p53 gets transported into the nucleus and activates cell-cycle checkpoint proteins; promote DNA repair, cellular senescence, and apoptosis. The failure of p53 structural mutants to restrict abnormal malignant growth mainly stems from their inability to get efficiently transported into the nucleus. Therefore the biological consequence of mutant p53 mis-localization/cytoplasmic retention becomes significant as it accelerates tumor cell proliferation, migration, and invasion with the loss of tumor suppressive activity.In conclusion, the inferences drawn from our study confirm the role of G-actin in p53 nuclear transport event, though it was not completely explored whether G-actin only delivers p53 in the perinucleus; or it also dictates the final entry of p53 inside nucleus. A report by Liang and Clarke demonstrates that once the p53 cargo reaches the perinuclear region, it interacts with importin receptors, probably through recognition of one or more nuclear localization signal Overall the present study emphasizes on the role of G-actin in guiding p53 transport towards the nucleus, which needs further study to unveil the contribution of other protein transporters associated with this event. Parallel to G-actin mediated traffic, other interactions mediated by importin-α/β at the nuclear membrane, also need to be explored with an effort to explain mutant p53 cytoplasmic retention.+/+/p53−/−) and lung Carcinoma cell line H1299 (p53−/−) were purchased from ATCC, USA. The cell lines were cultured in DMEM or RPMI, supplemented with 10% heat-inactivated fetal bovine serum , L-glutamine (2 mM), sodium pyruvate (100 μg/ml), non-essential amino acids (100 μM), streptomycin (100 μg/ml), and penicillin . Primary antibodies used are p53 , α-GFP , α-Tubulin , GAPDH and Histone-3 . Precision plus protein dual color standards (Bio-Rad) was used to detect the molecular weight of proteins in SDS-PAGE. Cyt-D, Lat-B, and Jas purchased from Calbiochem, USA was used at 5 μM, 1 μM, and 50 nM concentration respectively followed by Dox  for 45 minutes. We made working stocks of all drugs .A549 (lung carcinoma) cell line was purchased from National Centre for Cell Sciences, Pune. Human colon carcinoma HCT116 2, 1 mM Na-EDTA, 1 mM Na-EGTA, and 1 mM DTT) supplemented with protease and phosphatase cocktail inhibitorFor whole cell lysates, cells were homogenized in buffer . The Slurry was centrifuged at 30,000 g for 30 min, 4 °C. Pellet (0.38 gm) was re-suspended in 1 ml buffer G. 2 M KCl, 2 M MgCl2 and KCl were subsequently added and homogenized at 4 °C. Supernatant was spun at 100000 g, 2 h, 4 °C. The pellet was rinsed with 50 μl buffer G. Pellet was re-suspended into 2 ml buffer G and dounce homogenized. Dounced slurry was then dialyzed. Dialyzed actin was spun at 100000 g, 2 h, 4 °C. Supernatant, the ultimate G-actin was collected in a dialysis bag and stored in dialyzing G-buffer at 4 °C.Actin was purified from muscle acetone powder of rabbit purchased from Sigma-Aldrich (M6890)To purify His6-p53, BL21(DE3) harboring pET15-p53 vector was cultured at 37 °C until reaching A600 = 0.6, induced with IPTG (0.5 mM) and grown for another 3 h at 30° Cells were grown on coverslips and fixed with 3.7%(v/v) paraformaldehyde for 20 min at RT, followed by permeabilization with 0.05%(w/v) Triton-X-100 for 5 minutes. Next, cells were blocked with 3%(w/v) BSA in PBS for 2 h. Primary antibodies (1:100) were diluted in 1%BSA in PBS and incubated for overnight at 4 °C. Cells were stained with Alexa-488 phalloidin and/or Alexa-594 DnaSe1  for staining F-actin and/or G-actin, respectively, according to the manufacturer’s protocol. Secondary antibodies used , were diluted in 1%BSA and incubated for 2 h at 4 °C. Nucleus was stained with DAPI . Images were acquired in Olympus fluoview confocal microscope/BX61 using 63×/100× objective. Laser intensities and detector gains were maintained at the same level during all imaging sessions. Z-stack images with an average step size of 0.35 μ were captured and 3D-projections of each stack processed for background correction. Image calculations and colocalization analysis have been done using Olympus fluoview and ImageJ software.PDB code: 4MZR for p53, fragment: 94-356 and 3HBT for G-actin) were used for protein-protein docking. Different docking algorithms; ZDOCK version 3.0.245+ ions. The system was minimized for 500 of minimization cycles, first, 250 cycles using steepest descent minimization and next 250 cycles using conjugate gradient minimization. Energy minimized system was used further for production simulation of 50 ns. The parameters set for simulations are as follows: calculation was performed for each 2 fs time step, coordinates were recorded at each 10 ps time step, temperature was maintained at 300K using Langevin thermostat with the collision frequency of 1ps−1 5457Crystal structures (PDB code: 1YCS). The ability of ZDOCK to reproduce crystal structures was evaluated with the following procedure. First, the guest (BP2) and host (p53DBD) molecules in the complex crystal structure (1YCS) were separated, and the guest structure was then docked back into the host structure by ZDOCK. Of many ZDOCK-generated guest-host complexes, only the top models with the strongest interaction energy (according to the energy scoring function) were compared to the corresponding crystal structure of the complex. In this comparison, the individual docked poses were superimposed on the crystal structure 1YCS, and the root mean square deviation (RMSD) values for all atoms were calculated. RMSD is frequently used to measure the quality of reproduction of a crystallographic binding pose by a computational method, such as docking1YCS and p53:G-actin complex. Knowledge of interface residue was also included in docking calculation to increase the likelihood of specified interaction. Data from mutational studies provide critical information for interaction, i.e. the complex does not form if certain residues are mutatedTo establish docking benchmark for delineating the robustness of our p53:G-actin model, we took crystal structure of the complex p53DBD with BP2  except where otherwise indicated. Comparison of multiple experimental groups was performed by 2-way ANOVA followed by a post-hoc Bonferroni modification of multiple comparison t-test. Data were analyzed and, when appropriate, the significance of the differences between mean values was determined by a Student’s t-test. Results were considered significant at p ≤ 0.05.How to cite this article: Saha, T. et al. G-actin guides p53 nuclear transport: potential contribution of monomeric actin in altered localization of mutant p53. Sci. Rep.6, 32626; doi: 10.1038/srep32626 (2016).


N215S patients and compare this to patients with other mutations.Fabry disease (FD) results from X-linked inheritance of a mutation in the GLA gene, encoding for alpha galactosidase A, and is characterized by heterogeneous clinical manifestations. Two phenotypes have been described “Classic” and “late onset” which cannot be predicted exclusively by genotype. The latter has been considered an attenuated form of the disease often affecting a single organ system commonly the heart. Recent studies have demonstrated that cardiac outcomes are similar in patients with classic and late onset mutations. In this study we investigate the relationship between clinical heterogeneity and plasma lyso-Gb3 in a large single centre cohort of N215S mutation  and 167 non-N215S mutations . The Mainz severity score index (MSSI) was used as an index of overall disease severity. Cardiac function and morphology were assessed by electrocardiogram and echocardiogram. Left ventricular mass was calculated using the Devereux formula and the left ventricular mass index (LVMI) calculated to adjust for height (g/m2.7). The presence of white matter lesions was assessed by cerebral MRI or computed tomography (CT). GFR was measured by radio-isotope (chromium-EDTA) method and adjusted for patient height (ml/min/m2.7), and urinary protein quantification was undertaken by 24 hour urine collection. Plasma globotriaosylsphingosine (lyso-Gb3) was analysed prior to ERT in 84 patients.In this single-centre, retrospective, cross-sectional study we analysed a cohort of 251 FD patients: 84 N215S patients showed later symptom onset , later development of left ventricular hypertrophy (LVH)  vs. 64 (N215S) years, p< 0.0001), later development of proteinuria  vs 71 years (N215S), p< 0.0001), later occurrence of cerebrovascular events (stroke/ Transient Ischaemic Attacks (TIA); median survival without stroke: 74 years (non-N215S) vs. not reached (N215S), p< 0.02), later decline in renal function to GFR <60 ml/min/1.73m2  vs. 72 (N215S) years, p< 0.01), and greater overall survival  vs. 66 (non-N215S) years, p< 0.0006). Lyso-Gb3 was found to be less elevated in N215S compared to non-N215S male and female patients. However, the N215S population eventually reached an overall severity measured by MSSI comparable to the non-N215S without equivalent elevation of lyso-Gb3 . In addition, N215S patients showed strong correlations between lyso-Gb3 levels and LVMI, GFR, and MSSI. These associations became stronger when we investigated individuals’ life time exposure to lyso-Gb3 : MSSI , LVMI , and GFR .N215S mutation results in a late onset phenotype involving the heart and other organs. Correlations between clinical manifestations and plasma lyso-Gb3 variations in this group suggest a Fabry-relevant disease mechanism for the heterogeneity observed in this group.These results demonstrate that the  GLA gene *age) we multiplied a patient’s lyso-Gb3 level by age at time of diagnosis.Statistical analysis was performed using Excel 2010, Microsoft® and GraphPad Prism version 5 (GraphPad®). For comparisons between two groups, t-tests were used for normally distributed data, Mann-Whitney U test if the data was not normally distributed and Fisher’s exact test to compare between proportions. Retrospectively collected data on events (development of stroke or transient ischaemic attack (TIA), development of stage III CKD (GFR <60ml/min/1.73m2), presence of WML, development of proteinuria, development of left ventricular mass indexed to height (LVMI) ≥ 50g/m2.7 in males and ≥ 48g/ m2.7 in females, and death) were used to assess the event free survival by using Kaplan-Meyer curves. Survival time was defined as the interval described in years between birth and event or last follow-up. A p value <0.05 was considered significant.N215S mutation. Most of these patients have been diagnosed in the 21st Century, with only 3/84 patients (3.6%) of patients diagnosed prior to 2000 having the N215S mutation compared to 81/84 (96.4%) diagnosed 2000 onwards. 22/43 (51.2%) of index males had the N215S mutation.The cohort comprised 251 patients  , 156 female (62.2%) ) from 96 different family pedigrees. Population characteristics are given in N215S index males had a significantly older age of symptom onset compared to those with non-N215S mutations  Cardiac: shortness of breath, palpitations, syncope, abnormal ECG, chest pain, fatigue, murmur, cardiomegaly found on screening, and cardiac arrest. (2) Renal: proteinuric CKD, haematuria, hypertension, renal failure, proteinuria during pregnancy, and persistent proteinuria post pregnancy. (3) Dermatological: rash and angiokeratomas. (4) Neurological: pain, migraine, and TIA. (5) Strokes: single episodes and recurrent strokes. (6) Ophthalmological: sore eye, cornea verticillata, and eye deposits. (7) Gastrointestinal: irritable bowel syndrome (IBS) .N215S mutation presented predominately with later onset cardiac manifestations in males. In 18 out of 22 index N215S patients the initial disease manifestations were cardiac, either symptomatic  or the incidental finding of LVH during pre-operative assessments or insurance medicals (7 patients)  with ESRF. In contrast, acroparesthesia and/or angiokeratoma were the commonest initial symptoms in non-N215S index males, occurring in 17 out of 21 patients . In three the initial symptoms were cardiac  and one proteinuria .The atients) . In two N215S patients (MSSI < 20) were significantly older compared to non-N215S . Moderate (MSSI = 20–40) and severe (MSSI≥ 40) patients were also older in the N215S group (63.7 years (n = 17) vs. 35.6 years (n = 45) p< 0.0001 and 71 (n = 1) vs. 29.7 years (n = 2), p = ns, respectively).Both groups attained similar overall severity scores, but at different ages. Mild non-N215S population compared with only two (8.3%) in N215S patients (p = 0.0004), one presenting with proteinuria in pregnancy aged 25 years and the other with cardiac hypertrophy aged 68. Whilst non-N215S women were diagnosed, on average, 8.4 years after males , N215S women were diagnosed 12.8 years before males with the same mutation  due to family screening of younger generations. Only non-N215S females reached severe overall MSSI severity scores (MSSI ≥ 40), both at baseline and during follow-up , only 11/47, 23.4% of N215S females were on treatment compared to 62/109, 56.9% in the non-N215S group (p = 0.0001). Indeed, N215S females received the least proportion of any concomitant medication when compared to the rest of the cohort  and 51.1 years in N215S (n = 37), p <0.0001. The mean age at latest follow up for non-N215S males was 44.1 years  and 56.3 years for N215S males . There were 10 deaths amongst male FD patients, 7 in non-N215S males  and 3 N215S males . Only 2 females passed away during the follow up, one carrying the G361R mutation  and the other harboured a H46Y mutation (she had a stroke at 65 years old). Overall, survival was significantly greater in N215S males than non-N215S males  at a mean age of 26.1 years in 5S males .2.7 in females and > 50 g/m2.7 in males). 29 patients were missing echocardiogram at baseline .Cardiac evaluation at baseline included ECG and echocardiogram  and 49 (29.3%) non-N215S  had LVH on echocardiogram. 1 male non-N215S had LVH by voltage criteria (LVMI data missing). Furthermore, 2 male N215S patients and 3 non-N215S  were found to have LVH at baseline but their LVMI data was not available.24 (28.6%) N215S females had developed LVH at baseline and they were aged 68, 72, 78 and 42 years old. Cardiac hypertrophy was present in all except for 8 male non-N215S patients (R301Q (aged 14), A143T (aged 1), P205T (aged 3), exon 1 deletion (aged 13), c.700_702 del GAT deletion (aged 27), G361R (aged 11), L372P (aged 19), and R49L (aged 28)). The patients with c.700_702 del GAT deletion, L372P, and R49L have subsequently developed conduction abnormalities. However, only 30/109 of non-N215S female patients exhibited cardiac hypertrophy, at a mean age of 43.3 (range 10–75) years old.Of the 6 patients one presented with ESRF at age 25, two teenagers had conduction abnormalities on ECG and showed no symptoms, 1 required an ICD after developing a Non-Sustained Ventricular Tachycardia (NSVT) with pre-syncope, and 1 had acroparesthesia, gastrointestinal symptoms and a few WML on brain MRI. Only 4 2.7 in males occurred at a younger age for the non-N215S population  inserted for arrhythmias at a median age of 69 years (range 51–79), whereas only 5 (8.8%) non-N215S men required one at a median age of 56 years  classic men. Furthermore, 5 (13.5%) N215S men had type 2 diabetes compared to only 1 (1.8%) non-N215S man  215S man . In the 5S woman . HyperteN215S males, whereas angiotensin converting enzyme inhibitors (ACEI) / angiotensin receptor blockers (ARB) were in non-N215S males. However, for females, all of these four concomitant medications were more common in the non-N215S population  and N215S  males had proteinuria at baseline  have all had or are awaiting renal transplants. One further non-N215S male patient was diagnosed on family screening having been symptomatic with acroparesthesia since age 7, had a GFR of 33 at diagnosis aged 34 and has progressed to end stage renal disease (ESRD). The other N215S patient was diagnosed on family screening aged 73, but had a history of proteinuria of undefined aetiology first detected at age 56 and a partial nephrectomy many years previous for tuberculosis.The three index males whose initial disease manifestations were renal  at an earlier age but this age difference was not significant  than N215S males  compared to N215S  and N215S  males but were found at a significantly younger age in non-N215S  were more common in N215S mutation had a significantly lower baseline age-adjusted FOS-MSSI severity score  and higher leukocyte α-Gal A activity . They also showed a trend towards higher plasma enzyme activity compared to non-N215S .Males with the GLA mutation, there was marked variation in enzyme activity amongst N215S patients, with enzyme activity correlating weakly with overall disease severity  and demonstrated weak correlation with severity in the non-N215S females only (data not shown).α-Gal A activity was higher in females regardless of N215S patients but not the non-N215S patients .Lyso-Gb3 was found to be elevated in both  females . At basel groups . On averterparts . Lyso-GbN215S population reached an overall severity measured by MSSI comparable to the non-N215S without equivalent elevation of lyso- Gb3  were found to have the non-N215S mutations in the GLA gene. 59 different mutations comprised the latter mutation group including deletions, nonsense, and missense mutations .Patients who harboured N215S males. N215S males reached similar total MSSI scores to that of non-N215S males but, on average, 25.5 years later. Males with N215S attained similar overall MSSI scores and degree of LVMI to non-N215S males despite higher residual enzyme activity and lower plasma lyso-Gb3 levels. Liao et al. reported similar differences in lyso-Gb3 levels between classical and late onset patients, and similar associations between lyso-Gb3, age and LVMI in their Taiwanese patients carrying the late onset mutation IVS4 + 919G>A [There were no differences in LVMI between mutation groups at baseline, however, at the time of the first assessment the mean age was 25 years older in the + 919G>A . This ma+ 919G>A . Other s+ 919G>A , inducin+ 919G>A  and Notc+ 919G>A .N215S males and classical females had similar lyso-Gb3 levels [non-N215S patients, the plasma lyso-Gb3 levels of N215S males showed strong correlations with LVMI, GFR and MSSI. Indeed, these associations became even stronger when we investigated the individual’s life time exposure to lyso-Gb3, considering the impact of age.Plasma lyso-Gb3 levels appeared to be useful to differentiate between phenotypes in males, however 3 levels . DespiteN215S patients’ clinical outcomes and overall disease severity (measured by MSSI), LVMI and GFR. Furthermore, for N215S patients, strong significant correlations of clinical manifestations with life time exposure to lyso-Gb3 suggests that the cumulative exposure is crucial for the disease progression. Contrary to previous suggestions [Our results demonstrated that both age and lyso-Gb3 are major factors influencing gestions , 44 thisN215S patients were evident for non-N215S male patients, consistent with the results of Rombach et al. who investigated these associations in 37 classic male Fabry patients [None of the correlations between plasma lyso-Gb3 and clinical manifestations found in patients . The absLower levels of plasma lyso-Gb3 were found in treated patients, implying an effect of ERT; however, this is a cross-sectional analysis and could reflect a treatment bias therefore further longitudinal studies are required.N215S index cases were mostly males, and women were generally diagnosed through cascade screening. Regardless of the mutation, female patients showed higher enzyme activity, lower lyso-Gb3 levels, and received, proportionally, less treatment (either ERT or concomitant medications). The ratio of females to males in our N215S cohort is only 1.3 to 1 which is lower than the expected 2 to 1 for an X-linked disease, and may suggest that N215S female patients are underdiagnosed due to their mild phenotype. Even in classic men clinical diagnosis of FD can be difficult due to frequent misinterpretation of early symptoms, and often patients present with a long consultation history. Hsu et al. reported that cardiac disease can progress silently, without showing significant clinical symptoms [symptoms . Family N215S cohort of patients to our total cohort of non-N215S patients including the full range of genotypes some of which are also late onset thereby avoiding the bias of selecting only mutations previously described to have classic phenotype. We report data prior to the initiation of therapy for clinical parameters in all patients however a limitation is that lyso-Gb3 at baseline was only available for a subset in which the analysis is performed. Similarly, whilst genotyping was available in all patients a full profile of enzyme activities was not available for every patient. For the small number of patients diagnosed prior to 1999 there is a lapse in time between date of diagnosis and baseline assessment as patient were not immediately seen in the specialist centre.This is a cross sectional baseline study of a large number of patients with a single mutations compared to other patients without that mutation in our cohort all investigated and managed according to a common protocol. We identify a number of strengths including the standardised follow up and completeness of the genotyping and clinical data. A further strength is that we have chosen to compare the N215S patients v non-N215S patients were in receipt of ERT although a smaller proportion of N215S females were on treatment reflecting their overall milder phenotype which would have been a confounder in any analysis of ERT response. Similarly, the sample size lacked the necessary power to detect statistically significant differences in ERT effect by genotype.We present event and survival follow up for the genotype-defined cohorts as a whole and have not further analysed by exposure to enzyme replacement therapy which was outside of the scope of this study. After baseline assessment similar proportions of male According to our local protocols, during the majority of the follow up period echocardiography with its potential limitations of variability rather than MRI was used to measure LVMI.N215S mutation exhibit a distinct phenotype with later onset but not exclusively cardiac manifestations, with some patients presenting with renal disease. Contrary to previous reports, lyso-Gb3 is an important parameter in the assessment of patients with the N215S mutation. In particular severity and clinical manifestations correlated with life time exposure to plasma lyso-Gb3 in the N215S group and may prove useful in prognostication and therapeutic decision making.Patients with the S1 Fignon-N215S  and females non-N215S , (b) leukocyte α-Gal A activity for non-N215S males  and non-N215S females . (E-H) LVMI and: (a) plasma α-Gal A activity for males  and non-N215S females , (b) leukocyte α-Gal A activity for non-N215S males  and non-N215S females ; ns = not significant.(A-D) GFR and: (a) plasma α-Gal A activity for males (TIF)Click here for additional data file.S2 FigN215S (n = 36), (B) non-N215S (n = 49), and females: (C) N215S (n = 46), non-N215S (n = 102); ns = not significant.plasma α-Gal A activity and age for males: (A) (TIF)Click here for additional data file.S3 FigN215S males  and non-N215S females ; ns = not significant.Plasma globotriaosylsphingosine (Lyso-Gb3) and age for Non-(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.